b y t V khoa hc v o to

Kim nghim dc phm

(Sch dng o to dc s i hc)
M s: .20.Z.08

Nh xut bn Y hc H ni - 2005

Ch bin PGS.TS. Trn T An Tham gia bin son PGS.TS. Trn T An CN. Trn Tch DS. Nguyn Vn Tuyn TS. Chu Th Lc ThS.Nguyn Th Kiu Anh Tham gia t chc bn tho ThS. Ph Vn Thm

Bn quyn thuc B Y t (V Khoa hc v o to)

Li ni u

Thc hin Ngh nh 43/2000/N-CP ngy 30/8/2000 ca Chnh ph quy nh chi tit v hng dn trin khai Lut Gio dc, B Gio dc & o to v B Y t ph duyt, ban hnh chng trnh khung cho o to Dc s i hc. B Y t t chc thm nh sch v ti liu dy hc cc mn c s v chuyn mn theo chng trnh mi nhm tng bc xy dng b sch chun trong cng tc o to dc s i hc ca Ngnh Y t. Kim nghim l mt khu quan trng trong h thng qun l cht lng ton din ca dc phm. N c mt trong cc cng on sn xut (kim nghim nguyn liu, bn thnh phm v kim nghim sn phm cui cng), trong tn tr, lu thng v s dng thuc. Cc k thut c s dng trong kim nghim rt phong ph v a dng thuc cc lnh vc vt l, ho hc v sinh hc. Chnh v vy cc kin thc c bn cu 3 b mn ny - c bit l cc mn hc chuyn ngnh nh ho phn tch, ho l, vi sinh - thc s l c s cho mn hc kim nghim dc phm cn phi c nghin cu trc. Tuy nhin, vic la chn mt k thut no cho nh gi cht lng mt nguyn liu hoc ch phm thuc khng ch ph thuc vo c im ca k thut phn tch m cn ph thuc vo c im ca i tng phn tch l dc phm, cho nn cc kin thc v ho dc, dc liu, bo ch l rt cn thit cho kim nghim dc phm. Trong thc hnh kim nghim dc phm ngi ta s dng hu ht cc k thut phn tch, cc php o vt l nh o im chy, im si, ch s khc x, p sut thm thu,... cng l nhng k thut thng gp. Tuy nhin, trong gio trnh vt l nm th nht, cc ni dung ny c trnh by chi tit c l thuyt v thc hnh. V vy cc ni dung trn khng a vo chng trnh Kim nghim dc phm. Cc phng php ha hc v ha l c s dng rt ph bin: t loi thc hnh n gin nh chun th tch, so mu, n nhng loi i hi cc thit b hin i nh sc k, quang ph, khi ph ... Nhng vi thi lng l 2 n v hc trnh l thuyt cho sinh vin nm th 4 nn mc tiu mn hc c hn ch 2 vn : 1. Gii thch c nguyn l ca mt s phng php ho hc, ho l v vi sinh thng dng trong kim nghim. 2. Trnh by c h thng m bo cht lng thuc v v tr ca cng tc kim nghim trong h thng ny. Cun sch ny c chia lm 6 chng. Sau chng 1 gii thiu s lc v h thng m bo cht lng thuc v vai tr ca kim nghim, trong 3 chng tip theo trnh by 3 nhm phng php ho hc, ho l v sinh hc. C s l 3

thuyt ca cc phng php kim nghim ny c nghin cu trong cc mn c s nh: phn tch, ho l, vi sinh. y ch yu gii thiu nguyn tc ng dng ca chng nh: hiu chun thit b, k thut xc nh nng , gim thiu sai s... trong kim nghim. Do hn ch ca thi lng mn hc nn ch cp n mt s phng php thng dng nh: chun mi trng khan, quang ph phn t, HPLC. Chng 5 trnh by s lc ni dung kim nghim cc dng bo ch: thuc vin, thuc tim, thuc dng ngoi ... Nhm mc ch cung cp cho sinh vin mt s kin thc c bn v tnh bn vng ca thuc, lm c s gii thch nhng bt thng c th gp trong qu trnh kim nghim nh gi cht lng, chng ti gii thiu thm rt s lc chng cui cng: n nh v tui th ca thuc. Kt qu phn tch nh gi cht lng l sn phm ca thc hnh Kim nghim dc phm. V vy sn phm ny cng nh bao loi sn phm khc lu hnh trong x hi phi c cht lng tha mn nhu cu ngi tiu dng. m bo cht lng ca kt qu, cn thc hin nhiu bin php ng b. l: Trang thit b phng th nghim, K nng ca kim nghim vin, Qui trnh k thut kim nghim.

Thc hnh tt phng th nghim (GLP) l c s thc hin cc bin php nu trn. Nhng ni dung ny s c gii thiu trong chng trnh cao hc thuc chuyn ngnh Kim nghim dc phm v c cht. V cch tip cn ni dung, trong cun sch khng nhc li qui trnh k thut c trong Dc in m cung cp nhng kin thc cn thit sinh vin c th gii thch c qui trnh v thc hnh kim nghim cho kt qu tin cy.
Sch Kim nghim dc phm c cc ging vin B mn Ho Phn tch Trng i hc Dc H Ni bin son. Sch c Hi ng chuyn mn thm nh sch giao khoa v ti liu dy hc chuyn ngnh Dc ca B Y t thm nh v c B Y t ban hnh lm ti liu dy hc chnh thc ca Ngnh Y t trong giai on hin nay. V Khoa hc v o to xin chn thnh cm n cc ging vin B mn Ho Phn tch Trng i hc Dc tham gia bin son cun sch ny. V l ln u xut bn nn chc chn cn nhiu thiu st, chng ti mong nhn c kin ng gp ca ng nghip v sinh vin cun sch ngy cng hon thin hn.
V khoa hc v o to B Y t

Mc lc
Li ni u Chng 1 3

i cng
Trn Tch 1.1. Cht lng thuc v m bo cht lng 1.1.1. Thuc v yu cu cht lng 1.1.2. Kim tra cht lng thuc 1.1.3. H thng t chc kim tra cht lng thuc 1.2. Cng tc tiu chun ho 1.2.1. Khi nim 1.2.2. Cng tc xy dng tiu chun 1.2.3. Cng tc p dng tiu chun trong thc t 1.2.4. Gii thiu Dc in Vit Nam 1.3. Kim nghim thuc theo tiu chun 1.3.1. Ly mu kim tra 1.3.2. Tin hnh kim nghim 1.3.3. Ni dung chnh ca thc hnh tt phng kim nghim (GLP) Ti liu tham kho Cu hi t l ng gi Chng 2 9 9 12 14 16 16 18 22 23 26 26 31 34 36 36

Kim nghim thuc bng cc phng php ho hc

*

Trn Tch, Trn T An 37 48 48

2.1. 2.2.

Cc phn ng nh tnh Th gii hn cc tp cht trong thuc 2.2.1. Mc ch

*Phn 2.1 & 2.2. Trn Tch, phn 2.3. & 2.4.: Trn T An

2.2.2. Phng php xc nh gii hn tp cht trong thuc 2.2.3. Mt s thuc th trong cc phn ng ho hc xc nh gii hn tp cht 2.3. Chun acid - base trong mi trng khan 2.3.1. Vai tr ca dung mi 2.3.2. Khi nim pH 2.3.3. Xc nh im tng ng 2.3.4. ng dng kim nghim thuc 2.4. Xc nh hm lng nc bng thuc thi Karl fischer 2.4.1. Nguyn tc 2.4.2. Pha ch v xc nh chun 2.4.3. Xc nh im tng ng 2.4.4. ng dng 2.5. 2.6. nh lng mt s cht hu c a chc bng thuc th periodat ng dng cp ion trong kim nghim thuc Ti liu tham kho Cu hi t l ng gi Chng 3

48 50 51 51 52 53 54 58 58 58 59 59 60 61 65 66

cc phng php ho l trong kim nghim thuc

Nguyn Vn Tuyn 3.1. Phng php quang ph phn t 3.1.1. Quang ph hp th UV VIS 3.1.2. Quang ph hng ngoi (IR) 3.1.3. Quang ph hunh quang 3.2. Phng php sc k lng hiu nng cao (HPLC) 3.2.1. Cc thng s c trng ca qu trnh sc k 3.2.2. My HPLC 3.2.3. Cc k thut HPLC 3.2.4. Hng dn chn k thut HPLC 3.2.5. Chun ho ct HPLC 6 68 68 79 82 84 84 86 88 94 100

3.2.6. nh lng bng phng php HPLC 3.2.7. Cc phng php nh lng Ti liu tham kho Cu hi t lng gi Chng 4

102 104 111 111

Kim nghim thuc bng phng php sinh hc

Chu Th Lc 4.1. M u 4.1.1. Nguyn tc 4.1.2. Cht chun 4.1.3. nh gi kt qu 4.2. 4.3. Kim nghim thuc bng phng php th trn ng vt Kim nghim thuc bng phng php th vi sinh vt 4.3.1. i cng v vi sinh vt 4.3.2. Mi trng nui cy vi sinh vt 4.3.3. Th v trng 4.3.4. Th gii hn vi sinh vt 4.3.5. Xc nh hot lc khng sinh bng phng php th vi sinh vt Ti liu tham kho Cu hi t lng gi Chng 5 115 115 116 116 116 117 117 121 124 128 131 137 138

Kim nghim cc dng bo ch

Nguyn Th Kiu Anh 5.1. 5.2. 5.3. 5.4. 5.5. 5.6. 5.7. Kim nghim thuc bt Kim nghim thuc vin nang Kim nghim thuc vin nn Kim nghim thuc tim, thuc tim truyn Kim nghim thuc nh mt Kim nghim thuc ung dng lng Kim nghim thuc m 140 145 147 149 153 154 157 7

5.8. 5.9. 5.10

Kim nghim thuc n, thuc trng Th ho tan ca vin nn v vin nang Th r ca vin nn v vin nang Ti liu tham kho Cu hi t lng gi Chng 6

159 160 166 169 169

n nh v tui th ca thuc
Trn T An 6.1. 6.2. Qu trnh pht trin nghin cu n nh ca thuc i cng v n nh ca thuc 6.2.1. nh ngha 6.2.2. Mt s thut ng lin quan 6.2.3. Mc tiu nh gi n nh 6.2.4. Tiu chun nh gi n nh 6.2.5. Phn vng kh hu 6.3. ng ho hc dung dch 6.3.1. Bc ca phn ng 6.3.2. nh hng ca nhit 6.4. Xc nh n nh ca thuc 6.4.1. Ly mu 6.4.2. Phng php th cp tc 6.4.3. Phng php th di hn 6.4.4. Phng php phn tch nh gi kt qu 6.5. Cc dc cht km bn vng Ti liu tham kho Cu hi t lng gi 171 172 172 173 174 175 176 177 177 181 182 182 183 185 185 186 188 189

Chng 1

i cng

Mc tiu hc tp

1. Trnh by c h thng m bo cht lng thuc v v tr ca cng tc kim nghim trong h thng ny. 2. Trnh by c cc nhim v ch yu ca cng tc kim nghim.
1.1. Cht lng thuc v m bo cht lng 1.1.1. Thuc v yu cu cht lng Khi nim v thuc: Theo T chc y t th gii, thuc l mt cht hay mt hn hp cc cht c sn xut em bn, cung cp bn hay gii thiu s dng nhm mc ch: iu tr, lm gim, phng hay chn on bnh tt, tnh trng c th bt thng hoc triu chng bnh; khi phc, hiu chnh, thay i chc nng hu c ca c th ngi (hay ng vt - thuc th y).

Vn dng vo Vit Nam, trong "iu l thuc phng bnh, cha bnh" ban hnh 24/1/1991 qui nh: Thuc l nhng sn phm c ngun gc t ng vt, thc vt, khong vt, hay sinh hc c sn xut dng cho ngi nhm: Phng bnh, cha bnh Phc hi, iu chnh chc nng c th Lm gim triu chng bnh Chn on bnh Phc hi hoc nng cao sc kho Lm mt cm gic mt b phn hay ton thn Lm nh hng qu trnh sinh sn Lm thay i hnh dng c th. Vt liu dng trong khoa rng, bng bng, ch khu y t, cng c coi l thuc. Thuc l u hnh trn th trng a phn l cc tn dc v thuc y hc c truyn (l cc thuc c sn xut theo phng php y hc c truyn). Trong c nhiu thuc di dng bit dc (bit dc l nhng thuc mang tn ring cn gi l tn thng mi ring ca mt c s sn xut hay mt 9

hng sn xut ln u t cho n v c php a ra th trng ang c bo h quyn s hu cng nghip).

Cht lng thuc v yu cu cht lng: Cht lng ca mt thuc l tng hp cc tnh cht c trng ca thuc (th d; c cha ng cc thnh phn theo t l qui nh, c tinh khit theo yu cu, ng gi c nhn qui nh) c th hin mt mc ph hp vi nhng yu cu k thut nh trc tu theo iu kin xc nh v kinh t, k thut, x hi nhm m bo cho thuc t cc mc tiu sau:

C hiu lc phng bnh v cha bnh Khng c hoc t c tc dng c hi n nh v cht lng trong thi hn xc nh Tin dng v d bo qun. Thuc l sn phm hng ho c bit, c quan h trc tip n sc kho cng ng, n cht lng v hiu qu ca vic phng bnh, cha bnh. V th thuc phi c bo m cht lng trong ton b qu trnh sn xut t nguyn liu cho n l thnh phm, trong qu trnh bo qun, lu thng phn phi n ngi s dng. Mc tiu ca m bo cht lng trn ch c coi l t khi no thuc p ng c cc yu cu c bn sau: Thuc c cha ng cc thnh phn theo t l qui nh ca cng thc c ng k v c cp php (nh tnh, nh lng). Thuc c php sn xut v sn xut theo ng cc qui trnh ng k v c php. C tinh khit t yu cu qui nh. Thuc c ng gi trong cc ng v bao gi vi nhn thch hp v ng qui cch ng k. Thuc c bo qun, phn phi, qun l theo qui nh cht lng ca thuc c duy tr trong sut tui th ng k hay thi hn bo hnh. t cc mc tiu trn, cn phi c nhiu yu t, trong 3 yu t c bn phi c l: Thc hnh tt sn xut thuc (GMP) Thc hnh tt phng kim nghim thuc (GLP) Thc hnh tt bo qun thuc (GSP) 1.1.1.1. Thc hnh tt sn xut thuc (Good Manufacture Practice) Bao gm nhng qui nh cht ch v chi tit v mi mt ca qu trnh sn xut nh: T chc, nhn s, c s v tin nghi, my mc, trang thit b, kim 10

tra nguyn ph liu, bao b, ng gi, kim tra bn thnh phm, thnh phm cui cng, nhm sn xut ra c thuc theo d kin v t tiu chun k thut t ra. Mun vy, phi thc hin c cc yu cu c bn sau: Tt c cc quy trnh sn xut phi c qui nh r rng v chc chn c kh nng t mc ch ra. Cc cn b, nhn vin phi c o to t yu cu; c s v din tch ph hp, cc trang thit b v phng tin phc v, nguyn liu, ph liu, bao b, ng gi, nhn, ng quy cch v theo yu cu ra. Trong sut qu trnh sn xut phi c s kim tra, theo di, ghi chp y chng minh rng tt c cc giai on ca qui trnh sn xut u c thc hin nghim chnh, c cht lng v s lng sn phm ph hp vi qui nh. C cc h s sn xut v phn phi d dng xem xt lch s ca mt l thuc no . C mt h thng t chc cn thit khi cn c th thu hi bt k l thuc no cp pht hoc bn ra.
1.1.1.2. Thc hnh tt phng kim nghim thuc (Good Laboratory Practice)

Bao gm nhng quy nh cht ch v chi tit cc yu t tham gia vo qu trnh kim tra cht lng thuc, nhm m bo kt qu c chnh xc, ng, khch quan. thc hnh tt qu trnh kim tra cht lng thuc, cn phi p ng cc yu cu c bn sau y: Cn b c o to chu o, c t chc tt, c trnh cao, c thc trch nhim. C s vt cht, trang thit b tt, p ng yu cu k thut. Ho cht, thuc th, cht chun tt. iu kin v sinh tt Ly mu, lu mu tt Kt qu th nghim tt, ghi chp x l s liu tt. H s lu tr tt. Vn ny s c cp k hn phn nghip v ca cng tc kim nghim.
1.1.1.3. Thc hnh tt bo qun thuc (Good storage practice)

Bao gm nhng quy nh cht ch v nghim ngt cho vic tn tr, iu kin bo qun, xut nhp nguyn ph liu, bao b, ch phm, nhn thuc... Tt c nhm gim st kim tra cht ch thuc m bo cht lng n 11

ngi s dng. Mun thc hnh tn tr thuc tt cn l u lm tt cc yu cu sau: Cn b phi c o to ph hp, c trch nhim Nh ca v phng tin bo qun tt iu kin v sinh tt Thc hin tt qui trnh bo qun: nguyn liu, bn sn phm, sn phm, bao b, ng gi Quy trnh bo qun, vn chuyn phi c tun th nghim chnh. 1.1.2. Kim tra cht lng thuc (Drug quality control)
1.1.2.1. Khi nim

Kim tra cht lng thuc hay kim nghim thuc l vic s dng cc phng php phn tch: l hc, ho hc, ho l, sinh vt, vi sinh vt, qui nh xc nhn mt thuc hay mt nguyn liu lm thuc c t hay khng t tiu chun qui nh. Ni mt cch c th l kim tra cht lng thuc nhm tr li cc cu hi: y c phi l thuc cn kim tra khng? C m bo hot lc hay hm lng ng k v c duyt ? C t tinh khit theo yu cu hay khng? C b phn hu hay bin cht hay khng? bao gi, nhn c ng qui cch khng? Nh vy, mc tiu c bn ca cng tc kim tra cht lng thuc l: ngi s dng dng c thuc m bo cht lng, t hiu qu s dng cao. Pht hin thuc khng t tiu chun, thuc gi, thuc km phm cht x l v khng cho php lu hnh trn th trng. Trong ngnh y t quy nh: Tt c cc thuc v nguyn liu lm thuc u phi c kim nghim v xc nh cht lng, nu t tiu chun qui nh mi c a vo s dng. Bi vy thuc phi c kim tra cht lng mt cch nghim ngt, cht ch, bo m cho thuc t cht lng trong mi hot ng sn xut, lu thng phn phi, xut nhp khu, qun l v s dng thuc.
1.1.2.2. Cc yu t nh hng n cht lng thuc

C nhiu yu t, trong c th l: Nhng ri ro hoc nhm ln trong sn xut, do khng chp hnh nhng qui nh ca GMP. 12

 Nhng sn phm do v tnh hay c trong sn xut to ra sn phm c hm lng thp hn qui nh. Do la chn cng thc bo ch cng nh k thut bo ch cha ng nn thuc b phn hu bin cht. Qu trnh bo qun cha tt. Do li ch c nhn bt chnh lm hng gi la ngi tiu dng gy ra tc hi nghim trng
1.1.2.3. Khi nim v thuc t v khng t tiu chun, thuc gi mo, thuc km phm cht Thuc t tiu chun (thuc bo m cht lng): L thuc p ng y cc yu cu k thut tiu chun ra (hay thuc p ng y cc mc cht lng trong tiu chun cht lng ng k). Thuc khng t tiu chun: L thuc khng p ng y cc yu cu k thut tiu chun ra. Mc khng t c th l mt hay mt s ch tiu. Thuc khng t tiu chun thuc loi thuc km cht lng. Thuc gi: Theo qui nh ca T chc y t th gii, thuc gi l ch phm c sn xut khng ng vi nhn kha cnh nhn dng hay ngun gc thuc, vi s c v mang tnh cht la o ca nh sn xut. Sn xut sai thnh phn cng thc ng k, khng c hay khng hm lng hot cht, hoc c ng gi trong cc bao b gi mo. Nh vy, c th ni thuc gi l nhng sn xut thuc ca ngi sn xut mang la o, gian ln c th da vo mt s biu hin pht hin:

Thuc khng c hoc c t dc cht Thuc c cha dc cht khc vi tn dc cht ghi trn nhn Nhn, bao gi ging hay gn ging vi nhn, bao gi ca mt thuc khc
Thuc km phm cht: L thuc khng t tiu chun k thut m trc n t. Mc khng t tiu chun c nhiu hnh thi khc nhau. Tt c cc nguyn nhn gy ra c th xc minh c bng cc phng php khoa hc, k thut cho php. Cc nguyn nhn c th l:

Do k thut sn xut, bo qun khng ng, do thuc t bin cht. Do bao gi khng t tiu chun, nn a tp cht vo thuc. 13

 Do tui th (hn dng) ht Do nguyn ph liu khng t tiu chun Do tc ng ca mi trng: nhit , nh sng, m 1.1.3. H thng t chc qun l, kim tra cht lng thuc Nh nc giao cho B Y t chu trch nhim qun l ton din cht lng thuc. V vy, h thng t chc, qun l, kim tra cht lng thuc ca nghnh y t c chia lm 3 phn: H thng qun l cht lng thuc; H thng kim tra cht lng thuc; H thng thanh tra Dc.
1.1.3.1. H thng qun l cht lng thuc Cc qun l Dc Vit Nam: L c quan c B Y t u quyn thc hin cc nhim v trong lnh vc qun l nh nc v cht lng thuc:

Xy dng quy hoch, k hoch v qun l cht lng thuc v t chc k hoch c ph duyt. Xy dng cc vn bn php quy v tiu chun v cht lng thuc B ban hnh, hng dn kim tra vic thc hin cc vn bn trn. Qun l vic ng k tiu chun c s. Cung cp thng tin khoa hc k thut lin quan n m bo cht lng thuc. Ch o, gim st h thng kim tra cht lng thuc trn ton quc. Kim tra v cp giy chng nhn; c s sn xut t tiu chun Thc hnh tt sn xut thuc v phng kim nghim t tiu chun Thc hnh tt kim nghim thuc. T chc hng dn nghip v cho cn b qun l cht lng thuc ca ngnh y t, t chc o to v bi dng nghip v v tiu chun - o lng - cht lng thuc. Phi hp vi thanh tra B Y t thc hin chc nng kim tra, thanh tra nh nc v cht lng thuc v x l vi phm php lut v cht lng thuc theo thm quyn.
C quan qun l nh nc v cht lng thuc a phng: S Y t ch o qun l ton din v cht lng ca thuc a phng (thng u quyn cho phng nghip v Dc) c nhim v:

Ph bin, hng dn v t chc thc hin cc vn bn php lut v qun l cht lng thuc ti a phng. Thc hin chc nng kim tra, thanh tra nh nc v cht lng thuc v x l vi phm v cht lng thuc trong phm vi a phng. 14

1.1.3.2. H thng kim tra cht lng thuc C quan kim tra cht lng thuc ca nh nc v thuc

Trung ng l Vin Kim nghim thuc Quc gia ( H Ni) v Phn vin Kim nghim ( TP. H Ch Minh). Gip B Y t qun l ch o cng tc kim tra cht lng thuc trong ton quc v mt k thut. C nhim v: + Nghin cu xy dng tiu chun Vit Nam v thuc. + Kim tra xc nh cht lng thuc lu hnh trn th trng. + Thm tra k thut, gip B xt duyt cc tiu chun kim nghim xt cp ng k sn xut v lu hnh thuc Vit Nam. + Pht hnh cc cht chun v cht i chiu dng trong kim nghim. + Lm trng ti v cht lng khi c tranh chp khiu ni v cht lng thuc. + Tham gia o to cn b lm cng tc kim nghim . + T vn v chnh sch cht lng thuc quc gia. + Xy dng tiu chun phng kim nghim thuc t tiu chun v gip , kim tra cng nhn cc phng kim nghim thuc trong c nc. + Kim tra vic kim nghim thuc theo tiu chun trong phm vi ton quc. tnh, thnh ph trc thuc Trung ng l Trung tm kim nghim dc phm, m phm. L c quan trc thuc S y t, c nhim v nh Vin Kim nghim nhng gii hn trong phm vi tnh, thnh ph.
H thng t kim tra cht lng cc c s (phng kim nghim): Cng tc kim tra cht lng thuc c thc hin tt c cc c s c lin quan ti thuc. Tu theo qui m ca c s sn xut (x nghip, cng ty trung ng hoc a phng, cc c s nh), kinh doanh (cng ty, ca hng) bnh vin (trung ng, a phng) m lp phng kim nghim hay t kim nghim t kim tra cht lng thuc.Vi cc c s sn xut, bt buc phi c b phn t kim tra cht lng. B phn ny phi c kh nng kim nghim xc nh cht lng thuc c sn xut c s mnh theo tiu chun duyt. Phi kim nghim, theo di c cht lng ca thuc trong sut qu trnh sn xut t nguyn liu, bn thnh phm, thnh phm. Phi lp h s theo di tng l sn phm. C s sn xut phi chu trch nhim v thuc do c s sn xut ra.

Cc c s bo qun, phn phi thuc phi c b phn t kim nghim (vi cc cng ty ln) hoc kim tra, kim sot qun l, nh gi cht lng thuc, theo di cht lng trong qu trnh bo qun, cung cp h s cht lng cho n v s dng thuc. 15

Cc bnh vin, ty theo quy m ln, nh cng phi c b phn kim nghim cc thuc t pha ch, kim tra, kim sot cht lng thuc trc khi phn phi n ngi s dng.
1.1.3.3. H thng thanh tra dc

Cng cc c quan qun l nh nc v cht lng thuc, thc hin chc nng kim tra, thanh tra nh nc v cht lng thuc c t chc t trung ng n a phng.
1.2. Cng tc tiu chun ho 1.2.1. Khi nim 1.2.1.1. Mt s nh ngha

Tiu chun ho l mt lnh vc hot ng bao gm hai ni dung: Xy dng ra cc tiu chun v p dng cc tiu chun trong thc t nhm a cc hot ng ca x hi (c bit trong lnh vc sn xut v kinh doanh) i vo n np t c hiu qu chung c li nht cho mi ngi v cho x hi. Tiu chun l nhng qui nh thng nht v hp l c trnh dng mt vn bn hoc mt th thc nht nh do mt c quan quyn ban hnh bt buc p dng cho nhng ni c lin quan. cch khc tiu chun l mt vn bn mang tnh php ch trong nhng qui nh thng nht v hp l bt buc p dng nhm m lng cho mt sn phm no . by di c thm Ni mt ra bo cht

Cht lng sn phm l mt tp hp cc ch tiu c trng th hin tnh nng s dng nhm tho mn yu cu c xc nh trc cho mt sn phm trong nhng iu kin xc nh ph hp hon cnh kinh t, khoa hc k thut, x hi. i vi thuc, tiu chun l mt vn bn khoa hc k thut mang tnh php ch trong qui nh: qui cch, ch tiu yu cu k thut, phng php th, ng gi, ghi nhn, bo qun v cc vn khc lin quan n vic nh gi cht lng ca mt thuc. y l c s cc cc c quan kim nghim (hoc ngi kim nghim) tin hnh thc nghim, nh gi kt qu, cng b kt qu (bng phiu kim nghim) nh gi cht lng thuc l t hay khng t v c c php lu hnh (hoc s dng) hay khng. V mt lch s, cng tc tiu chun ho gn lin vi lch s sn xut ca loi ngi, ca cc phng thc sn xut ca tng ch x hi khc nhau. Nhng hnh thc s khai ca tiu chun ho c t thi c i, nhng pht trin c tnh t chc rng ri trn phm vi quc t th ch c t u th k 20.
1.2.1.2. i tng ca cng tc tiu chun ho

Bao gm rt rng, hu nh trn tt c mi lnh vc, v d: 16

 Cc my mc, dng c, trang thit b cng ngh Nguyn, nhin vt liu Nng lm hi sn, hng tiu dng Cc nguyn tc, phng php, th tc, cc vn t chc, qun l Thut ng, k hiu, o lng Sn phm v bn sn phm. Trong ngnh Dc, mi hot ng sn xut, kinh doanh, s dng thuc, mi vn c lin quan n cc i tng nu trn u phi tiu chun ho.
1.2.1.3. Ni dung chnh ca mt tiu chun v thuc

Tiu chun v thuc l mt tiu chun ton din, bao gm rt nhiu ni dung th hin c mc cht lng ca thuc c to ra v duy tr cht lng cho ti khi s dng hoc ht tui th (hn dng) ca thuc. Ni dung chnh ca mt tiu chun v thuc (nguyn liu hay thnh phm) thng gm cc mc sau: Tiu : Trong nu r tn nguyn liu hoc thnh phm, tn n v ban hnh tiu chun, loi tiu chun, hiu lc thi hnh. Yu cu k thut: L loi tiu chun rt quan trng v n ra mc cht lng hp l ca sn phm (mi yu cu k thut gi l mt ch tiu hay tiu ch, l yu t cu thnh ca yu cu k thut) v l c s cho vic lp k hoch sn xut (v s lng, cht lng), vic thc hin k hoch, vic qun l (kinh t, k thut, gi c, k kt hp ng, khiu ni). Phng php th: Tiu chun ny bao gi cng phi qui nh km theo cc ch tiu yu cu k thut. y l phn khng th thiu v n chnh l qui trnh th nghim hay qui trnh phn tch m t chi tit ton b qu trnh thc hnh thc hin xem mt ch tiu no ca yu cu k thut c t hay khng t yu cu t ra. ng gi, ghi nhn, bo qun: iu ny phi c qui nh r v tt c cc thuc lu hnh trn th trng phi c nhn thuc n n v ng gi nh nht. Nhn thuc phi: c ni dung cn thit gip nhn bit c thuc, cch s dng, trnh nhm ln, tn c s sn xut, c s ng k, s l sn xut, s kim sot, hn dng, iu kin bo qun.
1.2.1.4. Cc cp tiu chun v thuc Trc y c 3 cp tiu chun:

Tiu chun Vit Nam (tiu chun quc gia, Dc in Vit Nam) TCVN. Tiu chun ngnh y t Vit Nam. 17

Hai cp ny c hiu lc v phm vi p dng trong c nc. Tiu chun c s (TC hoc TCZ): do cc c s sn xut bin son, c hiu lc trong phm vi qui nh ca cc cp qun l.

Hin nay, ch cn 2 cp tiu chun l: Tiu chun Dc in Vit Nam v tiu chun c c (theo quyt nh s 2412/1998/Q-BYT ngy 15/09/1998).

Tiu chun c s l tiu chun do c s sn xut bin son, p dng i vi cc sn phm do c s sn xut ra. C hai loi tiu chun c s: Tiu chun c s ca nhng sn phm lu hnh th trng: phi ng k vi c quan c thm quyn. Cc mc tiu chun cht lng khng c thp hn cc mc quy nh trong tiu chun Dc in Vit Nam. Tiu chun c s ca cc thuc pha ch trong n v (khng lu hnh trn th trng): do th trng n v xt duyt v ban hnh.
1.2.2. Cng tc xy dng tiu chun

Tiu chun Vit Nam v thuc c nh nc y quyn cho B Y t t chc bin son, xt duyt v ban hnh sau khi ng k ti cc tiu chun o lng cht lng nh nc Vit Nam, cc tiu chun ny c tp hp trong mt b sch gi l Dc in Vit Nam (Hi ng Dc in c B Y t giao trch nhim ny). Mt s thuc cha c TCVN s s dng TC do cc c s sn xut bin son v c cp c thm quyn duyt.
1.2.2.1. Th tc xy dng tiu chun

Thng c tin hnh qua cc giai on sau: Chun b ti liu, kho st thc t, lp cng. Xy dng d tho tiu chun ln th nht gm cc vic: Tham kho ti liu, xut cc ch tiu v phng php th, kho st thc t v sn xut v s dng sn phm. Sn xut th, lm thc nghim phn tch cc tiu chun d kin, vit d tho tiu chun v thuyt minh, lp d tho bin php p dng (th d sa i hoc xut cc qui nh k thut, qui ch th tc trong tin hnh sn xut theo GMP). Thng qua d tho c s. Hon thnh d tho tiu chun sau khi tin hnh cc vic: Ly kin gp (cn th t chc hi tho). Thu thp gp , sa cha, hon chnh h s gi ti c quan qun l v xt duyt. Xt duyt v ban hnh tiu chun: C quan qun l cp tiu chun no th xt duyt v ban hnh tiu chun . Trc khi xt duyt, tiu chun phi c gi qua c quan thm tra k thut (Vin kim nghim 18

v phn vin kim nghim nu thuc trung ng; Trung tm kim nghim tnh, thnh ph nu thuc a phng) v cc c quan php ch (Cc qun l Dc trung ng v Phng nghip v dc a phng). Th trng B Y t hoc gim c S Y t k duyt sau khi tham kho cc c quan thm tra. Vic xem xt v sa i tiu chun cng c tin hnh nh th tc xy dng tiu chun. H s tiu chun: Bao gm tt c cc vn bn khoa hc k thut, kho st nghin cu, cng vn trnh duyt, quyt nh xt duyt.
1.2.2.2. Phng php xy dng tiu chun v yu cu k thut

Khi xy dng tiu chun v yu cu k thut cn ch ti vic kt hp gia cc loi ch tiu phn nh ht c cht lng ca thuc ph hp vi iu kin k thut, kinh t v thc t: Cc ch tiu phn nh v cng dng ca thuc (th hin tinh khit v hm lng). Ch tiu phn nh v mc tin cy v an ton ( c, bn, hn dng). Tiu chun v tm sinh l (dng thuc - qua cc ng vo c th). Ch tiu v thm m (hnh thc ng gi, trnh by p, bo qun).
Cc ni dung chnh ca yu cu k thut: Thng thng cc ni dung chnh ca yu cu k thut ca mt tiu chun v thuc bao gm:

Cng thc pha ch: Phi trnh by cng thc pha ch ca thuc, ghi r tn nguyn liu, ph liu vi s lng ghi bng s v bng ch, cng tiu chun ca chng (v d: t dc t tiu chun Dc dng). Cht lng thnh phm: Phi nu r v hnh thc cng nh tnh cht cm quan, mc tinh khit nh: Hnh dng, th cht, mu sc, mi v, cc c im c bit, bn c hc (vi thuc vin), tan r, sai s khi lng, sai s th tch. Yu cu v nh tnh, th tinh khit Yu cu v hm lng.
Phng php xy dng cc ch tiu: Da trn cc ni dung chnh ca yu cu k thut, cn c vo thc t, iu kin k thut, la chn ra cc ch tiu thch hp, khng qu nhiu hoc qu t nhng ni ln c trng cht lng thuc, ph hp vi iu kin c th. Sau phi xy dng c mc cho cc ch tiu trn. Mc ch tiu l gi tr c th hay l khong gi tr m thuc phi t c. xy dng mc ch tiu c tt, trc ht nn xem xt:

Nu loi ch tiu ny c sn trong cc qui nh th ch vic kim tra, xem xt li, p dng hoc xut (th d: nu l thuc vin, khi lng trung bnh vin qui nh sai x%; nu l thuc nc, th tch sai y%). 19

 Nu loi ch tiu ny cha c trong qui nh th phi lm thc nghim, t a ra s liu cho ph hp. Khi xy dng mc phi c tin hnh lm th vi mu t nht ly t 3 l sn xut th hay 3 l sn phm c qu trnh sn xut n nh; tt nht nn s dng phng php thng k (xt sai s, chnh xc, khong tin cy). Gii hn tin cy tm c qua th nghim , cn cn c thm vo cc iu kin kinh t, thc t (kh nng sn xut, trnh k thut, tiu chun dung sai chung) m qui nh mc ch tiu a vo tiu chun. Nu sn phm ly nhiu l hoc c sn xut nhiu c s th nn chn ch tiu trung bnh tin tin

x ( tt )

x (tt) =
Trong :

x + x max 2

x : ch tiu trung bnh ca nhiu c s t c

xmax : ch tiu cao nht mt c s t c.
1.2.2.3. Phng php xy dng tiu chun v phng php th: Cc loi qui trnh v phng php th: Mc ch chnh ca vic tiu chun ho mt phng php th l chn cho c mt qui trnh th hay qui trnh phn tch sao cho khi p dng s cho mt kt qu gn vi gi tr thc nht. Trong kim nghim thuc, cc loi qui trnh th thng c chia thnh 3 loi:

Cc php th nh tnh: l cc php th nhm chng minh rng cht cn phn tch c mt trong mu em th. Php th c thc hin bng cch tin hnh cc phn ng nh tnh, o cc thng s c trng cc cht cn th (tnh cht ph UV-VIS, IR, sc k) hoc so snh vi cht chun i chiu. Cc php th v tinh khit: L cc php th nhm chng minh rng mu em th t (hay khng t) v mc tinh khit. Php th c thc hin bng cch s dng mt s tnh cht c hiu (mu sc, c) so snh nh lng tp cht c trong mu th v mu chun t xc nh c gii hn ca tp cht c trong mu em phn tch. Cc php th nh lng: L nhng php th nhm xc nh hm lng ca mu th (hoc thnh phn chnh ca mu) hay hm lng ca cc hot cht c trong mu em th. Cc php th ny c tin hnh bng cc phng php nh lng ho hc, ho l, vt l, sinh hc 20

 Cc yu cu cht lng i vi mt phng php th: c th hin mt s im chnh sau:

* C tnh tin tin: Th hin ng, chnh xc, tnh chn lc, c hiu. + ng: Mt phng php c coi l c ng cao ngha l khi p dng s cho kt qu gn vi gi tr thc nht tc l sai s

100% cng nh cng tt (trong x : gi tr trung bnh xc

nh c; : gi tr thc ). Mun c gi tr thc so snh, phi c mu chun bit trc hoc mt mu in hnh c sn xut theo mt phng php m bo ng nht v i din cho sn phm. ng ca phng php cng cn c biu th bng t l thu hi x 100% (: l hm lng cht chun cho vo; x : l hm lng xc nh c). T l thu hi cng cao (nhng khng c vt qu 100%), ng cng tt. + chnh xc: C chnh xc p ng c yu cu. chnh xc cho bit s ph hp gia cc kt qu (hay dao ng) ca cc php xc nh song song, thng c biu th bng lch chun S hoc lch chun tng i RSD :

S =

xi x n 1

hoc RSD % =

S X

100 %

(n: s th nghim; xi : kt qu xc nh ln th i; ca n th nghim).

x : kt qu trung

bnh

+ Tnh chn lc - c hiu: Phng php cho php xc nh ng cht cn phn tch v t chu nh hng bi s c mt ca cc cht khc trong mu th. + C tnh cht tuyn tnh: i vi trng hp khi xc nh kt qu phn tch da vo ng chun. ng chun l ng biu din y = f(x). Trong x l nng dung dch, y l i lng tn hiu o (mt quang, din tch pic sc k, chiu cao sng cc ph) ph thuc vo x. Tnh cht tuyn tnh c biu th bng cch tnh h s hi qui theo phng php bnh phng ti thiu. Nu h s ny cng gn 1 th hm y cng tuyn tnh.

21

* C tnh thc t: Phng php th a ra phi ph hp vi iu kin thc t, c tnh kh thi cao (ph hp trang thit b, my, k thut, ho cht, thuc th, trnh con ngi ). * C tnh kinh t: Phng php th a ra t tn km m vn p ng cc yu cu nu trn. * C tnh an ton cao: An ton lao ng v bo v sc khe (t dng ho cht c hi, trnh c cc thao tc k thut phc tp nguy him ).
Kinh nghim tin hnh xy dng phng php th:

Phi da trn yu cu k thut chn xy dng phng php th cho thch hp. Tham kho cc ti liu, cc tiu chun tng t c. Kt hp vi t nghin cu, la chn. Phi ly mu v tin hnh lm thc nghim. Vit chng trnh d tho c km bn thuyt minh, a ra cc bng kt qu thc nghim, x l kt qu, ti liu tham kho.
1.2.3. Cng tc p dng tiu chun trong thc t 1.2.3.1. Mc ch ngha

p dng tiu chun l mt mt cng tc ca ton b cng tc tiu chun ho. Qua p dng : Kim chng li cc kt qu nghin cu khi xy dng tiu chun (mc ch tiu c ph hp khng? Phng php th ng hay sai? ). Xc nh hiu qu kinh t ca tiu chun (tiu chun c kch thch cho sn xut, kch thch tiu th). Trn c s tip tc sa i, hon thin nng cao cht lng ca tiu chun. Ngn chn vic a cc thuc khng t tiu chun ra lu hnh, s dng. Pht hin thuc gi, thuc km cht lng, pht hin nguyn nhn vi phm v tm cc bin php khc phc.
1.2.3.2. Cc hnh thc p dng tiu chun trong thc t p dng tiu chun trong sn xut: Ngha l phi thc hin ng cc tiu chun (k thut, th tc, nguyn tc v cc qui nh c lin quan) sn xut ra thuc t tiu chun. Th d: Phi kim tra x l nguyn vt liu t tiu chun mi a vo sn xut; trong qu trnh sn xut phi kim tra; tt c cc cng on, 100% l sn xut ti c s; kim tra thu nhn sn phm

22

 p dng tiu chun trong kim tra cht lng: Ngha l tin hnh thc nghim tt c cc ch tiu yu cu k thut theo ng phng php th nu nh gi xem thuc c t tiu chun hay khng. 1.2.3.3 Cc cng vic phi thc hin Ph bin, gii thch, cung cp tiu chun tu theo cp qun l:

Cc c quan chc nng: B Y t, S Y t chu trch nhim cung cp tiu chun sau khi duyt. Cc c quan k thut: Vin Kim nghim, Trung tm Kim nghim tnh, thnh ph hng dn gii thch v thi hnh vic p dng tiu chun.
Lp k hoch cc bin php p dng tiu chun:

V t chc: Qui nh trch nhim, quyn hn ca cc cp qun l, cc b phn thc hin. Qui nh v con ngi (phi c o to c th p ng c vic thc hin tiu chun ). V k thut: Sot xt li trang thit b, ho cht, cht chun, thuc th, nh xng, phng th nghim p ng y theo yu cu ca tiu chun ra.
Kim tra p dng tiu chun: Cng vic ny c tin hnh thng xuyn cc c s hoc h thng kim tra nh nc: Vin Kim nghim, Trung tm Kim nghim, thanh tra Dc Ni dung kim tra bao gm:

Kim tra c s vt cht ca cng tc kim nghim: Ti liu, trang thit b, phng th nghim, ho cht, thuc th Kim tra nguyn liu, bn thnh phm, thnh phm v tt c cc thuc ang lu hnh trn th trng.
1.2.3.4. Sa i, sot xt li tiu chun

c tin hnh khi thy tiu chun khng cn ph hp.

1.2.4. Gii thiu Dc in Vit Nam 1.2.4.1. Mt s nt chung v Dc in Vit Nam Dc in Vit Nam l mt ti liu bao gm:

Tp hp cc tiu chun nh nc (TCVN) v thuc (ho dc v cc ch phm, huyt thanh v vaccin, dc liu, ch phm ng dc). Mi tiu chun cn c gi l mt chuyn lun. 23

 Nhng qui nh chung trong cng tc kim nghim, gii thiu cc phng php kim nghim chung, cc ho cht, thuc th, thuc chun, ch th dng phn tch v nh gi cht lng thuc. Mt s ph lc, cc bng tra cu

Dc in Vit Nam c gi tn theo ln xut bn (ging cc nc khc), tun theo qui tc ln sau ph nhn ln trc : Dc in Vit Nam I: gm 638 chuyn lun tn dc v 284 chuyn lun ng dc (tp 1 in 1970, tp 1 b sung in 1977, tp 2 ng dc in ln 1 vo 1983 ). Dc in Vit nam II: gm 357 chuyn lun tn dc, 64 chuyn lun ng dc, v 32 chuyn lun v vaccin (tp 1 in 1990, tp 2 in 1991 v tp 3 in 1994). Dc in Vit nam III: gm 342 chuyn lun ho dc, 276 chuyn lun v dc liu, 37 chuyn lun v ch phm ng dc, 47 chuyn lun ch phm sinh hc v 500 chuyn lun v ho cht thuc th. (Qu trnh bin son chun b t 1995-2000, d tho in ly kin 2000, in chnh thc 2002).

1.2.4.2. Mt s qui nh chung khi s dng Dc in Vit Nam (trong cng tc kim nghim thuc)

C nhiu qui nh, di y nu tm tt ni dung chnh ca 10 qui nh: 1. Tn cc chuyn lun: ly tn chnh l tn Vit Nam, sau tn Vit Nam l tn Latin v tn thng dng khc nu c. 2. n v o lng: s dng theo ng yu cu ca c quan o lng nh nc Vit Nam. 3. Khi nim cn chnh xc ngha l cn trn cn phn tch c nhy n 0,1 mg (0,0001 g). Khi nim ly khong c ngha l ly mt lng vi chnh khng qu 10% so vi yu cu. Khi nim n khi lng khng i ngha l x l ch phm n khi no sai gia 2 ln k tip nhau < 0,5 mg. 4. Nng phn trm khng c ch dn g coi l cch biu din theo % KL/TT. Cn cc trng hp khc s ghi c th. 5. Khi nim alc ol khng c ch dn g c ngha l alcol cha khong 96% (TT/TT) ethanol (C2H6O). Ethanol khng c ch dn g khc ngha l ethanol tuyt i. 6. tan : Qui c: Mt cht l d tan : Khi ho 1g cht 24 di 1ml dung mi

Rt tan : Tan : Hi tan : Kh tan : Rt kh tan :

-nt-nt-nt-nt-nt-

1 - 10ml > 10-30

ml

-nt-nt-nt-nt-nt-nt-

> 30-100ml > 100-1000ml >1000-10.000ml >10.000ml

Thc t khng tan : -nt-

7. V nhit : Dng thang bch phn k hiu oC. Khi khng ghi c th, qui c : Nhit chun: Nhit thng: Nc m: Nc nng: Nc cch thu: 20oC 20-30oC 40-50oC 70-80oC 98-100oC

Nhit trong th v mt khi l ng do lm kh cho php hiu l: 100 oC 2 oC. Nhit ni bo qun : Rt lnh: Lnh: Mt: Nhit phng : Nhit phng c iu nhit : Nng: Rt nng: Nung : thm: <-10 oC 2-10 oC 10-20 oC 20-35 oC 20-25 oC 35-40 oC > 40 oC 400 oC 600 oC

trng: 900oC 8. V phng php: C th dng phng php hay phng tin khc vi qui nh trong Dc in hoc TC nhng vi iu kin cc kt qu c chnh xc tng ng nhau. Nu cc kt qu khc nhau th coi phng php hoc phng tin qui nh trong Dc in hay TC l chnh thc. 9. V hm lng: Nu trong chuyn lun khng ghi gii hn trn th c ngha l khng c qu 101,0%.

25

10. Khi th tinh khit, nu pht hin thy tp cht l khng ghi trong chuyn lun th vn phi ghi vo kt qu th.
1.3. Kim nghim thuc theo tiu chun

Nh trn nu, kim nghim thuc l vic tin hnh phn tch mt mu thuc i din cho l thuc bng cc phng php ho hc, l hc, ho l, sinh hc c qui nh xem thuc t hay khng t tiu chun t quyt nh xem c c php lu hnh hoc s dng hay khng. s nh gi ny chnh xc, i hi phi lm tt 3 vic sau: ly mu kim nghim, thc hnh phn tch, nh gi kt qu v vit phiu tr li (phiu kim nghim, phiu phn tch).
1.3.1. Ly mu kim nghim 1.3.1.1. Mt s khi nim

L thuc: L mt lng thuc xc nh ca cng mt loi sn phm c sn xut trong mt chu k nht nh p ng yu cu GMP, c coi l ng nht v c ghi bng s l ca c s sn xut trn nhn cc bao b. Tng th: L ton b cc n v sn phm c xt. Tu theo tng trng hp tng th c th l mt l, mt s l hay mt qu trnh sn xut. n v bao gi: L dng bao gi sn phm lp li trong l (thng, hm, hp). n v ng gi: L dng c ng gi trc tip sn phm (chai ng thuc vin, v thuc ). n v sn phm: L i tng qui c hoc c th ca mt lng sn phm nht nh (vin thuc, ng thuc, 1g, 1kg ). Mu: L mt s n v sn phm ly t tng th th v c dng lm c s c nhng thng tin quyt nh v tng th . S n v sn phm c trong l gi l c l, s n v c trong mu gi l c mu. Mu ban u: L mt lng sn phm ca l thuc c ly trong mt ln mt hay nhiu n v bao gi. Mi bao gi ly mt ln. Mu ring: L mt lng sn phm c ly t nhng mu ban u c gp li v trn u ca mt bao gi. Mu chung: L mt lng sn phm c ly t nhng mu ring ca tng n v bao gi gp li v trn u. Mu trung bnh th nghim: L mt lng sn phm c ly ra t mu chung dng tin hnh cc php th qui nh (k c lm li). 26

 Mu lu: c ly t mu trung bnh th nghim hay t mu ban u tng ng vi lng mu th. Mu lu dng lu li khi cn thit hoc lm cc th nghim trng ti.
1.3.1.2. Qui nh v ly mu

Ly mu l mt tp hp cc thao tc nhm ly ra mt lng mu thuc i din kim tra cht lng. Do vy kt lun v mu thuc mang tnh php l, cn phi tun th mt cch nghim ngt cc qui nh v th tc ly mu nh sau:
i tng ly mu:

Vi h thng t kim tra: L cc nguyn liu dng lm thuc, bao b ng gi, sn phm trung gian, sn phm cha ng gi, thnh phm. Vi h thng qun l nh nc: Thuc v cc nguyn liu lm thuc ang trong qu trnh lu thng hoc tn tr trong kho.
Cc trng hp ly mu:

Trng hp t kim tra cht lng: Phi ly mu kim tra ton b cc l thuc ti cc c s sn xut, lu tr, phn phi. Vi cc c s sn xut thuc, yu cu 100% s l phi c kim tra. Vic ly mu do cn b chuyn mn ca phng kim tra cht lng sn phm (KCS) tin hnh, c s chng kin ca cn b n v ly mu. Th trng n v cn c vo qui nh chung c th c nhng qui nh c th hn cho ph hp vi tnh hnh ca c s. Trng hp kim tra gim st cht lng hoc thanh tra: u tin ly mu kim tra v gim st l cc thuc cha bnh, c gi tr kinh t cao, c cht lng khng n nh v c bit l c nghi ng v hm lng hoc hiu lc tc dng. Ly mu kim tra gim st cht lng ca cc c s sn xut (ly 10% s l sn xut trong nm) hoc ly theo qui nh ca B Y t, S Y t. Ly mu thanh tra t xut trong nhng trng hp c thng tin v cht lng thuc xu, thuc khng an ton, t hiu lc v c bit l thuc gi hay thuc km phm cht. Vic ly mu c thc hin bi cc thanh tra vin hoc cc cn b c giy u nhim ca c quan kim tra, thanh tra v c s chng kin ca cn b c s.
Cc iu kin cn lu khi ly mu:

Ni ly mu: Ti ni cha sn phm, mi trng xung quanh khng c gy nhim bn hoc tc ng lm thay i tnh cht ca mu v ngc li khng mu tc ng xu n mi trng. Ngi ly mu: Phi l ngi c chuyn mn nht nh v p ng c yu cu ca qu trnh ly mu. 27

 Phi quan st kim tra s b l hng (phn loi nu cn), nhn xt v phi ghi vo bin bn ly mu. Dng c ly mu: Sch, kh, p ng yu cu cn ly mu. ng mu: p ng yu cu ly mu (sch, khng lm hng mu, kh, c nhn ghi chp y ). Thao tc ly mu: Phi thn trng, t m, quan st cn thn Phng thc ly mu: Ngi ly mu phi t tay ly mu, ghi nhn, lm bin bn, ng gi nim phong bo m v bo qun mu. c bit lu phi ly ch k xc nhn ca n v c ly mu.
1.3.1.3. Tin hnh ly mu

S ly mu: Vic ly mu phi bo m cho c tnh khch quan, i din cho c cht lng ca thuc cn ly kim tra. V vy, phi ly theo hng dn ca qui trnh (hnh 1.1).
L thuc

Mu ring

Mu chung

Mu trung bnh th nghim

Mu lu ti c s

Mu gi (hoc ly) kim nghim

Mu lu thanh tra

Mu lu ti b phn kim nghim

Mu lm kim nghim

Hnh 1.1. S qui trnh ly mu, lu mu

28

T l sn xut ly ra cc n v bao gi mt cch ngu nhin, c mu ban u ly theo ch dn phn sau. Trn u cc mu ban u v gp thnh nhng mu ring ca tng n v bao gi. Trn u cc mu ring thnh mu chung. T mu chung ly ra mt lng mu trung bnh th nghim. T mu trung bnh th nghim ly ra thnh cc mu lu v mu th kim nghim

Sau khi ly mu xong, ngi ly mu t tay dn nhn nim phong, bao gi (phi c ch k xc nhn ca ngi ly mu v c s c ly mu) v bin bn ly mu (cng phi c ch k xc nhn). Ly mu c th: Cn c vo l thuc phi ly mu, xem xt phn loi tin hnh ly nh sau:
Ly mu thuc c phn liu (l sn phm thuc dng thuc c phn liu). T l sn phm ly ra cc n v bao gi mt cch ngu nhin bt k: Cc gi c ly ra phi c lp vi d kin ca ngi ly mu (khng nn la chn theo cm quan ly mu xu hay mu tt). L thuc phi ng nht, hp l v s lng hay khi lng (Th d khng qu 500.000 vin vi thuc vin, khng qu 50.000 ng vi dng ng ).

S bao gi trong l ly ra to mu ban u tnh theo cng thc : n = 0,4 N Trong : n: s bao gi ly ra N: s n v bao gi cui cng trong l (th d vi thuc tim: cc ng ng trong hp giy, cc hp giy ng trong hm th n v bao gi cui cng l hm). Ch : + Khi tnh theo cng thc trn, nu phn thp phn nh hn 0,5 b qua, nu ln hn 0,5 th tng thm mt n v. + Khi N > 100 lu nmax 30 + Khi N < 100 c th dng bng:
N 1 -10 11 - 40 41 - 80 81 - 100 n 1 2 3 4

29

 Ly mu sn phm l cht rn (ht, bt, vin): 1. Trng hp mt bao gi:

+ Trc khi ly, xem xt sn phm c ng nht khng, nu khng ng nht phi chn ring ra tng loi v ly theo cc loi . + Trng hp sn phm l ht, cc, tr trng hp phi xc nh c ht, cn tt c phi c nghin nh thnh bt (khng c lm nh hng ti tnh cht ca sn phm). + Ly mu ban u 3 v tr khc nhau: trn, gia, di sau trn thnh mu chung. + Dn u lng mu chung thnh lp phng hnh vung dy khng qu 2 cm, chia mu thnh 2 ng cho, b 2 phn i din, trn u 2 phn cn li v chia tip cho n khi lng mu cn li tng ng vi 2-4 ln mu th cn ly, l mu trung bnh th nghim. + Chia mu trung bnh th nghim thnh cc mu lu v mu kim nghim.
B B

2. i vi thuc vin ch ng trong mt bao gi: cng ly mu ba v tr khc nhau trong bao gi, chai, l sau trn thnh mu chung, mu trung bnh th nghim nh trn. 3. Trng hp sn phm ng thnh nhiu bao gi: ly mu ban u theo cng thc: n = 0,4 N nu trn.
Ly mu l sn phm lng: 1. Trng hp mt bao gi: Nu sn phm l ng nht th ly mu bt k v tr no cng c. Nu khng ng nht, trc khi ly mu phi khuy u, sau mi ly mu.

2. Trng hp nhiu bao gi : ly theo cng thc : n = 0,4 N Nu l nhng chai l nh th c th ly ht th tch.
Ly mu l cc sn phm thuc m, bt nho: Tin hnh ly mu nh cc sn phm lng, rn nhng ch khuy k, trn u c hn hp ng nht, sau mi ly mu.

Ly mu thnh phm cha ng gi l: Thng cc thnh phm ny cha trong cc bao gi ln chuyn ch n c s ng gi l. Nu l sn phm ch c 1 - 2 bao gi th m c 2 bao gi; 30

nu l sn phm c t 3 bao gi tr ln th m 3 bao gi. Ly t nht 3 mu ban u 3 v tr khc nhau ca mi bao gi. Sau trn cc mu ban u li vi nhau to mu th nghim. Bao gi v dn nhn: Sau khi ly mu v cho vo ng, ngi ly mu bao gi, dn nhn v nim phong mu, lm bin bn ly mu. Lu phi c ch k xc nhn ca c s c ly mu nhn nim phong v bin bn ly mu.
2.3.1. Tin hnh kim nghim 2.3.1.1. Nhn mu

B phn nhn mu ca c quan kim nghim phi kim tra xem mu c p ng cc yu cu sau khng: Mu phi c ly theo ng cc th tc qui nh trn. Mu phi c ng gi nim phong v c nhn ghi cc thng tin cn thit (nhn gc, tn thuc, s l sn xut, tn tiu chun yu cu kim tra ). Cc mu do thanh tra ly v phi c km bin bn ly mu. Cc mu gi phi km cng vn hoc giy gii thiu. Nu mu xin php sn xut phi km cc ti liu theo qui nh thuc xin ng k sn xut. Nu mu nhn qua ng bu in, phi kim tra k nim phong sau bo li cho ni gi mu, ch sau khi nhn c kin tr li ca ni gi mu mi tin hnh kim nghim.
2.3.1.2. Kim nghim, x l kt qu

Cng vic ny do b phn k thut thc hin. Thng thng gm cc ni dung sau : Chun b ti liu: theo TCVN hoc TC Chun b dng c, ho cht, my p ng yu cu m tiu chun qui nh. B tr th nghim mt cch hp l c mu lm v khng lm nhim bn hoc bin cht mu cn th. Tin hnh cc th nghim phn tch theo tiu chun. Ngi lm kim nghim phi c cun s ghi chp y cc s liu khi tin hnh th nghim, s ny gi l S tay kim nghim vin. S tay kim nghim vin c coi l chng t gc ca cc s liu sau ny cng b trn phiu tr li kt qu kim nghim (gi l phiu kim nghim). X l cc s liu thc nghim quyt nh xem cc ch tiu th theo tiu chun t hay khng t yu cu. 31

2.3.1.3. Vit phiu tr li kt qu

Bng phiu kim nghim hay phiu phn tch. Phiu kim nghim l vn bn php l ca cc t chc kim tra cht lng thuc, xc nhn kt qu kim nghim theo ti liu k thut hp php ca mt mu thuc. Phiu phn tch l vn bn php l xc nhn kt qu phn tch ca mt hay nhiu tiu ch trong tiu chun k thut ca mt mu thuc. Do vy, sau khi hon thnh cc th nghim v x l s liu nh gi kt qu, kim nghim vin phi vit vo phiu tr li ni b (cha phi phiu chnh thc), k tn chu trch nhim v a cho cn b ph trch phng duyt li, trc khi a phng chc nng trnh lnh o duyt ln cui, sau tr li chnh thc bng phiu ca c quan kim nghim (gi l phiu kim nghim hay phiu phn tch). Phiu kim nghim ch cn c ch k v con du ca gim c c quan kim nghim hoc n v. Cu ch vit trong phiu kim nghim phi r rng, chnh xc, gn, y v thng nht. Ni dung chnh ca mt phiu kim nghim phi c: Phn tiu (bao gm tn c quan kim nghim, s phiu kim nghim, tn mu kim nghim, l lch mu kim nghim), cc ch tiu th v kt qu, kt lun cui cng v mu thuc kim nghim Di y l mu thng dng ca mt phiu kim nghim (xem trang sau).
2.3.1.4. Lu mu kim nghim ti c quan kim nghim

Mu lu phi c nh s cng vi s ng k mu th cng loi, nhng c nhn ring vi ch mu lu v bo qun trong iu kin theo qui nh chung, mu ny c s dng n trong trng hp c tranh chp v kt qu cng b ( phiu kim nghim). Thng thng, mu lu ly t mt phn mu ly th, do vy s lng ly phi ging nh mu ly th.

32

B Y t Tn c quan kim nghim thuc a ch c quan

Cng ho x hi ch ngha Vit Nam c lp - T do - Hnh phc

Phiu kim nghim

Mu kim nghim: Vin nang Cephalexin 500mg Ni sn xut: Cng ty Dc phm X S l, hn dng:. S ng k:. Ngi v ni gi mu: Yu cu kim nghim (ghi r ni dung, s, ngy, thng, nm ca cng vn hay giy t km theo): Ngy thng, nm nhn mu: S ng k KN: Ngi nhn mu:. Th theo; Dc in Vit Nam III Tnh trng mu khi nhn v m nim phong kim nghim: ........................................................................................................................ Yu cu
1. 2. 3. 4. 5. 6. Tnh cht: Nang nhn bng, khng mo m, bt thuc bn trong ng nht. nh tnh: Ch phm phi c phn ng c trng ca cephalexin N c: Khng c qa 10% ng u khi lng: Khi lng trung bnh vin 7,5% ho tan: Khng t hn 80% cephalexin (C16H17O4S) ghi trn nhn c ho tan trong 45 pht. nh lng: Hm lng cephalexin khan (C16H17O4S) t 92,5110% so vi lng ghi trn nhn.

Kt qu
t ng t (6,75%) t t (86,5%) t (98,6%)

Kt lun: Mu th ny t yu cu kim tra cht lng theo tiu chun Dc in Vit Nam III. H Ni, ngy thng . nm .. Th trng c quan (K v ng du)

33

Cc mu lu phi c gi li theo ng thi gian qui nh. Cc mu c hn dng phi lu tip 3 thng k t khi ht hn dng. Khi ht thi gian lu, c quan lp bin bn x l theo qui ch. S sch, phiu kim nghim phi lu gi t nht l 3 nm. Khi ht hn lu, mun hu phi c gim c c quan duyt.
2.3.2. Ni dung chnh ca thc hnh tt phng kim nghim (GLP)

Mc ch c bn ca GLP l nhm xy dng c mt n v lm cng tc kim nghim p ng y mi yu cu ca cng tc kim tra cht lng thuc ra, m bo rng kt qu cc php phn tch thu c l c tnh chn lc cao, chnh xc v ng n, c tnh php l. ng thi gip cho vic tra cu v tm c nhanh chng ngun gc ca cc sai st xy ra khi gp phi. V vy, GLP l nhng qui nh nhm m bo thuc c cht lng tt trong qu trnh sn xut, tn tr ti kho v lu thng phn phi n tay ngi s dng. C th l nhng qui nh cht ch v c chun ho cho mt c s kim nghim phi tun th v mi mt: nhn s, t chc, c s vt cht, trang thit b, ho cht thuc th, qui trnh th nghim, cc iu khon kim tra, bo co kt qu, lu gi s liu
2.3.2.1. V t chc v nhn s

Mt phng kim nghim thuc phi c cc b phn chuyn mn, hu cn, ng k v lu tr mu ... c chc nng nhim v r rng, c xy dng v c ngi c thm quyn ban hnh. Cc nhn vin phi c o to v hun luyn v chuyn mn c th thc hin c cc nhim v c giao c tinh thn trch nhim cao. Cc nhn vin phi mc trang phc theo qui nh, phi c kim tra sc kho, khng lm nhim bn vo cc php th v cc dng c, h thng th nghim...
2.3.2.2. V c s vt cht chung

Phi c din tch lm vic ph hp vi yu cu t ra: C din tch, c qui m,kch thc xy dng thch hp cho php sp xp trt t, hp l cc dng c, trang thit b. C mi trng tt v: chiu sng, thng gi, nhit , m Phi m bo tnh bit lp: c s th nghim phi khng b nhim bn do mi trng hoc cc b phn ln cn khc, bo m v sinh theo qui nh. Bo m cng tc an ton, cc phng tin bo h lao ng, s dng v bo qun cht c hi, x l cht thi ... 34

2.3.2.3. Trang thit b

Phi c trang b y cc trang thit b p ng thch hp cc yu cu th nghim. Cc trang thit b ny cn c la chn, chun ho, c cht lng. Thit b phi t v tr thch hp tin cho s dng, vn hnh,kim tra, v sinh, bo dng. nh k phi c kim tra, chun ho, bo dng. Phi c ni qui vn hnh, hng dn s dng, c c nhn chu trch nhim. 2.3.2.4. C s vt cht cho cc php th Tt c cc php th phi c qui trnh c vit chi tit v c chun ho.Mi ho cht, thuc th, cht chun phi p ng ng cc yu cu qui nh. Thuc th, ho cht, cc dung dch b bin cht hay qu hn khng c php s dng.
2.3.2.5. Qui nh v nguyn tc, c s cho cc php th v nh tnh, th tinh khit, nh lng v hm lng, hay hot lc ca thuc, bn vng ca thuc, qui nh v mu th, thuc th v cht i chiu 2.3.2.6. Qui nh v qui trnh v hng dn th nghim

Qui trnh v hng dn th nghim phi r rng, chnh xc, c chun ho v c lnh o duyt thng qua. Kim nghim vin tin hnh th nghim phi bo co mt cch chi tit nhng hin tng gp phi trong qu trnh th nghim vi ph trch.
2.3.2.7. Qui trnh v bo co kt qu

Phi bo co y cho thy r : mc ch ca php th nghim, ni th nghim, tn mu th, ngy th nghim, phng php p dng th, ngi th nghim, cc phng php thng k p dng phn tch s liu, kt qu th nghim v bn lun, kt lun, k xc nhn, ni lu tr mu lu, h s, ti liu

35

Ti liu tham kho

1. B Y t (2002). Dc in Vit Nam III. NXB Y hc. H Ni. 2. B Y t (2002). Cc vn bn qun l nh nc trong lnh vc Dc. NXB Y hc. H Ni. 3. ng Vn Ho (2001). Gio trnh kim nghim thuc. i hc Y Dc Thnh ph H Ch Minh. 4. Lin hip cc x nghip Dc Vit Nam (1990). Hng dn thc hnh sn xut thuc tt (sch dch). NXB Ngoi Vn. H Ni.

Cu hi t lng gi
1.1. Mc tiu ca cng tc kim tra cht lng thuc? Cc yu cu c bn t mc tiu trn ? 1.2. Ni dung chnh ca cng tc kim tra cht lng thuc? iu kin thuc c a vo lu thng, phn phi, s dng? Cc yu t nh hng n cht lng thuc ? 1.3. Th no l thuc t v khng t tiu chun? Thuc gi mo? Thuc km phm cht? 1.4. Trnh by h thng t chc kim tra cht lng thuc ca Vit Nam? 1.5. Ni dung chnh ca cng tc tiu chun ho? Ni dung chnh ca mt tiu chun v thuc? 1.6. Trnh by phn g php xy dng tiu chun v yu cu k thut? v phn g php th? Yu cu cht ln g i vi mt ph ng php th ? 1.7. Ni dung chnh ca cng tc p dng tiu chun trong thc t? 1.8. Trnh by mt s qui nh chung khi s dng Dc in Vit Nam (dng trong cng tc kim nghim thuc)? 1.9. Cch tin hnh ly mu kim nghim? 1.10. Trnh by cc nhim v ch yu khi tin hnh kim nghim? 1.11. Ni dung chnh ca thc hnh tt phng kim nghim thuc?

36

Chng 2

kim nghim thuc bng cc phng php ha hc

Mc tiu hc tp

3. Trnh by c cch nh tnh v xc nh gii hn tp cht trong thuc. 4. Gii thch c k thut nh lng cc acid, base v cc loi mui trong mi trng khan. 5. Trnh by c cch s dng thuc th Karl Fischer xc nh hm lng nc trong cc mu phn tch rn, trong dung mi hu c. 6. Vit c phng trnh phn ng nh lng mt s cht hu c (polyol, v amino alcol) bng thuc th periodat. 7. Phn tch c ng dng ca cp ion trong kim nghim thuc.
2.1. Cc phn ng nh tnh 2.1.1. Acetat

Phn ng vi cc acid (mnh hn acid acetic) gii phng acid acetic c mi chua: CH 3COO + H+ = CH3COOH

Vi dung dch FeCl3 long cho phc mu [Fe3(OH)2(CH3COO)6]+. Khi pha long v un si s cho kt ta mu : [Fe3(OH)2(CH3COO)6] + + 4H2O = 3Fe(OH)2CH3COO + 3CH3COOH + H +

Vi H2SO4 c v C2H5OH to ra este etyl acetat c mi thm:

H SO dc 4 CH 3 COOH + OHC 2 H 5 2 CH 3 COOC 2 H 5 + H 2 O

2.1.2. Amoni (mui) B phn hu khi un nng vi dung dch NaOH, gii phng kh NH3:
+

NH4

OH

NH3

H2O

Nhn bit NH3 bng cc du hiu: Mi khai, lm xanh giy qu tm t, lm hng giy tm phenolphtalein. 37

 Thuc th Nessler (dung dch kim ca mui Kaliiodomercurat K2[HgI4] phn ng vi NH3 cho ta mu (lng nh cho dung dch mu vng):
+

NH4

+ OH

=
Hg

NH3

H2O

NH3 + 2K 2 HgI4 + 3KOH = O

2.1.3. Arseniat

NH2 I + 7KI + 2H2O Hg

Phn ng vi acid hypophosphor hoc dung dch hypophosphit (thuc th Bugo hay Tile): to ra kt ta As nguyn t c mu nu:

4AsO 3 + 5H PO + 6H +
4 3 2

4As nu + 5H PO + 3H O
3 4 2

Phn ng vi AgNO3: to kt ta nu Ag3AsO4, ta ny khng tan trong CH3COOH, tan trong HNO3, dung dch amoniac. Phn ng vi hn hp magnesi (MgCl2 + NH4OH + NH4Cl ): Cho kt ta tinh th trng magnesi amonarseniat : AsO43+ Mg2+ + NH4+ = MgNH4AsO4
trng

2.1.4. Arsenit Phn ng Tile:

4AsO33+ 3H 3PO2 + 12H+ = 4As nu + 3H3PO4 + 6H2O

Phn ng vi AgNO3: to ra kt ta trng hi vng Ag3AsO3, ta ny tan trong HNO3 , trong dung dch amoniac. Phn ng kh bng hydro mi sinh (do Zn trong mi trng acid to ra) : cc hp cht ca Arsen (c AsO3 3 v AsO43 ) u thnh AsH3 dng kh: AsO33+ 3Zn + 9H+ = AsH3 + 3Zn2+ + 3H2O C th nhn ra AsH3 bng: Cho tc dng vi AgNO3: AsH3 + 6AgNO3

3HNO3 + AsAg3.3AgNO3 (mu vng)

38

Sn phm mu vng ny d b thu phn to ra Ag en: AsAg3.3AsNO3 + H2O H3AsO3 + 3HNO3 + 6Ag en

Cho tc dng vi HgCl2: to thnh hp cht c mu t vng sang nu: AsH2(HgCl); AsH(HgCl)2; As(HgCl)3; As2Hg3 Phn ng vi CuSO ta ng xanh lc: 4: cho H AsO + CuSO = hydroarsenit CuHAsO mu + H SO
3 3 4 3 xanh lc 2 4

Nu thm NaOH v un nng s c kt ta mu ca Cu2O: 2CuHAsO3 + 6NaOH = Na3AsO4 + Cu2O + 4H2O + NaAsO3 (Phn ng ny dng phn bit gia AsO43-v AsO33- ).
2.1.5. Bc (mui) Phn ng vi HCl cho ta trng AgCl, ta khng tan trong HNO3 nhng tan trong dung dch amoniac:

Ag+ + Cl AgCl + 2NH 3

AgCl trng Ag(NH 3)2+ + Cl

Phn ng vi formol trong mi trng kim b kh thnh Ag c mu en (phn ng trng gng): Ag+ + NH4OH Ag2O
HCHO

Ag(NH3)2+ + Ag + HCOOH

2.1.6. Barbiturat

Khi un nng vi kim c, vng ureid b m gii phng ra cc sn phm khc nhau: NH CO O C NH CO C R2 R1 NaOH + H2O O C NH2 NH
2

COONa + R1 C R2 COONa

NH2 O C NH2

to + H2O

2NH3

CO2

To phn ng phc c mu vi cc ion kim loi nh Cu2+, Co2+

39

2.1.7. Bari (mui) Phn ng t cho mu ngn la xanh lc hi vng. Phn ng vi H2SO4 cho kt ta BaSO4 mu trng, khng tan trong cc acid v c. 2.1.8. Bismuth (mui) Phn ng thu phn:

Bi

3+

Cl-

H2O

BiOCl trng +

2H+

Tc dng vi dung dch kim: cho ta Bi(OH)3 mu trng, khi un nng vi nc ng mu vng:
3+

Bi

+ OH- = Bi(OH)3 trng +

(BiO)OH vng

H2O

Phn ng vi S2-: cho ta nu en Bi2S3 Phn ng vi thioure: trong mi trng acid cho mu vng da cam hay vng xanh l cy (nhiu cho kt ta): [Bi (H2N CS NH2)3 ](NO3)2: vng da

cam [Bi (H2N CS NH2)3 ]Cl3: vng xanh l cy

2.1.9. Borat

Hn hp mui borat vi ethanol (hoc methanol) v H2SO4 c s to ra ester trietyl borat, em t chy cho ngn la mu lc:
+ Na2B4O 7.6 H2O + 2 H

2 Na+ + 4H3BO3 + 5 H2O OC2H5

H3BO3 + 2 C 2H5OH

H+

OC2H5 OC2H5

3 H2O

Trong mi trng acid, borat chuyn thnh acid boric, acid boric phn ng vi giy ngh (hoc cn ngh) cho mu nu , sau tm t bng dung dch kim long (amoniac hoc natrihydroxyd) mu nu chuyn thnh

mu lam hay lc (do s to phc ca cucumin trong ngh vi acid boric). 40

2.1.10. Bromid Phn ng vi AgNO3: cho ta vng nht AgBr, ta ny kh tan trong dung dch amoniac 10M. Phn ng oxy ho Br- thnh Br bng: PbO + CH COOH hoc KMnO +
2 2 3 4

H2SO4. Nhn bit Br2 bng cch chit vo cloroform c mu vng hoc nu: 2Br + PbO2 + 4H+ = Br 2 + Pb2+ + 2H 2O 10Br + 2MnO
4

+ 16H+ = 5Br + 2Mn2+ + 8H O

2 2

2.1.11. Calci (mui)

Phn ng vi amoni oxalat: trong mi trng trung tnh hoc CH3COOH long cho kt ta mu trng, kt ta ny d tan trong cc acid v c :
2+ +

Ca

+ (NH4)2C 2O4

= CaC2O4 trng

+ 2 NH4

Phn ng vi Kali ferocyanid: trong mi trng NH4Cl cho ta mu trng:

2+

Ca

K4 [(Fe(CN))6] + 2 NH4 = Ca(NH4)2[Fe(CN)6] trng

+ 4K

2.1.12. Ch (mui) Phn ng vi dung dch KI: cho kt ta mu vng, tan trong KI tha:
2+

Pb

2I -

PbI 2 vng

PbI2

2I -

2-

PbI4

Ta PbI2 tan trong nc nng, khi ngui kt ta tr li.

Phn ng vi dung dch K2 CrO4 cho ta mu vng, ta d tan trong HCl v NaOH: Pb2+ + K CrO
2

PbCrO vng
4

2K+

2.1.13 Citrat (C6H5O7 3)

3

Cho ta vi Ca khi un nng. Ta ny d tan trong dung dch CH COOH6M.

++

41

Phn ng to thnh acid acetondicarboxylic: khi un nng vi H2SO4 c (hay dung dch KMnO4), acid citric s b oxi ho thnh acid acetondicarbonic CO(CH2COOH)2. Acid ny to ta vi mui Hg2+.

2.1.14. Clorat Khng kt ta vi dung dch AgNO3. un nng vi dung dch HCl, H2SO4 long s b phn hu thnh Cl2 bay ra:

3HClO3 = HClO4 + Cl2 + 2O2 + H2O Tc dng vi NaNO 2: kh thnh Cl: ClO3+ 3NO2 = Cl + 3NO3
2.1.15. Clorid

Phn ng vi AgNO3 : cho kt ta AgCl mu trng, ta ny tan trong dung dch amoniac v kt ta tr li trong HNO3

AgCl

+ 2NH3

Ag(NH3)2 Cl

+ HNO3

AgCl

Phn ng vi KMnO4 trong mi trng acid: mt mu KMnO4:

2MnO4 + 10Cl + 17H+

2Mn2+ + 5Cl2 + 8H2O

Nhn bit Cl2 do c mi c bit hoc lm xanh giy tm h tinh bt c KI: Cl 2 + 2I

2.1.16. ng (mui)

2Cl + I2

(I2 lm xanh h tinh bt)

Phn ng vi K4[Fe(CN)6] Cho ta mu nu khng tan trong acid acetic: Cu2+ + K [Fe(CN) ] = CuK [Fe(CN) ]
4 6 2 6 nu

+ 2K+

2+ Phn ng vi dung dch amoniac: cho ta mui base mu xanh Cu2(OH)2 ,

mui ny tan trong amoniac d thnh phc mu xanh Cu(NH3)4 2CuSO4 + 2NH4OH = (NH4)2SO4 + Cu2(OH)2SO4

2+

Cu2(OH)2SO4 + (NH4)2SO4 + 6NH3 = Cu(NH3)42+ + 2SO42 + 2H2O

2.1.17. Ethanol Tc dng vi acid acetic (mi trng H2SO4) to ra ethyl acetat c mi thm:

42

C2H5OH + CH3COOH

C2H5COOCH3 + H2O

Tc dng vi dung dch I2 trong mi trng kim to ta mu vng iodoform (CHI3) c mi c bit: CH3CH2OH + 4I2 + 6NaOH = CHI3 + 5NaI + HCOONa + 5H2O

2.1.18. Iodid

Phn ng vi AgNO3 cho ta mu vng AgI, ta ny khng tan trong amoniac. Phn ng vi Fe3+: 2I + 2Fe3+ = I2 + 2Fe2+ I2 gii phng ra c th chit vo lp cloroform c mu tm .

2.1.19. Kali (mui ) Phn ng ngn ph c =mu 768 nm vla: 404mui nm ).Kali t cho ngn la mu tm (vch quang

Phn ng vi natri hexanitrocobantat : Cho kt ta mu vng (trong mi trng CH3COOH long):

2 K+

Na3[Co(NO2)6]

+ K Na[Co(NO ) ] vng + Na 2 2 6

2.1.20. Km ( mui )

Tc dng vi dung dch NaOH cho ta trng Zn(OH)2, ta ny tan trong kim d thnh mui Zincat, khi thm Na2S s cho kt ta trng ZnS: Zn2+ + 2OH = Zn(OH) 2 trng Zn(OH)2 + 2OH = ZnO2 2+ 2H2O

ZnO22+ Na2S + 2H2O = ZnS trng + 2NaOH + 2H2O

2.1.21. Magnesi (mui)

Phn ng vi dinatrihydrophosphat trong mi trng (NH4Cl + NH4OH) cho kt ta mu trng Magnesi amoniphosphat, soi trn knh hin vi c hnh l dng x:
2+ 2+

Mg

+ HPO4

+ NH4

+ OH + 5 H2O =

MgNH4PO4.6H2O trng

43

2.1.22. Natri (mui)

Phn mu la: Mui Natri t cho ngn la mu vng (vch quang ng ph c =ngn 589 nm). Phn ng vi Kalidihydro antimonat: cho kt ta mu trng (trong mi trng trung tnh hoc acid nh).

Na

+ KH2SbO4 trng

NaH2SbO4

Phn ng vi km uranyl acetat : cho kt ta mu vng natri km uranyl acetat (trong mi trng CH3COOH long) :

3 (UO 2)(CH3COO)2. 2 H2O =

+ Zn(CH3COO)2 + CH3COOH + Na+

+ H + NaZn(UO2)3(CH3COO)9.6H2O vng

(C th dng mui magnesi cng c).

2.1.23. Nhm (mui)

Vi thuc th alizarin S to hp cht ni phc c mu .

2.1.24. Nitrat

Phn ng vi FeSO4 + H2SO4 c: to ra NO, Fe2+ d s kt hp vi NO to thnh st (II) nitrososulfat c mu nu:

8 FeSO4 +

2NO3 + 3 H2SO4 + 2 H+ 3Fe2(SO4)3 + 4H2O + = 2[FeNO]SO4

Phn ng vi nitrobenzen: trong mi trng H2SO4 c to ra mdinitrobenzen, hp cht ny phn ng vi aceton trong mi trng kim to thnh phc c mu tm (Phn ng Janovsky):
NO2 + NO 3 H2SO4 NO2

NO2 H NO2 + CH3COCH

3

NaOH

CH2COCH3 NO2

Na NO2 NO2

44

2.1.25. Oxalat

Phn ng vi CaCl2 cho kt ta trng CaC2O4, ta ny khng tan trong cc acid v c, khng tan trong acid acetic long. Lm mt mu dung dch KMnO4 trong mi trng acid : 2MnO4 + 5C2O4 2 + 8H+ = 2Mn2+ + 10CO2 + 4H2O

2.1.26. Peroxyd Phn ng vi K2Cr2O7 trong mi trng acid:

Cr2O72+ 4H 2O2 + 2H+

= 2H2Cr2O6 + 3H2O (acid pecromic)

Acid pecromic c mu xanh c chit bng ether (acid ny d b phn hu trong mi trng nc).
Phn ng vi KI gii phng I2 c mu :

H 2O 2 + 2I + 2H+ =
2.1.27. Phosphat

I2 + 2H2O

Phn ng vi (NH4 )2 MoO4 trong mi trng HNO3: cho ta mu vng (lng t cho dung dch mu vng) amoniphosphomolipdat (NH4)3[PMo12O40]:
[NH4]3P[Mo12O40] + 21NH4NO3 + 12H2O

H3PO4 + 12(NH4)2MoO4 + 21HNO3

Phn ng vi AgNO3: cho kt ta bc phosphat mu vng, ta ny d tan trong acid v c v dung dch amoniac:
+ = AgPO4 vng 3 + + 3Ag PO4

2.1.28. Salicylat (C 6H 4OHCOO) Phn ng vi dung dch FeCl long cho phc mu tm Fe(OH) C H O
3 2 7 5

Phn ng vi HCl long cho ta acid salicylic C6H4OHCOOH (c nung chy 156oC - 161oC).

2.1.29. St (II)

Phn ng vi dung dch kalifericyanid to thnh ta c mu xanh lam (nng nh cho dung dch keo mu xanh l). 45

Khng tan trong dung dch HCl 2M: 3Fe 2+ + 2K [Fe(CN) ] = Fe [Fe(CN) ] xanh + 6K+
3 6 3 6 2

2.1.30. St (III)

Phn ng vi KCNS to phc mu Fe(CNS)x (3 x)+; (x t 1-6). Phc ny chit c trong ether, alcol (hoc mt mu khi thm Hg2+) Fe 3+ + x(CNS) Fe(CNS)4 + Hg
2+

= Fe(CNS)x (3x)+ () = Hg(CNS)4

2

+ Fe3+

Phn ng vi kaliferocyanid to thnh ta xanh lam khng tan trong dung dch HCl 2M : 4Fe3+ + 3K [Fe(CN) ] = Fe [Fe(CN) ] xanh + 12K+
4 6 4 6 3

2.1.31. Silicat

Hp cht ca Silic khi un vi H2SO4 c v NaF (hay CaF2) trong chn bch kim hay ch s to ra tetraflorid silic SiF4 dng hi (hoc hexaflorosilicat SiF6), d b thu phn to ra ta l acid metasilisic H2SiO3 (hoc acid octosilisic H4SiO4). Phn ng vi amonimolipdat trong mi trng HNO3 cho ta mu vng : H2SiO3 + 12(NH4)2MoO4 + 20 HNO3 = (NH4)3[SiMo12O40] + 20 NH4NO3 + 11H2O (amonisilicomolipdat)

2.1.32. Stibi (mui)

Cho phn ng vi natrisulfid (Na2S) to thnh ta Sb2S3 hoc Sb2S5 c mu vng cam. Cc ta ny tan trong dung dch sulfid d hoc polysulfd kim hay amoni cho cc mui SbS5 4 hoc SbS . Ghi ch: Thng ho tan cc dng ca mui Stibi bng dung dch natrikalitartrat, khi Stibi di dng mui ni phc tartrat kp K v Sb d tan: KOOCCHOH - CHOH-COOSbO. Sau cho phn ng to Sb2S3 hoc Sb2S5.
2.1.33. Sulfat

Cho phn ng kt ta vi dung dch BaCl2 (to ta BaSO4) mu trng, ta ny khng phn ng vi I2, SnCl2 ). 46

2.1.34. Sulfid (S2) Tc dng vi HCl long gii phng kh H2S c mi thi Phn ng vi Pb2+ to kt ta mu en PbS. 2.1.35. Bisulfit v sulfit (HSO3 v SO32) Tc dng vi dung dch HCl gii phng kh SO2 c mi c bit. Lm mt mu I2 :

SO32+ I2 + H 2O = H 2 SO4 + 2I
2.1.36. Tartrat (C4H4O6 2 v HC4H4O6)

Khi phn ng vi H2SO4 c + resorcin: acid tartric gii phng ra v phn hu thnh aldehyd glycolic OHH2CCHO, cht ny s to mu tm m vi resorcin.
2.1.37. Thiosulfat

Phn ng lm mt mu dung dch I2; S2O32+ I2 = S4O62+ 2I

Tc dng vi HCl cho kt ta S v gii phng kh SO2:

S2O32+ 2H+ = S + SO2 + H2O

Tc dng vi AgNO3 cho kt ta vng chuyn dn sang en:

S2O32+ 2Ag+ = Ag2S2O3vng Ag2Sen

2.1.38. Thu ngn (I v II) Phn ng hn hng vi ng: Hg2+ + Cu = Hg + Cu2+

Khi lm, trn ming ng s thy vt hn hng sng bng, t nng vt bng s mt (v Hg bay i).
Vi KI:

Hg

2+

+ 2I-

= HgI2

+ 2 I-

HgI 4 (khng mu)

2+

+ 2 I-

2-

Hg2 + 2I

= Hg2I2 vng lc

HgI4

Hg en

47

2.2. Th gii hn cc tp cht trong thuc 2.2.1. Mc ch

Xc nh gii hn tp cht trong thuc thc cht l th tinh khit ca thuc nhm xc nh phm cht ca thuc. Nu thuc cng tinh khit th hiu qu tc dng cng cao. Cc tp cht trong thuc mc d rt nh nhng n c th: Gy tc hi cho sc kho (th d tp cht bari tan, arsen, ch ). Gy hin tng tng k ho hc, nh hng n phm cht hay bn vng cu thuc. Mt s tp cht c th khng c tc dng c hi nhng li l nhng cht xc tc y nhanh qu trnh phn hu thuc (th d: cc vt kim loi, m ). Mt s tp cht khng gy hi, khng gy tng k ho hc, khng lm phn hu thuc, khng gy phn ng ho hc nhng n biu th cho mc sch (hay mc tinh ch cha ) ca thuc. Khi bit mc tinh khit ca thuc (c bit trong trng hp khng t yu cu) cho php xem xt cc ngun gc gy ra cc tp cht ny v tm bin php khc phc. Cc nguyn nhn c th l: + Nguyn liu, ph liu hoc bn thnh phm dng sn xut thuc cha tinh khit. + Qui trnh sn xut qui nh khng c thc hin nghim chnh. + nh hng ca cc dng c s dng. + Phng php sn xut cha tt. + Trong qu trnh bo qun, cc phn ng ph do nhiu yu t nh: mi trng, vn v sinh, cht bo qun lm pht sinh cc tp cht. + Do dng gian ln ca ngi sn xut Bi vy, TCVN (Dc in) v TC thng qui nh cho php mi thuc ch c c nhng lng rt nh cc tp cht nht nh bo m cho thuc c sch nht nh tc l thuc c cht lng, t hiu qu tc dng cao nht.
2.2.2. Phng php xc nh gii hn tp cht trong thuc 2.2.2.1. Phng php xc nh

Xc nh gii hn tp cht trong thuc tc l xc nh xem cc tp cht c vt qu gii hn cho php hay khng, cc phn ng th tp cht c tnh cht bn nh lng v c thc hin bng phng php so snh: Ly hai bnh (thng l 2 ng nghim) thc hin phn ng. 48

Bnh 1: Ly mt th tch dung dch thuc em th. Bnh 2: Ly mt th tch dung dch mu. (Dung dch mu l dung dch c cha tp cht cn th vi s lng cho php). Sau tin hnh song song phn ng th tp cht vi cng mt thuc th. So snh kt qu phn ng hai bnh (thng l so mu hoc so c) t xc nh c gii hn tp cht cn th c trong mu thuc em th. Trong qu trnh thc hnh cn phi theo cc qui nh sau: Nc v nhng ho cht, thuc th s dng khng c c tp cht ang cn th. Khi pha dung dch mu phi s dng cn phn tch v dng c th tch chnh xc. Hai bnh phn ng so snh phi ging nhau: bng thu tinh khng mu, c ng knh bng nhau, dy nh nhau Khi so snh, quan st c th nhn t trn xung, quan st mu th nhn ngang trn nn trng. Phi cho cc thuc th vo hai bnh phn ng ging nhau v: thi gian, s lng v th tch cui. Khi phn tch, nu pht hin c mt tp cht l th phi ghi li v bo co.
2.2.2.2. Pha cc dung dch mu

pha dung dch mu ca mt tp no , ch cn cn lng chnh xc cht tinh khit ca tp (cht gc) pha vo mt th tch xc nh theo tnh ton ta s c mu tp chun c nng xc nh (thng biu th theo mg/ml; % hoc phn triu).
Th d: Pha dung dch mu Cl- nh sau :

Dung dch A: Cn chnh xc 0,8238g NaCl tinh khit ho tan trong nc v thm nc cho 1 lt (dng bnh nh mc). c dung dch c cha:

35,5 0,8238 = 0,0005 g Cl /1ml 58,5 1000

Dung dch B (dung dch mu chun em th): Ly chnh xc 1ml 00 dung dch A pha long bng nc cho 100,0 ml. Dung dch ny c cha

0,0005 = 0,000005g Cl / ml (tng ng 0,005 mg Cl/ ml hay 100

dung dch 0,0005% hoc 5 phn triu).

49

2.2.2.3. Pha dung dch th

pha, gi thit mu em kim tra c cha mt lng tp cht cho php ti a, t tnh h s pha long thch hp, sau tin hnh pha theo tnh ton ny. Th d: Pha dch ): th tp Cl trong paracetamol (theo tiu chun Cl khng dung c qu 0,01% V dung dch mu Cl khi em th l dung dch c cha 0,0005% (hay 5 phn triu). Do h s pha long dung dch th s l

0,01 0,0005 = 20 Ta c

cch pha nh sau: Cn 1,000 g paracetamol ho tan trong nc cho 20,00 ml, lc. Ly 10,00 ml dch lc em th v so snh vi 10, 00 ml dung dch mu chun Cl 0,0005%. (Thng trong Dc in hoc TC c ghi r cch pha dung dch th l da trn c s tnh ny).
2.2.3. Mt s thuc th trong cc phn ng ho hc xc nh gii hn tp cht
Ion cn th (tp cht) ClSO42Thuc th AgNO3 BaCl2 Sn phm Hin tng quan st Ta trng Ta trng

AgCl BaSO4

NH4+

Hg
Nessler

O Hg

NH

Mu vng (nu nhiu c nu )

Ca2+

(NH4)2C2O4

CaC2O4 AsH
3

Ta trng Giy tm HgCl2 chuyn t vng sang nu

Arsen

Zn + HCl

- Na2S (H2S) Kim loi nng - thioacetamid - acid mercaptoSt acetic - acid sunfosalicylic PbS en hoc nu

- Feri mercaptoacetat

Mu hng nu hay vng

Ferisulfisalicylat

50

Al/3 N

8-hydroxyquinolin Nhm ( pH 6) 8-hydroxyquinolin ( pH 10)

Mu vng rm (tan trong CHCl3)

(oxyquinolat nhm) Mu vng (tan trong CHCl3) Mu vng Ta trng

Magnesi

Oxyquinolat Mg

Phosphat Km

Sulphomolybdic K4[Fe(CN)6]

(NH4)3H4[P(Mo2O7)6] K2Zn3[Fe(CN)6]2

2.3. Chun acid - base trong mi trng khan

Chun trong mi trng khan da trn phn ng trung ho gia acid v base. Cho n nay c th thng k 4 thuyt chnh pht trin khi nim acid - base: Thuyt in ly acid - base trong mi trng nc ca Arrhenius - Ostwald Thuyt proton ca Bronsted-Lowry, Thuyt in t ca Lewis, Thuyt acid - base tng qut ca Usanovich. Vi mc ch gii thch ng dng cc phn ng acid - base trong nghim thuc, chng ta x dng thuyt proton ca Bronsted - Lowry. thuyt ny, acid v base to ra nhng cp acid - base lin hp, chng nhau mt proton. Phn ng acid - base l phn ng gia 1 acid v 1 thuc hai cp acid - base lin hp: acid 1/ base1 v acid 2/ base 2. Acid 1 + base 2 Acid 2 + base 1 kim Theo khc base

Cp th hai c th l phn t cht tan hoc phn t dung mi. Phn ng acid - base l phn ng cho nhn proton.
2.3.1. Vai tr ca dung mi

Trong phn ng acid - base, dung mi c th tc ng theo 2 hng.

Solvat ha cht tan: Nu dung mi c tnh acid, n lm tng tnh base ca cht tan B

51

V d: B + HS BH+ + S-

+ R NH + CH COOH R N H + CH COO 3 2 3 3
Ngc li nu dung mi c tnh base, ns tng tnh acid ca cht tan HA HA + S A + S+H Trong cc dung mi tr (dung mi khng cho hoc khng nhn proton), qu trnh solvat ha c thc hin do c ch khc (lin kt hydro, phc , lc Van der Waals).
Tc ng ln qu trnh in ly ca cp ion: Trong dung mi c hng s in mi ln (n c, formamid) hu ht cp ion to ra do qu trnh solvat ha cht tan u phn ly thnh cc ion t do.

Ngc li, trong dung mi c hng s in mi b, cc ion ch yu tn ti di dng cp ion. Qu trnh in ly ca cp ion do hng s quyt nh. C th phn chia s b nh sau: > 50 nh nc, formamid, dimetylsulfoxid: acid v base tn ti ch yu di dng ion t do . < 30 nh ethanol, aceton: tn ti nhiu cp ion < 10 nh benzen, cloroform, acid acetic: tn ti cp ion l ch yu. Cn lu l hng s ch tc ng ln qu trnh in ly ca cp ion. Nu trong phn ng acid - base khng to ra cc ion c in tch ngc du, do khng to cp ion th hng s t tc ng ln qu trnh in ly. Phng trnh tng qut m t qa trnh ion ha (th hin hng s Ki) v qu trnh in ly (th hin hng s Kd) ca cht tan HA trong dung mi S c th hin nh sau: ki HA + S HS+A solvat ha
2.3.2. Khi nim pH

kd HS+ + A in ly

Trong dung dch nc, ngi ta nh ngha pH = - lgaH+ Trong dung mi khan, ngi ta xc nh pH biu kin. 52

Thang ny lin quan n hng s t in ly KS ca dung mi. HS + HS H2S+ + S

H 2S
+

H2S + l ion lionium, S- l anionlyat, pH biu kin l pH = - lg a a V d: 2 CH3COOH CH3COOH2+ + CH3COOH 2S

+

. aS = KS

KS = [CH3COOH2 + ] [CH 3COO-] 2C2H5OH C2H5OH2 + + C2H5O -

KS = [C2H5OH2 + ] [C 2H 5O-] di ca thang pH biu kin tu thuc vo hng s t in ly ca dung mi. V d: Methanol: pKS== 17 Ethanol: pKS = 20 Acid acetic: pKS = 6,35 2.3.3. Xc nh im tng ng nh lng trong mi trng khan thng c thc hin bng cch chun . pht hin im tng ng thng dng 2 phng php:
Ch th mu pH: Theo di s i mu ca ch th. Thng dng tm tinh th, tm metyl. Ngoi ra cn dng cc loi ch th mu hn hp. Ch th o th: y in cc so snh c th l in cc calomel hoc bc clorid, in cc ch th thng l in cc thu tinh (nu c th, dng dung dch trong bu thu tinh c cng dung mi vi mi trng chun ).

c kt qu tin cy khi chun trong mi trng khan cn lu : Khi xc nh im tng ng theo di s thay i ca in th, khng phi ca pH, Cn trung ho dung mi trc khi chun , Cn x l in cc ch th ph hp. 53

2.3.4. ng dng kim nghim thuc

Chun trong mi trng khan c p dng khi: Cht phn tch khng ho tan trong nc. Trong kim nghim thuc, thng gp cc acid v base c khi lng phn t ln t tan trong nc. Sc acid, base qu yu trong nc nn kh pht hin im tng ng. Cc acid, base a chc c cc hng s in ly trong nc t khc bit nhau. Sau y s gii thiu cc loi dung mi, dung dch chun trong nh lng cc acid v base.
2.3.4.1. nh lng acid

Cc cht hu c c tnh acid yu thng c chun bng base trong mi trng khan nh: Cc acid carboxylic, Dn xut enol, imid, sulfonamid, Dn xut th phenol nh polyclorophenol, polynitrophenol. Hn hp cc cht c tnh acid hoc acid a chc .
Dung mi Thng chn dung mi c tnh base tng tnh acid ca cht phn tch nh: pyridin, dimetylformamid (DMFA). Ngoi ra tert - butanol thng c dng lm dung mi cho chun acid carboxylic, dn xut ca phenol. Dung dch chun Thng dng cc dung dch chun base nh:

KOH trong alcol (thng dng trong methanol), Metylat kim loi kim nh natri, kali, Tetraalkyl amonium hydroxyd: thng dng tert - Bu4NOH trong hn hp dung mi benzen - methanol (95:5). Khi dng cc dung dch chun ny cn lu : Dung dch chun kim loi kim gy sai s base cho in cc thu tinh khi chun o th. Dung dch chun R4NOH l base mnh, mnh hn dung dch hydroxyd kim nh KOH, cho nn c th chun cc acid rt yu. Tuy nhin cc dung dch ny c 2 nhc im: + c do c benzen + Pha ch mt nhiu thi gian, kh bo qun (d phn ng vi CO2 ca khng kh). 54

 Cht chun Cc cht chun acid thng dng l:

Acid benzoic: C6H5COOH, E = M = 122,12 Acid succinic (CH2COOH)2, E = M/2 = 59,05 Acid sulfamic NH2SO3H, E = M = 97,09 Kalihydrophtalat, E = M = 204,22.
Phn ng chun Ly dung mi in hnh l pyridin

HA + C5H5N

C5H5N + HA

chun cc enol, thiol, ngi ta thng thm AgNO3 vo mi trng RSH + C 5 H 5 N + AgNO3
2.3.4.2. nh lng cc base hu c
+ RSAg + C 5 H 5 N HNO3 -

Cc alcaloid v base nit tng hp thng c chun bng cc acid chun trong dung mi acid.
Dung mi tng tnh base ca cht phn tch ngi ta dng cc dung mi acid nh acid acetic khan.

Acid acetic khan thng c x dng ph bin nht v t c, r tin. Dng anhydrid acetic cng hay dng v c th nh lng cc base rt yu. Hn na bc nhy th im tng ng trong anhydrid acetic thng ln hn trong acid acetic. Tuy nhin anhydrid acetic c nhc im l d acetyl ha cc amin bc nht v bc hai nht l khi un nng. (CH3CO)2O + RNH2 CH3CONHR + CH3COOH

Trong trng hp nh lng amin khng dng dung mi ny. Ngoi acid acetic, acetonitril c dng nh lng cc base vi dung dch chun acid percloric trong 1,4 dioxan.
Dung dch chun Dung dch acid percloric trong acid acetic khan thng c dng nhiu nht. Dung dch ny pha ch t acid percloric thng mi 72% (kl/kl). V vy khi pha ch phi thm anhydrid acetic loi nc v 48 gi trc khi dng.

Ngoi acid acetic khan, cn dng 1,4 dioxan pha dung dch chun acid percloric. Dung dch ny km n nh, d chuyn thnh mu nu trong qu trnh bo qun. V vy ch pha v s dng khi cn thit. 55

 Cht chun Thng dng kali hydrophtalat. Phn ng chun Thng c vit thnh 2 giai on

Solvat ha RNH 2 + CH 3 COOH Trung ho RNH3+CH3COO- + HClO4

RNH 3+CH 3COO-

RN+H3Cl-O4 + CH3COOH

Nu cht cn nh lng t tan, phi un nng. Nhng cn thn trng v c th acetyl ha chc amin I hoc amin II. i khi ngi ta thm acid formic vo dung mi acid acetic khan thay cho un nng lm tng tan (do hng s ca dung mi tng ln). 2.3.4.3. nh lng cc mui Nhiu dc cht l mui ca cc base hu c. Chn phng php nh lng chng trong acid acetic tu thuc vo anion to mui vi base.
Mui ca cc acid yu hn acid acetic BH+ YY- l anion propionat, maleat, benzoat, salicylat,... Trong trng hp ny mui BH+Y- c chun trc tip nh mt base bng HClO4:

Anion Y- c trung ho bng HClO 4. V vy phn ng khng c hiu.

Mui ca acid mnh hn acid acetic BH+XX- l cc halogenid, anion sulfat. Ngi ta phn ra hai trng hp:

Mui halogenid (thng hydroclorid, hydrobromid) Cch n gin nht l nh lng X- nh Y- trong trng hp trn. Nhng nu X l base rt yu s khng phn ng ton lng vi HClO 4. gii quyt kh khn ny, ngi ta dng 2 phng php: + Phng php Pifer - Wollish Thm vo mi trng mt lng d Hg(II) acetat gii phng ion acetat. Trong mi trng acid acetic, n l base mnh c chun bng HClO4 2BH+X- + Hg(CH3COO)2
+

2CH3COO-BH+ + HgX2
+ -

2CH3COO BH + 2HClO4

2CH3COOH + 2BH ClO4

y nh lng mui BH+X- thng qua anion CH 3 COO- (thay cho anion X-) tng t Y- trong trng hp trn. 56

+ Phng php Billon Ho tan mui trong ethanol, BH+ trong dung mi ny l acid tng i mnh, c th c trung ho bng NaOH 0,1N. Phn ng nh lng tri qua 2 giai on u tin thm mt lng d HCl chuyn ht base B (nu c trong BH X ) sang dng mui
+ -

B + HCl

BH+ + Cl-

Hn hp BH+ X- v HCl d c trung ho bng NaOH HCl + NaOH BH+ X- + NaOH NaCl + H2O NaX + B + H 2O

Pht hin im tng ng bng chun o th: c 2 bc nhy th ng vi trung ho HCl d v BH+X- trong mu phn tch. Nhc im chnh ca phng php ny l s chnh lch sc acid ca HCl v BH+X- phi ln c 2 bc nhy. Mt khc B l base khng qu yu trong ethanol. Mui sulfat H2SO4 c th to nhiu dng mui vi base hu c n chc (stricnin, atropin) hoc a chc (quinin). + Mui dng BH+HSO - y anion HSO - l base kh mnh c th nh
4 4

lng bng HClO4 trong acid acetic. + Nu base hu c to mui trung tnh: dng (RN)2H2SO4 hoc (RNH+)2SO42- nh strycnin sulfat, atropin sulfat (RNH+) SO
2 24

+ HClO

RNH+ClO

+ RNH+HSO

Vi quinin bisulfat cng xy ra tng t R (NH+) SO 2- + HClO (NH+ HSO -)R (NH+ . ClO -)
2 4 4 4 4

1 mol HClO4 ng vi 1 mol mui sulfat + Mui khng trung tnh nh quinin sulfat basic
22 4

R(NH+N) SO

+ 3 HClO

(N+H ClO -)R(NH+ HSO ) + R(NH+ClO -)

4 4

4 2

1 mol mui cn 3 mol HClO4 trung ho. Mui tetraalkyl amonium bc bn Cation R4N+ c th lin kt vi hydroxyd, halogenid (X-), phosphat. Cc anion tham gia vo phn ng trung ho nh mt base. 57

Vi hydroxyd: trung ho trc tip bng HClO4

+ Vi halogenid: thm Hg (II) acetat nh trong phng php Pifer Wollish.

2.4. Xc nh hm lng nc bng thuc th karl fischer

C nhiu k thut xc nh hm lng nc trong cht rn, trong dung mi hu c. y gii thiu phng php dng thuc th Karl Fischer da vo tnh cht oxy ha ca iod.
2.4.1. Nguyn tc

Thuc th ny gm c iod, SO2, methanol v pyridin. Hn hp phn ng vi nc theo hai phng trnh sau: C5H5N.I2 + C5H5N.SO2 + C5H5N + H2O C5H5N. SO3 + CH3OH 2C5H5N.HI + C5H5N.SO3 C5H5N(H)SO4CH3

Hn hp c lng ln pyridin nn cc cht tham gia phn ng v sn phm u tn ti di dng phc. Phn ng u tiu th 1 phn t nc. Phn ng th 2 xy ra khi c d CH3OH l cn thit, quyt nh s thnh cng ca chun v phc SO3 cng phn ng vi nc C5H5N. SO3 + H2O C5H5N(H)SO4H

y l phn ng ph. loi b phn ng ny, ngi ta dng methanol d trong hn hp.
2.4.2. Pha ch v xc nh chun Pha ch: C ch phn ng ch r mt phn t iod oxy ho mt phn t SO2 tiu th mt phn t nc trong mi trng c d pyridin v methanol. Khi pha thuc th ngi ta dng mt lng d SO2 v pyridin. V vy phn ng ca thuc th vi nc do lng I2 quyt nh. chun ca thuc th thng 2 n 5 mg H2O/ ml. Lng SO2 d gp 2 ln, pyridin gp 3 - 4 ln.

chun ca thuc th gim dn trong qu trnh bo qun. V vy thng ch pha ch trc 1 - 2 ngy. C trng hp pha thnh hai dung dch: Dung dch A: SO2 v pyridin trong methanol, Dung dch B: I2 trong methanol khan. Khi dng trn 1 th tch A vi 1 th tch B. 58

 Xc nh chun ca thuc th: c 2 cch. 1. Xc nh hm lng nc di 1%, ngi ta chn mt ha cht c hm lng nc kt tinh xc nh, sy kh loi m. Cho thuc th tc dng vi ha cht ri tnh ra ng lng. Mui natri tartrat dihydrat (C4H4Na2. 2H2O) hay c la chn.

2. Xc nh hm lng nc trn 1%, xc nh hm lng nc theo dung dch chun nc/ methanol.
2.4.3. Xc nh im tng ng

Hai cch ph bin xc nh im tng ng ca phn ng nh lng: 1. Theo lng tha ca iod khi nc phn ng ht. S i mu do tha thuc th. 2. Chun amper vi 2 in cc platin (chun n im dng). Mt s nh sn xut cho ra i cc dng c chun t ng dng thuc th Karl Fischer.
2.4.4. ng dng

Thuc th Karl Fischer c dng xc nh hm lng nc trong nhiu dng mu khc nhau. Da vo c im ca mu ngi ta xy dng qui trnh phn tch cho ph hp. Nu mu d tan trong methanol, ngi ta dng chun trc tip nc trong cc cht hu c nh acid, alcol, ester, anhydrid, k c cc mui ngm nc. Nu mu t tan trong thuc th, ngi ta dng chun tha tr: cho mt lng tha thuc th. Sau thi gian phn ng thch hp, xc nh lng d bng dung dch chun nc trong methanol. Mt ci tin khc l chit hi lu nc trong mu bng methanol khan. Xc nh lng nc chit xut c bng chun trc tip. K thut ny rt thch hp cho nh lng nc hp th, nc kt tinh. Cn lu l mt s phn ng ha hc cn tr phng php Karl Fischer do to thnh nc sau phn ng: Cc hp cht carbonyl tc dng vi methanol: R CHO + 2CH3OH R CH (OCH3)2 + H2O

Oxyd kim loi phn ng vi HI MO + 2 HI MI2 + H2O 59

Ngoi ra cc cht oxy ha kh cng thng cn tr phng php Karl Fischer v cht oxy ha s phn ng vi iodid l sn phm ca thuc th, cn cht kh phn ng vi iod ca thuc th.
2.5. nh lng mt s cht hu c a chc bng thuc th periodat 2.5.1. Nguyn tc y l phng php nh lng da vo tnh cht oxy ha ca cp I7+/ I5+. Tu theo mc hydrat ha ca I2O7 ta c 2 dng acid:

I2O7 + H2O I2O7 + 5H2O

2HIO4 : acid metaperiodic 2H5IO6 : acid paraperiodic

Trong dung dch nc cc acid v ion periodic nm trng thi cn bng, H4IO6 - , IO 4 -, H 3 IO6 2-. Cp oxy ha kh periodic H5IO6 + H+ + 2e IO3- + 3H2O

Th chun khong 1,6 V. Periodat l cht oxy ha mnh

Phn ng oxy ha ca periodat vi cc cht hu c c d bo theo 4 nguyn tc sau:

B gy lin kt C C c mang 2 nhm chc cnh nhau nh: chc C = O, - NH2, - OH. Nguyn t C c nhm chc OH b oxy ha thnh aldehyd hoc ceton . Nguyn t C c nhm chc ceton c chuyn thnh acid COOH. Nguyn t C c nhm - NH2 s chuyn thnh aldehyd ng thi to ra NH3. Ba phn ng sau minh ho 4 nguyn tc trn H3C CO CO CH3 + HIO4 + H2O HIO3 + 2 CH3COOH

2H3C CO CH(OH) CH3 + H2O + HIO4 H2C CH2 + HIO4 HO NH2

HIO3 + CH3COOH + CH3CHO

2HCHO + NH3 + HIO3

Trong cc phn ng trn, ngi ta thng nh lng bng cch cho tha thuc th HIO4 vo mu phn tch. Sau khi phn ng kt thc cho lng d As2O3 v dung dch KI. Iod gii phng c chun bng dung dch Na2S2O3. Song song lm mt mu trng. l nguyn tc ca phng php Fleury. C trng hp ngi ta dng phng php iod xc nh nng periodat ch vi lng d KI, khng cn dng As2O3.

60

Dung dch chun Nu phn ng oxy ha thc hin trong mi trng acid nh dng dung dch H5IO6. Nu trong mi trng acid mnh dng dung dch NaIO4 hoc Na3 H2IO6 (thng pha trong dung dch H2SO4). nh lng t hng tin hnh theo cch gin tip, c hiu chnh vi mu trng.
2.5.2. ng dng

Ngi ta t quan tm n ng dng ca chun periodat cho nh lng cc cht kh v c bi c nhiu thuc th oxy ha d thc hin, khng t tin nh Cr2O72-, MnO4-, I2. Ngc li ngi ta ch n phn ng ca periodat vi cc cht hu c a chc do tnh cht oxy ha chn lc ca n. Trc y cc phn ng ny c dng trong phn tch cu trc. Hin nay, tnh oxy ha ca periodat c s dng trong phn tch nh lng.

C th dng thuc th periodat nh lng nhiu cht hu c nh:

Cc .diol: etylenglycol, propradiol 1,2 Polyol : phn ng tng qut Cn lu l :

H2COH (CHOH)n CH2OH + (n+1) HIO4 2HCHO + (n) HCOOH + (n+1)HIO3+ H2O

+ S phn t formadehyd to thnh bng s chc alcol bc nht. + S phn t acid formic to thnh bng s chc alcol bc 2. Vi cc aldose ta c th vit:
CH2OH(CHOH)nCHO+(n+1)HIO4 (n+1)HIO3+HCHO + (n+1)HCOOH

Cc diamin nh etylen diamin, amino - alcol nh ephedrin. C th minh ho ng dng ca phng php periodat thng qua nh lng glycerin ca Dc in Php X + Dng HIO4 c d oxy ha ht glycerin + Thm propylenglycol loi ht HIO4 d + Dng dung dch chun NaOH trung ha ht acid to thnh sau phn ng (HIO3 v HCOOH) + Song song lm mt mu trng. Tnh kt qu da vo lng NaOH dng cho mu phn tch v mu th.
2.6. ng dng cp ion trong kim nghim thuc

K thut to cp ion c trin khai u tin trong phng php chit lng - lng vi mc tiu tng hiu sut chit t dung dch nc ca mt s ion. 61

Sau , k thut ny c ng dng nhanh chng nh lng nhiu dc cht mang tnh acid hoc base di dng phn tch th tch hoc o quang. Hin nay, k thut to cp ion xm nhp vo lnh vc sc k khng ch nh gi cht lng thuc m c trong nghin cu dc ng hc v phn tch ha sinh. Trong phm vi gio trnh kim nghim thuc, chng ti ch trnh by s lc khi nim v cp ion v nguyn tc ng dng ca n.
2.6.1. nh ngha cp ion

Cp ion c hnh thnh do tng tc tnh in gia 2 ion c in tch tri du: 1 ion dng v 1 ion m. Theo thuyt lin hp Bjerrum (nm 1926) th hai ion solvat ha c in tch tri du khi chuyn ng nhit trong dung dch s tin li gn nhau do lc ht Couloms. n mt khong cch no gia 2 ion c gi l khong cch c trng q, chng to thnh mt tiu phn ng hc chuyn ng t do nh cc ion. Tiu phn ng hc l cp ion (ion pairs). Vi cc cht in ly i xng (1 -1, 2 2, ... ) cp ion to thnh khng mang in tch. Vi cc cht in ly khng i xng (1 - 2, 1 3, 2 3, ... ) cp ion c in tch nh hn in tch ca cc ion ban u. Trong c hai trng hp dn in ca dung dch s gim xung. Khong cch c trng q ni trn c xc nh bi t l gia nng lng to cp ion v nng lng chuyn ng nhit trung bnh tch ring cc ion. Gi tr q c tnh theo phng trnh sau:

za zk q=
Trong :

2kT

za v zk l in tch ca anion v cation, e l in tch ca in t k l hng s Boltzmann l hng s in mi ca dung dch, T l nhit tuyt i (0K) Khong cch q ph thuc in tch zion ca ion hng s : nu in tch cng ln v cng nh th q vo cng tng, cp to rav cng nhiu. 0 Vi cht in ly 1 -1 trong dung dch nc 25 C th q = 3,6 A0 (1A0 = 10-8 cm). iu c ngha l trong dung dch nc, cht in ly 1-1 ho tan, v d mui KCl, khi 2 ion K+ v Cl- khong cch q < 3,6 A0 s to thnh cp ion. Chng chuyn ng t do trong dung dch nh cc ion K+, Cl- v phn t khng in ly. Cp ion trung ho in tch li do va chm lm gim bt lp v hydrat ha trong dung dch nc nn d chuyn sang ho tan trong dung mi hu c. 62

2.6.2. Cc yu t nh hng n s to thnh cp ion

Ngoi yu t in tch z v hng s , s to thnh cp ion cn chu nh hng ca 2 yu t sau:

c im ca ion Nu ion cng ln v cng s nc, cng t b hydrat ha trong dung dch nc th cng d b solvat ha bi cc phn t dung mi hu c. pH ca dung dch nc c th to cp ion t cc cht mang tnh acid, base th pH dung dch phi bo m tn ti c cation (t base) v anion (t acid).

V d: Cc alcaloid v base nit tng hp c th mang in tch dng khi cng proton: R NH2 + H+ Chng c th to cp ion pH acid. Vi cc cht c tnh acid, chng to ra anion cng ty theo pH: R- COOH RCOO- + H+
+ +

R NH 3

to ra cp ion R N H 3 O OCR ' cn c pH thch hp.

2.6.3. ng dng

K thut to cp ion c ng dng nhiu trong phn tch th tch, o quang v sc k.

2.6.3.1. Phn tch th tch

Thng dng cch chun hai pha. V d: chun metoclopramid hydroclorid (MetH+Cl-) bng dung dch chun natri laurylsulfat (Na+LS-) trong h dung mi nc metylen clorid (CH2Cl2) vi ch th metyl (MIn). Khi anion LS- phn ng vi cation MetH+ (to cp ion MetH+ LS-), anion ny s kt hp vi ch th to cp ion MInH+ LS- tan trong dung mi CH 2 Cl 2 . Pha ny chuyn t mu vng (do c phn t ch th MIn ha tan) sang mu (mu ca cp ion MInH+ LS-).

63

Met H+ClH2O MIn + H+

Cl- + Met H+ MInH+ LS- + Na+ NaLS

CH2Cl2

Met H+LS-

MInH+LS-

Nhiu alcaloid v base tng hp c chun theo nguyn tc ny.

2.6.3.2. Chit o quang

y l phng php thng c dng nh lng bng o hp th ca cc cht c tnh acid hoc base.
Chit cc base dng ch th mu acid: Cc ch th ny l i ion to cp vi base mang in tch dng (khi cng hp proton) pH thch hp. Cc ch th thng dng thuc nhm azoic (metyl da cam, tropeolin 00, ...), bromophenol, bromothymol, ...

Chit cp ion bng cloroform v o quang. Cc alcaloid v base tng hp c nh lng theo nguyn tc ny. V d: Alcaloid: morphin, codein, cocain, atropin, quinin, ... Base tng hp: novocain, quinolon, ... Mui amonium bc bn: tetraetyl amonium clorid (Et4NCl), tetrabutyl amonium iodid (Bu4NI), ...
Chit cc acid hu c Cc acid ny khi phn ly thnh anion s to cp ion vi cc cation l cc ch th mu base (lc malachit, tm tinh th, rodamin v dn xut ...).

Cc dn xut phenol v acid hu c c chit di dng cp ion v o quang nh: Dn xut phenol: 2,4 dinitrophenol, acid picric. Dn xut ca acid benzoic nh: acid 3,5 dinitrobenzoic, acid 2 nitrobenzoic. Dn xut ca acid salicylic nh: acid 4 - nitro salicylic, acid 3,5 dinitrosalicylic. Mui alkyl sulfat: lauryl sulfat, dodecyl sulfat. Mui acid hu c mch thng nh: oleat, stearat. 64

2.6.3.3. Sc k lng hiu nng cao (HPLC)

Khi xy dng chng trnh HPLC phn tch cc dc cht, thng thng ngi ta s dng k thut to cp ion. V d, nh lng cc cht hu c mang in tch dng, ngi ta thng thm vo pha ng cc cht c th to ra anion to cp nh natri lauryl sulfat, natri heptansulfonat, natri octansulfonat. Vn ny s c cp c th hn mn hc cc phng php sc k trong chng trnh cao hc.

Ti liu tham kho

1. B Y t (2002). Dc in Vit Nam III. NXB Y hc, H Ni. 2. H Nh Ph (1971). Kim nghim thuc. NXB Y hc, H Ni. 3. Phm Hi Tng, Phm Gia Hu (1987). Ho hc phn tch. NXB Y hc, H Ni. 4. Nguyn B Hip (1988). Kim nghim Dc Phm. NXB KHKT, H Ni. 5. Block (1959). Phn tch nh tnh (phn II phn ng cation) Bn dch ting Vit. NXB Gio dc, H Ni. 6. British pharmacopeia BP 2001 The United States of Pharmacopeia USP XXIV (2000). 7. Conors K. A (1982). A Textbook of Pharmaceutical Analysis, third edition, John Wiley & Sons, Inc, pp 3 - 92. 8. Delvordre - Steinmetz A. C., Prognon P. (1992). Protomtrie en milieu aqueux et non aqueux dans Analyse pratique du mdicament. Coordonateur D. Pradeau, Editions Mdicales internationales, Paris, pp 288 - 344. 9. Le Hoang M. D, Prognon (1992). Oxydo - reductimtrie, dans Analyse pratique du mdicament. Coordonateur D. Pradeau, Edition Mediales internationales, Paris, pp. 352 - 385. 10. Skoog D. A, West D. M, Holler F. J (1988). Fundamentals of Analytical chemistry. Sounders College Publishing, pp 233 - 344. 11. The Merck Index (1996). 12th edition. 12. The United States of Pharmacopeia USP XXIV (2000).

65

cu hi t lng gi
2.1. Gii thch phn ng th nh tnh ca: Cc ion: Amoni; Nitrat; Phosphat; Arseniat v arsenit; Thu ngn; St (II v III) Phn t: Ethanol 2.2. Gii thch mc ch ca vic th gii hn tp cht trong thuc. 2.3. Trnh by phng php xc nh gii hn tp cht trong thuc 2.4. Cc cch pha dung dch mu v dung dch th xc nh gii hn tp cht trong thuc. 2.5. Trong dung dch nc, amin l mt base yu, nhng trong dung mi acid acetic, n l mt base mnh. Ti sao ? 2.6. Pha dung dch chun acid percloric bng cch ly 17,0 ml acid acid percloric c 72% (kl/kl) pha va thnh 1000 ml dung dch acid acetic khan. Tnh s ml anhydrid acetic cn thit phn ng ht vi lng nc c trong 17 ml acid percloric c dng. Bit khi lng ring ca acid percloric l 1,60 g/ ml v ca anhydrid acetic l 1,02 g/ ml. (42,3 ml) 2.7. Vit phng trnh phn ng gii thch qu trnh nh lng acid barbituric trong pyridin vi s c mt ca AgNO3. Dung dch chun l KOH/ methanol. Nu cht cn nh lng l mui Na barbiturat, qu trnh phn ng c g khc khng ? 2.8. Ly 20 vin phenobarbital c khi lng 6,025g, nghin mn, cn 2,000 g ho tan trong DMFA, chun bng dung dch Lithi metylat 0,1000N ht 8,50ml. Tnh khi l ng trung bnh ca phenobarbital trong vin, bit M = 232,2 (29,8 mg/ vin). 2.9. Cn 0,6120 g diphenhydramin hydroclorid (M = 291,8) ho tan trong acid acetic bng. Thm 15 ml thu ngn acetat 3,2% v chun bng dung dch chun HClO4 0,1145N ht 17,12 ml. Tnh t l % ca dc cht trong mu (93,45%). 2.10. So snh nh gi 2 phng php Pifer - Wollish v Billon trong nh lng mui halogenid. 2.11. Ha tan 0,5404g mui vo methanol v nh mc thnh 50,00ml (dung dch A). Ly 5,00ml dung dch A thm 20,0ml methanol v chun vi thuc th Karl Fischer ht 9,00ml. Khi xc nh nc trong methanol trn dng ht 0,80ml thuc th Karl Fischer cho 25,0ml. Tnh chun ca thuc th Karl Fischer (6,21 mg H2O/ml).

66

2.12. Cn 0,2310 g mu phn tch ha tan vo 10,00 ml methanol ( bi tp 2.11) v chun bng thuc th Karl Fischer ht 2,40 ml. Tnh t l % nc trong mu phn tch trn (5,59%). 2.13. Vit phn ng xc nh nng dung dch chun H5IO6 theo phng php iod. 2.14. Ch r s mol ca mi sn phm to thnh khi 1 mol cc cht sau phn ng vi HIO4. Vit phn ng CH2OH(CHOH)4 CH2OH CH3 CH CHO NH2 CH3 CH CHO CH3 CH CO CH3 NH2 C6H5 CH CH2 CH2OH OH OH

2.15. V s gii thch nh lng HIO4 d theo phng php Fleury. 2.16. C g khc nhau gia phn t trung ha in v cp ion (xt v cu to v tnh cht). 2.17. Ti sao nhiu cp ion c th chit c (t dung dch nc) bng dung mi hu c t phn cc? 2.18. Gii thch ti sao pH dung dch l yu t quan trng nht n s hnh thnh cp ion? 2.19. Cetyl pyridin clorid l mt base nit bc bn c th to cp ion vi ch th vng metyl. Cp ion ny d tan trong cloroform. Anh (ch) xut nguyn tc chit o quang nh lng base ny. 2.20. Mt phng php dc in nh lng cetyl pyridin clorid di dng chun hai pha bng dung dch chun natri lauryl sulfat 0,004M vi ch th vng metyl (bi 2.19). Hy v s gii thch chun ny.

67

Chng 3

cc phng php ho l trong kim nghim thuc

Mc tiu hc tp

Gii thch c cch hiu chun my quang ph t ngoi - kh kin. Trnh by c cc phng php nh lng bng quang ph t ngoi kh kin. Gii thch v vn dng c k thut sc k pha o v pha o cp ion trong cng tc kim nghim thuc. Trnh by c cc phng php nh lng bng sc k lng hiu nng cao: phng php chun ngoi, chun ni.
3.1. Phng php quang ph phn t

Phng hunh quang dc in tinh khit cc

php quang ph phn t: Ph UV-VIS, ph hng ngoi, ph ng vai tr quan trng trong kim nghim thuc. Hu ht cc p dng phng php ny trong nh tnh, nh lng v th thuc v ch phm.

3.1.1. Quang ph hp th UV.Vis

3.1.1.1. hp th
Khi cho bc x n sc i qua mt mi trng c cha cht hp th th hp th ca bc x t l vi nng ca cht hp th v chiu dy ca mi trng hp th (dung dch cht hp th). Mi quan h ny tun theo nh lut Lambert Beer v c biu din bng phng trnh sau:

1 A = lg = lg I = K.C.L T I
0

(3.1)

Trong : T: truyn qua I0: cng nh sng n sc ti I: cng nh sng n sc sau khi truyn qua dung dch. K: l h s hp th ph thuc , thay i theo cch biu th nng L: l chiu dy ca lp dung dch C: nng cht tan trong dung dch 68

 Trng hp C tnh bng mol/l, L tnh bng cm A = . L . C (3.2)

Khi C = 1 mol/l v L = 1 cm th A = (h s hp th phn t ) c trng cho bn cht ca cht tan trong dung dch, n ch ph thuc vo bc sng. nh lut Lambert-Beer ch ng khi dng bc x n sc.

A =

1 % 1 cm

. L .C

1 % 1 cm

A L .C

3.3

Trng hp nng C tnh theo %(Kl/TT) v bng 1%, L = 1cm th: E (1%, 1cm ) l h s hp th ring, n c trng cho mi cht. Trong phn tch kim nghim hay dng E (1%, 1cm). 3.1.1.2. iu kin p dng nh lut Lambert-Beer nh sng phi n sc, Khong nng phi thch hp: nh lut Lambert-Beer ch ng trong mt gii hn nht nh ca nng . Dung dch phi trong sut. Cht th phi bn trong dung dch v bn di tc dng ca nh sng UV-VIS. 3.1.1.3. My quang ph My quang ph thch hp cho vic o ph vng t ngoi v kh kin bao gm mt h quang hc c kh nng to nh sng n sc trong vng t 200 n 800nm v mt thit b thch hp o hp th. Ngun sng cho vng t ngoi: n deuteri hoc hydro, Ngun sng cho vng kh kin: n tungsten. Hai cc o (cuvet) dng cha dung dch th v dung dch so snh phi c c tnh quang hc nh nhau. my loi 2 chm tia, cuvet dung mi c t bn c chm tia so snh (reference beam) i qua. Cuvet thch anh (quartz): dng o vng t ngoi v kh kin. Cuvet thu tinh: ch o vng kh kin. 3.1.1.4. Hiu chun my quang ph (Calibration) Trong nh tnh v nh lng cc cht bng phng php quang ph UV-VIS, vic chun ho my ng vai tr quan trng, c bit l trong k thut nh lng bng o ph trc tip. 69

Hu ht dc in cc nc (Vit Nam, Anh, n , Nht Bn, Chu u,...) u yu cu kim tra 6 ch tiu k thut ca my quang ph t ngoi kh kin nh sau: Kim tra thang di sng: Bng cch s dng cc i hp th ca dung dch holmium perchlorate. Vch ph ca n ngun hydro, n deuteri hoc cc vch ph ca n hi thu ngn c ch ra di y vi dung sai cho php vng t ngoi l + 1nm, vng kh kin l + 3nm:
241,15 nm 253,70 nm 287,15 nm 302,25 nm 313,16 nm 334,15 361,50 365,48 (H0) (Hg) (H0) (Hg) (Hg) (Hg) (H0) (Hg) 404,66 nm 435,83 nm 486,00 nm 486,10 nm 536,30 nm 546,07 nm 576,96 nm 579,07 nm (Hg) (Hg) (D) (H) (H0) (Hg) (H0) (Hg)

y l phng php kim tra ca DVN, D Anh, D n 1996, D Chu u 1998. D Nht XII s dng bc x ca n Hg p sut thp bc sng 253,65 nm, 365,02 nm, 435,84 nm v 546,07 nm hoc bc x ca n deuteri 486,00 v 656,1 nm. D Trung Quc 1997: s dng cc vch ph sau y ca n thu ngn:
237,83nm 313,16nm 435,83nm ; ; ; 253,65nm 334,15nm 546,97nm ; ; ; 275,28nm 365,02nm 576,96nm ; ; ; 296,73nm 404,66nm

Cc cc i hp th ca dung dch holmium perchlorat khi dng n hydro: 379,79; 456,13 v 656,28 nm. n deuteri: 486,02 v 656,10nm. Dng knh holmium: c cc cc i hp th : 279,4; 287,5; 333,7; 360,9; 460,0; 484,5; 536,2 v 637,5nm. Kim tra hp th kim tra hp th, a s cc dc in dng dung dch kali dicromat trong acid sulfuric (VN, Anh, Chu u, Nht, Trung quc...). M hay dng dung dch kali cromat trong kali hydroxyd. 70

Di y l mt s phng php chun ha hp th ca mt s dc in: o hp th ca dung dch kali dicromat cc di sng ch nh bng 3.1. Trong bng mi di sng c mt gi tr chnh xc ca hp th ring E(1%, 1cm) v cc gi hn cho php.
Bng 3.1. hp th ring ca dung dch kali dicromat
di sng nm 235 257 313 350 E(1%, 1cm) 124,5 144,0 48,6 106,6 Gii hn cho php 122,9 126,2 142,4 145,7 47,6 50,3 104,9 109,2

Pha ch dung dch kali dicromat: Dc in VN, D Anh, D Chu u, Nht XII, Trung Quc qui nh nh sau: Ho tan 57,0 n 63,0 mg kali dicromat sy kh n khi lng khng i 1300C trong acid sulfuric 0,005 M/l vi th tch va 1000ml. Dc in VN, Chu u cn cho thm bng hp th ca dung dch kali dicromat cha ng 60,06mg K2CrO7 trong va 1000ml acid sulfuric 0,005M/l, dng cuvet dy 1cm (bng 3.2.).
Bng 3.2. hp th ca dung dch kali dicromat
di sng (nm) 235 257 313 350 hp th 0,748 0,864 0,292 0,640

Gii hn nh sng lc (stray light) nh sng lc l nh sng khng i vo mu th m i thng vo detector.

nh sng lc c th c pht hin di sng cho vi knh lc hoc cc dung dch thch hp. D VN, Anh, Chu u, n qui nh : kim tra nh sng lc nh sau: o hp th ca dung dch kali clorid (TT) 1,2% trong cuvet 1cm = 200nm, so snh vi nc ct, phi ln hn 2,0. D Trung Quc (1997): kim tra nh sng lc vi dung dch natri iodid 1,00% v dung dch natri nitrit 5,00% (KL/TT). 71

 truyn qua ca cc dung dch ny c o so snh vi nc ct trong cuvet thch anh 1cm phi nh hn 0,8% bc sng 220nm v 380nm. phn gii (cho phn tch nh tnh) Khi chuyn lun yu cu, tin hnh o phn gii ca my nh sau: ghi ph ca dung dch toluen (TT) 0,02% trong hexan (TT) (v/v). Gi tr ti thiu ca t s gia cc i hp th 269 nm v cc tiu hp th 266 nm c qui nh trong chuyn lun ring.
rng gii ph ngun (cho phn tch nh lng) trnh sai s gy ra do rng gii ph ngun, khi s dng my c rng gii ph thay i di sng chn th rng ca gii ph phi nh so vi na rng ca bng hp th. Song rng ny phi ln thu c gi tr cao ca cng nh sng Io v vic thu hp rng gii ph phi khng lm cho hp th tng ln. Cuvet Dung sai ca chiu dy lp dung dch ca cc cuvet l + 0,005cm. Khi c np y vi cng mt dung mi, cc cuvet c nh cha dung dch th v dung dch so snh phi c truyn qua nh nhau. Nu iu ny khng c p ng th phi c s hiu chnh thch hp.

Cc cuvet phi c lm sch v chm sc cn thn trnh b xc. C th bo qun chng trong cn tuyt i trnh mc. 3.1.1.5. ng dng ph UV-Vis trong kim nghim thuc
nh tnh v th tinh khit Cc cc i hp th l c trng nh tnh ca cc cht.

V d: Da vo cc i hp th: Trong dung dch nc + Vitamin B12 c 3 cc i hp th cc bc sng: 278 nm + 1 nm 361 nm + 1 nm 548 nm + 2 nm + Vitamin B2 c 4 cc i hp th cc bc sng: 223nm 375 nm 267nm 444nm + Cloramphenicol c 1 cc i hp th bc sng: 278 nm; 72

+ Phenoxymethyl penicillin c 2 cc i hp th 268 nm v 274nm. T s gia cc cc i hp th cng l i lng c trng cho nh tnh cc cht. V d: + Vitamin B12 (USP XXII) : C t s A361/ A278 = 1,79 - 1,90 + Phenoxymethyl penicillin (BP.80): A268/ A274 = 1,20 - 1,25 H s match: h s ny nh gi s tng ng gia 2 ph, mt ph chun v mt ph th. H s match (Mat) c tnh theo biu thc sau:

10 3 [ x. y ( x. y ]2 ()x )2 Mat = y x2
Trong :

()

y )2

(3.4)

x v y l hp th ca ph th nht v ph th hai cng mt bc sng. n l s im chn 2 ph. Cch nh gi Mat: + < 900: hai ph khc nhau. + 900 - 990: hai ph c nhng im tng ng cn cn nhc khi kt lun. + > 990: hai ph tng t nhau. + Xp x 1000: hai ph ging nhau hon ton.
nh lng Chn iu kin xy dng qui trnh nh lng

xy dng qui trnh nh lng, chng ta cn kho st chn cc iu kin thch hp . Chn bc sng hoc knh lc : + Chn bc sng ng vi cc cc i hp th, khi ng chun c dc ln nht, tc l vi cng mt sai s ca mt quang th sai s ca nng C v bc sng l nh nht.

+ i vi my dng knh lc (quang k): Mu ca knh lc v mu dung dch phi ph nhau. Nhng cng c trng hp c th chn knh lc khc. 73

 Chn khong nng thch hp : Khong nng c quan h tuyn tnh vi hp th. Chn cc iu kin khc : + Loi tr nh hng ca cht l: chit cht cn nh lng ra khi hn hp phc tp (cc dng bo ch). Dng mu trng c cc thnh phn nh dung dch th nhng khng c cht cn nh lng. + Chn pH v dung mi thch hp + Thc hin phn ng mu: s hp th ca cc cht l, thuc th cng c th nh hng ti kt qu nh lng. V vy trong qu trnh lm phn ng to mu phi ch ti iu kin v cc thnh phn tham gia phn ng.
Cc phng php nh lng

Phng php o ph trc tip o hp th A ca dung dch, tnh nng C ca n da vo gi tr hp th ring (c trong cc bng tra cu).
1%

A
1% L = 1cm C = E1cm

A = E1cm .L.C

(3.5)

p dng phng php ny, cn phi chun ho my quang ph c v bc sng ln hp th. Phng php gin tip Cc phng php gin tip nh: phng php ng chun, so snh v thm chun tng t nh cc phng php ho l khc. c im ca phng php gin tip: + Phi c cht chun so snh, + C th khng cn phi chun my. Hu ht dc in cc nc u s dng c 2 phng php trc tip v gin tip. Ring dc in M (USP XXI, XXII, XXIII, XXIV) qui nh ch dng phng php so snh vi chun. Phng php so snh Theo nh lut Lambert-Beer, sau khi o hp th ca dung dch chun so snh (S) v dung dch mu th (X) ta c : A S = K . L . CS AX = K . L . CX y: + AS : l hp th ca dung dch chun c nng CS, 74

+ AX: l hp th ca dung dch mu th nng CX. V h s hp th K v b dy L ca cuvet l nh nhau nn ta c:

AX AX C X = C S AS = C S AS CX

(3.6)

Ch : Nng ca dung dch th CX v dung dch chun CS khng c chnh lch nhau qu nhiu. Cc nng ny cng gn nhau, kt qu cng chnh xc. Phng php thm chun so snh. Trong phng php quang ph, loi tr cc yu t nh hng gy sai s cho qu trnh nh lng: X l mu (chit xut), sai lch do thit b v ho cht thuc th..., ngi ta p dng phng php thm. * Nguyn tc tin hnh: Ly hai lng ging nhau ca mt mu th. Thm mt lng cht chun bit vo mt mu. Tin hnh x l c 2 mu trong cng iu kin (chit xut, pha long...), thu c 2 dung dch: Dung dch th v dung dch th thm chun. o hp th ca c 2 dung dch. Thu c: AX : hp th ca dung dch th. AX: hp th ca dung dch th thm chun. Vi CS l nng ca dung dch chun, ta tnh c nng CX theo phng trnh:

AX CX AX = ' ' AX C S + C X = C S AX AX CX

(3.7)

Phng php ng chun y l phng php cng hay dng trong phn tch quang ph.

Chun b mt dy chun khong 5 dung dch c cc nng cht chun CS khc nhau. o hp th AS ca dy chun v lp th ca A theo C o hp th AX ca dung dch mu th v da vo ng chun ta xc nh c nng mu th CX. Ch : Khi xy dng ng chun nn kho st khong tuyn tnh ca nng

vi hp th.

75

Trng hp dy chun khng tun theo nh lut Lambert-Beer (ng chun cong) th cn lm thm mt s im chun na vi cc nng gn nhau hn (khc nhau khng qu 10%). V th i qua cc v tr gn nht vi cc im thc nghim hoc da vo bng chun xy dng phng trnh hi qui tuyn tnh hay phi tuyn v tnh h s tng quan r. Nu r > 0,995 l tt.

AX

CX

Hnh 3.1. th ca phng php ng chun A = f (C)

Phng php thm ng chun Thm nhng th tch ging nhau ca dung dch th vo dy chun cha nhng lng khc nhau v chnh xc ca cht chun o hp th ca c dy r v ng chun quan h gia mt quang vi lng cht chun thm vo. Giao im ca ng chun vi trc honh (nng ) cho ta nng ca cht cn nh lng(hnh 3.2.)
A 0,3 0,2
dd th 0,1 A th thm chun

C dd th C chun thm vo

Hnh 3.2. th ca phng php thm ng chun

76

 K thut o quang vi sai theo bc sng Trong kim nghim cc dng thuc bo ch, trc tin phi qua cng on chit hot cht ra khi t dc. Dch chit kh trnh khi mang theo tp cht. Tp cht ny c th gy sai s cho qu trnh nh lng bng phng php o quang. loi tr sai s ny, ngi ta thng s dng k thut o quang vi sai: Trn ca cht. nghin cu, chn 2 bc sng 1 v 2, hiu s hp th Aph l ln nht Amax = A 1 A2 (3.8) Chun b mt dy dung dch chun vi cc nng khc nhau v o hp th A 2 bc sng 1 v 2. Kho st khong nng tun theo nh lut Lambert-Beer ca A, v th quan h A - C. o hp th A ca cht th 2 bc sng trn. Da vo th A - C suy ra nng cht th trong mu. Phng php nh lng hn hp Do hp th c tnh cng tnh nn nu c mt dung dch hn hp gm n cht th hp th ca hn hp bng tng hp th ca n cht ring r c cng nng nh trong hn hp. Ti mt bc sng xc nh ta c: Ahh = A1 + A2 + . . . + An = E1C1 + E2C2 + . . . + EnCn (3.9) thc hin phng php ny, cn phi bit h s hp th ring ca tng cht cc bc sng khc nhau. (Ej i = hp th ring ca cht th i j ) Khi s bc sng bng s cht ta c h n phng trnh vi n n s.
1 1 = E 1C A hh + E2 C 1 1

+ ... + E

2 = E 2C + E 2 2 C 2 + ... + A hh 1 1 .......... .......... .......... .......... .......... n n = E n C + E2 A hh C 1 1

2

(1 ) 2 En C n (2 )
1
n

......
n

(3.10)

n + ... + E n C

(n )

Gii h phng trnh ny s tm c nng ca cc thnh phn trong hn hp: C1 ; C2 ; . . . ; Cn Hin nay c nhiu chng trnh my tnh gii h phng trnh nhiu n s xc nh nng cc thnh phn trong dung dch. 77

Trng hp n gin nht l hn hp gm hai thnh phn, ta c: Ahh = A1 + A2 Chn 2 bc sng 1 v 2 v o hp th ca hn hp 2 bc sng .
1 = E 1C + E 1 C A hh 1 2 2 1 2 = E 2C + E 2 C A hh 1 2 2 1

Gii h phng trnh trn ta tm c:

A 1 E 2 A 2 E 1 C =
1 hh 2 hh 2

1 2 2 1 A hh E 1 A hh E 1 C2 = 1 2 = E1 E 2 E1 2 E21
Phng php ph o hm

Ph o hm l mt k thut bao gm s chuyn i ph hp th thnh ph o hm bc nht, bc hai hoc bc cao hn. * C s ca phng php ph o hm: Theo nh lut Lambert-Beer ta c : hp th A = E.C.L ( C: %,L: cm ) C th ly o hm bc 1, bc 2, hoc bc cao hn ca A i vi bc sng . A = f( ) (3.11) + Ph o hm bc nht l th ca tc bin thin ca hp th (dA/d) i vi bc sng . dA/d = dE/d .C.L (3.12)

+ Ph o hm bc hai l th ca cong ca ph hp th (d2A/d2) i vi bc sng. d2A/d2 = d2E/d2 .C.L (3.13) Do E l hng s v L khng i nn gi tr ca o hm ca A ch cn ph thuc tuyn tnh vi nng C ca dung dch. * Ph o hm ca hn hp nhiu cht : 78

Ging nh ph hp th, ph o hm cng c tnh cht cng tnh Ahh = A1 + A2 + ... + An = Ai ( i = 1 .... n ) (3.14) dAhh/d = dA1/d + dA2/d + ... + dAn/d 2 2 2 2 2 2 2 d2A /d = d A /d + d A /d + ... + d A /d (3.15)
hh 1 2 n

Theo ngha ton hc ca o hm th : Ti cc im cc tr, o hm bc 1 bng 0. Ti cc im un, o hm bc 2 bng 0. Ti gi tr 0 ca ph o hm ca cht ny c th gp gi tr khc 0 ca ph o hm ca cht kia. Nu hp th tun theo nh lut Lambert-Beer th o hm bc hai bc sng bt k no cng c lin h vi nng bi phng trnh sau :
2 2E d A d C.L = d2 d2

(3.16)

Ti mt bc sng xc nh, E l mt hng s . y: A: hp th bc sng E: hp th ring bc sng C: nng %(kl/tt ) ca cht hp th L: b dy ca lp dung dch cht hp th (cm) u im ca phng php ph o hm: Ph o hm c th gip nh lng ring bit c cc cht c cc cc i hp th gn nhau trong hn hp, trong khi phng php o ph thng thng khng th gii quyt c. Trong mt s trng hp, ph o hm gip xc nh c hm lng ca cht th trong s c mt ca cc tp cht gy cn tr. Hin nay phng php ph o hm c mt s nc a vo dc in: Anh, n 1996, Vit Nam.
3.1.2. Quang ph hng ngoi

3.1.2.1. M u Ph hng ngoi l phng php o s hp th bc x hng ngoi (IR) khi n i qua mt lp cht cn th, cc s sng khc nhau.

79

Vng bc x hng ngoi s dng trong cc my quang ph IR thng thng l 600-4000cm -1. Cc my hin nay c th m rng vng bc x (100-10.000cm-1). Trong phn t khi c nhm nguyn t no hp th nng lng v thay i trng thi dao ng th to nn mt di hp th trn ph IR. C mi tng quan gia nhm nguyn t v di hp th nn c th da vo s c mt ca di hp th nhn bit mt nhm chc no . Nhiu nhm chc c cc di ph hp th c trng. y l c s ca vic phn tch cu trc bng IR (bng 3.3.). Vic xc nh c s c mt ca cc nhm chc trong phn t gip chng ta c th s dng ph IR nh tnh mt cht. Trong kim nghim thuc hu ht cc dc in ch yu ch s dng IR vo vic nh tnh, t c dng trong nh lng.
Bng 3.3. Mt s pic hp th hng ngoi c trng
Pic hp th Nhm chc S sng (cm-1) O-H NH2 C-H C-H C N C CCOOR COOH C=O CONH2 C=CAr-O-R R-O-R Mch thng, vng thm Bc hai v bc ba Thm Mch thng Nitril Alkyn Ester Acid carboxylic Aldehyd v ceton Amid Alken Thm Mch thng 3600-3000 3600-3100 3150-3000 3000-2850 2400-2200 2260-2100 1750-1700 1740-1670 1740-1660 1720-1640 1670-1610 1300-1180 1160-1060 B c sng m 2,8-3,3 2,8-3,2 3,2-3,3 3,3-3,5 4,2-4,6 4,4-4,8 5,7-5,9 5,7-6,0 5,7-6,0 5,8-6,1 6,0-6,2 7,7-8,5 8,6-9,4

3.1.2.2. My My quang ph ghi ph trong vng hng ngoi bao gm mt h quang hc c kh nng cung cp nh sng n sc trong gii ph t 4000cm-1 n 670 cm -1 (2,5m-15m) hoc trong mt vi trng hp ti 200cm -1 (50m) v mt phng tin o t s gia cng nh sng truyn qua v nh sng ti. 80

3.1.2.3. ng dng ph hng ngoi trong nh tnh Phng php quang ph hng ngoi ch yu c ng dng trong nh tnh cc cht hu c. Vic nh tnh ny da trn 2 nguyn tc: So snh s ph hp gia ph cht th vi ph chun cho sn trong sch tra cu (atlas) hoc trong th vin ph lu gi trong my tnh. So snh s ph hp gia ph cht th vi ph ca ho cht chun c ghi trong cng iu kin. Phng php nh tnh bng ph hng ngoi ca hu ht cc dc in trn th gii u da trn hai nguyn tc ny.
nh tnh s dng cht chun so snh Chun b mu th v mu cht chun so snh ri o ph ca chng t

4000cm -1 n 670cm 1(2,5m - 15m) trong cng iu kin . Cc i hp th ph ca cht th phi tng ng vi ph ca cht chun v v tr v tr s.
nh tnh s dng ph chun trong atlas hoc th vin ph . c th so snh ph ca cht th vi ph chun tra cu trong atlas hoc th vin ph ca my tnh, trc ht chng ta cn phi chun ho my quang ph.

Tt c cc dc in u chun ho 2 thng s c bn ca my: phn gii v thang s sng. Tm tt cch lm nh sau: Chun ho phn gii: Ghi ph ca phim polystyren dy 0,05mm + Hiu s gia phn trm truyn qua cc tiu hp th A 2870cm-1 (3,48m) v cc i hp th B 2849cm -1 (3,51m) phi ln hn 18. + Hiu s gia phn trm truyn qua cc tiu hp th C 1589,5cm -1 (6,29m) v cc i hp th D 1583cm -1 (6,32m) phi ln hn 12. Chun ho thang s sng Cng c th chun ho thang s sng bng cch s dng phim polystyren. Phim ny c cc tiu truyn qua (cc i hp th) cc s sng (cm-1) (bng 3.4.).

81

Bng 3.4. Cc i hp th ca phim polystyren (cm-1)

3027,1 2924,0 2850,7 1944,0 1871,0 1801,6 1601,4 (+ 0,3 ) (+ 2 ) (+ 0,3 ) (+ 1 ) (+ 0,3 ) (+ 0,3 ) (+ 0,3 ) 1583,1 1181,4 1154,3 1069,1 1028,0 906,7 698,9 (+ 0,3 ) (+ 0,3 ) (+ 0,3 ) (+ 0,3 ) (+ 0,3 ) (+ 0,3 ) (+ 0,5 )

3.1.3. Quang ph hunh quang (Fluorometry)

3.1.3.1. M u Ph hunh quang l phng php ph pht x phn t. Sau khi hp th nng lng ca bc x t ngoi, kh kin hoc cc bc x in t khc (bc x kch thch), phn t b kch thch s tr li trng thi c bn v gii phng nng lng di dng bc x, c gi l bc x hunh quang. Cng hunh quang F ca mt dung dch long t l thun vi nng C (mol/l) trong iu kin xc nh khi cng v bc sng ca bc x kch thch l hng s.

y : K : hng s

F = K . I0 . . . C . L

(3.17)

I0 : cng ca bc x kch thch : hiu sut hunh quang S photon pht x = ------------------------- ( 0 < < 1 ) S photon hp th (3.18)

: H s hp th mol ca cht bc sng kch thch. Rt gn li ta c: F = K . C. y K = K . I 0 . . L . Vic o ph hunh quang ch nn tin hnh vi dung dch long C <10-4 trnh nh hng suy gim cng ca bc x pht ra. 3.1.3.2. My Ngun sng: thng dng n xenon (to nh sng =200-800nm), laser. n xenon cho ph bc x khng u nn km nhy hn ngun laser. 82

 B n sc: dng cch t Cuvet: thng dng thch anh (1 x 1cm) c 4 mt trong sut. 3.1.3.3. Chun ho my My quang ph hunh quang phi c chun ho thng xuyn vi cht chun. Tin hnh chun my theo ti liu hng dn ca hng sn xut my. 3.1.3.4. Phng php o Mu th lun lun c o so snh vi mu chun. Kim tra vng tuyn tnh ca cng hunh quang v iu chnh nhy ca thit b vi pha long thch hp ca dung dch chun . Ghi cng hunh quang ca dung dch th, dung dch chun v cc mu trng tng ng ca chng. Tnh ton nng ca dung dch th :

I X I OX
y:

(3.19)

C X = CS

I S I OS

CX: nng ca dung dch th CS : nng ca dung dch chun IX : tr s o c ca dung dch th IS : tr s o c ca dung dch chun I0X v I0S : tr s o c ca cc mu trng tng ng. Vng trong cng hunh quang t l thun vi nng ca cht thng rt hp, cho nn t s (IX - I0X)/ (IS - I0S) phi khng c < 0,50 v khng > 2,0. Nu t s khng nm trong khong trn th phi iu chnh nng v tin hnh o li. Trong mt s trng hp, c th s dng cch o so snh vi chun c nh. Th d: dng knh hunh quang hoc mt cht pht hunh quang khc. Nu cng hunh quang khng t l nghim ngt vi nng th nn s dng phng php th chun tng quan gia cng v nng . nhy ca my c th c kim tra bng mt dung dch bn ca cht hunh quang khc vi di sng kch thch v pht x tng t nh di ca cht th, thay th cho cht chun. V d: Quinin trong H2SO4 long cho hunh quang mu xanh, 83

 Natri fluorescein cho hunh quang xanh l cy, Rhodamin B cho hunh quang mu . tinh khit ca dung mi c th nh hng ng k ti cng hunh quang. Nu cn nn ct li trong my ct bng thu tinh trc khi dng. S c mt ca cc tiu phn keo c th gy nn s tn x nh sng, cho nn cn phi loi b chng bng ly tm hoc lc bng mng lc thu tinh xp. Oxy ho tan trong dung dch lm gim cng hunh quang rt mnh, cn phi ui n bng cch sc kh tr qua dung dch.
3.2. Phng php sc k lng hiu nng cao

(High- Performance Liquid Chromatography- HPLC) Sc k lng hiu nng cao i khi cn c gi l sc k lng p sut cao (High-Pressure) l k thut phn tch da trn c s ca s phn tch cc cht trn mt pha tnh cha trong ct, nh dng di chuyn ca pha ng lng di p sut cao. Sc k lng da trn c ch hp ph, phn b, trao i ion hay loi c l tu thuc vo loi pha tnh s dng. Khi phn tch sc k, cc cht c ha tan trong dung mi thch hp v hu ht s phn tch u xy ra nhit thng. Chnh v th m cc thuc khng bn vi nhit khng b phn hy khi sc k. Sc k thng c hon thnh trong mt thi gian ngn (khong 30 pht). Ch nhng thnh phn c h s chn lc khc nhau mi c th phn tch c bng HPLC. Ngy nay HPLC v ang c s dng nhiu trong lnh vc phn tch ho hc ni chung cng nh trong kim tra cht lng thuc v phn tch sinh dc hc ni ring. Trong phn tch thuc bng phng php sc k, phn ln cc dc in u s dng sc k phn b. Nn chng ti ch yu cp ti phng php ny.
3.2.1. Cc thng s c trng ca qu trnh sc k

H s dung lng k (Capacity factor)

Hnh 3.3. Minh ha cc thng s sc k

84

' k =K
y: K : h s phn b Vs : th tch pha tnh Vm: th tch pha ng Qs: lng cht trong pha tnh Qm: lng cht trong pha ng tR : thi gian lu tR : thi gian lu hiu chnh t0: thi gian cht Cn chn ct, pha ng ... sao cho k nm trong khong ti u : 1 < k< 8.
H s chn lc :

'

Vs QS t R t R t o = to = to V m = Qm

(3.20)

k ' t RB = k 'B = t RA A
Qui c y B l cht b lu gi mnh hn A nn >1 tch ring 2 cht thng chn 1,05 < < 2,0 H s i xng ca pic F:

(3.21)

F=

W 2a

(3.22)

y: W: Chiu rng pic o 1/20 chiu cao pic, a: Khong cch t ng vung gc h t nh pic n mp ng cong pha trc ti v tr 1/20 chiu cao pic.

Hnh 3.4: Xc nh h s i xng

85

S a l thuyt v hiu lc ct N: Hiu lc ct c o bng thng s: S a l thuyt N ca ct

N = 16
y:

2 tR

t2 2 = 5,54 W R 1/ 2

(3.23)

W: Chiu rng o y pic, W 1/2: Chiu rng pic o na chiu cao pic.
phn gii Rs:

RS = )
Vi:

2(t RB t RA )

1,18(t RB t RA W1/ 2 B

3.24

WB + W A

+ W1/ 2 A

tRB, tRA: Thi gian lu ca 2 pic lin k nhau (B v A), W B , WA: rng pic o cc y pic, W 1/2B, W 1/2A: rng pic o na chiu cao pic. Cc gi tr: tRB, tRA, W B, W A , W 1/2B , W 1/2A phi tnh theo cng mt n v. Yu cu RS > 1, gi tr ti u RS = 1,5.
3.2.2. My HPLC

Hnh 3.5: S nguyn l ca my HPLC.

1- Bnh dung mi 2- Bm 3- Tim mu 4- Ct 5- Detector 6- Phn mm x l 7- Bnh nc thi

86

My sc k lng hiu nng cao gm c cc b phn sau: Bnh cha pha ng, bm y pha ng qua h thng sc k p sut cao, h tim mu a mu vo pha ng, ct sc k, detector, my tnh hay my tch phn hoc my ghi. H bm H bm HPLC c chc nng to p sut cao y pha ng t bnh dung mi qua h thng sc k. H bm hin i hin nay c iu khin bng my tnh c th lp chng trnh thay i t l ca cc thnh phn pha ng theo yu cu (sc k gradient). Bm HPLC cn phi p ng mt s yu cu : To c p sut cao 3000-6000 psi (250-500 atm), lu lng bm khong 0,1 n 10ml/pht, khng b n mn vi cc thnh phn pha ng, c tc bm khng i. H tim mu C th dng bm tim tim mu vo u ct. Phng php ph bin l dng van tim c vng cha mu (sample loop) c dung tch xc nh v chnh xc, c th thay i vng cha mu vi cc dung tch khc nhau. Mt s my HPLC hin i c h tim mu t ng c th lp trnh iu khin th tch mu, s ln tim v chu k ra vng cha mu.

Ct Ct c dng ph bin lm bng thp khng g, thng thng c chiu di

10-30cm, ng knh trong t 2- 5 mm, ht cht np (packings) c 5-10 m. Ct c ng knh trong ln hn c dng cho sc k iu ch. Ct c th c lm nng t hiu qu phn tch tt hn, nhng him khi tin hnh nhit trn 60o C v nhit c th lm suy gim hiu lc ct hoc lm pha ng bay hi. Cht np thng l silica gel hoc silica gel c bao mt lp mng hu c hoc lin kt ha hc vi cc nhm chc hu c. Bn cnh silica gel ngi ta cn dng cc cht liu khc nh: nhm oxyd, polyme xp, nha trao i ion....
Detector Detector l b phn pht hin v o cc tn hiu sinh ra khi c cht ra khi ct v cc tn hiu ny c ghi di dng pic trn sc .

Cc loi detector c dng hin nay: Detector t ngoi v kh kin (UV-VIS) c bc sng tu chn c dng ph bin nht. Detector hin i thuc loi ny l detector dy diod (diode array detector). Detector ny lun lun c t l tn hiu nhiu thp hn so vi detector UV-VIS bnh thng. 87

Cc loi detector khc : Detector o ch s khc x Detector hunh quang, in ho. 3.2.3. Cc k thut HPLC. C 4 k thut HPLC c bn: Sc k phn b, hp ph, trao i ion v sc k loi c (ry phn t). Trong phm vi cun sch, ch yu chng ti gii thiu v sc k phn b, v y l k thut HPLC c s dng nhiu nht trong kim nghim thuc. Cn cc k thut khc, ch c tnh cht gii thiu tm tt. Sc k phn b hiu nng cao (Partition Chromatography) Pha tnh Pha tnh trong sc k phn b bao gm mt lp mng pha lng hu c bao trn b mt ca cc tiu phn cht mang silica hoc cc cht liu khc. Cc tiu phn ny thng c ng knh t 3 n 10 m, c ht c th tng n 50 m hoc ln hn cho sc k iu ch. Sc k dng loi pha tnh ny c gi l sc k lng- lng (Liquid - liquid Chromatography - LLC). Nhng kiu pha tnh ny c nhc im l b ra tri dn theo dng pha ng. Kt qu, hiu lc ct b gim dn trong qu trnh s dng. khc phc nhc im trn, ngi ta s dng k thut sc k pha lin kt (Bonded- Phase Chromatography - BPC). Trong k thut ny, pha tnh c lin kt ha hc vi cht mang. K thut ny nhanh chng tr thnh k thut ph bin nht trong HPLC. Trong loi pha tnh ny, cc nhm chc hu c lin kt vi b mt ca cc tiu phn silica qua cc nhm silanol. Tnh phn cc ca loi pha tnh ny ph thuc vo tnh phn cc ca cc nhm chc lin kt, chng trong vng t nhm khng phn cc octadecyl silan n nhm rt phn cc nitril. Di y l mt s pha lin kt thng dng: Octyl Octadecyl Phenyl Cyanopropyl Diol = Si- (CH2))7- CH3 = Si- (CH2))17- CH3 = Si- (CH2)n- C6H5 = Si- (CH2)3- CN C8 C18 C6H5 CN NH2

Aminopropyl = Si- (CH2)3- NH2

= Si- (CH2)3- OCH (OH) - CH2- OH

H bao gm pha tnh phn cc v pha ng khng phn cc c gi l sc k pha thun. Ngc li, pha ng phn cc cn pha tnh khng phn cc, c gi l sc k pha o. Khi s dng silica, nhm oxyd hoc polyme xp th cc cht c phn tch theo c ch hp ph nn c gi l sc k hp ph. 88

 Nu pha tnh l nha trao i ion th gi l sc k trao i ion . Nu pha tnh l polyme xp nh dextran..., ta c sc k loi c (sc k ry phn t Size Exclution Chromatography). Pha ng i lc ca mt thnh phn i vi pha tnh hay ni mt cch khc, thi gian lu gi ca n trn ct c iu khin bng cch thay i phn cc ca pha ng. Pha ng c th l dung mi n hay hn hp ca 2, 3 hay 4 thnh phn. Ngi ta c th thay i phn cc ca pha ng bng cch thay i t l ca cc thnh phn dung mi trong hn hp. K thut thay i lin tc thnh phn dung mi trong thi gian chy sc k c gi l ra gii gradient hoc chng trnh ha dung mi. Tu thuc vo vic s dng pha ng v pha tnh ngi ta chia sc k phn b thnh 2 loi (sc k pha thun v sc k pha o): * Sc k pha thun (normal phase): Trong k thut ny ngi ta s dng cht np ct (packings) gm cc nhm phn cc lin kt vi cht mang nh nhm alkylnitril [-(CH2)nCN ] v alkylamin [-(CH 2)nNH2 ]. Dung mi s dng l dung mi ca sc k hp ph (sc k lng rn - LSC). K thut ny, s dng pha tnh phn cc hn pha ng, c gi l sc k phn b pha thun. S phn tch t c bng sc k pha thun cng tng t nh LSC. Song pha tnh trong sc k pha lin kt c u im hn pha tnh trong LSC: . Ct nhanh t cn bng nn rt thun li cho phn tch gradient. . Ct thch hp vi nhiu dung mi v d ti sinh. . Ct rt bn. . Ct alkylamin c th c chc nng nh mt ct trao i anion yu. Pha ng trong sc k pha thun ni chung khng phn cc, thng dng hn hp ca cc hydrocarbon mch thng nh pentan, hexan, heptan, isootan. Cc dn cht halogen dy bo cng c s dng nh: diclorometan, dicloroetan, butyl clorid v cloroform. C th thm vo cc pha khan nc ny 1- 2% acid acetic hoc phosphoric ngn cn s ion ha ca cc cht nh: cc phenol, acid carboxylic. Ngc li, s ion ha ca cc base yu c th c km li bng cch thm mt lng nh amoniac hoc mt amin khc (0,1- 1%). Ngoi ra cng c th thm vo pha ng nhng cht ci bin hu c (organic modifier) khc nh cc alcohol, tetrahydrofuran. Ni chung, vic ui kh ha tan khng thnh vn vi dung mi pha thun. Trong k thut sc k pha thun cc cht khng phn cc s c ra gii sm. Th t ra gii s chm dn theo chiu tng ca phn cc ca cc thnh phn trong mu th. 89

V d: Tch hn hp steorid bng sc k pha thun. iu kin sc k: - Ct: MicroPak NH2 2,2 x 150 mm, 5 m. - Pha ng: hexan cha 0,2% isopropanol. - Tc dng: 1 ml/ pht. - Detector: UV 254 nm

pht
Hnh 3.6: Sc tch cc steroid
1- Progesterone 2- Androstendione 3- 4- pregnon-20--ol-one 4- 17--hydroxyprogesterone 5- Andrenosterone 6- Cortisone acetat 7- Cortisone 8- Hydrocortisone

* Sc k pha o (reversed phase) : Trong kim nghim thuc bng phng php HPLC, sc k phn b pha o c s dng nhiu nht. Trong k thut ny pha tnh bao gm cc nhm khng phn cc nh octadecyl (C18), octyl (C8) hay phenyl (C6H5). Pha ng l nhng dung mi phn cc nh: nc, methanol (MeOH), acetonitril (ACN). Tm li, pha ng phn cc hn pha tnh. chn lc ca k thut ny khc bit ng k vi qu trnh tch trn pha thun. Ngi ta thy rng, mt cht cng tan tt trong nc th n c ra gii khi ct cng nhanh. Quy tc ra gii ny ngc vi pha thun. Vi cc cht c ra gii qu sm trong sc k pha thun, chng s rt kh tch ri khi nhau. Trong khi , pha o cc thnh phn ny s c ra gii vo gn cui ca qu trnh sc k lm cho vic tch d dng hn (c ngha l k ln hn). 90

Khi mt pic nh c ra gii sau mt pic ln th thng rt kh nh lng bng sc k pha thun. Tuy nhin dy ra gii c th o ngc li bng cch dng sc k pha o. Khi pic nh c th c xc nh chnh xc hn. Thnh phn u tin ca pha ng trong sc k pha o l nc. Nhng dung mi ha ln c vi nc nh methanol, ethanol (EtOH), acetonitril, dioxan, tetrahydrofuran (THF) v dimethylformamid c thm vo iu chnh phn cc ca pha ng. Mt s thnh phn khc cng c th c thm vo pha ng nh: cc acid, base, m , cht din hot. Cp dung mi c s dng ph bin nht trong sc k pha o l: H2O - MeOH v H2O ACN. iu d nhn thy, MeOH c nhc im l nht cao ca n c th lm gim hiu lc ct. Trong khi ACN c nht thp hn, thch hp hn vi cc cht khng phn cc v th n thng c dng nhiu hn MeOH. Thi gian lu s ngn hn nu nng ACN bng nng MeOH. Ch : Khi pha ng c thm cc mui v c hoc cc cht hot ng b mt, nn lc n trc khi dng v c th c cn khng tan trong nc gy bn ct. Vic ui kh rt quan trng vi pha ng pha o. V th t ra gii: Trong sc k pha o cc cht phn cc ra trc, cc cht t v khng phn cc ra sau. V d: Tch hn hp morphin, codein v heroin bng HPLC pha o (hnh 3.7). iu kin sc k: - Ct: - Pha ng: - Tc dng: - Detector: Zorbax Rx, Sb- C 8, 4,6 x 150 mm, 5m. ACN/KH2PO4 25 mM, pH 3,5 7/ 93. 1,5 ml/pht UV 254 nm

Pht

Hnh 3.7: Sc tch morphin, codein, heroin.

1 - Morphin 2 - Codein 3 - Heroin.

91

Sc k cp ion (Ion-Pair Chromatography IPC) Sc k cp ion da trn c s s to thnh cp t ion cn phn tch mang in v tc nhn to cp mang in tri du (i ion).

X n + Qn = [ XQ ]hc
+

Nhn chung, IPC thc cht l HPLC pha o. Dung mi: Chnh l dung mi trong pha o, thng phi hp H2OMeOH v H2O-ACN. im quan trng nht l phi m bo rng thuc th to cp ion tan ht trong pha ng trnh kt ta trong h thng. i ion: i ion thng l cc amin bc 4 hoc cc mui sulfonat. Cn phi iu chnh pH ca pha ng vi m ti gi tr thch hp. ng thi phi kim tra tan ca m trong dung mi trc khi bm qua h sc k. Ct: Dng cc cht np pha o vi cc nhm alkyl C2, C8, v C18.... V d: Tch hn hp vitamin tan trong nc bng HPLC cp ion pha o. iu kin sc k: - Mu th: - Ct: - Pha ng: - Tc dng: - Detector: Vitamin tan trong nc. Zorbax SB C 8, 4,6 x 150 mm, 5 m. ACN/ MeOH/ diethylamin 2%/ acid acetic 2% (2/ 3/ 4/ 28) v 1,1 g Na heptan sulfonat/ lit. 1 ml/ pht. UV 280 nm

DAD1 A, Sig = 2801 , 4 Ref = 360,100 Mu

100 88 66 48 28 0 0 1 2 3 4 5 6 7 8 n

1
2.151

2
3.272

4 3
5.583 7.059

Hnh 3.8: Sc tch hn hp vitamin tan trong nc.

1. Vitamin PP 3. Vitamin B2 2. Vitamin B6 4. Vitamin B1

92

Sc k hp ph hiu nng cao (Liquid Solid Chromatography LSC). L k thut pht trin sm nht v c dng ph bin. Trong k thut ny, cht cn phn tch b gi trn b mt pha tnh (cht hp ph) v b dung mi y ra (phn hp ph). Pha tnh c th l silicagel, nhm oxyd ... nhng silicagel c dng nhiu hn. Pha ng thng l nhng dung mi t hoc khng phn cc. Khi ra gii, cc cht khng phn cc s ra trc, cc cht cng phn cc cng ra chm. V d: Tch hn hp steroid acetat bng HPLC hp ph iu kin sc k: - Mu th: - Ct: - Pha ng: - Tc dng: - Detector: Hn hp steroid acetat LiChrosorb Si 60 4,6 x 250 mm, 7 m. Chloroform/ methanol 95,5/ 0,5. 1,5 ml/ pht 254 nm

Hnh 3.9: Sc tch hn hp steroid acetat

1. 11- Deoxycorticosteron 2. Cortison 3. 9 - Fluorohydrocortison acetat 4. Hydrocortison 5. Prednisolon

93

Sc k trao i ion (Ion Exchange Chromatography IEC) Sc k trao i ion c dng phn tch cc thnh phn ion ha tan trong nc, c phn t lng nh hn 1500. Pha tnh l cc nha trao i ion di dng bt mn. l nhng hp cht hu c cao phn t c cha nhm chc c kh nng trao i. Nha c nhm hot ng mang in tch m, dng tch cc cht base nh cc amin. Nha c nhm hot ng tch in dng phn tch cc cht c nhm mang in m, nh cc nhm phosphat, sulfonat, carboxylat. pH ca pha ng, nhit , loi ion, nng ion, v cc cht ci bin hu c nh hng n cn bng, nhng c th iu chnh c thu c kt qu tch mong mun. Sc k loi c (Size Exclusion Chromatography SEC) Phng php SEC c ng dng ch yu phn tch cc cht c phn t lng (MW) ln hn 2000. Pha tnh l nhng ht xp ca silicagel hay polyme. Khi sc k, cc phn t ca cc cht phn tch c lc theo c ca khi lng phn t. Khi cho hn hp cn phn tch qua ct, ta thy: Cc phn t c MW qu ln, chy nhanh qua ct, chng khng b lu gi. Cc phn t c MW nh hn th b lu gi trn ct. Khi ra gii chng s ln lt ra khi ct vi thi gian lu tng theo chiu gim ca khi lng phn t.
3.2.4. Hng dn chn k thut HPLC

Trong HPLC, c 4 k thut sc k c bn: phn b, hp ph, trao i ion v sc k loi c. Mun chn mt k thut sc k thch hp phn tch mt hn hp, phi da vo cc c tnh l, ha hc ca cc cht thnh phn: + Tnh tan: tan hay khng tan trong nc. + Phn t lng (MW). + Tnh phn cc: ion ha hay khng ion ha. Hnh 3.10. trnh by s chn k thut HPLC.Tt nhin, y cha phi l mt ch dn tht chnh xc m ch l mt nh hng s b. Nu phn t lng ln hn 2000: nn s dng sc k ry phn t. Dng dung mi l nc nu mu th tan trong nc. Nu mu tan trong dung mi hu c, th dng dung mi hu c lm pha ng. Pha tnh l Sephadex (dextran) hoc Bondagel Series E th pha ng l nc. Cn Styragel hoc gel MicroPak TSK th dng pha ng hu c. Nu phn t lng di 2000: trc ht phi xc nh xem mu c tan trong nc hay khng. 94

+ Nu mu tan hoc t tan trong nc th c th s dng sc k trao i ion hoc phn b pha o. + Nu tan c tng ln khi thm acid hoc kim th dng k thut trao i ion. + Nu tan khng b nh hng bi acid hoc kim v dung dch nc ca n hon ton trung tnh th nn chn sc k phn b pha o. + Nu mu khng tan trong nc th nn s dng sc k phn b pha thun hoc LSC. + tch cc ng phn, nn dng LSC. + tch cc ng ng, nn dng sc k phn b. Cn phi nhc li rng nhng thng tin dn ch l nhng gi ban u, khng mang tnh bt buc. Tt nhin, m bo vic phn tch sc k c thnh cng nh kim nghim cn phi tin hnh kho st bng thc nghim.

95

 tan mu th

Tnh cht mu th

K thut SK

Ct

Hexan Alcol

Pha o

C18 SiCN

- Pha thun - Hp ph

C18 CN C8 C3 C18PTH Amin

MW < 2000

Khng ion ha

Pha o

C18 Amin

Pha o cp ion Nc mu Th Ion ha

C18 - IP

SAX SCX Trao i ion Spherogel Amin

Pha o Peptid Loi c (ry phn t)

C18 C8 USG SEC 2000

MW > 2000

Tan/ nc

SEC, pha nc

Khng tan/nc

SEC, pha hu c

Hnh 3.10: S hng dn chn k thut HPLC

96

Bng 3.5: iu kin phn tch mt s thuc bng HPLC

Tn thuc Acenocumarol Ct RP - 2 Pha ng ACN/ H2O/CH3COOH Detector 305 Mu th Huyt tng Huyt thanh Siro ho

Acephyllin

C - 18

MeOH/ NaH2PO 4 0,01 M

280

Acetaminophen Guaifenesin Dextromethorphan Acetaminophen Aspirin Caffein Codein Phenacetin Salicylamid Acetaminophen P aminophenol Acetazolamide

C 18

HCOONH4/ MeOH/H2O 10/ 450/ 540 (pH 4,2)

254

C 18

MeOH/ NaH2PO4 0,01 M 19/ 81 pH 2,3

254

Vin nn

Phenyl

TBA phosphat 0,005 M /ACN 85/15 pH 7,5

254

Vin nn

C - 18

ACN / acetat 0,05 M

254

Huyt tng, nc tiu Nc tiu

Acetazolamide

Silica

Hexan/CHCl3/MeOH/AcOH 65 /25 / 10 / 0,25

254

Acid aminocaproic Acid 5- aminosalicylic Acyclovir

C8 C 18 C 18

MeOH / H2O / H 3PO4 40 / 60 / 0,1 MeOH / H2O / H 3PO4 40 / 60 / 0,1 NaOAc 0,005 M, Na heptansulfonat 0,0025 M, pH 6,5 H2O / MeOH / AcOH 85 / 15 / 0,5

345 345 300; 418

Huyt thanh Huyt tng Huyt tng Nc tiu

Amoxicilin

C 18

395; 485

Amoxicilin Ampicilin Antipyrin

C-8

KH2PO4 0,067 M (pH 4,6) / MeOH 425 / 75

225

Huyt tng Nc tiu

C 18

ACN / AcOH 1% 35/65

254

Huyt tng

Apomorphin Acid ascorbic Aspirin

Phenyl C 18 Silica

MeOH / ACN / m pH 3,5 cha Na laurylsulfat H3PO4 0,8% trong nc Heptan/ AcOH 95/ 5

273 254 300

Huyt thanh Nc tiu Vin nn

97

Tn thuc Aspirin Acid salicylic Acid salicyluric Acid gentisic Atenolol

Ct C- 18

Pha ng ACN/ AcOH 1% 20/ 80

Detector 280

Mu th Huyt tng, nc tiu

Cyano

Acid heptansulfuric/ triethanolamin 0,2 M / MeOH 2/100/ 1900 MeOH/ NaH2PO 4 0,1 M 60/ 40

220

Huyt tng Huyt thanh Huyt thanh Dung dch M

Barbiturat

C 18

240

Benzocain

C 18

MeOH/ NaH2PO4 0,1 M 60/ 40

240

Benzocain Acid benzoic Acid salicylic Benzoyl peroxyd Beta methason Sodium phosphat Bromazepam

C 18 C 18

MeOH/ ACN/ H 2O MeOH/ KH2PO4 10/ 90 pH 6,2

254 ; 294 254

C 18 C 18

ACN/ H 2O 50/ 50 MeOH/ KH2PO4 0,09 M 60/ 40 254

M Dung dch

C 18

MeOH/ ACN/ H 2O (c cha 20 ml TBAH 10%/ lit 20/ 300/ 70

230

Huyt tng Huyt thanh Dng phn liu Huyt tng Huyt tng Huyt tng Huyt tng Huyt tng Nc tiu

Caffein

Silica

THF/ CH 2Cl2 20/ 80

272

Camphor

Silica

Heptan/ CHCl3 60/ 40

254

Captopril Carbamazepin

C 18 C - 18

MeOH/ K3PO4 (pH 6,5) 52/ 48 52/ 48 ACN/ H2O 50/50

340 288

Chloramphenicol

C8

MeOH/ H3PO 4 0,05 % 40/ 60

280

Chlodiazepoxide

C 18

ACN/ NH4CO3 0,1%

260

Chloroquin

C 18

MeOH/ H2O/ AcOH 80/ 19/ 1 Na heptansufonat

340

Chlopheniramin

Cyano

ACN/ MeOH/ phosphat 0,015 M (pH 6,6) 25/ 25/ 50

254

Chlopromazin

Cyano

Acetat 0,05M(pH 6,5)/ ACN

254

Huyt tng

98

Tn thuc Chlorzoxazon Cimetidin Cimetidin

Ct C 18 C 18 C 18 MeOH/ H2O 60/40 MeOH/ H2O 60/40 ACN/ H 2O/ NH 4OH 1000/ 50/ 1

Pha ng

Detector 254 180

Mu th Huyt tng Huyt tng Huyt tng

Codein Cortisone v hydrocortison Cortisone v hydrocortison Dexamethason Dextromethophan

C 18 C 18 Silica C 18 C 18

ACN/ H 2 O 375/ 625 0,005 M Na dioctylsulfonat, pH 3,3 ACN/ H2O 375/ 625 0,005 M Na dioctylsulfonat, pH 3,3 Hexan/ ethyl acetat 60/ 50 KH2PO4 0,01 M/ MeOH 50/ 50 ACN/ m acetat (pH 4,3) 35/ 65

254 254 360 254 220

Dng phn liu Dng phn liu Huyt thanh Dng phn liu Huyt tng Vin nn Huyt thanh Vin nn Huyt thanh Dng phn liu Vin nn Huyt thanh Vin nn Huyt thanh, nc tiu Nc tiu

Diazepam Vitamin D3 Estrogen Fluorouracil

C 18 C 18 C 18 C 18

MeOH/ H2O 65/ 35 H2O/ MeOH 1/ 9 MeOH/ H2O 60/ 40 MeOH/ acetat 0,01 M (pH 4) 6/ 94

254 254 280 270

Acid folic

C 18

H2O/ MeOH (pH 7,0) 76/ 24 0,015 M phosphat & 0,3 % TBA hydroxyd

280

Furosemid Gentamicin

C 18 C 18

ACN/ Na acetat (pH 5,0) 40/60 ACN/ CH 2Cl2/ H2O/ MeOH 80/ 10/ 8/ 4

254 230

Griseofulvin Heptaminol

Cyano C8

MeOH/ H2O 40/ 60 ACN/ AcNa 0,05% chnh ti pH 6,1 45/ 55

254 338

Heroin

C 18

ACN/ phosphat 0,015 M 100/ 300 (pH 3,0)

135

Histamin

C 18

NaCl 0,1 M/ MeOH 55/ 45 pH 3,0

350

Huyt tng Vin nn

Hydrochlothiazid

C 18

MeOH/ AcOH 0,01 M. 15/ 85

228

Hydrocortison

Silica

CHCl3/ isooctan/ MeOH/ H 2O 48,5/ 48,5/ 2,9/ 0,12

254

Huyt tng

99

Tn thuc Ibuprofen Ketoprofen Ketoprofen Lidocain Methyldopa

Ct C 18 C8 Silica Phenyl C 18

Pha ng ACN/ AcOH 0,05 M 40/ 60 H2O/ MeOH 85/ 15 CH 2Cl2/ hexan 60/ 40 ACN/ H 3PO4 0,006% 30 / 70 MeOH/ H2O 14/ 86 Na octansulfonat 0,006 M, pH 6,4

Detector 230 255 254 200 GCE + 0,5 V 313

Mu th Huyt tng Huyt tng Huyt tng Huyt tng Huyt tng Huyt tng Huyt tng, nc tiu Dng tim Huyt tng Huyt tng

Mebendasol

C 18

ACN/ phosphat 0,05M (pH 6) 27/ 73

Metronidazol

C 18

ACN/ phosphat 10-5 M pH 4 8/ 92

322

Morphine Acid Nalidixic Naphazolin Tetrahydrozolin

C 18 C 18 C 18

MeOH/ NaH 2PO4 60/ 40 MeOH/ H2O 70/ 30 MeOH/ m citrat pH 2,2 300/ 700

254 313 255

3.2.5. Chun ho ct sc k lng hiu nng cao Mc ch:

Mc ch ca vic chun ho ny l kim tra hiu nng ca cc ct HPLC v xc nh mt cch khch quan xem khi no chng cn phi thay th. Tt c cc ct pha o (C18, , C8) v cc ct silica ni chung mi v c u cn c chun ho. Cc thng s ca ct HPLC cn chun ho : + H s dung lng : k.

+ Hiu lc ct (s a l thuyt) : N H s i xng :F i vi cc ct mi: Cn chun ho ngay khi mi nhn khng nh cht lng ca chng. i vi ct ang s dng: Nu trong thi gian s dng, nhn thy hnh dng ca pic b xu hn trc v hiu lc ca ct khng tho mn cho vic phn tch th nn chun ho li ct v ghi ngy thng thc hin vo bng theo di. 100 +

 Chun ho ct sc k pha o

Chun b dung dch cc cht chun: Pha mt hn hp dung dch cc cht chun trong methanol 60%(v/v) vi cc nng nh sau: Uracil Phenol Anisol 0,02 mg/l 1,00 mg/l 1,50 mg/l iu kin sc k: Pha ng Tc dng Th tch tim Detector : methanol - nc (60 : 40) : 1ml/pht : 20 L : UV 254 nm

Hnh 3.11: Sc tch hn hp uracil, phenol, anisol.

1. Uracil 2. Phenol 3. Anisol

Tnh ton:

H s dung lng (k) k = (tA t0)/t0 t0 : thi gian lu ca uracil tA : thi gian lu ca anisol Hiu lc ct (N) N = 5,54. t2A / W21/2 tA : thi gian lu ca anisol W 1/2 : chiu rng pic o na chiu cao ca pic anisol 101

 H s i xng (F) F = W A / 2a y: W A : chiu rng pic anisol o 1/20 chiu cao pic a: khong cch t ng vung gc h t nh pic n mp ng cong pha trc ca pic ti 1/20 chiu cao pic. Chun ho ct sc k pha thun Chun b dung dch: Ho tan khong 0,1 0.2 ml n-pentan v 20mg acetophenon trong 100ml hexan c cha 0,5%(v/v) methanol. iu kin sc k : Pha ng Tc dng : hexan - methanol (99,5 : 0,5) : 1,0 ml/pht

Th tch tim : 20 L Detector : UV 254 nm Tnh ton tng t chun ho ct pha o . Vi: tAC : thi gian lu ca acetophenon t0 : thi gian lu ca n-pentan W 1/2 : chiu rng pic o na chiu cao ca pic acetophenon
Bng 3.6. Cc tiu chun chp nhn cho ct
Ct mi H s dung lng (k ) Hiu lc ct (N) H s i xng (F) 2,0 3000 a l thuyt hin nay > 10.000 0,9 2,0 Ct s dng < 0,7 1000 a 0,9 2, 0

3.2.6. nh lng bng phng php HPLC

Qu trnh nh lng bng HPLC c th chia thnh 4 bc. Mi bc u c nh hng trc tip n kt qu nh lng. Cc bc ny l : 102 Ly mu th Tin hnh sc k o tn hiu detector Phng php nh lng

Ly mu

Sai s do ly mu c th tng ln t nht 3 khu: Ly mu i din, bo qun mu v x l mu. gim sai s, vic ly mu phi tn trng trit cc nguyn tc c qui nh i vi tng loi i t ng mu c th ly c i din. Qu trnh bo qun mu cng c th dn n sai s: Vi mu th l cht bay hi, cht c th phn ng c vi hi nc hoc khng kh. Cc thnh phn vi lng c th b hp ph hoc phn hp ph bi bnh cha trong thi gian bo qun. C th tin hnh cc phn ng ho hc trn mu th to dn cht c nhy cao hn c th o c bng detector ca my. Cch ny c p dng i vi nhng cht c p ng rt nh hoc hon ton khng c p ng vi detector o hp th quang hc. tin hnh tch sc k Qu trnh chy sc k cn ch : C th xy ra s phn hu ca cht th trong khi phn tch. C th xut hin pic l trn sc do dung mi dng ho tan mu th c cha tp cht. V vy cn phi kim tra bng sc k cc vt tp . Chun b mu th: Dung mi dng ho tan mu th phi c xem xt cn thn . L tng nht l dng pha ng lm dung mi ho tan n. Tr trng hp mu th kh tan trong dung mi ny th phi tm dung mi khc thch hp hn. Dung mi phi p ng cc yu cu sau : + tinh khit cao khng c pic l, + C th ho ln c vi dung mi ra gii (pha ng), + Cho p ng rt nh vi detector, + Dung mi v dung dch th phi c lc qua mng lc 0,45m. Tim mu: C 2 cch tim mu vo ct: + Dng bm tim v + Van tim mu th tch xc nh. Dng van tim mu cho kt qu chnh xc hn (+1%) so vi dng bm tim ( 1 - 2%).
o tn hiu detector

Detector c dng ph bin nht trong HPLC l detector UV-Vis. 103

 Detector s dng phi p ng mt s yu cu sau : + Hot ng c vng tuyn tnh ca n, + C hp th nh i vi dung mi ( nhiu ng nn thp), + Trnh lt khng kh vo ct v detector, + Gi sch cell v lm sch n thng xuyn. Tn hiu detector: Tn hiu detector o c khi cht ra khi ct sc k c my ghi li di dng pic. my sc k c in, vic o din tch hoc chiu cao pic c thc hin bng tay. Cn cc my sc k hin i vic o c ny c t ng ho nh my tch phn, my tnh.
3.2.7. Cc phng php nh lng

Tt c cc phng php nh lng bng sc k u da trn nguyn tc: Nng ca cht t l vi chiu cao hoc din tch pic ca n. C 4 phng php nh lng c s dng trong sc k :
Phng php chun ngoi (External Standard)

Phng php chun ngoi l phng php nh lng c bn, trong c 2 mu chun v th u c tin hnh sc k trong cng iu kin. So snh din tch (hoc chiu cao) pic ca mu th vi din tch (hoc chiu cao) pic ca mu chun s tnh c nng ca cc cht trong mu th. C th s dng phng php chun ho 1 im hoc nhiu im. * Chun ho 1 im: Chn nng ca mu chun xp x vi nng ca mu th. Tnh nng mu th theo cng thc:

SX c X = CS S
S

y: CX CS : nng mu th : nng cht chun

SX (HX): din tch (chiu cao) ca pic mu th SS (HS): din tch (chiu cao) ca pic mu chun 104

* Chun ho nhiu im: Tin hnh qua cc bc sau : Chun b mt dy chun vi cc nng tng dn ri tin hnh sc k. Cc p ng thu c l cc din tch hoc chiu cao pic mi im chun. V th chun biu din s tng quan gia din tch S (hoc chiu cao H) pic vi nng ca cht chun (C), xem hnh 3.12. S dng on tuyn tnh ca ng chun tnh ton nng ca cht cn xc nh. C th thc hin vic tnh ton ny theo 2 cch : p d kin din tch (hoc chiu cao) pic ca cht th vo ng chun s suy ra c nng ca n .

Hnh 3.12. th phng php chun ngoi

+ Xy dng phng trnh hi qui tuyn tnh m t quan h gia din tch (hoc chiu cao) pic vi nng ca cht cn xc nh. Y = a + bCx Trong : Y: Din tch pic a : Giao im ca ng chun vi trc tung. b : dc ca ng chun. Cx : Nng ca cht th . Da vo phng trnh hi quy ny ta tnh c nng cht th . 3.25

C =
X

a Y b

3.26

Ch : ln ca din tch (hoc chiu cao) pic mu th phi nm trong on tuyn tnh ca ng chun. 105

Phng php chun ni (Internal standard).

Trong nh lng bng phng php sc k, in di ni chung cng nh HPLC ni ring, phng php chun ngoi (ESTD) t ra c nhiu nhc im trong phn tch cc mu phi x l phc tp (chit tch...) v c bit l cc mu va phc tp va c hm lng thp ca cht cn nh lng. V d nh cc mu huyt tng trong nghin cu dc ng hc. Phng php chun ni (ISTD) gip khc phc c nhng nhc im trn ca ESTD. Ngoi ra, n cn c th gip cc tiu ha c nhng sai s gy nn do my mc v k thut. K thut chun ni c th c tm tt nh sau: Ngi ta thm vo c mu chun ln mu th nhng lng bng nhau ca mt cht tinh khit, ri tin hnh sc k trong cng iu kin. Cht c thm ny gi l chun ni. T nhng d kin v: din tch (hoc chiu cao) pic v lng (hoc nng ) ca chun, chun ni v mu th, c th xc nh c hm lng ca thnh phn cn nh lng trong mu th mt cch chnh xc. C mt s yu cu t ra vi cht chun ni: Trong cng iu kin sc k, cht chun ni phi c tch hon ton v c thi gian l u gn vi thi gian l u ca cht cn phn tch trong mu th . C cu trc ho hc tng t nh cht th . C nng xp x vi nng ca cht th . Khng phn ng vi bt k thnh phn no ca mu th . Phi c tinh khit cao v d kim . V rng p ng (response) ca cht chun v chun ni vi detector khng cng nhy, nn trc ht cn phi xc nh h s p ng (response factors) hiu chnh trong tnh kt qu. * Xc nh h s p ng FX : Chun b mt hn hp c cha nhng lng (hoc nng ) bit ca cht chun v chun ni ri chy sc k. Sc thu c s cho ta bit cc d liu v din tch ca cc pic. Trong phng php chun ni, ngi ta thy c mi tng quan gia t s ca khi lng (hoc nng ) ca chun v chun ni vi t s din tch ca 2 pic. V h s p ng c tnh theo phng trnh sau: Tnh theo nng ta c:

F =
X

mC

m IS

S C = mC .S IS S IS m IS S C

(3.27)

FX =
106

CC .S IS C IS S C

(3.28)

 y: - mC , mIS ln lt l khi lng ca cht chun v chun ni. - CC , CIS ln lt l nng ca chun v chun ni. - SC , SIS ln lt l din tch pic chun v chun ni. Cc sai s s c cc tiu ha nu h s FX xp x n v, c ngha l cht chun v chun ni c cng p ng vi detector. Tuy nhin trong thc t iu ny thng kh t c hon ho. * nh lng thnh phn trong mu th. Phng php chun 1 im: Chun ni c thm vo c hai, mu chun v mu th, ri tin hnh sc k. Lng hoc nng ca thnh phn trong mu th c tnh nh sau:

ST mT =
Tnh theo nng ta c:

S IS

m IS FX

(3.29)

CT =

ST C F S IS IS X

(3.30)

Phng php chun ha nhiu im: Chun b mt dy chun c cha nhng lng (hoc nng ) cht chun khc nhau nhng tt c cng cha mt lng (hoc nng ) chun ni. Sau khi sc k v thu c cc d kin din tich, tin hnh v ng chun (hnh 3.13.) biu din s tng quan gia t s din tch (hoc chiu cao) pic ca chun trn chun ni (SS/ Sis) vi t s ca nng chun ngoi trn chun ni (CS/CIS).
SS/Sis

SX/ SIS

CX/ CIS

CS/CIS

Hnh 3.13. Phng php ng chun s dng chun ni

107

Song song tin hnh sc k mu th cng c thm chun ni vi lng (hoc nng ) nh thang chun. Tnh t s din tch (hoc chiu cao) pic ca cht th trn din tch (hoc chiu cao) pic chun ni (ST/ Sis) ri da vo ng chun s tm c nng ca cht th (CT).
Phng php thm chun (Standard addition)

K thut ny phi hp phng php chun ngoi v chun ni. u im ca k thut thm chun l c chnh xc cao v n loi tr c sai s do cc yu t nh hng, c bit l nh hng ca qu trnh x l mu (chit xut, tinh ch cc cht t cc dng bo ch...) K thut so snh tin hnh nh sau: X l mu th ri tin hnh sc k. Thm vo mu th nhng lng bit ca cc cht chun tng ng vi cc thnh phn c trong mu th ri li tin hnh x l mu v sc k trong cng iu kin. Nng cha bit CX ca mu th c tnh da vo s chnh lch nng C (lng cht chun thm vo) v s tng ca din tch (hoc chiu cao) pic S theo cng thc:

cX = S X

C S

C th tnh ton nng CX ca mu th theo mt cch khc: Tin hnh sc k mt mu th v mu th c thm chun nh trn. S dng mt pic khng mun nh lng ca mu th nh l mt chun ni. Th d: Tin hnh sc k mt mu th c 3 thnh phn, thu c sc (a), sc ny c 3 pic c nh s l 1; 2 v 3. Gi s khng mun nh lng thnh phn th nht. nh l ng thnh phn th hai v ba, chng ta thm nhng l ng bit ca hai thnh phn ny vo hn hp ri tin hnh sc k li, thu c sc (Hnh 3.14).

Hnh 3.14: Sc nh lng, phi hp phng php chun ngoi v ni.

108

Bng cch s dng pic khng mun xc nh ca mu th nh l mt chun ni trong phng php thm ny, chng ta s cc tiu ho c cc sai s gy nn trong qu trnh nh lng nh trnh by trn. Trong trng hp mu th khng cha pic khng mun xc nh th c th thm mt cht chun ni vo mu th ri tin hnh phn tch nh trn. Tnh kt qu: Da vo din tch pic (S) hoc chiu cao (h). Tnh nng ca thnh phn th hai: Ra Rb = S2a/S1a ( sc a ) = S2b /S1b ( sc b )

Rthm = Rb - Ra Yth = (Ythm). (Ra)/Rthm

Nng ca thnh phn th ba: Cch tnh tng t . K thut thm ng chun: Nguyn tc: Chun b mt dy hn hp gm cc lng mu th ging nhau v cc cht chun (tng ng vi cc thnh phn cn xc nh) vi lng tng dn. X l mu ri tin hnh sc k. Dng ng chun tng quan gia din tch (S) hoc chiu cao (H)ca pic tng (th + chun) vi lng hoc nng ca cht chun thm (C). Giao im ca ng chun ko di vi trc honh chnh l nng ca cht cn xc nh.

S (H)

dd th SX CX thm chun

C chun thm vo

Hnh 3.15: th phng php thm ng chun

109

 phng php chun ho din tch (Area Normalization)

Nguyn tc: Hm lng phn trm ca mt cht trong hn hp nhiu thnh phn c tnh bng t l phn trm din tch pic ca n so vi tng din tch ca tt c cc pic thnh phn trn sc . Phng php ny yu cu tt c cc thnh phn u c ra gii v c pht hin. Tt c cc thnh phn u c p ng detector (response) nh nhau. K thut ny c s dng nhiu trong sc k kh v thng c p ng nh nhau detector ion ho ngn la (FID). Trong khi li ch ca k thut ny trong HPLC b hn ch v p ng nh nhau l iu thiu chc chn. V d : Phn tch sc k mt hn hp gm 3 thnh phn : X, Y, Z. Hm lng phn trm ca X c tnh nh sau :

%X =

.SX .100 S .100 = X n S X + SY + S Z S

i =1 i

Trong phng php trn nu xt n p ng khc nhau ca detector th cn phi xc nh cc h s p ng i vi mi cht hiu chnh s sai khc . H s p lin h vi cht chun theo cng thc:

FX =
y:

S S .C X . f S S X .C S

SS v SX : din tch ca pic chun v pic th CS v CX : nng ca cht chun v cht th fS : h s hiu chnh ca chun. Phng trnh tnh hm lng % ca X s l:

%X =

S X .FX .100 S X FX + SY FY + S Z FZ

Trong mt s trng hp c th ng dng phng php chun ho din tch pic nh lng cc tp cht trong thuc. 110

Ti liu tham kho

1. Trn T An, Nguyn Vn Tuyn (1984). Bi ging kim nghim c cht. NXB Y hc. Tr. 85 - 90 2. Dc in Vit Nam III . Ph lc 3 , 4 . PL . 75 - PL. 85 . 3. Phm Gia Hu, Trn T An (1998). Ho Phn tch II. i hc Dc H Ni. Tr. 4-32; 55 104. 4. Vin kim nghim (1995). Tp hun k thut kim nghim thuc bng phng php phn tch dng c. Tr. .28 30. 5. B.P 2001. Appendix II, Appendix III. A 130 - A.145 6. Bureau of food and drugs (1998). Training course on drug and cosmetic quality control. BFAD. Philippines . P.p. 95 107. 7. European Pharmacopoeia (1997). P. p. 25 32 8. Indian Pharmacopoeia 1996. P.p .A.72 A77 9. Johnson E. and Stevenson R. (1978). Basic liquid chromatography. Copyright by Varian Associates. 10. Munson J.W. (1984). High - performance Liquid Chromatography: Instrumentation and pharmaceutical applications in pharmaceutical analysis. Marcel Dekker Inc. P.p. 16 - 136. 11. Pharmacopoeia of the peoples Republic of China 1977. P.p. A16 A19. 12. Skoog D.A. - West D.M.West, Holler F.J (1988). Fundamentals of Analytical Chemistry 5th . ed. Saunders college Publishing. P.p. 557 - 598. 13. U.S.P. 24 . P.p. 1920 - 1924; 1992 - 1997.

cu hi t lng gi

3.1. a/. Mt dung dch ca mt cht thuc c nng 6,4.10-5 M, o hp th ca dung dch ny bc sng 255 nm, dng cuvet 1cm thu c mt quang A = 0,847. Tnh h s hp th phn t ca cht 255 nm ? b/. Cn chnh xc 10,0 mg ca thuc ni trn (c phn t lng Mw 200,0), em ho tan trong nc va 1 lt. o hp th 255nm vi cuvet 1cm c A = 0,556. 111

Hy tnh tinh khit ca mu thuc. p s: a/. = 1,32 . 104 b/. 84,2% 3.2. Poldin methylsulfat c cc i hp th 257 nm. Chun b 5 dung dch ca n trong methanol vi nng di y v o hp th ca mi dung dch = 257nm, cuvet 1cm. a/ H ny c tun theo nh lut Lambert-Beer khng ? b/ Tnh A1% 1cm 257 nm. c/ Tnh h s hp th phn t . Bit Mw ca poldin sulfat l 451.
C(mg/ml) 0,1 0,2 0,3 0,4 0,5 A257 0,105 0,207 0,318 0,420 0,529

d/ Ho tan 50,0mg Poldin methylsulfat trong methanol trong mt bnh nh mc dung tch 1 lt, thm methanol ti vch. o hp th ca dung dch ny 257nm, dng cuvet 2 cm, c A = 0,946. Gi thit rng nhng tp cht c mt khng hp th nh sng bc sng ny. Hy tnh % tinh khit ca mu th. p s : b/ A1% 1cm = 10,5; c/ = 474; d/ 90,2% 3.3. Tolbutamid c Mw 270,4; = 703 262 nm. Nu mt vin nn tolbutamid c ho tan trong nc v ho long ti 2500ml th hp th ca n A= 0,520 262 nm, cuvet 1cm. Hy tnh hm lng ca tolbutamid trong vin ?
p s : 500mg

3.4. S hnh thnh ph hunh quang v ng dng ca phng php ph hunh quang trong phn tch ? 3.5. ng dng quang ph hng ngoi trong nh tnh cc cht hu c ? 3.6. Tnh nng ca dung dch Viatamin B12 bit rng khi pha long 200 ln v o hp th 361 nm c A = 0,450. Cho A (1%, 1cm) ca B12 361nm = 207. 3.7. Sc k pha thun v pha o l g ? Cho v d v pha tnh v pha ng dng trong sc k pha thun v pha o. 112

3.8. Thi gian lu tng i ca codein, heroin, methadon, morphin v propoxyphen l: 1,00; 1,89; 0,55; 1,16 v 5,90. Bit tR ca codein l 6 pht. Tnh thi gian lu ca cc cht khc? 3.9. D kin sau thu c bng sc k trn ct di 40cm.
Tn cht Cht khng l u gi A B C tR (pht) 1,9 10,0 10,9 13,4 W1/2(pht) 0,76 0,82 1,06

Hy tnh : a/. S a l thuyt trung bnh ca ct ? b/. Chiu cao ca a ? c/. phn gii cho: + B v A + B v C + A v C 3.10. Cho ct sc k di 30 cm. Khi chy sc k tch hn hp 2 cht thu c kt qu:
Cht Cht khng l u gi A B tR (pht) 1,30 16,40 17,63 Chiu rng y pic W(pht) 1,11 1,21

Hy tnh : phn gii ca ct S a l thuyt trung bnh ca ct, chiu cao a. Nu chng ta mun phn gii trong tch 2 cht A v B l RS(m) = 1,5 th: a/. Yu cu ct phi c s a l thuyt l bao nhiu ? b/. Nu khng thay i 2 pha, chiu di ct s phi l bao nhiu ? 3.11. Khi chy sc k mt hn hp 2 cht A v B thu c thi gian lu ln lt l: 10,0 v 10,9 trn ct di 40cm. Mt cht khng lu gi qua ct 1,9 pht. Chiu rng y pic ca A v B l 1,52pht v 1,64 pht. Cho bit th tch pha tnh VS = 19,6ml v th tch pha ng V M = 62,6ml. 113

Hy tnh : a/. H s dung lng k cho mi cht ? b/. H s phn b cho mi cht ? c/. H s chn lc cho A v B ? 3.12. Hy so snh sc k lng hiu nng cao vi sc k ct c in ? 3.13. Trnh by v s khc nhau gia sc k trao i ion v sc k loi c (sc k trn gel) ? 3.14. Hy trnh by v cc loi bm s dng trong HPLC, u, nhc im ca mi loi ? 3.15. Nguyn tc hot ng ca detector dn nhit v ion ho ngn la ? 3.16. Hy trnh by v phng php s dng chun ni trong nh lng bng sc k lng hiu nng cao ? 3.17. Hy ni v phng php thm chun trong nh lng bng HPLC ?

114

Chng 4.

Kim nghim thuc bng phng php sinh hc

Mc tiu hc tp 1. Vit c nhng c im c bn v hnh thi, tnh cht nui cy ca vi khun, nm mc, nm men p dng trong th nghim vi sinh vt. 2. Bit phng php lm mt mi trng nui cy vi sinh vt v nu c cc phng php tit trng. 3. Trnh by c mc ch, nguyn tc, phng php th v trng v m s lng vi sinh vt trong 1 gam (1 ml) dc phm. Nu c vai tr ca phng php sinh hc trong kim nghim cht khng sinh, v trnh by c th nghim nh lng cht khng sinh bng phng php khuch tn. 4.1. M u Trong ngnh Dc c nhiu phng php khc nhau kim tra cht lng ca thuc nh phng php ho hc, phng php vt l, phng php sinh hc. Phng php ho, l tin hnh nhanh chnh xc nhng ch p dng c vi cc cht c thnh phn ho hc bit v khng nh gi c bn cht sinh hc ca mt dc phm. xc nh cc c tnh sinh hc theo yu cu ca mt s dc phm nh: hot lc tc dng ca cht khng sinh, s v trng, nhim khun, c tnh bt thng ca mt s loi thuc, hay hiu lc v tnh an ton ca cc vaccin ... ngi ta phi dng phng php sinh hc. 4.1.1. Nguyn tc Phng php sinh hc da trn nguyn tc: So snh hiu lc tc dng hoc cc c tnh ring ca cht th vi cht chun tng ng trong cng mt iu kin v thi gian th nghim. Trong lnh vc kim nghim thuc, hai loi th nghim sinh hc c p dng nhiu nht l: Kim nghim thuc bng cc phng php th trn ng vt. Kim nghim thuc bng cc th nghim vi sinh vt. 115

4.1.2. Cht chun Trong th nghim sinh hc cht chun l mt yu t quan trng nh gi cht lng cht th. Cht chun c chia lm hai loi: Cht chun gc: l nhng cht ng nht c tinh khit cao. Cht chun gc thng c lm cc vin nghin cu quc gia hoc quc t ring v cht chun sinh hc. Cht chun th cp: cng l cht c tinh khit cao, c hot tnh sinh hc c xc nh theo cht chun gc quc t tng ng.

Cht chun phi c bo qun trong cc ng thu tinh nhit thch hp tu theo mu (thng nhit < 5oC) trong iu kin kh, trnh nh sng. 4.1.3. nh gi kt qu Th nghim sinh hc thng c thi gian th nghim ko di v ph thuc vo nhiu yu t nh tnh cht p ng ca sinh vt th nghim, ngi lm th nghim, cc iu kin th nghim. Cc yu t ny thng khng n nh. V vy kt qu th nghim sinh hc phi c nh gi bng ton thng k. chnh xc ca php th c th hin bng gii hn tin cy. 4.2. Kim nghim thuc bng phng php th trn ng vt 4.2.1. Nguyn tc Kim nghim thuc bng cc php th trn ng vt da trn s p ng ca ng vt th nghim i vi cc ch phm c a vo c th mt liu lng theo quy nh ca tng th nghim nh gi cht lng ca ch phm cn th. 4.2.2. ng vt th nghim ng vt dng trong th nghim phi ng u, thun khit v ni ging, kho mnh khng nhim bnh, khng c thai v c nui dng y . Mi th nghim c nhu cu khc nhau v ging, trng lng, tui ca ng vt. Cht lng ca ng vt quyt nh chnh xc ca php th. 4.2.3. Th in vivo v in vitro Th nghim c thc hin trn c th ng vt sng gi l th in vivo. Ngi ta da trn cc thng s nh nhit c th, nhp tim, in tm , in no , s thay i huyt p, phn x h thn kinh hoc t l sng, cht ca ng vt th nghim nh gi tc dng v cht lng ca thuc. Php th c th c tin hnh trn cc c quan c lp ca ng vt, nh tim, t cung, rut, mu gi l th in vitro.

116

4.2.4. Liu (Dose) Liu l lng ch phm th a vo c th ng vt mt ln cho tng mc ch th nghim. V d: Liu LD0, LD50, LD100, MLD, liu th cht h p, liu th cht gy st. 4.2.5. Cc th nghim trn ng vt c p dng trong kim nghim thuc Trong cc dc in thng c cc th nghim sau y c thc hin bng cc php th trn ng vt nh: Th c tnh bt thng Th cht h huyt p Th cht gy st nh lng cc hormon: gonadorelin, corticotrophin, insulin, oxytocin, menotrophin, ... Kim tra tnh an ton ca vaccin v sinh phm. Xc nh hiu lc ca cc vaccin v antitoxin.

4.3. Kim nghim thuc bng cc php th vi sinh vt 4.3.1. i cng v vi sinh vt Vi sinh vt l nhng c th sng c kch thc rt nh m mt thng khng th nhn thy c. Nu cn quan st hnh dng ca chng phi dng knh hin vi. Trong t nhin, vi sinh vt tn ti rt phong ph v a dng, chng ng vai tr tch cc vo vng tun hon vt cht. Nhiu loi vi sinh vt c ng dng trong cc lnh vc y t, cng nghip, nng nghip nh cc vi sinh vt c kh nng ln men ru, sinh tng hp khng sinh, vitamin, acid amin, vi sinh vt c nh m thc vt Trong qu trnh hot ng sng, bn cnh nhng c tnh c ch, vi sinh vt cng gy nhiu tc hi cho ngi, ng vt, thc vt nh: lm bin i cht lng thuc, hng thc phm, mt s c kh nng gy bnh hoc sinh c t c hi. phc v cho vic kim nghim thuc bng cc th nghim vi sinh vt, ta cn tm hiu mt s c im chnh ca hai nhm vi sinh vt l vi khun v vi nm. 4.3.1.1. Vi khun (Bacteria) c im: Vi khun l nhng vi sinh vt n bo c cu to t bo tin nhn (Procaryote), c kch thc rt nh. ng knh t bo phn ln thay i trong khong 0,2 2,0m, chiu di t 2 - 8m. Vi khun c nhiu hnh dng khc nhau, nh hnh cu, hnh que, xon, du phy. Vi khun ch sinh sn v tnh, 117

mt s to bo t. Mi t bo vi khun ch c mt bo t. Mt s vi khun c kh nng di ng nh s c mt ca mt hoc nhiu lng (flagella). Phn loi: Vic phn loi vi khun rt phc tp, phi da vo nhiu c im, hnh thi, sinh l, sinh ho chia vi khun thnh cc h, chi, loi khc nhau. Vi mc ch phc v cho cng tc kim nghim thuc, ta khng i su vo nghin cu phn loi, nhng cn tm hiu vi khun, theo cc nhm da trn hnh th, tnh cht bt mu thuc nhum Gram v kh nng h hp ca chng. Theo hnh th: + Cu khun (Coccus): Vi khun hnh cu c th ng ring r (Micrococcus), thnh tng m (Staphylococcus), hoc chui (Streptococcus), hay xp thnh tng i (Diplococcus). + Trc khun (Bacillus): Vi khun hnh que ngn ng ring l hay thnh chui (Bacillus anthracis) hoc hnh que hai u trn (Escherichia coli). + Xon khun (Spirillum): Vi khun hnh l xo nh: Treponema Pallidum. + Phy khun (Vibrio): Vi khun hnh du phy nh Vibrio cholerae. Theo tnh cht bt mu thuc nhum Gram. + Vi khun c mu tm sau khi nhum Gram: Vi khun Gram + + Vi khun c mu sau khi nhum Gram: Vi khun Gram Theo c tnh ca qu trnh h hp: + S dng oxy t do trong qu trnh h hp: Vi khun hiu kh. + Pht trin c c trong iu kin hiu kh v k kh, c qu trnh h hp nitrat: vi khun k kh khng bt buc. + Ch sng trong iu kin k kh, c qu trnh h hp sulfat: Vi khun k kh bt buc.

Sinh sn ca vi khun:
log N

3 4 2

thi gian

Hnh 4.1: ng cong sinh trng ca vi khun

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 Vi khun sinh sn bng cch phn i t bo. S phn chia t bo xy ra rt nhanh. Trong iu kin mi trng thch hp v khng c cc yu t km hm th mt t bo vi khun sau 6 gi c th sinh ra 250.000 t bo mi. Tuy nhin, s nhn ln ca vi khun khng phi l v tn, n cn ph thuc vo nhiu yu t. Trong mi trng nui cy, s sinh sn ca vi khun sau mt thi gian nht nh s b ngng li v nhiu nguyn nhn nh: thc n b ht dn, hoc vi khun c th tit ra nhng cht km hm s pht trin ca chng. Tc pht trin ca vi khun trong mi trng nui cy tnh thay i theo thi gian v tun theo mt quy lut nht nh bao gm 4 pha: Pha lag, pha logarit, pha n nh v pha t vong (Hnh 4.1). S pht trin ca vi khun trong mi trng lng c th quan st sau khong 18 - 24 gi nui cy. Chng c th lm c mi trng, to vng trn b mt hoc lng cn y ng nghim. Trn cc mi trng c, khun lc ca cc vi khun thng nh hn khun lc ca vi nm, b mt nhn, bng, t hoc nhn nheo, ... Khun lc ca vi khun to sc t vi cc mu nh: trng (Staphylococcus albus), vng (Staphylococcus aureus), hng (Micrococcus), xanh (Pseudomonas aeruginosa), ... Mi vi khun c c tnh ring v hnh dng, kch thc, mu sc khun lc. Cc c tnh ny gip cho vic xc nh vi khun trong qu trnh kim nghim dc phm. 4.3.1.2. Vi nm (Microfungi)
c im:

Vi nm c cu to t bo nhn tht (Eucaryote). T bo vi nm rt nh. Mun quan st cn dng knh hin vi. Nm khng c cht dip lc, sng hoi sinh hoc k sinh, sinh sn v tnh hoc hu tnh. Vi nm bao gm hai loi l nm men v nm mc. Trong kim nghim vi sinh vt, cn pht hin hai loi ny c trong dc phm. Nm men (Yeast): Nm men c cu to n bo, sinh sn ch yu bng ny chi. T bo nm men c kch thc, hnh dng khc nhau tu loi. Chng c th hnh cu, bu dc, hnh qu chanh, hnh ng Khun lc nm men bao gm nhiu c th thng thuc mt loi pht trin t mt c th m to thnh mt khi. Khun lc nm men thng to hn khun lc vi khun, b mt c np nhn hoc trn nhn, khng to si. Nm men c s dng nhiu trong cng nghip thc phm nh lm bnh m, bia, ru Nhng nhiu nm men gy bnh hoc lm hng thc phm, thuc. 119

Nm mc (Mold): Nm mc c cu to si, sinh sn bng bo t, sng hoi sinh, chng thng pht trin trn b mt c cht di dng nhng lp hnh si, mng nhn hoc khi si bng. Si nm rt nh, ng knh trung bnh 5m, chiu di c th vi chc centimet. Si nm c vch ngn hoc khng c vch ngn. Ton b si nm v cc nhnh pht trin t mt bo t nm ri an kt nhau thnh mt khi gi l h si nm. Trn mi trng thch nui cy, h si nm pht trin thnh mt khi c tit din hnh trn hoc gn trn gi l khun lc. Khun lc c c trng bi mu sc ca si nm v ca bo t. B mt khun lc c th mt, dng ht, dng si hoc xp Nm sinh sn bng bo t: Bo t v tnh hoc bo t hu tnh . S sinh sn v tnh v hu tnh lun an kt nhau trong qu trnh sinh trng ca nm. V vy, nm pht trin rt nhanh trn b mt cc c cht. Nm mc thng gy ra nhng bin i v mu sc, mi v, cht lng ca thuc. Mt s sinh cc c t (Mycotoxin) c hi cho ngi v ng vt.

4.3.1.3. S nh hng ca cc yu t ngoi cnh i vi qu trnh pht trin ca vi sinh vt Sinh trng v trao i cht ca vi sinh vt lin quan cht ch n cc iu kin ca mi trng bn ngoi. Cc iu kin ny bao gm hng lot cc yu t khc nhau, tc ng qua li vi nhau. a s cc yu t u c mt c tnh tc dng chung biu hin 3 im hot ng: Ti thiu, ti u, cc i. Khi mt yu t c tc dng ti u, vi sinh vt pht trin vi tc cc i. Nu yu t ny c tc dng cc i , vi sinh vt ngng sinh trng v thng cht. Cc yu t bn ngoi c nh hng n i sng ca vi sinh vt l vt l, ho hc v sinh hc, trong cc yu t vt l l ng ch nht. Yu t vt l bao gm nhit , m v nh sng: Nhit : Nhit l yu t quan trng nht i vi i sng vi sinh vt. Mi loi vi sinh vt c mt gii hn nhit pht trin thch hp. Ni chung i vi vi sinh vt, nhit pht trin thng t 15 45oC. nhit cao s lm thay i qu trnh trao i cht ca vi sinh vt, vi sinh vt b cht. Cc t bo sinh dng th ng b cht nhit 60oC/20 - 30 pht. Cc bo t ch b tiu dit nhit 120oC/ 30 40 pht. Tnh cht ny c ng dng trong vic tit trng. Nhit thp ch c tc dng km hm s pht trin ca vi sinh vt (tr vi sinh vt a lnh). 120

m:

Hu ht cc qu trnh sng ca vi sinh vt c lin quan n nc. Khi thiu nc xy ra hin tng loi nc khi t bo vi sinh vt, trao i cht b gim, t bo s cht. V vy, bo qun dc phm, dc liu trnh khi tc ng ca vi sinh vt cn c mt gii hn m nht nh. nh sng: nh sng mt tri gm cc tia bc x nh: tia t ngoi, hng ngoi, tia gamma c tc dng ph hu t bo vi sinh vt, c bit l tia t ngoi. Bc x UV bc sng khong 260nm, c tc dng dit khun mnh nht. Di nh hng ca tia UV, vi sinh vt b cht hoc t bin tu theo liu lng. ngn nga tc hi ca vi sinh vt i vi thuc cc tc nhn vt l trn cn c vn dng trong qu trnh sn xut v bo qun dc phm, nhm hn ch ti a s lng vi sinh vt gy nhim ban u. ng thi cc ch phm dc phi c quy nh gii hn vi sinh vt cho php. 4.3.2. Mi trng nui cy vi sinh vt Mi trng nui cy l nhng cht dinh dng thch hp nhm m bo cho vi sinh vt sinh trng v pht trin. Mi trng cn c 3 iu kin sau: y cht dinh dng theo yu cu th nghim, c pH trong khong quy nh v phi v trng. Mi trng gm 3 loi: Mi trng t nhin: nguyn liu c ngun gc t ng vt hay thc vt (nh cao tht, cao men, pepton, tinh bt). Thnh phn c th thay i tu theo ngun gc nguyn liu. Mi trng tng hp: bao gm cc ho cht thun khit c quy nh v thng ho tan trong nc. Mi trng bn tng hp: trong thnh phn mi trng c c cc nguyn liu t nhin v tng hp.

Pha ch mi trng l mt khu rt quan trng trong cc th nghim vi sinh vt. chnh xc ca kt qu th nghim ph thuc rt nhiu vo cht lng mi trng. 4.3.2.1. Phng php pha ch mi trng

Khi pha ch mi trng cn tin hnh qua 6 bc sau: Chun b dng c ho cht:

Dng c pha ch mi trng tt nht l bng men hoc thu tinh. Cc dng c phi ra sch, hoc tit trng nng trc khi s dng. 121

Nguyn liu pha ch mi trng phi m bo cht lng, ho cht phi tinh khit. Nu l cc dng bt hoc tinh th phi kh, khng i mu, khng chy nc. Cn ong nguyn liu: Cc nguyn liu phi c cn ong chnh xc, nht l nhng ho cht hoc nguyn t vi lng c th gy c ch vi khun (mui mt, st) phi c cn bng cn phn tch. Ho tan nguyn liu: Thng dng nc ct hoc nc kh khong pha mi trng. Cc ho cht c ho tan nng hoc lnh tu theo tnh cht ca chng. Mi trng khng c thch nn ho tan lnh hoc nng nh. Mi trng c thch cn un cho thch tan hon ton sau mi cho cc thnh phn khc vo. iu chnh pH: Khi iu chnh pH ca mi trng nn thc hin nhit 45 - 50oC pH t b thay i sau khi tit trng. Cc dung dch NaOH 1N v HCl 1N thng c s dng iu chnh pH. Sau khi iu chnh pH, cn b sung nc cho th tch quy nh. Lm trong mi trng Cc mi trng lng (bao gm cc cht ho tan) phi trong d quan st s pht trin ca vi sinh vt. Sau khi ho tan cc cht, nu mi trng c cn phi lc qua vi gc hoc giy. ng ng tit trng: Mi trng c cho vo ng nghim bnh nn hoc bnh cu, tu theo yu cu th nghim. Khi ng ng, khng c mi trng dnh vo ming ng hoc bnh. Mi trng cn phi c tit trng ngay sau khi ng gi. Nu lu, tp khun s pht trin lm hng mi trng. Cc mi trng thng thng c tit trng 110oC/30 pht hoc 120oC/ 20 pht. Mi trng c cc cht d b ph hu bi nhit, cn tit trng nhit thp bng phng php Tyndall, Pasteur, hoc dng lc vi khun. Mi trng c ly ra khi ni hp ngay sau khi tit trng xong. Nu lu trong ni hp mi trng b chuyn mu v gim cht lng. Pha ch mi trng t hn hp bt mi trng ch sn: nhiu nc trn th gii c cc loi mi trng di dng bt kh cha y cc thnh phn theo yu cu, cc mi trng ny c lm t nguyn liu, ho cht tinh khit nn cht lng mi trng m bo v n nh. Khi lm th nghim mi trng c pha vi nc theo t l quy nh, nhng phi 122

dng nc mi ct hoc nc kh khong, trung tnh pha ch. Nu bt mi trng c cn phi kim tra li pH sau khi lm mi trng. 4.3.2.2. Bo qun mi trng Mi trng bt kh c gi 10 - 12oC trong iu kin kh, trnh nh sng. Mi trng pha ch c bo qun 4 10oC trong 1 2 thng tu theo thnh phn mi trng.

4.3.2.3. Cc phng php tit trng Tit trng l mt qu trnh lm cho mt vt hoc mt sn phm khng cn vi sinh vt sng c. Tit trng c thc hin bng phng php vt l, ho hc. Chn phng php tit trng ph thuc vo tnh cht l ho v bn vng ca mi trng. Tit trng bng nhit kh: Phng php ny dng tit trng cc dng c th nghim bn vi nhit nh bng, bng, vi, gc, dng c thu tinh iu kin tit trng l: 180oC/ 30 pht hoc 170oC/ 1 gi, hoc 160oC/2 gi. Cc dng c thu tinh ng mi trng phi c tit trng kh trc khi dng. Tit trng bng hi nc: Phng php dng nhit t thng c dng tit trng mi trng nui cy v cc dng c phu thut. Mi trng thng c tit trng bng ni hp 121oC/ 15 pht. Cc mi trng d b hng bi nhit nh mi trng c ng, sa, bia, mu, albumin cn tit trng nhit thp bng cc phng php sau: Tit trng gin on (phng php Tyndall): Mi trng c hp 3 - 4 ln nhit khng qu 100oC trong 30 - 40 pht, cch nhau 24 gi. Gia hai ln hp cho mi trng vo 28 32oC/ 24 gi cho bao t ny mm. Cc bo t sng st ny mm s b tiu dit ln hp tip theo. Kh trng nhit thp (phng php Pasteur): un cch thu mi trng 60oC/ 30 pht, hoc 73oC/ 15 pht sau lm lnh t ngt di 10oC. Phng php ny khng dit c bo t. Phng php lc:

Phng php lc c dng tit trng cc cht d b ph hu bi nhit. Cho cht lng chy qua mng lc c kch thc l lc 0,22m. Phn 123

chy qua phu c ng trong cc dng c v trng. Thit b lc v mng lc phi c tit trng trc khi dng. Phng php dng tia bc x: Tia t ngoi (UV) c dng nhiu nht tit trng cc bung pha ch, t cy vi sinh vt. n t ngoi phi c chiu trc tip, thng gc vi ni lm th nghim v liu lng chiu phi vi din tch bung. Tia UV t c tc dng dit nm, v vy khi kh trng bung pha ch cn phi hp thm phng php dng ho cht kh nm. 4.3.3. Th v trng 4.3.3.1. Mc ch Th v trng nhm mc ch pht hin s c mt ca vi khun, vi nm trong cc ch phm nh dch tim truyn, mt s loi thuc tim, thuc tra mt v cc dng c y t m theo tiu chun ring cn phi v trng. 4.3.3.2. Nguyn tc Vi sinh vt c trong ch phm th s pht trin trn cc mi trng dinh dng thch hp, chng lm c mi trng lng to vng trn b mt hoc lng cn y ng nghim. Trn mi trng c vi khun, vi nm mc thnh cc khun lc c trng. 4.3.3.3. Mi trng Trong nhiu dc in thng dng mi trng Thioglycolat lng pht hin vi khun hiu kh, k kh v mi trng Casein u tng lng pht hin vi khun, vi nm. Tuy nhin, c th dng cc mi trng khc cho th nghim, vi iu kin cc mi trng ny thch hp cho s pht trin ca loi vi sinh vt cn pht hin. V d c th dng mi trng canh thang cao tht pepton pht hin vi khun hiu kh; mi trng Wilson - Blair pht hin vi khun k kh; mi trng Sabouraud lng cho s pht hin vi nm. Kim tra cht lng mi trng: v trng: Ly 1-2 ng (hoc bnh) mi trng mi lm 30 - 35oC t nht trong 4 ngy i vi mi trng pht hin vi khun, v 25 - 28oC t nht trong 7 ngy vi mi trng pht hin vi nm. Sau thi gian nui cy, cc ng mi trng khng c c vi sinh vt mc (c th cc ng mi trng song song vi cc ng c cy cht th) Kh nng dinh dng: Cy vo mi ng mi trng thch hp khong 100 t bo cc vi sinh vt sau: 124

+ Vi khun hiu kh: Staphylococcus aureus Bacillus subtilis Pseudomonas aeruginosa + Vi khun k kh: Clostridium sporogenes + Vi nm: Candida albicans Aspergillus niger ng th vi khun c nui cy 30 - 35oC/ 3 ngy, ng th vi nm 25 - 28oC/ 5 ngy. Sau thi gian nui cy vi sinh vt phi pht trin tt trn cc mi trng. 4.3.3.4. Ly mu th Trong th v trng, c th coi ton b s ng hoc l thuc c kh trng hoc c phn b v trng trong cng mt iu kin l mt l thuc. Thng thng, khi mt l c t hn 100 n v, ly 3 n v kim nghim. Nu nhiu hn th c 50 n v ly thm mt n v nhng khng qu 10. Tuy nhin, khi ly mu cng cn phi da vo tiu chun ngnh hoc tiu chun c s ca tng sn phm ly mu cho thch hp. 4.3.3.5. Kim tra tc dng c ch vi sinh vt ca ch phm th Mt s thuc trong qu trnh sn xut c thm cc cht bo qun. Nhng cht ny c th nh h ng n s pht hin vi sinh vt c trong ch phm. Mt ch phm cha bit c tc dng c ch hay khng th cn phi kim tra tc dng c ch i vi cc vi sinh vt sau: Ly t nht 2 ng ca mi loi mi trng pht hin vi khun hiu kh, k kh, vi nm, cy vo mt trong hai ng ch phm cn th. Cy khong 100 t bo (0,1 ml nh dch vi sinh vt c pha khong nng thch hp) Staphylococcus aureus (vi khun hiu kh), Clostridium sporogenes (vi khun k kh), Candida albicans (vi nm) vo c hai ng ca cc mi trng tng ng. Nui cy 30 - 35oC/4 ngy i vi vi khun v 25 28oC/7 ngy i vi vi nm. Trong thi gian nui cy, nu vi sinh vt pht trin ging nhau (mc nhanh v phong ph) trong ci ng chng v ng kim tra, ch phm th c coi l khng c tc dng c ch. 125

Nu cc ng c cht th, vi sinh vt pht trin yu hoc khng pht trin so vi ng khng c cht th, ch phm c cht c ch. Tc dng c ch ca cht th phi c loi b bng cch pha long, trung ho, hoc dng phng php mng lc. 4.3.3.6. Phng php th Th v trng c th c thc hin theo hai phng php tu theo tnh cht ca mu th: Phng php lc qua mng lc vi khun . Phng php nui cy trc tip. Phng php dng mng lc: Thit b lc thng bng thu tinh, thp khng r hoc nha gm hai b phn c th tho ri, gia c li mng lc. Mng lc c thnh phn l nitrat celleulose thng dng lc nc, du, v dung dch alcol yu. Mng ecetat cellulose lc cc dung dch alcol mnh. L mng lc c nhiu kch thc khc nhau, trong th v trng thng dng mng c ng knh khong 50mm v ng knh l mng lc 0,45m. Thit b lc v mng lc phi c tit trng trc khi th nghim . Dung dch cht th chy qua mng lc, cc vi sinh vt c gi li trn mng, cy mng lc vo cc mi trng thch hp pht hin vi khun, vi nm. Phng php mng lc kim nghim c cc thc c tc dng c ch vi sinh vt, c bit l thuc khng sinh, nhng i hi thit b tt v iu kin v trng cao. Phng php nui cy trc tip: Phng php nui cy trc tip c k thut n gin, nhng kh nng pht hin vi sinh vt gim khi s lng vi sinh vt c t v phn phi trong mt th tch cht th ln. Phng php ny khng thc hin c vi cc ch phm c tc dng c ch v cc cht khng sinh, v khi th nghim cht th c cy trc tip vo cc mi trng nui cy thch hp cho cc vi khun, vi nm. Lng mu th dng trong th nghim: Lng mu th cn cy vo cc mi trng tu thuc vo tng loi mu (bng 4.1.). Cht lng l du hay dung dch du phi thm vo mi trng nui cy 1% tween 80 hoc cc cht nh ho khc vi nng thch hp. Mu th l dng m hay kem c ho long vo dung dch pepton 0,1% v trng theo t l 1/10 trc khi cy vo mi trng, (dung dch pepton, cn thm tween 80 vi t l 1ml/ 1lt). 126

K thut th: Mu th l dc phm:

Dng dng c v trng cy trc tip ch phm th vo cc mi trng pht hin vi khun, vi nm theo s lng quy nh. Cht rn l dng bt c th cho trc tip vo mi trng hoc lm thnh dung dch hay nh dch 1% sau cy vo mi trng.
Bng 4.1. Lng mu th dng cho th nghim nui cy trc tip
Lng ch phm trong mt n v ng gi - Cht lng: Th tch V < 1ml 1 ml V< 4 ml 4 ml V < 20ml 20ml V < 50ml 50ml V< 100ml V 100ml - Cht rn: Khi lng P< 50mg Ton b mt n v ng gi 1/2 khi l ng ca mt n v ng gi 50mg < P< 200mg 100mg 80 20 Ton b mt ng 1/2 ng 2ml 5ml 10ml Thng 10% 10 15 20 40 80 100 Lng ti thiu cho mt mi trng nui cy Th tch mi trng (ml)

40

P 200mg

Mu th l bng gc, ch khu phu thut nu kch thc v hnh dng cho php, nhng ton b mu th vo 100ml mi trng. Mu th l dy truyn dch: Cho dung dch pepton 0,1% v trng chy qua thu c t nht 15ml v cy vo 100ml mi trng. Mi trng pht hin vi khun c nui cy 30 - 35oC t nht trong 4 ngy, v 25 - 28oC t nht trong 7 ngy i vi vi nm. (Mi loi mi trng lm 2 - 3 ng th song song). Nhn nh kt qu: Mu th c coi l v trng nu sau thi gian nui cy khng c vi khun, vi nm pht trin. Nu c 1 hoc nhiu ng c vi sinh vt mc cn lm li ln th hai (Trc khi lm li th nghim ln 2 cn phn lp xc nh c tnh hnh thi ca vi sinh vt cc ng th dng tnh): 127

 Nu khng c vi sinh vt mu v trng. Nu c vi sinh vt ging vi ln mt mu th khng v trng. Nu c vi sinh vt khc vi ln mt th nghim c lm li ln 3 vi s lng mu gp i: + Khng c vi sinh vt pht trin mu v trng. + C vi sinh vt mc mu khng v trng. 4.3.4. Th gii hn vi sinh vt Cc dc phm trong qu trnh sn xut thng b nhim vi khun, vi nm do cc nguyn nhn nh: Do bn cht nguyn liu, nu nguyn liu c ngun gc t ng vt, thc vt th mc nhim khun cao hn nhiu so vi cc ho cht. Cc t dc nh tinh bt, ng, mt l mi trng cha nhiu vi sinh vt, v vy d gy nhim bn cho thuc. C s sn xut, trang thit b, bao b ng gi, ngi sn xut cng l nguyn nhn gy nhim khun. Dng bo ch nh vin hon mm c m cao thng to iu kin cho vi sinh vt pht trin hn vin nn, vin hon cng.

V vy, th gii hn vi sinh vt l mt th nghim bt buc cho cc dc phm t nguyn liu n thnh phm khng c tit trng trong qu trnh sn xut. 4.3.4.1. Mc ch Th nhim vi sinh vt nhm mc ch xc nh gii hn ti a ca s lng vi khun hiu kh, vi nm c trong 1g (hoc 1ml) ch phm th. ng thi pht hin cc vi khun ch im v sinh quy nh khng c c trong thuc l: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, cc loi Salmonella, vi khun k kh Clostridia. 4.3.4.2. Nguyn tc Php th c da trn nguyn tc: m s vi khun hiu kh, nm mc, nm men c trong dc phm c th hin bng cc khun lc c trng trn a thch dinh dng thch hp. Cn c vo cc c tnh hnh thi, sinh l, sinh ho ca tng loi vi khun xc nh vi khun gy bnh. Trn c s kt qu th nghim nh gi cht lng ca thuc theo tiu chun dc in hoc tiu chun c s. 128

4.3.4.3. Phng php th Mi trng: a s cc dc in thng dng mi trng thch casein u tng m vi khun hiu kh, mi trng thch Sabouraud - khng sinh m vi nm. Cng c th dng mi trng thch thng th vi khun hiu kh v mi trng ny rt thch hp cho s pht trin ca a s vi khun hiu kh (theo Dc in Trung Quc 1997) Chun b mu th: Mu th theo quy nh chung c ly 10g (hoc 10 ml) th nghim. Mu th l cht rn hay cht lng c th lm thnh dung dch hay nh dch trong nc, c pha long vo dung dch m phosphat pH =7,2 hoc dung dch NaCl 0,9% c nng 10-1 sau pha cc nng tip theo 10-2, 10-3 tu theo yu cu th nghim. Mu th l cht lng khng ho ln vo nc nh dng du, kem, hoc thuc m. Cn ch to nh dch bng cch thm mt lng cht nh ho v trng thch hp nh tween 20, tween 80, lm nng nh 45oC to mt nh dch ng nht. Kim tra cht c ch: Kt qu th nghim s khng c gi tr nu trong mu th c cht bo qun hoc cc thnh phn c nh hng n s pht trin ca vi sinh vt. V vy, cn kim tra cht c ch trc khi m s lng vi sinh vt. Vi sinh vt ch th: Staphylococcus aureus (i din vi khun G +). Escherichia coli (i din vi khun G -). Candida albicans (i din vi nm). Cc chng trn c nui cy trong cc mi trng dinh dng thch hp sau 18 - 24 gi (i vi vi khun) v 24 n 48 gi i vi vi nm, c lm thnh nh dch c khong 100 t bo/ ml. Cch tin hnh: a th 1: Cho 1ml cht th nng thch hp. a th 2: 1 ml cht th + 1ml nh dch vi sinh vt. a chng: 1ml nc ct v trng + 1ml nh dch vi sinh vt Cho vo mi a 15 - 20 ml mi trng dinh dng thch hp ngui di 45oC. 30 - 35oC/ 24 - 48 gi i vi vi khun v 25 - 28oC/48 - 72 gi i vi C. albicans. 129

 Nhn xt kt qu: m s khun lc vi sinh vt trn cc a th nghim. Gi A l s khun lc a 1 Gi B l s khun lc a 2 Gi C l s khun lc a chng Nu B A + C mu th khng c cht c ch. Nu B A hoc B << A + C mu th c cht c ch vi sinh vt. Cht c ch phi c x l bng cch tng th tch mi trng, trung ho cht c ch bng cc cht trung ho thch hp nh tween 20 4%. Nu mu c cht c ch qu mnh phi th bng phng php mng lc. * Kim tra cht c ch cng c th c thc hin theo cch sau: Cy khong 100 t bo cc vi khun vo cc ng mi trng chn lc khng c mu th v c mu th nng thch hp cho E. coli Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa. Mu th khng c tc dng c ch nu vi khun mc tt nh nhau c hai ng mi trng. m s lng vi sinh vt: i vi vi khun: Cho vo mi a petri 1ml cht th nng thch hp sao cho khng qu 300 khun lc trong 1ml. Thm 15 - 20ml mi trng thch casein - u tng hoc mi trng thch thng ngui di 45oC, xoay nh a cht th trn u vo mi trng. Nui cy 30 - 350C trong 1 - 2 ngy. i vi vi nm: Cho vo mi a petri 1ml cht th nng thch hp sao cho khng qu 100 khun lc vi nm trong 1ml. Thm 15-20ml thch Sabouraud + khng sinh. Nui cy 25 - 28oC trong 4 - 5 ngy. Th nghim c thc hin vi 1 hay 2 nng pha long cui cng. Mi nng c lm 2 - 3 a th ly gi tr trung bnh. Sau thi gian nui cy, cc a c s khun lc vi khun nh hn 300 v vi nm nh hn 100 c m tnh s lng. Tnh kt qu: Tng s vi khun hiu kh, vi nm trong 1g (1ml) c tnh theo cng thc:

X=

A 1k 1 + A 2 k 2 2

(Php th thc hin vi 2 nng ). 130

A1 A2

: S khun lc vi sinh vt trung bnh nng pha long k1. : S khun lc vi sinh vt trung bnh nng pha long k2.

k1, k2 : pha long. Cc dc in c quy nh: mu th c t hn 10 vi sinh vt/1g (1ml) nu khng c khun lc mc trn a th nng 10-1. 4.3.5. Xc nh hot lc cht khng sinh bng cc php th vi sinh vt 4.3.5.1. Mc ch Trong lnh vc kim nghim, nh lng khng sinh ngi ta c th s dng cc phng php khc nhau. Mt s cht khng sinh c th c nh lng bng phng php ho hc, phng php ha l. Tuy nhin, phng php sinh hc vn ng vai tr quan trng trong vic nh gi hot tnh sinh hc ca thuc v kim tra c s gim hay mt hot lc ca cht khng sinh m cc phng php l, ho khng thc hin c. Cc cht khng sinh c cu trc phc tp hoc thnh phn c tc dng gm mt hn hp nhiu thnh phn, thng phi dng th nghim vi sinh vt xc nh hot lc ca thuc. Php th trn vi sinh vt cn cho bit nhy cm ca vi khun gy bnh i vi cc cht khng sinh c th, v mc khng thuc ca chng. Trn c s c th chn khng sinh thch hp cho bnh nhn trong iu tr.

4.3.5.2. Nguyn tc Hot lc ca mt cht khng sinh c xc nh bng cch so snh kh nng c ch ca chng vi sinh vt ch th, bi nhng nng bit ca khng sinh th (cha bit hot lc) v nng bit ca khng sinh chun ( bit r hot lc). 4.3.5.3. Chng ch th Chng ch th l chng vi sinh vt thun khit, nhy cm i vi mt cht khng sinh, chng ch th thng c phn lp t cc bo tng ging Quc gia, c bo qun trong ng ng kh hoc trong mi trng thch hp 4 - 10oC. Tu theo dc in ca mi nc, mi cht khng sinh c th c mt vi chng ch th khc nhau.
Ch to nh dch vi sinh vt khng c bo t:

Cc chng vi khun: Staphylococcus aureus Sarcina lutea 131

Escherichia coli Micrococcus luteus Klebsiella faecalis. Chng vi nm: Saccharomyces cerevisiae. i vi cc chng vi sinh vt khng c bo t, khi th nghim cn s dng chng sau 16 - 24 gi nui cy c cc chng ch th ang giai on pht trin logarit. Chng ch th l vi khun trc khi s dng c cy sang mi trng thch dinh dng (pepton 10g, cao tht 1,5g, cao men 3g, glucose 1g, thch 15g, nc ct 1000 ml). Sau 16 - 18 gi nui cy 35 - 37oC cc vi khun c lm thnh mt nh dch c nng thch hp vi NaCl 0,9% to vng v khun r rt trong phng php khuch tn v c c thch hp trong phng php o c. Chng ch th l vi nm c nui cy trn mi trng thch Sabouraud 28 - 30oC trong 24 gi v cng c lm thnh mt nh dch c nng thch hp. Ch to nh dch bo t: Cc chng ch th c bo t: Bacillus subtilis Bacillus pumilus Bacillus cereus Nu chng ch th c bo t nn ch to dng bo t s dng, v dng bo t c bo qun di 4oC c th tn ti trong mt thi gian rt di. Cc chng vi khun trn c cy vo bnh mi trng thch dinh dng c b mt thch rng (pepton 6g, cao tht 1,5g, cao men 3g, glucose 1g, K2HPO4 3,68g, KH2PO4 1,32g, thch 15g, nc ct 1000ml) Mi trng ny c thm MnSO4 vi hm lng 0,001g/lt thc y s hnh thnh bo t. Nui cy 35 - 37oC trong vng 7 ngy vi B. cereus nui cy 30oC. Dng nc ct v trng ra lp bo t trn b mt mi trng to mt nh dch. un cch thy nh dch ny 70oC/ 30 pht, pha long nh dch n nng 107 - 108 bo t trong 1ml. 4.3.5.4. Cht chun v n v hot lc
Cht chun:

Cht chun l nhng cht c tinh khit cao, c th dng cht chun gc hoc cht chun th cp c hot lc c xc nh theo mu chun quc t 132

tng ng. Cht chun c ng trong ng thy tinh hn kn bo qun kh, trnh nh sng, di 0oC. n v hot lc: Hot lc ca mt cht khng sinh c tnh bng n v hot lc quc t (vit tt l U hoc UI, IU). Mt n v hot lc l hot lc c trng ca mt lng nht nh cht khng sinh chun sinh hc quc t hoc ch phm i chiu sinh hc vi chun quc t. V d 1U gentamicin l hot lc ca 0,0056mg gentamicin. n v hot lc ca cc cht khng sinh c xc nh bi cc vin nghin cu ln ca t chc y t th gii v cht chun sinh hc. Hot lc ca cc cht khng sinh c th c th hin bng g hot lc. i vi cht khng sinh hon ton tinh khit, trong a s cc trng hp g hot lc tng ng vi g khi lng ca cht khng sinh. Khi cht khng sinh cha hon ton tinh khit, hoc l hn hp ca nhiu thnh phn tng t nhau v ho hc nhng khc nhau v hot tnh sinh hc th g hot lc khng nht thit tng ng vi g khi lng ca cht khng sinh. 4.3.5.5. Mi trng v dung mi Ty theo cht khng sinh v vi sinh vt ch th chn cc mi trng thch hp cho th nghim. Dung mi v cc cht pha long khng c nh hng n s pht trin ca vi sinh vt. Cc cht pha long nh: nc ct, dung dch HCl, ethanol, methanol, dung dch m c pH khc nhau c s dng tu thuc vo tan ca cc cht khng sinh. Cc vn ny cn tham kho dc in.

4.3.5.6. nh lng cht khng sinh bng phng php khuch tn C hai phng php vi sinh vt nh lng cht khng sinh l: Phng php o c: (Turbidimetric method) Phng php khuych tn: (Diffusion method)

Phng php khuych tn thng c s dng nhiu trong nh lng khng sinh.
Nguyn tc:

Cht khng sinh khuych tn vo mi trng dinh dng c cy vi sinh vt ch th, to cc vng c ch vi sinh vt c ng knh t l thun vi logarit nng tng ng. Hot lc ca cht th c so snh vi cht chun theo phng php thng k. 133

 Pha dung dch chun v dung dch th:

Pha chun: Cn mt lng cht chun thch hp ho vo dung mi c mt dung dch gc nng khong 1000 UI/ ml. T dung dch gc pha thnh 3 nng cui cng theo cp s nhn h s hai l s1, s2, s3 hoc hai nng s1, s2 (s1 < s2 < s3). Cht th c pha nh cht chun vi gi nh cht th c hot lc tng ng cht chun. Cht th c 3 nng cui cng l t1, t2, t3 (hoc t1, t2). Phng php khuch tn ch cho kt qu tt khi cht chun v cht th c cng bn cht ha hc. S khng ng nht gia chun v th lm thay i tnh song song ca hai ng thng biu th s tng quan gia ng knh vng c ch v logarit nng . V hai cht c hai h s khuch tn ring. V vy khng so snh cht khng sinh cc dng mui khc nhau nh erythromycin stearat v erythromycin propionat, oxytetracyclin v clotetracyclin. Khi pha dung dch chun v dung dch th cn lu : nu chn khong nng cui cng nh lng khng thch hp, hoc hot lc gi nh ca th qu xa hot lc ca chun, th s lm thay i tnh cht ca s tng quan gia ng knh vng c ch v logarit nng . Kt qu nh lng s khng chnh xc.
Tin hnh th nghim:

vo cc a Petri mt lng mi trng dinh dng c cy chng ch th to mt lp ng nht c dy t 2-5mm. Chiu dy lp thch mng hay dy qu, hoc khng ng u c th lm cho quan h gia ng knh vng c ch v logarit nng khng cn l ng thng. Cng c th lm 2 lp mi trng, nhng ch lp trn c cy vi sinh vt ch th. Chng ch th l loi khng c bo t, phi cy vo mi trng c ngui di 45oC, nu l dng c bo t nhit cho php khi cy chng l 65 - 70oC. Cn cy vo mi trng mt lng vi sinh vt ch th sao cho c vng c ch r nt vi kch thc ng knh thch hp tng ng vi cc nng khng sinh chn. Nu lng chng qu t, vng c ch s qu to, vi sinh vt mc tha tht, kh xc nh c vng c ch. Nu lng chng qu nhiu, vng c ch nh s kh o v km chnh xc. Cc a mi trng c kh nhit phng th nghim trong 30 pht trc khi dng. B mt thch t s lm cho vng c ch b nhe. i vi th nghim 3 liu: dng 6 ng tr bng thp khng r c kch thc nh nhau (chiu cao khong 10mm, ng knh trong khong 6mm). t cc ng tr ln b mt a thch theo s , cho cht th v chun vo cc ng tr vi mt lng bng nhau. C th thay ng tr bng cch c cc l trn a thch c ng knh t 6 - 8mm hoc dng cc khong giy tm khng sinh c dy thch hp, ng knh khong 6 mm.

134

 to vng c ch to nht cn thc hin giai on tin khuch tn bng cch cc a th nhit phng trong 2 gi cho khng sinh khuych tn vo mi trng (i vi colistin, polymyxin B thi gian khuych tn di hn t 3 4 gi). Sau thi gian khuych tn, cc a th c nhit thch hp trong 16 18 gi. Trong qu trnh nhit phi n nh v phi ng u mi ni. Cc a th nn mt lp trong t m sao cho v tr ca cc nng t v s c s khc nhau t nht v ngun nhit.

S1 T3 S2 T1 T2 S3

o ng knh vng c ch to bi cc nng chun v th bng thc o c chnh xc n 0,1mm. Mt th nghim c tin hnh vi 10 a th song song.
Tnh kt qu:

(Th nghim 3 liu) T l phn trm hot lc ca khng sinh th so vi chun c tnh theo cng thc:

4A R = Anti log 2 + log I (P = 0,95) 3B

A = (T1 + T2 + T3) (S1 + S2 + S3) B = (T3 T1) + (S3 S1) T1, T2, T3, S1, S2, S3 l tng gi tr ng knh vng v khun tnh bng mm ca cc nng t1, t2, t3, s1, s2, s3 . I l t s ca hai nng pha long k tip nhau, I = 2. Vy

4 A R = Anti log 2 + log 2. 3 B

A R = Anti log 2 + 0,4013. B

Hot lc ca mu th =

R hot lc chun 100

135

 Mun php th c gi tr, th nghim phi t c cc iu kin sau:

Hot lc ca cht th c gi thit gn st vi thc t kt qu c c nm trong phm vi ng cong chun. dc ca ng biu din logarit nng vi ng knh vng v khun phi c ngha.

Ta< ta .V

Tb >> tb .V
Cc ng biu din logarit nng ca cht th v cht chun vi ng knh vng v khun phi song song. Trong khong nng chn s ph thuc gia logarit ca nng vi ng knh vng v khun tng ng l tuyn tnh.

Tab < tab .V

Tc < tc .V Tac < tac .V

V: l tng ca cc hiu gi tr ng knh ln nht v nh nht ca mt nng . Ta = (T1 + T2 + T3) (S1 + S2 + S3) Tb = (T3 + S3) (T1 + S1) Tab = (S1 + T3 ) (S3 + T1) Tc = (S1 + S3 + T1 + T3) 2 (S2 + T2) Tac = (2S2 + T1 + T3) (2T2 + S1 + S3) ta, tb, tc, tab, tac l cc h s tra bng theo s vng v khun (n) do c ca mt nng . Gi tr t:
S vng v khun (n) 5 6 7 8 9 10 Gi tr t (P = 0,95) ta 0,80 0,80 0,81 0,82 0,83 0,84 tb 0,66 0,66 0,66 0,67 0,68 0,68 tc 1,14 1,14 1,14 1,16 1,17 1,19 tab 0,66 0,66 0,66 0,67 0,68 0,68 tac 1,14 1,14 1,14 1,16 1,17 1,19

136

 Xc nh gii hn tin cy:

lch chun:

S =
M

2 2 2,666.Hn.V n(A + 1,5B )

B2

Hn: H s vng o ph thuc vo n. n: S vng v khun o c ca mt nng . Gii hn tin cy:

M 1, 2 = 0,4013
Phn trm gii hn tin cy trn:

A 0,301S B

R1 = Antilog (2 + M1) Phn trm gii hn tin cy di; R2 = Antilog (2 + M2) Sai s th nghim:

e% =

R1 R 2R

2 100

Kt qu nh lng c gi tr vi e 5%. Gi tr Hn
N Hn 3 1,27 4 1,03 5 0,90 6 0,81 7 0,75 8 0,69 9 0,65 10 0,65

Ti liu tham kho

1. B y t (2002). Dc in Vit Nam III. NXB Y hc, H Ni 2. Nguyn Ln Dng v cng s (1997). Vi sinh vt hc. H Ni 3. British Pharmacopoeia 2001. 4. Collins C.H. and Patricia M.L (1976). Microbiological methods, London. 5. Pharmacopoeia of the Peoples Republic of China (1997). 6. The United States Pharmacopoeia XXIV (2000). 137

Cu hi t lng gi

4.1. Trnh by nhng c im chnh v hnh thi v tnh cht nui cy ca vi khun, nm mc, nm men? 4.2. Nu cch phn loi vi khun theo hnh th v c tnh h hp? 4.3. Phn tch nhng yu t ngoi cnh nh hng n s pht trin ca vi sinh vt? 4.4. Trnh by phng php lm mi trng nui cy vi sinh vt? 4.5. M t cc phng php tit trng? 4.6. Trnh by th nghim th v trng bng phng php nui cy trc tip? 4.7. Nu mc ch, nguyn tc ca th nghim th v trng v th gii hn vi sinh vt? 4.8. Trnh by th nghim m s lng vi sinh vt trong 1g (1ml) dc phm bng phng php a thch? 4.9. Vit tn nhng chng ch th c s dng th cht c ch trong th nghim th v trng v th gii hn vi sinh vt. Nu s khc nhau v chng ch th trong hai php th v gii thch? 4.10. Vai tr ca phng php sinh hc trong kim nghim cht khng sinh? 4.11. Trnh by phng php chun b nh dch vi sinh vt trong nh lng khng sinh.Ti sao i vi nhng chng ch th khng c bo t trc khi s dng cn phi cy truyn vo mi trng thch hp? 4.12. Cch pha dung dch chun v dung dch th trong nh lng khng sinh bng phng php khuych tn. Nu nhng im cn ch trong qu trnh pha cc dung dch ny? 4.13. M t cch tin hnh nh lng cht khng sinh bng phng php khuych tn? 4.14. Nu nhng nguyn nhn trong cc giai on lm th nghim nh hng n s chnh xc ca phng php khuych tn? 4.15. Nu nhng iu kin th nghim cn t c trong nh lng khng sinh php th c gi tr? 4.16. Vit cc cng thc tnh kt qu nh lng khng sinh vi th nghim ba liu v cng thc xc nh gii hn tin cy?

138

4.17. Trnh by phng php xc nh s lng ti a vi khun hiu kh, vi nm trong mt gam sp bi mi Lip Ice. Cho bit mu th c cht bo qun ring ca nh sn xut? 4.18. Trnh by phng php nh lng bt Gentamicin sulphat bng th nghim vi sinh vt( phng php khuych tn), vi ch th Bacillus pumilus NCTC 8241. Cho bit: Gentamicin sulphat chun c 680 U/ ml, dung mi pha long l nc. Ba nng l: S1= 4 U/ ml; S2=8 U/ ml; S3=16 U/ ml.

139

Chng 5.

kim nghim cc dng bo ch

Mc tiu hc tp: 1. Trnh by c cc yu cu k thut v phng php th nh gi cht lng cc dng bo ch: Thuc bt, thuc vin nn, vin nang, thuc tim, thuc tim truyn, thuc nh mt, thuc m, thuc ung dng lng, thuc n, thuc trng. 2. nh gi c kt qu kim nghim i vi mt mu kim nghim thnh phm c th ca cc dng bo ch trn. Khi tin hnh kim nghim mt mu ch phm thuc mt dng bo ch c th (thuc bt, vin nn, vin nang, thuc tim, ...) theo chuyn lun ring ca ch phm trong Dc in, thng thng yu cu ghi phn u ca chuyn lun l ch phm phi p ng cc yu cu chung ca dng bo ch v cc yu cu ring ca dc cht, ca ch phm nh tnh cht, nh tnh, nh lng, tp cht (nu c), ... Cn nu tiu chun kim nghim l tiu chun ca nh sn xut (TCCS) th trong tiu chun kim nghim c y yu cu k thut v phng php th c th cho ch phm , mt s tiu ch c trng cho dng bo ch ca ch phm c tin hnh th theo hng dn ca Dc in. Trong ti liu ny, chng ti cp ti yu cu k thut v phng php th ca cc dng bo ch thng dng nh thuc bt, thuc vin nn, vin nang, thuc tim, thuc tim truyn, thuc nh mt, thuc m, thuc ung dng lng, thuc n, thuc trng. Ti liu tham kho chnh c s dng l Dc in Vit Nam III, ng thi chng ti cng s dng Dc in ca mt s nc khc nh Dc in Anh, Dc in M, Dc in Trung Quc v mt s ti liu kim nghim cc dng bo ch khc. 5.1. Kim nghim thuc bt 5.1.1. Yu cu k thut v phng php th 5.1.1.1. Tnh cht Bt phi kh ti, khng b m, vn, mu sc ng nht. Mi v ty theo tng loi ch phm ca cc nh sn xut. Cch th: Tri mt lng bt va thnh mt lp mng trn mt t giy trng mn. Quan st mu sc bng mt thng, di nh sng t nhin. 140

5.1.1.2. m Cc thuc bt khng c cha hm lng nc qu 9,0%, tr ch dn khc. Cch th: theo Xc nh mt khi lng do lm kh - Ph lc 5.16 DVN III. 5.1.1.3. mn Thuc bt phi t mn quy nh trong chuyn lun ring. Nu khng c ch dn khc, php th ny dng cho tt c cc thuc bt kp, cc thuc bt dng p, thuc bt dng pha ch thuc dng cho mt, tai. Cc c bt c quy nh da vo cc s ca ry. Ry c li ry v li ry c th lm bng si kim loi hoc si cc vt liu thch hp khc c dt thnh nhng mt vung. Li ry dng ry bt thuc c phn loi bng nhng con s biu th kch thc l ry, quy nh tnh bng m theo bng 5.1. Cch th: Chn c ry thch hp theo qui nh ca tiu chun. Cn mt lng thuc bt, em ry qua ry c c qui nh. i vi bt th hoc na th: ly 25g -100 g bt. Cho vo ry thch hp, lc ry theo chiu ngang quay trn t nht 20 pht v ry ti khi xong. Cn chnh xc s lng cn li trn ry v s thu c trong hp hng. i vi bt na mn, mn hay rt mn: ly khng qu 25 g bt, cho vo ry thch hp, lc ry theo chiu ngang quay trn t nht 30 pht ri ry ti khi xong. Cn chnh xc s lng cn li trn ry v s thu c trong hp hng. i vi cht c du hay bt c xu hng bt mt ry th trong qu trnh ry thnh thong chi cn thn mt ry, tch ri nhng ng t li khi ry. Thuc bt t tiu chun v mn nu: Khi quy nh dng 1 ry xc nh c bt th khng c c di 97% khi lng thuc bt qua c c ry . Khi quy nh dng 2 ry xc nh c bt th mt ry ln trn ry kia v tin hnh ry; khng c c di 95% khi lng thuc bt qua ry c s ry cao hn v khng c qu 40% khi lng thuc bt qua ry c s ry thp hn.

141

Bng 5.1. Qui nh s ry

S ry (m) 1400 710 355 250 180 125 90 C mt ry (mm) 1,400 0,710 0,355 0,250 0,180 0,125 0,090 ng knh si ry (mm) 0,710 0,450 0,224 0,160 0,125 0,090 0,063

Cc k hiu quy nh c bt: Bt th (1400/355) Bt na th (710/250) Bt na mn (355/180) Bt mn (180/125) Bt rt mn (125/90)

5.1.1.4. ng u khi lng Nhng thuc bt khng quy nh th ng u hm lng th phi th ng u khi lng. Ring thuc bt pha tim hoc truyn tnh mch, nu khi lng nh hn hoc bng 40 mg th khng phi th ng u khi lng nhng phi t yu cu ng u hm lng. Cch th: Cn tng n v trong s 5 n v ng gi nh nht c ly bt k. Khi lng thuc phi nm trong gii hn cho php theo bng 5.2.
Bng 5.2. Gii hn cho php chnh lch khi lng i vi thuc bt
Khi lng ghi trn nhn Di hoc bng 0,50g Trn 0,50 - 1,50g Trn 1,50 - 6,00g Trn 6,00g Phn trm chnh lch 10 7 5 3

Tt c cc n v phi t qui nh trong bng trn. Nu c mt n v c khi lng lch ra ngoi quy nh ny th th li vi 5 n v khc, nu ln th li c qu mt n v khng t th l thuc khng t yu cu. 142

i vi cc ch phm ng gi trong hp, l th sau khi cn c v phi b ht thuc ra, dng bng lau sch thuc, cn v ri tnh theo lng thuc trong tng hp hoc l. chnh lch c tnh theo t l phn trm so vi khi lng trung bnh bt thuc trong mt n v ng gi. 5.1.1.5. ng u hm lng Tr khi c ch dn khc, php th ny p dng cho cc thuc bt ung, tim c trnh by trong cc n v ng gi mt liu c cha mt hoc nhiu hot cht, trong c cc hot cht c hm lng di 2 mg hoc di 2% (kl/kl) so vi khi lng thuc mt liu. Php th ng u hm lng c tin hnh sau php th nh lng v hm lng hot cht trong gii hn quy nh. Cch th: Ly 10 n v ng gi nh nht bt k, xc nh hm lng hot cht tng gi theo phng php nh lng ch dn trong chuyn lun. Cch nh gi: Ch phm em kim tra t yu cu php th nu khng qu mt n v c gi tr hm lng nm ngoi gii hn 85 - 115% ca hm lng trung bnh v khng c n v no nm ngoi gii hn 75 - 125% ca hm lng trung bnh. Ch phm khng t yu cu php th nu qu 3 n v c gi tr hm lng nm ngoi gii hn 85 - 115% ca hm lng trung bnh hoc 1 n v tr ln nm ngoi gii hn 75 - 125% ca hm lng Nu hai hoc ba n v c gi tr hm lng nm ngoi gii hn 85 -115% nhng trong gii hn 75 - 125% ca hm lng trung bnh th th li trn 20 n v khc, ly ngu nhin. Ch phm t yu cu, nu khng qu 3 n v trong tng s 30 n v em th c gi tr hm lng nm ngoi gii hn 85 115% ca hm lng trung bnh v khng c n v no c gi tr hm lng nm ngoi gii hn 75 - 125% ca hm lng trung bnh. 5.1.1.6. nh tnh Tin hnh nh tnh theo cc phng php c qui nh trong tiu chun, thuc bt phi cho cc phn ng ca cc hot cht c trong ch phm 5.1.1.7. nh lng Ly thuc ca 5 n v ng gi nh nht bt k, trn u. Tin hnh nh lng theo cc phng php c qui nh trong tiu chun, hm lng ca tng hot cht trong ch phm phi nm trong gii hn cho php theo bng 5.3. 143

5.1.1.8. Gii hn nhim khun Thuc bt c ngun gc dc liu phi t yu cu Gii hn nhim khun. Cch th: Nu khng c qui nh ring th tin hnh th v nh gi theo "Th gii hn vi sinh vt" - phn 4.3.4, trang 119-121.
Bng 5.3. Gii hn cho php v hm lng hot cht i vi thuc bt
Lng ghi trn nh n Ti 100 mg Trn 100 mg ti 1g Trn 1 g n 5 g Trn 5 g Phn trm chnh lch 15 10 5 1

5.1.2. Cc loi thuc bt 5.1.2.1. Thuc bt ung

Thuc bt ung phi p ng cc yu cu cht lng chung ca thuc bt. Thuc bt si bt phi t thm yu cu v tan. Cch th : Th mt l ng thuc bt t ng ng vi mt liu vo mt cc thu tinh c cha 200 ml nc nhit 15 -20oC, xut hin nhiu bt kh bay ra. Khi ht bt kh bay ra, thuc phi tan hon ton. Th nh vy vi 6 liu n. Mu th t yu cu nu mi liu th u tan trong vng 5 pht, tr khi c ch dn ring. 5.1.2.2. Thuc bt dng ngoi Thuc bt dng ngoi phi p ng cc yu cu cht lng chung ca thuc bt. Ngoi ra, phi t cc yu cu ring sau: v khun: thuc bt p, dng cho vt thng rng hoc trn da b tn thng nng, thuc bt dng cho mt phi v khun. Cch th: Nu khng c qui nh ring th tin hnh th v nh gi theo "Th v trng" - phn 4.3.3, trang 115-118. 5.1.2.3. Thuc bt pha tim Thuc bt pha tim p ng cc yu cu cht lng chung ca thuc bt v yu cu i vi thuc tim, thuc tim truyn dng bt nh: v khun, cht gy st ... Cch th: Theo cch th chuyn lun thuc tim. V d: Thuc tim benzylpenicilin - DVN III trang 32 - 33. 144

5.2. Kim nghim thuc vin nang 5.2.1. Yu cu k thut v phng php th 5.2.1.1. Tnh cht Nang cng hoc nang mm cha bt hoc cm hoc cht lng. Cch th: Th bng cm quan. 5.2.1.2. ng u khi lng Cn khi lng ca mt nang: Vi vin nang cng th tho ri hai na v nang thuc ra, dng bng lau sch v ri cn khi lng ca v; vi vin nang mm, ct m nang, bp thuc ra ht ri dng ether hoc dung mi hu c thch hp ra sch v nang, kh t nhin ti khi ht mi dung mi, cn khi lng v. Khi lng thuc trong nang l hiu gia khi lng nang thuc v v nang. Lm nh vy vi 19 nang khc c ly bt k. chnh lch khi lng ca tng vin vi khi lng trung bnh phi t theo bng 5.4. Nu c yu cu th ng u hm lng th khng phi th ng u khi lng.
Bng 5.4. Gii hn cho php chnh lch khi lng i vi vin nang
Khi lng trung bnh nang Nh hn 300 mg Ln hn hoc bng 300 mg Phn trm chnh lch 10 7,5

5.2.1.3. ng u hm lng Nghin mn bt thuc ring tng vin v tin hnh th v nh gi nh i vi thuc bt. 5.2.1.4. r Nu khng c qui nh ring th tin hnh th v nh gi theo "Php th r vin nn v vin nang" - phn 5.10, trang 154 - 157. Vin khng cn th r khi php th ha tan c thc hin. 5.2.1.5. ha tan Trong DVN III, nhiu ch phm thuc vin yu cu nh gi ha tan. Khi c yu cu s ch dn trong chuyn lun ring. Cch th: Tin hnh theo Phng php th tc ha tan ca vin nn v vin nang - phn 5.9, trang 160 - 165. 145

5.2.1.6. nh tnh Tin hnh nh tnh theo cc phng php c qui nh trong tiu chun, vin nang phi cho cc phn ng ca cc hot cht c trong ch phm 5.2.1.7. nh lng Cn thuc trong 20 vin, tnh khi lng trung bnh vin, lm ng nht bng cch nghin (i vi vin nang cha bt hoc cm) hoc trn u (i vi nang cha cht lng). Tin hnh nh lng theo cc phng php c qui nh trong tiu chun, hm lng ca tng hot cht trong ch phm phi nm trong gii hn cho php theo bng 5.5.
Bng 5.5. Gii hn cho php v hm lng i vi thuc vin nang, vin nn
Lng ghi trn nh n Ti 50 mg Trn 50 mg ti 100 mg Trn 100 mg Phn trm chnh lch 10 7,5 5

5.2.1.8. Tp cht (nu c) Khi c yu cu s ch dn trong chuyn lun ring. 5.2.2. Cc loi vin nang Cc loi vin nang phi tun theo yu cu chung ca thuc vin nang v yu cu ring i vi ty tng loi. Thuc nang cng phi t cc yu cu cht lng chung ca vin nang. 5.2.2.1. Thuc nang cng Thuc nang cng c v nang gm hai phn hnh tr lng kht vo nhau, mi phn c mt u kn, u kia h. Thuc ng trong nang thng dng rn nh bt hoc cm. V d: Vin nang Cephalexin - DVN III. Trang 53 - 54. 5.2.2.2. Thuc nang mm Thuc nang mm c v nang l mt khi mm vi cc hnh dng khc nhau. Thuc ng trong nang thng dng dung dch, hn dch hoc nh tng. V d: Vin nang mm vitamin A - DVN III. Trang 291 - 292.

146

5.2.2.3. Thuc nang tan trong rut Thuc nang tan trong rut l cc nang cng hoc nang mm c v nang bn vng vi dch d dy, ch tan trong dch rut; hoc cc nang c ng cm c bao lp ch tan trong dch rut. 5.2.2.4. Thuc nang gii phng hot cht c bit Thuc nang gii phng hot cht c bit l cc nang cng hoc nang mm c v nang hay thuc trong nang hoc c v nang v thuc trong nang c bo ch c bit, kim sot hay chng trnh ho tc hoc v tr gii phng hot cht trong c th. Thuc nang gii phng hot cht c bit phi t cc yu cu cht lng chung ca vin nang, ring r khng phi th v bt buc th ha tan theo yu cu ring ca tng chuyn lun. 5.3. Kim nghim thuc vin nn 5.3.1. Yu cu k thut v phng php th 5.3.1.1. Tnh cht Vin nn thng c dng hnh tr dt, hai y phng hoc cong, c th khc ch, k hiu hoc rnh. Cnh v thnh vin lnh ln. Mu sc ng nht. Cch th: Bng cm quan. 5.3.1.2. r Nu khng c qui nh ring th tin hnh th v nh gi theo "Php th r vin nn v vin nang" - phn 5.10, trang 154 - 157. Vin khng cn th r khi php th ha tan c thc hin. 5.3.1.3. ng u khi lng Cn chnh xc 20 vin bt k v xc nh khi lng trung bnh ca vin. Cn ring khi lng tng vin v so snh vi khi lng trung bnh, tnh lch theo t l phn trm ca khi lng trung bnh t tnh ra khong gii hn ca gi tr trung bnh. Khng c qu 2 vin c khi lng chnh lch qu khong gii hn ca khi lng trung bnh v khng c c vin no c chnh lch qu gp i lch tnh theo t l phn trm, theo bng 5.6.
Bng 5.6. Gii hn cho php chnh lch khi lng i vi vin nn
Khi lng trung bnh vin Ti 80 mg Trn 80 mg n 250 mg Trn 250 mg Phn trm chnh lch 10 7,5 5

147

Nu c yu cu th ng u hm lng th khng phi th ng u khi lng. 5.3.1.4. ng u hm lng Nghin mn ring tng vin v tin hnh th v nh gi nh i vi thuc bt. 5.3.1.5. ha tan Ch thc hin khi c yu cu c ch dn trong chuyn lun ring. Nu khng c qui nh ring th tin hnh th v nh gi theo "Php th ha tan ca vin nn v vin nang" - phn 5.9, trang 149 - 154. 5.3.1.6. nh tnh Tin hnh nh tnh theo cc phng php c qui nh trong tiu chun, vin nn phi cho cc phn ng ca cc hot cht c trong ch phm. 5.3.1.7. nh lng Cn 20 vin, xc nh khi lng trung bnh vin. Nghin mn. Tin hnh nh lng theo cc phng php c qui nh trong tiu chun, hm lng ca tng hot cht trong ch phm phi nm trong gii hn cho php, theo bng 5.5. 5.3.1.8. Tp cht (nu c) Khi c yu cu s ch dn trong chuyn lun ring. 5.3.2. Cc loi vin nn Cc loi vin nn phi tun theo yu cu chung ca thuc vin nn v yu cu ring i vi ty tng loi. 5.3.2.1. Vin nn khng bao (Uncoated tablets) Gm cc vin nn c bo ch bng cch nn cc ht gm dc cht, t dc. Vin nn loi ny khng cha mt thnh phn no thay i s gii phng hot cht trong ng tiu ha. Khi b gy vin nn mt lp v quan st bng knh lp th thy ng nht, khng c lp bao ngoi. 5.3.3.2. Vin bao (Coated tablets) Vin bao c b mt nhn, thng c mu, c nh bng. Ly mt phn vin b gy, quan st di knh lp, thy r nhn c bao bng mt lp hay nhiu lp.

148

5.3.2.3. Vin bao bn vi dch v d dy (Gastro - resistant tablets) Vin c bao bng lp bao bn vi dch v d dy nhng li tan c trong dch rut do s dng cc cht bao l cellacephate (cellulose acetat phtalate) v cc polymer. 5.3.2.4. Vin nn si bt (Effervescent tablets) L vin nn khng bao c cha cc acid v carbonat hoc hydrocarbonat, chng phn ng vi nhau rt nhanh khi c mt ca nc v gii phng CO2, ng thi ho tan hay phn tn hot cht trong nc trc khi dng. 5.3.2.5. Vin ngm (Tablets for use in the mouth) Vin ngm thng l vin nn khng bao c iu ch cc thnh phn v hot cht gii phng dn v tc dng ti ch, gii phng v hp th di li hoc cc phn khc trong ming. 5.3.2.6. Vin nn tan trong nc (Soluble tablets) Vin nn tan trong nc l vin nn khng bao, ha tan trong nc. Dung dch to thnh c th hi c nh. 5.3.3.7. Vin nn phn tn trong nc (Dispersible tablets) Vin nn phn tn trong nc l vin nn khng bao phn tn trong nc thnh cc tiu phn. 5.3.2.8. Vin nn thay i gii phng hot cht (Modified release tablets) Thng l vin bao hoc khng bao, c iu ch bng cch thm cc t dc c bit hoc iu ch theo phng php c bit nhm thay i tc gii phng hoc v tr gii phng ca thuc. Vin nn thay i gii phng hot cht thng khng yu cu th r m phi th tc ha tan. V d: Vin nn acid ascorbic - DVN III trang 6 Vin bao ibuprofen - DVN III trang 141. 5.4. Kim nghim thuc tim, thuc tim truyn Ch phm dng tim (parenteral preparations) c 5 loi l thuc tim (injections), thuc tim truyn (infusions), dung dch m c dng tim hoc truyn (concentrates for injections and infusions), thuc bt dng tim hoc truyn (powders for injections and infusions), thuc cy (implants). Trong phm vi ca chng trnh i hc, chng ti ch cp ti yu cu k thut v phng php kim nghim thuc tim v thuc tim truyn. 149

5.4.1. Tnh cht Dng dung dch nc hoc du, nh dch hoc dch treo, bt... ty theo tng ch phm. Cch th: Bng cm quan. 5.4.2. trong Dung dch tim khi kim tra bng mt thng iu kin qui nh phi trong v hu nh khng c tp c hc - Theo ph lc 8.9, mc B - DVN III. Dung dch thuc tim truyn tnh mch c liu truyn t 100ml tr ln phi p ng yu cu v gii hn kch thc v s lng cc tiu phn - Theo ph lc 8.9, mc A - DVN III. Nh tng phi khng thy du hiu ca s tch lp. Hn dch tim c th lng cn nhng phi phn tn ngay khi lc u v gi c s ng u khi ly liu ra khi ng thuc tim. 5.4.3. Mu sc Khng mu hoc c mu do hot cht ty theo tng chuyn lun. Tin hnh so vi mu mu - Ph lc 5.17 - DVN III. 5.4.4. pH pH phi nm trong gii hn qui nh. Tin hnh o pH bng my o pH - Ph lc 5.9 - DVN III. 5.4.5. v khun Thuc tim phi v khun. Cch th: Nu khng c qui nh ring th tin hnh th v nh gi theo "Th v trng" - phn 4.3.3, trang 115 - 118. 5.4.6. Ni c t vi khun Php th ni c t vi khun thc hin khi c yu cu c qui nh trong chuyn lun ring. Th theo Ph lc 10.3 - DVN III. Khi th ni c t vi khun th khng phi th cht gy st, tr khi c qui nh khc. 5.4.7. Cht gy st Thuc tim phi khng c cht gy st. Th theo Ph lc 10.5-DVN III. Php th ny c tin hnh i vi: 150

 Thuc tim ng liu n l c th tch t 15 ml tr ln v khng c qui nh php th ni c t. Thuc tim ng liu n l c th tch nh hn 15 ml nhng trn nhn c ghi "khng c cht gy st" v khng c qui nh php th ni c t.

5.4.8. Th tch (i vi thuc tim, tim truyn dng lng) Cch th: Thuc tim c thng bng vi nhit phng v phi c phn tn ng u trc khi th. i vi thuc tim c th tch nh hn hoc bng 5 ml: + Ly 6 ng thuc, trng bm tim bng 1 ng, th trn 5 ng. + Kim tra bng cm quan 5 ng thuc, thy cc ng th cha th tch ngang nhau. + Dng bm tim kh sch c dung tch khng ln hn 2,5 ln so vi th tch cn o, c gn kim tim thch hp. Ly thuc sao cho trong bm tim khng c bt kh v trong kim tim vn cha y thuc tim. Ln lt ly thuc ca tng ng tim theo cch . Tnh kt qu th tch trung bnh ca 5 ng. i vi thuc tim c th tch ln hn 5 ml, thuc tim truyn: + Ly 4 ng thuc, trng bm tim bng 1 ng, th trn 3 ng. + Cch tin hnh nh i vi thuc tim c th tch khng ln hn 5 ml. Tnh kt qu th tch trung bnh ca 3 ng. i vi thuc tim nhiu liu trong 1 l th th tch phi ln hn so vi s liu qui nh c ly ra. Th tch ca ch phm phi nm trong gii hn cho php theo bng 5.7.
Bng 5.7. Gii hn cho php v th tch ca thuc tim, thuc tim truyn
Th tch ghi trn nhn (ml) Ti 5 ml T 5ml ti 50 ml. Trn 50 ml Phn trm chnh lch + 15 + 10 +5

5.4.9. ng u khi lng (i vi ch phm dng bt) Loi b ht nhn, ra sch v lm kh bn ngoi, loi b ht cc nt v cn ngay khi lng c thuc v v; ly ht thuc ra, dng bng lau sch, nu cn th ra bng nc, sau ra bng ethanol 96% ri sy 100 - 1050C trong 1 gi; nu v ng khng chu c nhit th lm kh iu kin 151

thch hp v cn. Hiu s khi lng hai ln cn l khi lng ca thuc. Lm nh vy vi 9 n v khc ly bt k. Cho php khng qu mt n v c khi lng chnh lch khi bng 5.2. 5.4.10. ng u hm lng Tr khi c ch dn khc, ch phm c hm lng hot cht di 2 mg hoc di 2% (kl/kl) so vi khi lng thuc ca ton b. Thuc tim, thuc tim truyn dng bt ng gi c khi lng nh hn hoc bng 40 mg. Php th ng u hm lng c tin hnh sau php th nh lng v hm lng hot cht trong gii hn quy nh. Cch th: Ly 10 n v ng gi nh nht bt k, xc nh hm lng hot cht tng ng theo phng php ch dn trong chuyn lun. Cch nh gi: Ch phm t yu cu php th nu hm lng u nm trong gii hn 85 - 115% ca hm lng trung bnh. Ch phm khng t yu cu nu c qu 1 n v c gi tr hm lng nm ngoi gii hn 85 - 115% ca hm lng trung bnh hoc c mt n v c hm lng nm ngoi gii hn 75 - 125% ca hm lng trung bnh. Nu mt n v c gi tr hm lng nm ngoi gii hn 85 -115% nhng trong gii hn 75 - 125% ca hm lng trung bnh th th li trn 20 n v khc, ly ngu nhin. Ch phm t yu cu, nu khng qu 1 n v trong tng s 30 n v em th c gi tr hm lng nm ngoi gii hn 85 -115% ca hm lng trung bnh v khng c n v no c gi tr hm lng nm ngoi gii hn 75 - 125% ca hm lng trung bnh. 5.4.11. nh tnh Tin hnh nh tnh theo cc phng php c qui nh trong tiu chun, thuc tim v thuc tim truyn phi cho cc phn ng ca cc hot cht c trong ch phm theo tng chuyn lun. 5.4.12. nh lng Ly thuc trong s l thuc nh i vi php th th tch (ch phm dng lng) hoc nh i vi php th ng u khi lng (ch phm dng bt), trn ng nht. Tin hnh nh lng theo cc phng php c qui nh trong tiu chun, hm lng ca tng hot cht trong ch phm phi nm trong gii hn cho php theo bng 5.8.
Bng 5.8. Gii hn cho php v hm lng i vi thuc tim, thuc tim truyn
Lng ghi trn nh n Dng dung dch Dng bt Phn trm chnh lch 5 10

152

5.5. Kim nghim thuc nh mt 5.5.1. Tnh cht: Th cht, mu sc ty theo tng chuyn lun. Cch th: Bng cm quan. 5.5.2. trong Thuc nh mt dng dung dch phi trong, khng c cc tiu phn khi quan st bng mt thng v t yu cu v trong theo tng chuyn lun. Thuc nh mt dng hn dch c th lng ng, nhng khi lc phi phn tn d dng v ng nht trong ton khi. Cch xc nh: trong c xc nh bng cch so snh cc dung dch vi cc hn dch mu i chiu. Th theo ph lc 5.12 - DVN III. 5.5.3. Th tch Th tch thuc nh mt phi nm trong gii hn 100 - 110% so vi th tch ghi trn nhn i vi mi th tch ng gi. Cch th: Ly 5 n v ng gi bt k. Xc nh th tch ca tng n v bng bm tim chun hoc ng ong chun sch, kh, c chnh xc ph hp (Th tch khng ln hn 2,5 ln th tch cn xc nh). Th tch mi n v phi nm trong gii hn cho php. Nu c mt n v khng t th phi kim tra li ln th hai ging nh ln u. Nu ln th hai c qu mt n v khng t th l thuc khng t yu cu. 5.5.4. pH pH phi nm trong gii hn qui nh. o bng my o pH - Ph lc 5. 9 - DVN III. 5.5.5. v khun Thuc nh mt phi v khun. Cch th: Nu khng c qui nh ring th tin hnh th v nh gi theo "Th v trng" phn 4.3.3, trang 115 - 118. 5.5.6. Gii hn cc tiu phn Th nghim ny ch yu cu i vi thuc nh mt dng hn dch. Cho mt th tch ch phm thch hp vo cc o hay vt knh ca knh hin vi. Quan st bng knh hin vi mt din tch tng ng 10 g pha rn. 153

Kt qu: - Khng c c qu 20 tiu phn c kch thc ln hn 25 m. - Khng c c qu 2 tiu phn c kch th c ln hn 50 m. - Khng c c tiu phn no c kch thc ln hn 90 m. 5.5.7. nh tnh Tin hnh nh tnh theo cc phng php c qui nh trong tiu chun, thuc nh mt phi cho cc phn ng ca cc hot cht c trong ch phm. 5.5.8. nh lng Ly thuc ca 5 n v ng gi nh nht bt k, trn u. Tin hnh nh lng theo cc phng php c qui nh trong tiu chun, hm lng ca tng hot cht trong ch phm phi nm trong gii hn cho php qui nh trong chuyn lun. 5.6. Kim nghim thuc ung dng lng 5.6.1. Yu cu k thut v phng php th 5.6.1.1. Tnh cht Th cht, mu sc, mi v ty theo tng chuyn lun. Cch th: Bng cm quan. 5.6.1.2. trong Thuc ung dng dung dch phi t yu cu v trong theo tng chuyn lun. Cch xc nh: Ging nh i vi thuc nh mt. 5.6.1.3. Th tch Cch xc nh: Ging nh i vi thuc nh mt. Th tch thuc phi nm trong gii hn cho php theo bng 5.9. 5.6.1.4. pH Mt s ch phm c yu cu th pH phi nm trong gii hn qui nh. o bng my o pH - Ph lc 5.9 - DVN III.

154

Bng 5.9. Gii hn cho php th tch thuc ung dng lng
Dng thuc Thuc nc ung Th tch ghi trn nhn (ml) Ti 20 Trn 20 - 50 Trn 50 - 150 Trn 150 Siro Ti 100 Trn 100 - 250 Trn 250 Phn trm chnh lch + 10 +8 +6 +4 + 10 +8 +6

5.6.1.5. nhim khun nhim khun phi t gii hn qui nh cho ch phm theo tng chuyn lun. Cch th: Nu khng c qui nh ring th tin hnh th v nh gi theo "Th gii hn vi sinh vt" - phn 4.3.4, trang 119-121. 5.6.1.6. T trng T trng ca ch phm phi nm trong gii hn qui nh theo tng chuyn lun. Cch th: Theo Xc nh t trng cht lng - ph lc 5.15 - DVN III. 5.6.1.7. ng u hm lng Mt s ch phm yu cu th phi tin hnh php th ny. Cch th v nh gi ging nh i vi thuc bt. 5.6.1.8. nh tnh Tin hnh nh tnh theo cc phng php c qui nh trong tiu chun, ch phm thuc phi cho cc phn ng ca cc hot cht c trong ch phm. 5.6.1.9. nh lng Ly thuc ca 5 n v ng gi nh nht bt k, trn u. Tin hnh nh lng theo cc phng php c qui nh trong tiu chun, hm lng ca tng hot cht trong ch phm phi nm trong gii hn cho php theo bng 5.10. 5.6.2. Cc dng thuc ung dng lng Thuc ung dng lng phi t yu cu tiu chun k thut chung i vi thuc ung dng lng v yu cu ring ty tng chuyn lun. 155

Bng 5.10. Gii hn cho php v hm lng i vi thuc ung dng lng
Dng thuc Thuc c A, B Thuc thng Phn trm chnh lch 5 10

5.6.2.1. Cn thuc Cn thuc l nhng ch phm lng c iu ch bng cch ngm chit dc liu thc vt, ng vt hoc bng cch ha tan cao thuc, dc cht theo t l qui nh trong ethanol cc nng khc nhau. 5.6.2.2. Siro Siro l dung dch m c (khng di 60%) ca ng trng (Saccharose) trong nc, c cha cc dc cht hoc cc dch chit t dc liu v cc cht lm thm. 5.6.2.3. Potio Potio l dng thuc nc c v ngt, cha mt hay nhiu dc cht, dng ung tng tha. Potio c th dng hn dch hoc nh dch. Dung mi ca potio c th l nc, nc thm, nc hm hay nc sc dc liu. Potio thng cha 20% siro. 5.6.2.4. Dung dch ung (Oral solution) Dung dch ung l nhng ch phm lng trong sut, cha mt hay nhiu dc cht ha tan trong mt dung mi hay hn hp nhiu dung mi thch hp dng bng cch ung. Dung mi ca dung dch c th l nc, du, ethanol, glycerin... 5.6.2.5. Thuc ung git (Oral drop) Thuc ung git l thuc ung c dng vi th tch nh bng dng c phn liu l ng nh git. 5.6.3. Dung dch thuc dng ngoi dng lng Thuc dng ngoi dng lng l cc ch phm lng ng nht, thng l dung dch, cn thuc, hn dch hoc nh dch ca mt hay nhiu hot cht trong cht dn thch hp dng ngoi. Cc thuc dng ngoi dng lng phi t theo cc yu cu k thut chung ca thuc ung dng lng v ring tng dng thuc c nhng yu cu ring. Ring yu cu " nhim khun" khng phi lm. 156

5.7. Kim nghim thuc m 5.7.1. Yu cu k thut v phng php th 5.7.1.1. Tnh cht Th cht: thuc m phi mn, ng nht, khng cng li hoc tch lp iu kin thng, khng chy lng nhit 37oC v phi bt dnh c trn da hay nim mc khi bi. Mu sc, mi: ty tng ch phm. Cch th: Bng cm quan.

5.7.1.2. ng u khi lng Cch th v nh gi: Ging nh i vi thuc bt. chnh lch khi lng phi t theo bng 5.11.
Bng 5.11. Gii hn cho php chnh lch khi lng i vi thuc m
Khi lng ghi trn nhn (gam) Di 10,0 T 10,0 - 20,0 Trn 20,0 - 50,0 Trn 50,0 Phn trm chnh lch 15 10 8 5

5.7.1.3. ng nht Cc tiu phn phi phn tn ng u. Cch th: Ly 4 n v ng gi, mi n v khong 0,02 -0,03 g, tri ch phm ln 4 tiu bn, bn trn t mt phin knh. y mi phin knh bng mt phin knh khc v p mnh cho n khi to thnh mt vt c ng knh khong 2 cm. Quan st vt thu c bng mt thng, 3 trong 4 tiu bn khng c nhn thy cc tiu phn. Nu c cc tiu phn nhn thy trong phn ln s cc vt th phi lm li vi 8 n v ng gi. Trong s cc tiu bn ny, cc tiu phn cho php nhn thy khng c vt qu 2 tiu bn. 5.7.1.4. nh tnh Tin hnh nh tnh theo cc phng php c qui nh trong tiu chun, thuc m phi cho cc phn ng ca cc hot cht c trong ch phm. 5.7.1.5. nh lng Cn thuc trong 5 n v ng gi nh nht, tnh khi l ng trung bnh, trn ng nht. Cn mt lng ch phm nh ch dn trong chuyn lun, 157

tin hnh nh l ng. Hm lng hot cht phi nm trong gii hn qui nh theo bng 5.12.
Bng 5.12. Gii hn cho php v hm lng i vi thuc m
Nng hm lng ghi trn nhn Ti 200 mg Trn 200 mg - 1 g Trn 1 - 5 g Trn 5 g Phn trm chnh lch 15 10 7,5 3

5.7.2. Thuc m tra mt t cc yu cu ca thuc m v nhng yu cu ring ca thuc m tra mt nh sau: 5.7.2.1. v khun Thuc m tra mt phi v khun v khng c c Staphylococcus aureus v Pseudomonas aeruginosa. Cch th: Nu khng c qui nh ring th tin hnh th v nh gi theo "Th v trng" - phn 4.3.3, trang 115-118. 5.7.2.2. Cc phn t kim loi S lng cc phn t kim loi phi nm trong gii hn cho php. Cch th: Ly 10 ng thuc, bp ht thuc cha bn trong vo tng a petri ring c ng knh 6 cm, y bng, khng c cc vt xc v cc phn t l nhn thy c. y cc a, un nng n 80 -85oC trong 2 gi v cho thuc m phn tn ng u. Lm ngui v lm ng lnh thuc m, lt ngc mi a t ln bn soi ca knh hin vi thch hp; chiu sng t trn xung bng mt ngn n chiu t gc 45o so vi mt phng ca bn soi. Quan st v m cc phn t kim loi sng bng, ln hn 50 m bt k kch thc no. Khng c c qu mt ng trong 10 ng thuc em th cha nhiu hn 8 phn t v khng c qu 50 phn t tm thy trong 10 ng. Nu ch phm khng t ln th th nht th lm li ln th hai vi 20 ng thuc khc. Mu th c coi l t yu cu nu khng c qu 3 ng cha qu 8 phn t trong mi ng v tng s khng qu 150 phn t trong 30 ng th. 5.7.2.3. Gii hn kch thc cc phn t Khng c c phn t no ca thuc c kch thc ln hn 75 m. Cch th: Tri mt lng nh ch phm thnh mt lp mng trn bn soi ca knh hin vi, ph phin knh ln trn v soi. 158

5.8. Kim nghim thuc n, thuc trng 5.8.1. Tnh cht Thuc n c hnh nn hoc hnh con sut, khi lng 1 - 3 g, di 3- 4 cm. Thuc trng c hnh cu hoc hnh tri xoan, khi lng 2 - 4 g. Th cht, mu sc, mi t theo chuyn lun ring. Cch th: Bng cm quan. 5.8.2. ng u khi lng Cch th: Nh i vi vin nn nh gi kt qu: Khi lng ca mi vin ( mi khi lng) phi nm trong khong khi lng trung bnh 5%. Cho php khng qu 2 n v lch ra ngoi gii hn, nhng khng c c n v no lch gp hai ln gii hn cho php. 5.8.3. r r ca thuc n v thuc trng l kh nng tan r hoc mm ra ca thuc n v thuc trng trong mi trng th, bng thit b th r c qui nh trong thi gian nht nh c qui nh tng chuyn lun. Cch tin hnh: Th trn 3 vin, theo ph lc 8.5 - DVN III. Mu th c coi l t yu cu v r i vi thuc n: Ha tan hon ton; hoc Phn tn thnh nhng tiu phn v tp trung trn mt nc (cc cht m), chm xung y (bt khng tan) hay ha tan trong nc (thnh phn ha tan c); hoc Tr nn mm ra km theo bin dng m khng nht thit phi b phn tn hon ton thnh nhng tiu phn, nhng khi dng a thy tinh chc vo th phi khng c c nhn rn. Thuc n c t dc thn m phi r trong thi gian di 30 pht. Thuc n tan trong nc phi r trong thi gian di 60 pht. i vi vin nang t trc trng: V nang gelatin v ra v gii phng cc cht cha bn trong. Thi gian r phi trong vng 30 pht. i vi thuc trng: V nang gelatin v ra v gii phng cc cht cha bn trong. 159

Thi gian r phi trong vng 30 pht. i vi vin nn t m o: Khng c cn cn trn a kim loi; hoc Nu cn cn trn a kim loi th ch l khi mm hay rng xp, khi dng a thy tinh chc vo th phi khng c c nhn rn. Thi gian r phi trong vng 30 pht. 5.8.4. nh tnh Tin hnh nh tnh theo cc phng php c qui nh trong tiu chun, thuc n v thuc trng phi cho cc phn ng ca cc hot cht c trong ch phm. 5.8.5. nh lng Cn thuc trong 20 vin, tnh khi lng trung bnh vin, lm ng nht. Tin hnh theo phng php qui nh trong tiu chun, hm lng ca tng hot cht trong ch phm phi nm trong gii hn cho php nh bng 5.5. 5.9. php Th ho tan ca vin nn v vin nang (Dissolution test for tablets and capsules) 5.9.1. Khi nim ho tan ca mt ch phm l t l hot cht c gii phng ra khi dng thuc theo thi gian vi cc iu kin qui nh trong tng chuyn lun. Vi mi ch phm c cc qui nh c th v thit b th, mi trng ha tan, thi gian th nghim v phn trm hot cht c gii phng (Ch s Q). 5.9.2. Thit b Ty thit b c th c mt bnh hoc 6 - 8 bnh th. 5.9.2.1. Thit b kiu gi quay (Basket apparatus) Thit b bao gm: 1. Mt cc hnh tr C bng thu tinh borosilicat hoc bng cht liu trong sut thch hp, c y hnh bn cu v c dung tch 1000ml, hnh 5.1 v hnh 5.2. Ming bnh c vin rng, c y bng np c mt s l nh, trong c mt l tm ca np. 2. Mt ng c vi b phn iu tc c kh nng duy tr tc quay ca gi trong phm vi 4% tc t. ng c ny gn vi mt b phn khuy bao gm trc quay A v gi hnh ng tr B. Tr kim loi phi quay c mt cch nh nhng, khng b lc l nhiu trong khi quay. B phn gi gm c 2 phn: 160

 Phn np trn c mt l thot nh c gn vi trc. Np ny c lp

3 ci nhp n hi, hoc bng cch thch hp c th gi chc chn phn di ca gi ng trc vi trc ca bnh trong khi quay v c th tho phn di ra d dng khi cn cho ch phm th vo gi. Phn di tho lp c l mt gi hnh ng tr c lm bng vi ry kim loi, mp khu c hn lin, ng knh si dy l 0,254 mm, c cnh hnh vung ca l ry l 0,381 mm; gi hnh ng tr ny c vnh kim loi bao quanh y trn v y di. Gi c th c m mt lp vng kim loi dy 2,5 m khng b hng khi th trong mi trng acid. Khong cch t gi n y trong ca bnh lun c gi trong khong 23 - 27 mm trong qu trnh th. 3. Mt chu cch thu duy tr mi trng ho tan nhit 370C 0,5
0

C.

Hnh 5.1. Thit b th ha tan ca vin nn, vin nang ca Pharma TEST
1. B iu nhit 2. Cc ng mi trng ha tan 3. Cnh khuy 4. a y 5. Trc khuy 6. B phn iu khin

5.9.2.2. Thit b kiu cnh khuy (Paddle apparatus) Thit b ny ging nh thit b gi quay m t trn, ch khc l gi c thay bng cnh khuy D hnh 5.1 v hnh 5.3. Cnh khuy c lp t sao 161

cho i qua tm ca trc v cnh di ca n ngang bng vi mt y ca trc. Trc cnh khuy c lp t v tr sao cho khng lch qu 2 mm so vi trc ca bnh v cnh y ca cnh khuy cch mt y trong ca bnh t 23 - 27 mm. Thit b c vn hnh sao cho cnh khuy quay trn mt cch nh nhng v khng c rung ng r rt.

Hnh 5.2. Thit b kiu gi quay (kch thc tnh bng mm)

H. 5.3. Thit b kiu cnh khuy (kch thc tnh bng mm)

5.9.3. Cch th 5.9.3.1. Chun b mi trng ha tan Mi trng ho tan c ch dn trong tng chuyn lun ring. Nu l dung dch m th pH phi iu chnh sai khc khng qu 0,05 n v. Mi trng ho tan phi loi kh trc khi dng. Cho mt th tch quy nh mi trng ho tan ui kh vo bnh (th tch qui nh 1%). Lm m mi trng ho tan n nhit 37 0,5 oC. 5.9.3.2. Cho vin vo thit b th Nu khng c ch dn g khc th th ng thi trn 6 vin, cho mi vin vo mt bnh tr hoc mt gi quay. Khi dng thit b gi quay: Cho vin nn hay vin nang vo trong gi kh. H thp gi vo ng v tr ri cho gi quay.

162

 Khi dng thit b kiu cnh khuy: Cho vin nn hay vin nang chm xung y bnh trc khi cho cnh khuy quay; c th dng mt dy xon bng kim loi hay thu tinh gi cho vin thuc nm ngang di y bnh. Cn loi tr bt khng kh khi b mt vin. 5.9.3.3. Vn hnh thit b Vn hnh thit b ngay tc quay c ch dn trong chuyn lun ring. 5.9.3.4. Ly mu Tu theo ch dn chuyn lun m ly mu ra th pht th 45 hoc sau nhng khong thi gian quy nh, hoc ly mu ra lin tc; cho php chnh lch so vi thi gian qui nh l 2%. V tr ht mu khong gia b mt mi trng ho tan v mt trn ca gi quay hay cnh trn ca cnh khuy; im ny phi cch thnh bnh t nht 10 mm. Tr trng hp phn tch mu lin tc, o mu t ng khi mu ly ra sau khi phn tch li tr v bnh ho tan, cng nh trong trng hp ly mu phn tch mt ln; cn cc trng hp khc phi thm mt th tch mi trng ho tan bng th tch mu th ly nhit 37 0,5oC, hoc c th dng php tnh hiu chnh. 5.9.3.5. Xc nh lng hot cht Mu th ly ra c th c lc bng giy lc, mng lc hoc thit b thch hp. Dung dch thu c c th c dng ring bit hoc gp li xc nh phn trm hot cht gii phng. Xc nh lng hot cht cha trong dch lc bng phng php c ch dn trong chuyn lun ring. 5.9.3.6. nh gi kt qu Lng hot cht c gii phng ca c 6 vin th phi khng di 70% lng hot cht qui nh, tr khi c ch dn trong chuyn lun ring. Nu c 1 vin khng t th th vi 6 vin khc, ln ny c 6 vin u phi t yu cu (theo DVN III). Hoc phn trm dc cht gii phng so vi hm lng ghi trn nhn c nh gi theo bng 5.13 v bng 5.14 (theo USP 24). Nu v vin nang nh hng ti kt qu phn tch, ly t nht 6 vin nang, loi b ton lng thuc trong nang, ho tan v nang rng trong mt th tch mi trng ho tan c ch dn trong chuyn lun ri tnh hiu chnh. H s hiu chnh khng c ln hn 25% hm lng ghi trn nhn.

163

Bng 5.13. Gii hn v phn trm dc cht gii phng i vi vin nn, vin nang nh gi theo tng vin (Theo USP 24 - trang 1943)
Ln 1 S vin th 6 Yu cu kt qu (Phn trm hot cht ha tan) Mi vin phi Q + 5% Trung bnh ca 12 vin phi Q v khng c vin no < Q - 15% Trung bnh ca 24 vin phi Q v c di hai vin < Q - 15% v khng c vin no < Q - 25% Kt lun - Gii php t: Mu th t yu cu v ha tan. Dng th nghim. Khng t: Lm ln 2 t: Mu th t yu cu v ha tan. Dng th nghim. Khng t: Lm ln 3 t: Mu th t yu cu v ha tan. Khng t: Mu th khng t yu cu v ha tan.

12

Bng 5.14. Gii hn v phn trm dc cht gii phng i vi vin nn, vin nang nh gi theo cch gp dch lc ca cc vin th (Theo USP 24 - trang 1943)
Ln S vin th 6 Yu cu kt qu (Phn trm hot cht ha tan) Trung bnh ca 6 vin phi Q + 10% Trung bnh ca 12 vin phi Q + 5% 2 6 Kt lun - Gii php t: Mu th t yu cu v ha tan. Dng th nghim. Khng t: Lm ln 2 t: Mu th t yu cu v ha tan. Dng th nghim. Khng t: Lm ln 3 Trung bnh ca 24 vin phi Q . t: Mu th t yu cu v ha tan. Khng t: Mu th khng t yu cu v ha tan.

12

5.9.4. Th tc ha tan ca vin nn v vin nang bao tan rut Tin hnh cc bc nh trn. Ring mi trng v thi gian nh sau: Giai on 1: dng 1000 ml dung dch acid hydrocloric 0,1 M lm cht lng. Th trong 2 gi. nh gi kt qu: + Lng hot cht c gii phng ca c 6 vin th phi khng di 70% lng hot cht qui nh, tr khi c ch dn trong chuyn lun ring. Nu c 1 vin khng t th th vi 6 vin khc, ln ny c 6 vin u phi t yu cu (theo DVN III). Hoc Trong mi trng acid hydrocloric 0,1 M: theo bng 5.15. Trong mi trng m pH 6,8: nh gi nh bng 5.13 v 5.14. 164

Giai on 2: 1000ml dung dch m phosphat pH 6,8. Th trong 45 pht.

Bng 5.15. Gii hn v phn trm dc cht gii phng i vi vin bao, vin nang tan trong rut (mi trng th l HCl 0,1M) - Theo USP 24 - trang 1947
L 1 S vin th 6 Yu cu kt qu (Phn trm hot cht ha tan) Khng c vin no vt 10% so vi hm lng ghi trn nhn Trung bnh ca 12 vin phi nh hn 10% so vi hm lng ghi trn nhn Trung bnh ca 24 vin phi nh hn 10% so vi hm lng ghi trn nhn Kt lun - Gii php - t: Mu th t yu cu. Dng th nghim. - Khng t: Lm ln 2 - t: Mu th t yu cu. Dng th nghim. - Khng t: Lm ln 3 - t: Mu th t yu cu. - Khng t: Mu th khng t yu cu v ha tan.

12

V d: Vin nang Cephalexin (DVN III, trang 53 - 54) Thit b: Gi quay Mi trng ha tan: 1000 ml nc Tc : 100 vng/ pht Thi gian: 45 pht Ly v x l mu: Lc dung dch sau khi ha tan. Pha long dch lc bng nc c dung dch cephalexin c nng 25 g/ml. o hp th ca dung dch thu c bc sng 262 nm vi mu trng l nc. Song song lm mt mu chun cephalexin c cng nng . Yu cu: Khng c t hn 80% lng cephalexin (C16H17N3O4S) ghi trn nhn c ha tan trong 45 pht. Vin bao Ibuprofen (DVN III, trang141) Thit b: Cnh khuy Mi trng ha tan: 900 ml dung dch m phosphat pH 7,2 (Phi hp 250 ml dung dch KH2PO4 0,2 M vi 175 ml dung dch NaOH 0,2 M, thm nc va 1000 ml). Tc : 50 vng/ pht Thi gian: 60 pht Ly v x l mu: Lc dung dch sau khi ha tan. Pha long dch lc vi dung dch m phosphat pH 7,2 c dung dch ibuprofen c nng thch hp. o hp th ca dung dch thu c bc sng 221 nm trong cuvet thch anh c chiu dy 1 cm vi mu trng l dung dch m phosphat pH 7,2. Tnh hm lng ibuprofen theo A (1%, 1cm) l 449 bc sng 221 nm. 165

Yu cu: Khng c t hn 85% lng ibuprofen (C13H18O2) ghi trn nhn c ha tan trong 60 pht. 5.10. Th r ca vin nn v vin nang (Disintegration test for tablets and capsules) 5.10.1. Khi nim r ca vin nn, vin nang l kh nng tan r ca chng trong mi trng th theo qui nh (thng l nc), bng thit b th r c qui nh (My th r Erweka hay mt thit b tng ng) trong thi gian nht nh c qui nh tng chuyn lun. 5.10.2. Thit b Thit b bao gm: (Hnh 5.4, hnh 5.5) 1. Mt gi ng th thch hp, 6 ng thy tinh hnh tr c chiu di 75,0 - 80,0 mm, ng knh trong 21,5 mm v chiu dy thnh ng khong 2 mm. 2. Mt a hnh tr y trn mi ng, c ng knh 20,55 - 20,85 mm v chiu dy 9,35 - 9,65 mm, c lm bng cht do trong sut c t trng 1,18 1,20. a c 5 l hng, ng knh mi l l 2 mm, 1 l gia cn 4 l cn li t cch u nhau trn mt vng trn c bn knh 6 mm t tm a. C 4 rnh cch u nhau c ct bn cnh a sao cho mt trn a, rnh c chiu rng 9,5 mm, chiu su 2,55 mm; mt di ca a, rnh l hnh vung c cnh 1,6 mm. 3. Cc ng c gi v tr thng ng trong 6 l trng ca 2 tm nha cng trong sut c ng knh 90 mm v chiu dy 6 mm. Cc l cch u nhau v cch u tm. Mt di ca tm nha pha di c gn 1 tm li an bng cc si thp khng g c ng knh 0,635 mm v kch thc mt l l 2 mm. 4. Hai tm nha c c nh v cch u nhau 77,5 mm bng cc thanh kim loi c cht thng ng pha ngoi v mt thanh kim loi c gn cht vo tm tm nha pha trn, sao cho gi ng th c th lp rp vi b phn c vn hnh thit b chuyn ng ln xung mt khong t 50 - 60 mm vi tn sut 28 - 32 ln/ pht. 5. Gi ng th c nhng chm trong mt bnh thch hp, thng l cc thy tinh c dung tch 1000 ml cha cht lng c qui nh. Th tch cht lng phi sao cho khi gi ng th v tr cao nht th li kim loi phi di b mt cht lng t nht 15 mm v khi gi ng th v tr thp nht th li kim loi cch y cc thy tinh t nht 25 mm v ming cc ng thy tinh phi trn b mt cht lng. 6. Mt thit b ph hp (thng l chu cch thy) duy tr nhit ca cht lng l 370C 0,5 0C. 166

Hnh 5.4. Thit b o r ca vin nn, vin nang (chi tit)

5 4 2 3

Hnh 5.5. Thit b o r ca vin nn, vin nang

1. Ngun cung cp nhit 4. a y 2. B iu nhit 6. Gi 3. Cc ng mi trng th 7. B phn iu khin

5. ng hnh tr

167

5.10.3. Cch th 5.10.3.1. Chun b Cho mt th tch thch hp mi trng th (thng l nc) vo cc. Vn hnh my iu nhit nhit ca mi trng th t 370C 0,50C. Cho vo mi ng th mt vin nn hoc mt vin nang ri y a cht do vo tng ng.

5.10.3.2. Vn hnh thit b Nhng gi vo trong cc ng cht lng v vn hnh thit b theo thi gian qui nh. 5.10.3.3. nh gi kt qu Sau thi gian qui nh hoc khi thy cc vin r ht, ly gi ng th ra khi cc cht lng. Mu th c coi l t yu cu v r khi khng cn cn, tr nhng mnh v nang hoc v bao khng tan ca vin nn, cn li trn mt li ca thit b th hoc dnh vo b mt di ca a y. Nu cn cn th n ch l mt khi mm, khng c c nhn kh rn s thy c. Kt qu: Nu c 6 vin u r ht: Mu th t yu cu v r. Nu cn di hai vin cha r ht: Th li trn 12 vin na.

Ch phm t yu cu v r khi 16 trong s 18 vin th t r theo qui nh. 5.10.4. Th r ca vin nn v vin nang bao tan rut Tin hnh theo cc bc nh trn. Ring mi trng v thi gian nh sau: Giai on 1: mi trng th l acid hydrocloric 0,1M; trong 120 pht. Tt c cc vin th phi cn nguyn vn, khng th hin s gii phng hot cht. Giai on 2: mi trng th l dung dch m phosphat pH = 6,8; trong 60 pht. Nu c vin dnh a th th li vi 6 vin khc. nh gi kt qu: Nh i vi vin nn v vin nang. Cch pha m phosphat pH 6,8: Trn dung dch acid hydrocloric 0,1N v dung dch natri phosphat 0,2N theo t l 3: 1. iu chnh pH n 6,8 0,05 bng dung dch acid hydrocloric 2N hoc dung dch natri hydroxyd 2 N. 168

Ti liu tham kho

1. B Y t (2002). Dc in Vit Nam III. NXB Y hc. Tr. 6; 141 - 142; 254; PL. 15; PL. 18 -20; PL.131 - 133; PL. 136 -138. 2. ng Vn Ha (2001). Gio trnh kim nghim thuc. Trng H Y Dc TP H Ch Minh. 3. European Pharmacopoiea (2003), 4th edition, suppl. 4.3; 4.4. P.p. 3247 - 3250; 3367 - 3376. 4. Pharmacopoeia of The Peoples Republic of China (PPRC)1997, p.p. A5; A7; A51 - A56. 5. The Bristish Pharmacopoiea 2001, p.p. 1775 - 1824; A167; A233; A235; A236; A250; A252; A317. 6. The United States Pharmacopeia XXIV(2000), p.p. 1939; 1941 - 1951; 2000 - 2002; 2107 - 2118; 7. The United States Pharmacopeia XXVII (2004), p.p. 2108 2111; 2302 - 2312; 2577 - 2589. 8. Umesh Banakar. Pharmaceutical dissolution testing. Marcel Dekker 1992, p.p. 65 - 66; 71 - 72. 9. WHO, The International Pharmacopoiea (2003), 3rd edition, Vol. 5. P.p. 7- 28. 10. Brossard D. (1992). Les formes dermiques et les suppositoires dans L'analyse pratique du mdicament. Editions Mdicales Internationales, Paris. P.p. 971- 981. 11. Postaire E., Brossard D. (1992). Les formes liquides dans L'analyse pratique du mdicament. Editions Mdicales Internationales, Paris. P.p. 954 - 969.

Cu hi t lng gi
5.1. Trnh by tiu chun v k thut kim nghim chung ca thuc bt? 5.2. Trnh by tiu chun v k thut kim nghim chung ca vin nang? 5.3. Phn loi cc loi vin nang? Cho bit nhng yu cu ring cho tng loi nu c? 5.4. Trnh by tiu chun v k thut kim nghim chung ca thuc vin nn? 5.5. Phn loi cc loi vin nn? Cho bit cch th r i vi tng loi?

169

5.6. Trnh by tiu chun v k thut kim nghim chung ca thuc tim v thuc tim truyn? 5.7. Trnh by tiu chun v k thut kim nghim chung ca thuc nh mt? 5.8. Trnh by tiu chun v k thut kim nghim chung ca thuc m v thuc m tra mt? 5.9. Trnh by cch th tc ha tan v nh gi kt qu ca vin nn paracetamol 500 mg; vin nang cephalexin 500 mg v vin bao diclofenac 50 mg? 5.10. Anh (ch) hy cho bit cch th r v nh gi kt qu ca vin nn acid ascorbic 100 mg; vin nang cloxacilin 250 mg v vin bao cimetidin 400 mg ? 5.11. Anh (ch) hy xut tiu chun v qui trnh kim nghim cho vin nn A c hm lng hot cht l 1,5 mg/ vin. 5.12. So snh phng php th r ca vin nn ca Dc in Vit Nam I - tp 1 v Dc in Vit Nam III. 5.13. Ngi ta tin hnh nh lng paracetamol trong vin nn paracetamol 500 mg nh sau: Cn 20 vin c khi lng 14,6853g. Nghin mn. Cn 0,2205 g bt vin, ha tan trong va 200 ml dung mi. Lc u, lc. Pha long 1,00 ml dch lc thnh 100,0 ml trong bnh nh mc bng dung mi. Mt quang ca dung dch ny 257 nm l 0,501. Hi ch phm c t yu cu v hm lng khng? Bit paracetamol c A (1%, 1cm) bc sng 257 nm l 715. 5.14. Th ha tan ca vin nn ciprofloxacin 250 mg theo DVN III, sau khi dng khuy, pha long chnh xc mi trng ha tan trong mi cc th 50 ln. o hp th ca cc dung dch ny theo iu kin cho c kt qu ln lt tng ng vi cc cc 1; 2; 3; 4; 5 v 6 l 0,435; 0,346; 0,462; 0,378; 0,489 v 0,408. Hi ch phm c t yu cu v ha tan khng? Bit dung dch ciprofloxacin chun nng 5,5 g/ ml c hp th l 0,505 o trong cng iu kin.

170

Chng 6.

n nh v tui th ca thuc

Mc tiu hc tp 1. Trnh by c mc tiu v tiu chun nh gi n nh ca thuc. 2. Gii thch c nguyn tc xc nh n nh v cch tnh tui th ca thuc. Mt trong nhng yu cu quan trng nht i vi thuc phng v cha bnh l n nh v cht lng trong sut qu trnh bo qun t khi xut xng n khi ht hn dng. n nh ca thuc lin quan n qu trnh sn xut, phn phi v bo qun thuc. Khi nghin cu trin khai thuc mi hoc hon thin nng cao hiu lc ca mt thuc c x dng trong lm sng, nh sn xut cn nh gi n nh ca thuc trong iu kin bo qun xc nh, t tnh ra tui th ca n. Ngi phn phi, lu gi thuc phi duy tr c iu kin bo qun thuc qui nh m bo hn dng ca thuc. Vic nghin cu n nh ca thuc l mt qu trnh hon thin phng php v cho ti gn y, T chc Y t th gii (WHO) mi c vn bn chnh thc hng dn nghin cu n nh ca thuc. Dc in ca mt s nc c chuyn lun v vn ny. 6.1. Qu trnh pht trin nghin cu n nh Ngi ta c nhng ghi nhn ban u lin quan n bn vng ca thuc. Mt s ch phm kh bo qun , d phn hy nh aspirin, procain, mt s vitamin nh A, C. Ngc li, mt s ch phm khc li kh bn vng nh sulfonamid. T y khi nim v n nh ca thuc dn dn c hnh thnh. Nm 1948, tp ch Cng ngh Dc v M phm ca M ng mt kt qu nghin cu l th v vitamin A: bo qun vitamin A trong 5 tun l 420C s cho mt lng vitamin ny b phn hu t ng ng nh khi bo qun trong hai nm nhit phng. Kt qu nghin cu gi mt cch nh gi n nh. E.R. Garrett (1955) gi y l phng php th nghim cp tc. Cng trong thi gian ny ngi ta tin hnh mt php th n nh ca thuc (gang - testing): Mt nm ngi ta ly mu ngu nhin cc nh thuc mt ch phm no , tin hnh nh lng hot cht, nh gi s suy gim ca hm l ng. Trn c s kt qu nghin cu, cc nh bo ch M khuyn co i vi cc ch phm thuc cn: 171

Ghi hn s dng trn nhn thuc, t tiu chun dc in trong thi gian lu hnh.

u nhng nm 70 M, ngi ta thc hin mt chng trnh nghin cu n nh ca thuc, c nh gi thng k x l s liu. n nm 1975 Dc in M c a ra hn dng ca thuc. Tuy nhin Dc in ny cha qui nh phng php xc nh hn dng. Mi n nm 1984, c quan qun l thuc v thc phm M (FDA) mi c vn bn u tin v th n nh ca thuc. Vn bn ny c b sung trong ln xut bn th hai (1987) v hon chnh trong ln xut bn th ba (1994). Nm 1993 Hi ngh Quc t bn v hi nhp cho xut bn ti liu hng dn chi tit v th n nh ca thuc (Harmonized Tripative Guideline, Geneva). Vn bn hng dn ca FDA xut bn nm 1994 c ni dung tng t vi vn bn ho nhp 1993. Nm 1994, WHO xut bn ln u trn ti liu hng dn nghin cu n nh ca thuc. Ti liu ny c b sung ti bn nm 1998. Mt iu cn ghi nhn l: vic nghin cu n nh ca thuc ngy cng c y mnh v thu c kt qu kh quan l nh cc phng php phn tch xc nh hot cht v tp cht lin quan hoc tp cht phn hu c tin cy cao trong qu trnh sn xut cng nh bo qun v lu thng thuc. Bc t ph u tin l x dng quang ph UV-VIS nh lng hot cht v sc k lp mng pht hin tp cht phn hy. Tip n my sc k kh ri my sc k lng ra i gip cc nh phn tch xc nh c lng cht di ppm, phn tch nh lng c nhiu thnh phn trong hn hp, mt loi hnh phn tch hay gp trong nghin cu n nh ca thuc. Gn y vic p dng cc phng php phn tch hin i nh: nhiu x tia X, nhit vi phn,.... to ra nhiu thun li trong nghin cu n nh. Ni dung ca chng ny c bin son da ch yu vo ti liu hng dn ca WHO nm 1998, c tham kho thm mt s Dc in v mt vi ti liu khc. 6.2. i cng v n nh ca thuc 6.2.1. nh ngha Theo WHO, n nh ca thuc l kh nng ca nguyn liu hoc ch phm c bo qun trong iu kin xc nh c th gi c nhng c tnh vn c v ho l, vi sinh, sinh dc hc..... trong nhng gii hn nht nh. n nh ca thuc ph thuc vo nhiu yu t. C th phn chia cc yu t ny ra hai nhm: Cc yu t lin quan n mi trng nh: nhit , m, chiu sng, hm lng oxy cng cc yu t bn ngoi khc tc ng ln thuc. Cc yu t lin quan n thuc. l: 172

Tnh cht l ha ca hot cht v t dc c dng bo bo ch thuc. V d: dng tinh th, hm lng nc, tp cht trong nguyn liu... Dng bo ch ca thuc, Qui trnh sn xut thuc Nguyn liu cho ng, bao b, ng gi.

Nh vy n nh ca thuc ph thuc ch yu vo c im ca nguyn liu, qui trnh sn xut v iu kin mi trng. Vic a vo cng thc bo ch cc cht lm tng n nh ca thuc ch c chp nhn trong trng hp hu hn v phi c chng minh v mt khoa hc v thc t. 6.2.2. Mt s thut ng lin quan. Cc ti liu hng dn nghin cu n nh ca thuc cp n nhiu khi nim v thut ng. y s tm tt mt s thut ng chnh: Cc php th n nh (stability testings) l tp hp cc php th c thit k nhm thu c nhng thng tin v n nh ca ch phm. Trn c s nh ra tui th, hn dng iu kin ng gi v bo qun xc nh. Php th n nh di hn (long term (real time) stability testing) l nhng nghin cu thc nghim nh gi s thay i cc tnh cht ha l, sinh hc, sinh dc hc .... ca mt ch phm thuc trong qu trnh bo qun iu kin xc nh. Trn c s kt qu nghin cu ny xc nh hn dng v khuyn co iu kin bo qun. Php th n nh cp tc (accelerated stability testing) y l nghin cu thc nghim c b tr lm tng tc phn hy ha hc v thay i trng thi vt l ca thuc nh s thay i ca nhit , m, chiu sng,... Thng nghin cu c tin hnh nhit cao 35 400C, c khi ti 50 0C, m tng i 80 - 90% hoc hn. L sn xut (batch N0) l lng xc nh ca mt sn phm c sn xut theo mt qui trnh v c coi l ng nht. Trong trng hp sn xut lin tc, l tng ng vi mt phn nht nh ca qu trnh sn xut v c c trng bi tnh ng nht ca sn phm. Hn dng thuc (expiration date) y l thi im ht hn x dng ca thuc, c ngha l sau thi im ny ch phm thuc khng cn gi c cc tnh cht nh ng k trong tiu chun cht lng. Hn dng c xc nh theo l sn xut. Hn sn xut (manufacture date) y l thi im kt thc qu trnh sn xut l thuc. Hn ny c ghi thnh thng v nm cho tng l sn phm. C th tnh hn sn xut l thi 173

im thuc c a vo lu thng vi iu kin l khong thi gian gia thi im bt u sn xut v thi im a vo lu thng khng vt qu 1/ 20 ca thi hn bo qun. Thi hn bo qun (expiration dating period) l khong thi gian bo qun thuc trong iu kin xc nh m thuc vn gi c cc tnh cht ng k trong tiu chun cht lng. Da vo thi hn bo qun v hn sn xut tnh ra hn dng ca thuc. Ngi ta cng dng tui th (shelf life) ca thuc ng ngha vi thi hn bo qun thuc. iu kin bo qun chun ha (controlled storage condition) l iu kin bo qun trong kho thong kh, nhit trong khong 15 - 250C. Tu vng kh hu nhit c th ln ti 300C. Trong kho khng c mi l, khng c cht nhim, khng c nh sng trc tip chiu vo. Nhit ng hc trung bnh - NT (mean kinetic temperature) y l tr s nhit trung bnh c tnh ton cho tng vng kh hu da trn s phn b ca nhit theo thi gian. NT c dng nh gi tc ng ca nhit ln ng hc ca qu trnh phn hu ha hc lm gim n nh ca thuc. Tr s NT thng ln hn tr s trung bnh s hc. 6.2.3. Mc tiu nh gi n nh Theo hng dn ca WHO, nghin cu n nh ca thuc nhm 4 mc tiu chnh (bng 6.1) Giai on pht trin sn phm giai on ny, cc php th cp tc c thc hin nhm la chn cng thc bo ch thuc, qui trnh sn xut v bao gi thch hp. Sau khi c sn phm nh sn xut tip tc dng cc th nghim cp tc d bo n nh, s b nh gi tui th trong iu kin bo qun nh. Mt khc cc th nghim di hn cng bt u c trin khai phc v cho giai on tip theo.
Bng 6.1. Bn mc tiu cho nghin cu n nh
TT 1 2 3 4 Mc tiu Xy dng cng thc, k thut pha ch v bao gi Xc nh tui th v iu kin bo qun Khng nh bng thc nghim tui th thuc Phng php nghin cu Th nghim cp tc Th cp tc v di hn Th di hn Giai on p dng Pht trin sn phm Pht trin sn phm v lp h s ng k Lp h s ng k Thuc lu hnh trn th trng

Thm nh n nh Th cp tc v di hn lin quan n cng thc v qui trnh sn xut

174

 Giai on lp h s ng k Trn c s kt qu thc nghim cp tc v di hn, nh sn xut xc nh tui th v hn dng ca thuc iu kin bo qun v bao gi thch hp. Cc thng tin ny c ghi r trn nhn thuc v bao gi. Nh sn xut trnh vi c quan qun l thuc ton b h s khc xin php lu hnh thuc trn th trng. Giai on thuc lu hnh trn th trng Khi thuc lu hnh trn th trng, nh sn xut tip tc theo di n nh khng nh tui th ca thuc trong iu kin bo qun xut. bo m an ton v cht lng thuc lu hnh trn th trng, cc c quan qun l cng theo di n nh ca thuc thng qua vic thanh tra, kim tra qui trnh sn xut, ly mu thuc kim nghim. i vi mt ch phm c cp php lu hnh, nu c s thay i v cng thc, v qui trnh sn xut hoc qui cch ng gi,... nh sn xut phi nghin cu b sung v n nh v bo co vi c quan qun l thuc. 6.2.4. Tiu chun nh gi n nh C 5 tiu chun nh gi n nh ca thuc n nh ha hc Cc tnh cht ha hc (thnh phn nh tnh v nh lng) ca (cc) hot cht c mt trong ch phm nm trong mt gii hn cho php theo tiu chun cht lng. n nh vt l Cc c im vt l ca nguyn liu lm thuc nh: mu sc, trng thi tinh th, tan, im chy.... khng thay i. Cc c im ca ch phm nh mu sc, cng, r, ho tan dao ng trong khong gii hn cho php ca tiu chun cht lng. n nh vi sinh v trng hoc gii hn nhim khun ca ch phm phi p ng yu cu ca tiu chun. Nu ch phm c cha cht khng khun th hm lng ca n khng vt qu gii hn cho php. n nh iu tr Tc dng iu tr ca ch phm khng thay i n nh c tnh c tnh ca ch phm khng c tng ln trong sut qu trnh bo qun v lu hnh trn th trng.

175

6.2.5. Phn vng kh hu Tui th ca thuc ph thuc vo vng kh hu m thuc lu hnh. nghin cu n nh ngi ta chia th gii ra 4 vng kh hu: Vng 1: Kh hu n ho. l cc nc bc u, Anh, Canada, Nga. Vng 2: Kh hu nhit i c th c m cao nh M, Nht bn, cc nc nam u (Hy lp, B o Nha). Vng 3: Kh hu nng kh nh Iran, Irac, Sudan. Vng 4: Kh hu nng m. l mt s nc nam M (Brazil, Nicaragua,....), ng nam (Vit Nam, Philippin, Indonesia,...). Bng 6.2. tm tt gi tr trung bnh bn thng s ca 4 vng kh hu. Bng 6.3. cho ta iu kin bo qun ch phm trong nghin cu n nh di hn. iu lu l thng s nhit c chn ti thiu l 210C mc d vng c nhit thp hn tr s ny.
Bng 6.2. Nhit v m ca 4 vng kh hu
Vng 1 2 3 4 Tr s o c ngoi tri Nhit ( C) 10,9 17,0 24,4 26,5
0

Tr s o c trong kho Nhit (0C) 18,7 21,1 26,0 28,4 m (%) 45 52 54 70

m (%) 75 70 39 77

Bng 6.3. Thng s kh hu tnh ton v iu kin bo qun cho th di hn

Vng 1 2 3 4 Thng s kh hu tnh ton Nhit 20,0 21,5 26,4 26,7 NT 20,0 22,0 27,9 27,4 m ** 42 52 35 76 m bar * 9,9 13,5 11,9 26,6 iu kin bo qun Nhit 21 25 30 30 m ** 45 60 35 70

* p sut hi nc ring phn trong kh quyn ** m tng i tnh bng % WHO khuyn co cc nh sn xut: Nu ch phm c lu hnh vng 1 nn nghin cu n nh trong iu kin bo qun vng 2. Nu ch phm c lu hnh vng 3 hoc vng 4 nn nghin cu n nh trong iu kin bo qun vng 4.

176

Cn lu l vi mt s ch phm vic nghin cu n nh phi c tin hnh nhit di 00C, hoc iu kin lnh (+20C n 80C). Vi mt s khc i khi phi xem xt tc ng ca nh sng. 6.3. ng ha hc dung dch Di tc ng ca iu kin bn ngoi, nguyn liu v ch phm dn n b phn hu, hm lng hot cht gim dn theo thi gian. Qu trnh phn hu ng hc ny rt phc tp, bi v: Nhiu yu t tc ng ng thi, Nhiu loi tng tc c th din ra: tng tc vt l, phn ng ha hc, cc qu trnh sinh hc, Ch phm tn ti nhiu dng khc nhau: dung dch nc, dung dch du, nh dch, hn dch, bt rn,.... Ch phm thng c nhiu thnh phn: mt hoc nhiu hot cht, cc loi t dc.

Chnh v vy n nh ca thuc khng ng ngha vi ng ha hc. Trong qu trnh bo qun c th c mt s phn ng ha hc phn hu dc cht c m t bng phng php ng ha hc. V vy trc khi nghin cu n nh ca thuc chng ta n tp vi vn c bn ca ng ha hc dung dch. 6.3.1. Bc ca phn ng Tc ca mt phn ng c xc nh bng ba thng s: Nng ca cc cht phn ng, Nhit xy ra phn ng, S c mt ca cht xc tc, Cho mt phn ng: A+B P (6.1) Tc v ca phn ng c xc nh theo nh lut Vant Hoff:

v=

dC = k n m ( m + n ) [ A] [ B ] dt

(6.2)

C l nng ca cht nghin cu, cc mc vung l ch nng ca A v B, k l hng s tc (c lp vi nng C), n v m l bc ring ca A v B tng ng, n + m = p l bc tng cng ca phn ng. 177

Trong nghin cu n nh ngi ta quan tm n bc tng cng p. N c th l bc khng, bc nht hoc bc hai. 6.3.1.1. Phn ng bc khng

dC
v=

dt

= k 0

(6.3)

Phng trnh tc cho phn ng bc khng Tch phn phng trnh ta c

C = C0 k0t
hoc C0 (1 a) = k0 ta y a l phn s mol cn li thi im ta. thi im t1/ 2 , tc l Ct = 0,5C0 ta c C tt = 0
1/2

(6.4) (6.5)

(6.6)

2k 0 Thi gian bn hu t1/2 ph thuc vo nng ban u C0. Th nguyn ca hng s k l mol/ n v t.
Trong thc t c rt t phn ng ng ha hc bc khng. 6.3.1.2. Phn ng bc nht

dC dt

k 1C

( 6 .7 )

Tc phn ng ph thuc vo nng

ln
Dng tch phn

C C
0

= k1t

( 6 .8 )

ln a = k 1 t a
Phn s mol a cn li thi im ta

( 6 .9 )

Trong nghin cu ng hc phn hu thuc bc nht ngi ta lu tm thm thi im t0,9. l thi im m t s [C/ C0] = 0,9 kt1/2 = - 0,693 kt0,9 = -0,105 Th nguyn ca hng s k l t 178
-1

(6.10) (6.11)

Khi nghin cu n nh cn kim tra nng hot cht hoc tp cht phn hu. Nu trong h ch c mt tp cht phn hy, vic xc nh t s C/ Co kh n gin. Ngi ta c th xc nh nng hot cht hoc tp cht thi im t tnh hng s tc . Vn tr nn phc tp hn khi h to ra hai hay nhiu sn phm phn hu. Hai trng hp sau c th gp trong thc t nghin cu n nh . Phn ng bc nht k tip C trng hp phn ng phn hu ban u khng n nh. Ta c s phn ng sau: A k1 B k2 C (6.12)

Nu c ba cht trong h c xc nh th mi thi im tng s mol ca ba cht bng s mol ban u ca A. Cc phn ng din ra theo qui lut ng hc bc nht, ta c cc phng trnh tc :

d[ A ] k 1 [ A ] dt = d[B] dt = k2[B] + k 1 [A ]

(6 .13 ) (6.14 )

d[C ] k 2 [ B ] dt =
Gii cc phng trnh vi phn ny ta c [A] = A0 exp(k1t) [B] = A0 [k1/ (k2 k1)][exp(k1t) exp(k2t)]

( 6 .15 )

(6.16) (6.17)

[C ] = A

k 2 exp( k t ) + k 1 exp( k 1 2 t ) 0 1 k k k 2 k1 2 1

A0 l nng mol ban u ca A. tnh C, trong nhiu trng hp ngi ta tnh [C] mt thi im theo phng trnh [C] = A0 [A] [B] (6.19)

Bng 6.4 minh ho s quang phn ca cefotaxim bng hai phn ng bc nht k tip (Lerner 1988). 179

Bng 6.4. Quang phn ca Cefotaxim

Thi gian (gi) 0,00 0,25 0,50 0,75 1,00 1,50 2,00 3,00 4,00 Cefotaxim A (% mol) 100 82 70 55 43 28 20 10 5 B (% mol) 0 10 15 18 19 18 15 10 5 C (hiu s) 0 8 15 27 38 54 65 80 90

Ct C cui cng tnh theo phng trnh (6.19). Phn ng bc nht song song Nu A phn hu to ra hai sn phm ng thi l B v C A k1 Phng trnh tc c dng: d[A]/ dt = k1[A] k2[A] ln[A/ A0] = (k1 + k2)t (6.21) (6.22) B A k2 C (6.20)

thi im bt k t l ca hai sn phm to thnh B v C lun khng i [B] / [C] = k1/ k2 (6.23)

Trong thc t i khi cc phn ng k tip v song song din ra khng ng thi. Trong dung dch nc mui Cefotaxim natri phn hu theo s sau: Cefotaxim k2 Cefotaxim k1 Lacton k3 sn phm cui sn phm cui

180

Kt qu nghin cu ny (Fabre 1984) l mt v d minh ho s phc tp ca nghin cu ng hc phn hu thuc. 6.3.1.3. Phn ng bc hai Cho phn ng A + B Tc phn ng v = d[A]/ dt = k2[A][B] (6. 25) Nu nng ban u ca A v B l a v b, lng C to thnh thi im t l x th dx/ dt = k2(a x)(b x) hoc di dng tch phn: (6. 26) C (6. 24)

(a b)k2
2

b(a x ) t = ln a (b x )

(6.27 )

(a b)k t = ln

b + ln bx a

ax

R rng th nguyn ca k2 l (thi gian x nng )-1. Nu khi phn ng din ra a>>b, lng A tham gia phn ng coi nh khng ng k so vi lng ban u nn [A] coi nh khng i v phng trnh (6.25) c dng

v = k2[A][B] = k2[B]

(6. 28)

Trong trng hp ny, ta gi l phn ng bc nht gi. 6.3.2. nh hng ca nhit Hng s k trong phng trnh ng hc ph thuc vo nhit . Khi nhit tng, hng s k tng theo. Kt qu thc nghim cho thy: khi nhit tng ln 100C hng s tc k tng ln xp x 2 ln. nh lut Arrhenius Nm 1889 Arrhenius nhn thy kt qu thc nghim c th c biu din bng h thc kinh nghim: ln k = C1 C2/ T (6.29)

181

C1 v C2 l hai hng s v C2 lun lun dng. Phng trnh (6.29) ging phng trnh Vant Hoff di dng tch phn: ln k = C H0/ (RT) (6.30)

H0 l i lng khng ph thuc vo nhit . t C2 = E0/ R, c th vit phng trnh Arrherius di dng ln k = C1 E0/ (RT) (6.31)

E0 l nng lng hot ha c xc nh bng cch v th ln k theo 1/T (T l 0K = 0C + 273,15). C th vit phng trnh (6.31) di dng hm s m k = A exp(E0/ RT) y ln A = C1 6.4. Xc nh n nh ca thuc Khi nh gi n nh cn xem xt n cc yu t: Tnh cht v c im ca dc cht trong ch phm, Vng kh hu cho thuc lu hnh, Ti liu cng b lin quan n n nh ca thuc cn nghin cu. Nhng yu t ny s quyt nh s mu th, thi gian v tn s th nghim. 6.4.1. Ly mu Vi dc cht tng i n nh, cn ly hai mu hai l sn xut khc nhau. Nu dc cht km bn hoc c t ti liu cng b, cn ly ba mu ba l khc nhau. Cc mu ny cn ly i din cho qui trnh sn xut. i vi nghin cu n nh mt cch lin tc qui m cng nghip vic ly mu cc l c tin hnh theo chng trnh nh trc, v d: Vi cng thc n nh, c hai nm ly mu trn 1 l. Trong trng hp ngc li, mt nm ly mu trn 1 l. i vi cng thc nghin cu xong n nh, thng c 3 n 5 nm s kim tra li n nh mt ln. (6. 32)

182

6.4.2. Phng php th cp tc iu kin th Bng 6.5. tm tt iu kin th cho vng II v vng IV
Bng 6.5. iu kin th cp tc
Vng II IV Nhit (0C) 40 2 40 2 m (%) 75 5 75 5 Thi gian th (thng) 3 6

Vi cc ch phm c hot cht km bn, hoc c t ti liu nghin cu c cng b, thi gian th ko di hn 3 thng so vi qui nh. Ngi nghin cu c th la chn nhit cao hn trong thi gian ngn hn, v d 45 n 500C trong 3 thng vi m 75%. Nu trong qu trnh nghin cu, ch phm c nhng thay i quan trng th cn thc hin cc php th nghim b sung iu kin n ho hn, v d 30 20C, m (60 5)%. Nhng du hiu chng t c s thay i quan trng l: Gim hm lng hot cht t 5% tr ln so vi tr s ban u. C sn phm phn hu vi lng cao hn tr s cho php. pH nm ngoi gii hn qui nh. Tc ho tan ca 12 vin nn hoc vin nang thp hn gi tr ca tiu chun. Thay i c tnh vt l ca thuc nh: bin mu, tch pha, kh r,...

Vic th cp tc thng c tin hnh trong bung vi kh hu c th kim sot c nhit ( 20C) v m ( 5%). Mt s ch phm khng thch hp vi th nghim cp tc nh: ch phm sinh hc, thuc n, thuc trng,... V d: Nghin cu mt ch phm c hm lng theo cng thc l 100 mg. Th cp tc thi gian 9 thng ba nhit 350C, 450C v 550C. Kt qu c bng 6.6. Tnh hng s tc v tui th ca thuc khi bo qun 300C.

183

Bng 6.6. S thay i hm lng (mg) ca ch phm

Hm lng Thi gian (thng) 0 2 4 6 9 105 103 102 101 99 105 102 100 97 94 350C 450C Thi gian (thng) 0 1 2 3 4 5 105 102 99 97 94 91 Hm lng 550C

Gii: V th lnCt t(thng). Tnh hi qui bc nht, ta c hng s tc : - 350C: - 450C: - 550C: k35 = 0,00623 thng -1 (r = 0,993) k45 = 0,0123 thng -1 (r = 0,997) k55 = 0,0280 thng -1 (r = 0,997)

tnh tui th nhit 300C cn tnh k30 V th lnk 1/T (0K) theo phng trnh (6. 31)
k 0,0062 0,0123 0,0280 ln k 5,083 4,398 3,576 (1/ T). 1000 3,246 3,145 3,049

Nhit (0 C) 35 45 55

Tnh hi qui bc nht ln k = C1 E0/ RT = 19,7 7642 / T Tnh hng s k30 theo phng trnh (6.32) ln k30 = 19,7 7642 / (30 + 273,15) = 5,52 k
30

= 0,00411

184

Tui th ca thuc l khong thi gian hot cht cn li 90% so vi tr s ban u (phng trnh 6.11), nn: t 0,9 = 0,105/ k30 = 25,5 thng y l mt v d th n nh cp tc d bo tui th ca thuc. Tr s ny cn c khng nh bng phng php th di hn. 6.4.3. Phng php th di hn Theo hng dn ca WHO th iu kin thc nghim trong phng php th di hn phi gn vi iu kin bo qun thc t ca thuc, tc l vng kh hu m thuc d kin c lu hnh (bng 6.3). Tuy nhin mt s tc gi xut iu kin th di hn l nhit 25 20C, m tng i 60 5%. Nghin cu n nh di hn cn c tin hnh trong sut thi hn bo qun thuc. Thi gian kim tra hm lng trong mu: Nm u 3 thi im 0, 6 v 12 thng, T nm th 2: mt ln cho mt nm. Vi cng thc rt n nh ch cn kim tra hai ln: ln u sau 1 nm; ln th 2 cui hn dng. Vi ch phm km n nh, s ln kim tra nhiu hn: Nm th nht: 3 thng/ ln Nm th 2: 6 thng/ ln T nm th 3: 12 thng/ ln i vi ch phm c yu cu bo qun c bit nh: ch phm vaccin, hormon, thuc c hot cht rt khng n nh,... ngi nghin cu cn chn iu kin thch hp. Kt qu thc nghim c chp nhn nu: Khng c s thay i cc tnh cht vt l, ha hc, sinh hc. Ch phm vn p ng ng yu cu ca tiu chun. Phng php th di hn mt nhiu thi gian, nhng cho kt qu tin cy. Php th ny gip ngi nghin cu khng nh tui th d bo theo php th cp tc. 6.4.4. Phng php phn tch nh gi kt qu La chn phng php phn tch Vic la chn phng php nh lng hot cht v (hoc) tp cht phn hu ph thuc vo:

185

Tnh cht l ha ca hot cht, tp cht, Hm lng ca chng trong ch phm, Yu cu ca th nghim.

Cc phng php phn tch cn c thm nh: kim tra ng, lp li, ti hin, gii hn nh lng. Ring phng php xc nh tp cht lin quan, tp cht phn hu cn c kim tra tnh c hiu v nhy. X l s liu S liu nghin cu cn c x l thng k thu c thi hn bo qun tm thi. Thi hn ny c ngoi suy trn c s th n nh cp tc. Mt thi hn bo qun tm thi 24 thng c chp nhn khi: Hot cht c coi l n nh, Th nghim cp tc khng c thay i r rt, S liu hin c chng minh rng nhng cng thc tng t c thi hn bo qun 24 thng hoc hn. Nh sn xut s tin hnh th n nh di hn trong qu trnh bo qun xut.

Sau khi nh gi n nh, nh sn xut cn ghi r iu kin bo qun trn nhn thuc: Bo qun iu kin thng, Bo qun trong khong + 20C n 80C (t lnh), Bo qun di 80C (t lnh), Bo qun trong khong 50C n 200C (t ),

Bo qun di 180C (t ). iu kin thng theo WHO l: nhit 15 n 250C, c th n 300C, kh ro, khng c nh sng trc tip chiu vo. iu kin thng c th c qui nh theo tng quc gia. iu lu l nhng li ch gii nh: bo qun ni kh ro, bo qun trnh nh sng khng c s dng nhm mc ch che du s n nh km ca thuc. 6.5. Cc dc cht km bn vng Nm 1986 WHO thc hin ti :Nghin cu n nh cp tc ca cc dc cht thng dng trong iu kin nhit i. iu kin th cp tc: Bo qun 30 ngy 500C, m 100%. Nu khng c du hiu phn hu: tng nhit ln 700C trong thi gian tip theo 3 n 7 ngy.

186

Kt qu cho thy khong 100 dc cht cn c lu v n nh: chng c coi l cc cht km bn vng (bng 6.7)
Bng 6.7. Cc dc cht km bn vng
TT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Tn dc cht Acid acetyl salicylic Acid ascorbic Acid undecylenic Aminophyllin Amitryptilin, HCl Amoni clorid Amphotericin Ampicillin (mui natri) Ampicillin trihydrat Antimon tartrat Bc nitrat Bacitracin Bacitracin (mui km) Benzathin benzylpenicillin Benzylpenicillin (mui Na, K) Bephenium hydronaphtoat Calci gluconat Calci p.aminosalicylat Carbenicillin (mui Na) Cephalexin Cloral hydrat Cloramphenicol (Na succinat) Clorphenamin hydrogeno- maleat Clorpromazin, HCl Clortetracyclin, HCl Cloxacillin (mui Na) Codein phosphat Colecalciferol TT 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 Tn dc cht Hexyl resorcinol Hydralazin, HCl Hydrocortizon (Na succinat) Hyoscyamin sulfat Lidocain, HCl Melarsoprol Metrifonat Naloxon, HCl Natri calci edetat Natri lactat Natri nitrit Natri p. aminosalicylat Natri stibogluconat Neomycin sulfat Nystatin Orciprenalin sulfat Oxytetracyclin, HCl Paromomycin sulfat Penicillamin Pethidin, HCl Phenobarbital (mui Na) Phenoxymethyl penicillin (mui Na hoc K) Phentolamin mesilat Phenyl butazon Pilocarpin, HCl Pilocarpin nitrat Procain amid, HCl Procain benzyl penicillin

187

TT 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Dapson

Tn dc cht

TT 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 Procain, HCl

Tn dc cht

Dexametason (dng Na phosphat) Dicloxacillin (mui Na) Dietyl carbamazin (dihydrogeno citrat) Doxycylin hydrat Ephedrin Ephedrin sulfat Epinephrin Epinephrin hydro-geno tartrat Ergocalciferol Ergometrin hydrogenotartrat Ergotamin maleat Ergotamin tartrat Etho suximid Ethyl morphin, HCl Fe(II) sulffat Fluphenazin decanoat Fluphenazin, HCl Formaldehyd dung dch Gentamycin sulfat Guanethidin sulfat

Procarbazin, HCl Promazin, HCl Prometazin, HCl Pyridoxin , HCl Quinin, bisulfat Quinin, 2HCl Retinol (vitamin A) Salbutamol sulfat Sulfacetamid (mui Na) Sulfadiazin (mui Na) Sulfadimidin (mui Na) Tetracain , HCl Tetracyclin, HCl Thiamin, HCl Thiamin mononitrat Thiopental (mui Na) Thu ngn oxyd vng Tolbutamid Warfarin (mui Na)

ti liu tham kho

1. Bureau of Food & Drug (1998) Training course drug and cosmetic quality control, Filinvest Corporate city, Philippines. 2. Carstensen J.T. (1995) Drug stability Principles & Practices, 2nd edition, Marcel Dekker Inc. , USA. 188

3. Didier R. : Chimie minerale Technique et Documentation, Lavoisier, Ve edition, Paris. Bn dch ting Vit, tp II, NXB Gio dc, H ni 1997. 4. OMS (1998) Assurance de la qualit des produits pharmaceutiques, Vol 1, Genve. 5. Skoog D. A. , West D. M. , Holler F. J (1988) Fundamental of Analytical Chemistry, 5th edition, chapter 22, pp 578- 597 Saunders College Publishing.

cu hi t lng gi
6.1. Ti sao cn t vn nh gi n nh ca thuc. 6.2. Gii thch cc yu t tc ng n n nh ca thuc. 6.3. Phn tch php th n nh di hn v n nh cp tc. Ti sao cn tin hnh c hai php th ? 6.4. Nhng ch phm no khng thch hp vi php th cp tc? Ti sao? 6.5. Ti sao phi phn thnh 4 vng kh hu khi nghin cu tui th ca thuc. Theo anh (ch) vng kh hu no nh hng nhiu nht n n nh ca thuc? 6.6. Gii thch vai tr ca ng ha hc trong nh gi tui th ca thuc. 6.7. Trnh by nguyn tc s dng phng trnh Arrhenius tnh tui th ca thuc trong phng php th cp tc. 6.8. Nhng phn ng no thng xy ra lm gim n nh ca thuc? Cho v d. 6.9. Khi xc nh tui th ca thuc da vo s liu hm lng hot cht, cn thm nh: ng, chnh xc v gii hn nh lng. Gii thch? 6.10. Gii thch l do ti sao khi xc nh tp cht lin quan, tp cht phn hy cn kim tra tnh c hiu v nhy ca phng php thc nghim? 6.11. nh gi vai tr ca cc k thut phn tch trong nghin cu n nh ca thuc. 6.12. Ngi ta nh gi n nh ca mt ch phm vin nn hm lng 200 mg bng phng php lo ha cp tc trong thi hn 6 thng hai nhit 400C v 600C. S liu thc nghim c trnh by bng sau: 189

Hm lng (%) 400C Thi gian (thng) 0 1 2 3 4 5 6 102,1 101,5 101,1 100,6 100,1 99,5 98,8 102,1 101,6 101,1 99,5 98,9 98,3 97,6 600C

Hy tnh tui th ca vin nn 200 mg.

190