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VIN KIM NGHIM AN TON V SINH THC PHM QUC GIA

THM NH PHNG PHP


TRONG PHN TCH
HA HC V VI SINH VT












NH XUT BN KHOA HC V K THUT
H NI







Ch bin:
DS. Trn Cao Sn
Nhm bin son:
DS. Trn Cao Sn
PGS. TS. Phm Xun
TS. L Th Hng Ho
CN. Nguyn Thnh Trung
Hiu nh:
PGS. Phm Gia Hu
KS. Phm Thanh Nh


LI GII THIU

Cng vi s pht trin ca khoa hc cng ngh, kinh t - x hi vic
chm sc v bo v sc kho, nng cao cht lng cuc sng ca con ngi
ngy cng c quan tm, trong c vn an ton thc phm. Bo m
ngun thc phm an ton s gp phn bo v v ci thin sc kho, cht
lng cuc sng v ging ni dn tc. Tuy nhin, cng tc bo m an ton
thc phm lun l mt thch thc khng ch vi cc nc ang pht trin
m ngay c vi cc nc pht trin. S a dng ca cc chng loi thc
phm, cng ngh, cc cht ph gia, cc cht h tr ch bin, cc cht
nhim thc phm lun c ci tin, b sung l thch thc i vi h
thng qun l, kim sot an ton thc phm, c bit l i vi cc phng
kim nghim. Do , vic nng cao nng lc h thng phng kim nghim
an ton thc phm ng vai tr c bit quan trng trong cng tc qun l
an ton thc phm ca mi quc gia, nht l trong giai on hi nhp kinh
t ton cu hin nay.
Ti Vit Nam, trong nhng nm gn y cng tc bo m an ton
thc phm c ng v Nh nc c bit quan tm v gt hi c
nhiu thnh tu ng khch l. H thng php lut, qun l c hnh
thnh v kin ton, trong c h thng kim nghim an ton thc phm
m in hnh l s ra i ca Vin Kim nghim an ton v sinh thc phm
quc gia (theo Quyt nh s 376/Q-TTg ngy 23/3/2009) vi chc nng
l n v trng ti quc gia trong kim nghim thc phm. Tuy nhin, do
nhiu yu t v ngun lc u t cha p ng, h thng kim nghim thc
phm ca Vit Nam cn gp rt nhiu kh khn, ngay c cp Trung ng.
Bi v i vi h thng kim nghim thc phm, khng ch cn u t tho
ng v c s vt cht, trang thit b, con ngi m cn phi u t cc h
thng qun l cht lng, k thut (phng php) phn tch ph hp.
Hin nay a s cc phng kim nghim thc phm ca cc a
phng s dng cc phng php th nghim khc nhau, cha c chun
ho hoc khng thng nht nn rt kh nh gi kt qu hoc kt qu khng
bo m tin cy nh hng trc tip n cng tc qun l nh nc.
Nhm tng bc hng ti tiu chun ho cc phng kim nghim v
phng php kim nghim thc phm, Vin Kim nghim an ton v sinh
thc phm quc gia ban hnh cun ti liu Thm nh phng php trong
phn tch ho hc v vi sinh vt nh mt ti liu tham chiu cho cc phng
kim nghim thc phm. Ni dung cun ti liu gm hai phn chnh l thm
nh phng php phn tch ho hc v thm nh phng php phn tch vi
sinh vt vi nhng cch b tr th nghim v cch tnh c th c cc v d
thc tin. Ngoi ra cun ti liu cng gii thiu cch c lng khng
bo m o v cc chng trnh bo m cht lng kt qu th nghim m
cc phng kim nghim c th thc hin c.
Hy vng cun ti liu nh ny s t nhiu hu ch i vi bn c. Do
ln u tin bin son nn cun ti liu s khng trnh khi thiu st, ban
son tho rt mong nhn c s ng gp ca ng nghip xa gn.
Xin trn trng cm n.

TM. Nhm bin son
PGS.TS. Phm Xun
Vin trng
VIN KIM NGHIM AN TON
V SINH THC PHM QUC GIA

LI CM N

Xin by t lng bit n su sc n nhng ngi thy PGS. Phm Gia
Hu v KS. Phm Thanh Nh - nhng ngi trc tip h tr v ng gp
kin sa cha hon thin cun sch.
Xin chn thnh cm n nhng ngi thy, ngi bn, ng nghip
trong hi ng khoa hc thng qua ni dung cun sch PGS.TS. Trn
Chng Huyn, PGS.TS. T Th Tho, Trng i hc Quc gia H Ni;
PGS.TS. Thi Phan Qunh Nh, TS. Trn Vit Hng, Vin Kim nghim
thuc trung ng; TS. Trn ng Ninh, Cc Qun l cht lng Nng lm
sn v Thy sn; ThS. L Vn Giang, Cc An ton v sinh thc phm v
KS. Nguyn Th Vn Lan, Vin Kim nghim an ton v sinh thc phm
quc gia v nhng kin ng gp qu bu trong qu trnh bin son cun
sch.
Xin chn thnh cm n cc bn ng nghip ti Vin Kim nghim an
ton v sinh thc phm quc gia to iu kin h tr xut bn cun
sch.

Nhm bin son
MC LC
MC LC.................................................................................................. 7
DANH MC CH VIT TT................................................................... 9
Chng I: CC YU CU CHUNG........................................................ 10
1. Khi nim v thm nh phng php............................................... 10
2. Thm nh phng php tiu chun (method verification)................. 11
3. Thm nh phng php khng tiu chun (method validation) ........ 13
4. Thm nh li.................................................................................... 14
Chng II: THM NH PHNG PHP PHN TCH HA HC ..... 16
1. Tnh c hiu/chn lc...................................................................... 19
1.1. nh ngha ................................................................................. 19
1.2. Cch xc nh............................................................................. 20
1.3. Tnh c hiu/chn lc i vi phng php chun.................... 24
2. Khong tuyn tnh v ng chun................................................... 25
2.1. nh ngha ................................................................................. 25
2.2. Cch xc nh khong tuyn tnh................................................ 26
2.3. Xy dng ng chun .............................................................. 26
2.4. Cc lu khi xy dng ng chun ......................................... 30
2.5. Gii hn chp nhn ca ng chun ......................................... 31
3. Gii hn pht hin............................................................................. 32
3.1. nh ngha ................................................................................. 32
3.2. Cch xc nh............................................................................. 32
4. Gii hn nh lng.......................................................................... 36
4.1. nh ngha ................................................................................. 36
4.2. Cch xc nh............................................................................. 37
5. chnh xc ( ng v chm).................................................. 39
5.1. chm.................................................................................... 40
5.2. ng (trueness) ..................................................................... 47
6. n nh (hay vng/ chc chn) ca phng php................. 58
6.1. nh ngha ................................................................................. 58
6.2. Cch xc nh............................................................................. 59
Chng III: THM NH PHNG PHP PHN TCH VI SINH VT.....60
1. Cc yu cu chung ............................................................................ 60
1.1. Chun b thm nh .................................................................... 60
1.2. La chn thng s thm nh ..................................................... 62
2. Thm nh phng php tiu chun (method verification)................. 63
2.1. Phng php nh tnh ............................................................... 63
2.2. Phng php nh lng ............................................................ 67
Chng IV: C LNG KHNG M BO O....................... 74
1. Khi nim v khng m bo o .................................................. 74
2. Cc ngun gy ra khng m bo o............................................ 74
3. Cc cch nh gi khng m bo o........................................... 75
3.1. Cch 1: c lng khng m bo o theo hng dn ca
EURACHEM.................................................................................... 75
3.2. Cch 2: c lng khng m bo o t d liu phn tch mu
thc................................................................................................... 83
4. Cng b khng m bo o.......................................................... 85
4.1. Cch vit khng m bo o chun tng hp......................... 86
4.2. Cch vit khng m bo o m rng.................................... 86
Chng V: M BO CHT LNG KT QU TH NGHIM........ 88
1. Php th nghim lp li..................................................................... 89
2. Php th nghim ti lp..................................................................... 89
3. Php th nghim trn mu lu........................................................... 89
4. Php th nghim trn mu trng........................................................ 89
5. Php th nghim trn mu chun ...................................................... 90
6. Php th nghim trn mu thm........................................................ 90
7. S dng cc phng php khc nhau ................................................ 91
8. nh gi s ph hp ca h thng..................................................... 91
9. Tham gia cc chng trnh th nghim lin phng............................ 92
10. S dng biu kim sot .............................................................. 93
TI LIU THAM KHO....................................................................... 101
PH LC

DANH MC CH VIT TT
AOAC (Association of Official
Analytical Chemists)
Hip hi cc nh ha phn tch
chnh thc
ASTM (American Society for Testing
of Materials)
Hip hi php th M
CRM (Certified reference material) Mu chun c chng nhn
DAD (Diod array detector) Detector mng diod
GC-MS (Gas chromatography mass
spectrometry)
Sc k kh khi ph
HPLC (High performance liquid
chromatography)
Sc k lng hiu nng cao
HR (High resolution) phn gii cao
ICH (International Conference on
Harmonization)
Hi ng ha hp quc t
IP (Identification point) im nhn dng
ISO (International Standard
Organization)
T chc tiu chun quc t
LC-MS (Liquid chromatography
mass spectrometry)
Sc k lng khi ph
LOD (Limit of Detection) Gii hn pht hin
LOQ (Limit of Quantification) Gii hn nh lng
LR (Low resolution) phn gii thp
MRLs (Maximum residue Limits) Gii hn tn d ti a
QC (Quality control) Kim tra cht lng
S/N (Signal to noise ratio) T l tn hiu trn nhiu
SOP (Standard Operation
Procedure)
Quy trnh thao tc chun
TCVN Tiu chun Vit Nam
USFDA (United States Food and
Drug Administration)
Cc Dc phm v Thc phm
M
USP (United States Pharmacopoeia) Dc in M
10 CHNG I: CC YU CU CHUNG


Chng I: CC YU CU CHUNG
1. Khi nim v thm nh phng php
Thm nh phng php l s khng nh bng vic kim tra v cung
cp bng chng khch quan chng minh rng phng php p ng
c cc yu cu t ra (fitness for the purpose). Kt qu ca thm nh
phng php c th c s dng nh gi cht lng, tin cy ca kt
qu phn tch. Thm nh phng php phn tch l mt phn khng
th thiu nu mun c mt kt qu phn tch ng tin cy.
Hin nay nhiu thut ng khc nhau c s dng ch khi nim
trn, nh nh tr phng php, nh gi phng php, xc nhn gi tr s
dng ca phng php, ph duyt phng php. Tt c cc thut ng ny
u l cch gi khc nhau ca thm nh phng php (method validation).
Phng th nghim thng s dng nhiu phng php khc nhau.
Da vo ngun gc c th phn loi cc phng php thnh hai nhm:
- Cc phng php tiu chun: cc phng php th theo tiu chun
quc gia, quc t, hip hi khoa hc c chp nhn rng ri trn th gii
nh TCVN, ISO, ASTM, AOAC
- Cc phng php khng tiu chun hay phng php ni b (non-
standard/alternative/in-house method): l cc phng php do phng th
nghim t xy dng, phng php theo hng dn ca nh sn xut thit b,
phng php theo cc tp ch, ti liu chuyn ngnh...
Theo yu cu ca ISO 17025, phng php phn tch phi c thm
nh hoc thm nh li khi:
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 11
- Phng php p dng khng phi l phng php tiu chun (non-
standard method)
- Phng php do phng th nghim t xy dng mi trc khi a
vo s dng thnh thng quy.
- C s thay i v i tng p dng nm ngoi i tng p dng
ca phng php thm nh hoc phng php tiu chun.
- C s thay i cc iu kin thc hin phng php c thm
nh (v d: thit b phn tch vi cc c tnh khc bit, nn mu, ngi
phn tch ).
2. Thm nh phng php tiu chun (method verification)
Trc khi p dng mt phng php phn tch cn c cc chng minh
rng phng php p ng yu cu t ra, tc l phng php phi c
thm nh. Yu cu ny khng ch cho cc phng php th ni b m cn
cn cho cc phng php tiu chun. Vic thm nh phng php tiu
chun v phng php ni b c s khc nhau, do cn ch khi lp k
hoch thm nh.
C hai yu cu ch yu ca vic thm nh phng php tiu chun:
- Phi c kt qu thm nh ca phng php tiu chun, v kt qu
ny phi ph hp vi yu cu ca phng th nghim.
- Phng th nghim cn m bo c th t c cc thng s c
m t trong phng php tiu chun.
Theo yu cu ca ISO 17025, khi cc phng th nghim p dng cc
phng php tiu chun cn c h s nh gi cc iu kin c bn, cc
ngun lc theo yu cu ca phng php th v vic t c kt qu th
nghim c chnh xc nh phng php yu cu hoc nh mong mun
12 CHNG I: CC YU CU CHUNG

ca phng th nghim. i vi cc phng php th ban hnh m khng
c d liu v chnh xc th phng th nghim phi xc nh d liu
chnh xc ca php th da trn d liu nghin cu th nghim.
xy dng cc bc thm nh phng php tiu chun cn kim
tra:
1. Phng php c thm nh hay cha, thm nh ton b hay
mt phn?
2. Nn mu c ging nhau hay khng?
3. Khong lm vic ca phng php c ph hp vi yu cu ca
phng th nghim hay khng?
4. C cng loi thit b (hng sn xut, model) hay khng? Phng
php tiu chun c cho s dng cc loi thit b khc khng?
5. C nhng lu g c bit ca phng php tiu chun m phng
th nghim khng th p ng khng?
Nu mt trong cc yu t trn khng ph hp, th phng th nghim
cn thc hin cc php th nh gi li phng php. Cc kt qu nh
gi ny cn phi tng ng vi cc kt qu thm nh ca phng php
chun, nu khng cn phi thm nh li ton b phng php.
Vic nh gi bao gm:
1. Khng nh tnh trng y thit b, nhn vin, thuc th, mi
trng v cc iu kin khc thc hin php th.
2. Kim tra cc thng s c bn nht ca phng php, theo yu cu c
th ca tng lnh vc ha hc v vi sinh s c m t chi tit trong
cc chng sau.
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 13
3. Thm nh phng php khng tiu chun (method
validation)
i vi phng php khng tiu chun, vic thm nh phi tri qua
nhiu bc hn, bt u t qu trnh nghin cu kho st phng php, ti
u ha phng php n khi hon thin phng php. Thm nh phng
php l mt yu cu bt buc phi thc hin i km vi vic pht trin
phng php mi v p dng cc phng php khng tiu chun vo thc
hin thnh thng quy. Cc bc tin hnh thm nh bao gm:
1. Xy dng SOP d kin (theo cc ti liu tham kho hoc theo cc
nghin cu xy dng phng php mi)
2. Xy dng cng (k hoch) thm nh bao gm:
a. Xc nh thi gian v ngi thc hin
b. Cht cn phn tch: tn cht, d on hm lng trong mu
c. Xc nh i tng thm nh: nn mu
d. Xc nh mc ch cn phi t: yu cu v gii hn cho
php (nu c), cn t LOD, LOQ, chnh xc... bao nhiu?
e. Xc nh cc thng s cn thm nh v khong chp nhn
f. Xc nh cc th nghim cn thc hin
3. Kim tra cc iu kin cn cho cng vic thm nh
a. Cc yu cu v trang thit b
b. Ha cht, thuc th
c. Mu th nghim
4. Thc hin thm nh
14 CHNG I: CC YU CU CHUNG

a. Cc php th thm nh s b
b. Thay i cc thng s ca phng php (nu cn)
c. Thc hin thm nh hon thin
5. Hon thin SOP ca phng php
6. Bo co thm nh: cn c cc thng tin sau
a. Tn ngi thm nh, thi gian thm nh
b. Tm tt phng php: nguyn l, thit b, ha cht, quy trnh,
cc lu ...
c. Cc kt qu thm nh
d. Cc yu cu cn p ng a phng php vo thc hin
thng xuyn (routine): kim tra tnh tng thch ca h
thng, mu QC, c lng khng m bo o ca kt
qu...
e. Xc nh cc thng s v thi gian cn thm nh li
f. Ti liu tham kho (nu c)
g. Kt lun v xut (nu c)
h. K tn, ngy ca ngi lm bo co
i. K ph duyt ca ngi c thm quyn
4. Thm nh li
Cng vic thm nh khng phi ch thc hin mt ln khi pht trin
phng php ban u m cn thc hin trong sut qu trnh p dng. V a
s cc iu kin thc hin phng php c s thay i trong sut qu trnh
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 15
p dng. V d nh c s thay i hoc m rng i tng p dng, thay i
a im phng th nghim, thay i nhn vin, thay i thit b (p dng
trn cc thit b cng loi khc), thay i cc iu kin v tin nghi mi
trng, thay i v dung mi ha cht thuc th, nhng thay i nh khc
(v d nhit ct phn tch, pH pha ng...). Trong trng hp kt qu
phn tch mu kim tra (QC) hoc kt qu nh gi s ph hp ca h thng
nm ngoi gii hn cho php th phng php cng cn phi thm nh li.
Phng th nghim nn phi hp qu trnh tnh n nh ( vng) vi qu
trnh thm nh li cc phng php phn tch hng ngy (routine method).
Cc thng s cn thm nh li ph thuc vo mc nh hng ca
cc thay i n cc thng s ca phng php. Thng thng tin hnh
thm nh cc thng s c bn nht nh trong trng hp thm nh cc
phng php tiu chun. Tuy nhin nu nhng kt qu thm nh ny c s
sai khc nhiu so vi kt qu thm nh ban u th cng cn thc hin thm
nh li ton b.
Cc chng sau s gii thiu chi tit cc khi nim, cc cch b tr th
nghim thm nh phng php phn tch ha hc v vi sinh, cng nh
cc cch nhm m bo cht lng kt qu th nghim.
16 CHNG II: THM NH PHNG PHP PHN TCH HA HC


Chng II: THM NH PHNG PHP
PHN TCH HA HC
Nh nu trong chng I, thm nh phng php l mt cng vic
rt kh khn, nhm chn, v tn km tuy nhin li l mt ni dung quan
trng nh hng n chnh xc ca kt qu phn tch. Cn cn nhc mc
ch yu cu ca tng phng php v ngun lc la chn thng s thm
nh cho ph hp.
Theo cc quy nh ca USFDA, AOAC, USP v ICH, i vi cc
phng php phn tch ha hc cc thng s cn thm nh bao gm:
- Tnh c hiu, tnh chn lc; (Specifility/Selectivity)
- Khong tuyn tnh v ng chun; (Linearity and Calibration
curve)
- Gii hn pht hin; (Limit of Detection LOD)
- Gii hn nh lng; (Limit of Quantitation LOQ)
- ng; (Trueness)
- chm; (Precision)
- vng (n nh) ca phng php; (Robustness/Ruggedness)
Vic la chn cc thng s thm nh ty thuc vo k thut p dng
trong phng php, yu cu ca phng php, iu kin v ngun lc ca
phng th nghim... Tng trng hp c th cc thng s thm nh c th
c s khc nhau.
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 17
Mi phng php phn tch ha hc c mt mc ch nht nh, t
vic xc nh mt cht nng thp n xc nh tinh khit ca mt
cht. d dng cho vic la chn cc thng s thm nh, cc phng
php c th c chia lm bn nhm ty thuc vo mc ch ca chng:
- Cc phng php phn tch nh tnh;
- Cc phng php phn tch vi lng;
- Cc phng php phn tch a lng;
- Xc nh gii hn tp cht (th tinh khit).
Cc yu cu v thng s cn thm nh cho tng loi phng php
c m t bng 1 (phng php khng tiu chun) v bng 2 (phng
php tiu chun). Ngi thc hin thm nh cn la chn ng cch thm
nh cho tng thng s v lp k hoch cho tng ni dung.
Bng 1: La chn thng s thm nh phng php khng tiu chun
Cc thng s thm nh
Phn tch
nh tnh
Phn tch
vi lng
Phn tch
a lng
Xc nh gii
hn tp cht
ng
(Trueness)
+ +
chm
(Precision)
+ +
c hiu, chn lc
(Specification/Selectivity)
+ + + +
LOD + + +/- +
18 CHNG II: THM NH PHNG PHP PHN TCH HA HC

(Limit of detection)
LOQ
(Limit of quantitation)
+ +/-
tuyn tnh
(Linearity/range)
+ +
vng (n nh)
(Ruggedness/Robustness)
+ +
(+) Cn thc hin thm nh (-) Khng cn thc hin thm nh
Bng 2: La chn thng s thm nh phng php tiu chun
Cc thng s nh gi
Phn tch
nh tnh
Phn tch
vi lng
Phn tch
a lng
Xc nh
gii hn tp
cht
ng
(Trueness)
+ +
chm
(Precision)
+ +
c hiu, chn lc
(Specification/Selectivity)

LOD
(Limit of detection)
+ +
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 19
LOQ
(Limit of quantitation)
+
tuyn tnh
(Linearity/range)

vng (n nh)
(Ruggedness/Robustness)

(+) Cn thc hin thm nh (-) Khng cn thc hin thm nh
1. Tnh c hiu/chn lc
1.1. nh ngha
* Tnh c hiu: L kh nng pht hin c cht phn tch khi c mt
cc tp cht khc nh cc tin cht, cc cht chuyn ha, cc cht tng t,
tp cht.... C th, trong php phn tch nh tnh l phi chng minh
c kt qu l dng tnh khi c mt cht phn tch, m tnh khi khng c
mt n, ng thi kt qu phi l m tnh khi c mt cc cht khc c cu
trc gn ging cht phn tch. Trong php phn tch nh lng, l kh nng
xc nh chnh xc cht phn tch trong mu khi b nh hng ca tt c cc
yu t khc, nhm hng n kt qu chnh xc.
Tnh c hiu thng lin quan n vic xc nh ch mt cht phn
tch.
* Tnh chn lc: L khi nim rng hn tnh c hiu, lin quan n
vic phn tch mt s hoc nhiu cht chung mt quy trnh. Nu cht cn
xc nh phn bit r vi cc cht khc th phng php phn tch c tnh
chn lc.
Nh vy, tnh chn lc c th bao trm c tnh c hiu. Do cc
phng php phn tch thng c nhiu cht cng xut hin nn khi nim
tnh chn lc thng mang tnh khi qut hn.
20 CHNG II: THM NH PHNG PHP PHN TCH HA HC

1.2. Cch xc nh
1.2.1. Trng hp chung
xc nh tnh c hiu/chn lc ca phng php nh tnh, nh
lng cn b tr cc th nghim nh sau:
- Phn tch cc mu trng, lp li ti thiu 6 ln i vi tng loi nn
mu. Mu trng phi khng c cho tn hiu phn tch. Nu mu
trng c hn 10% dng tnh hoc xut hin tn hiu th cn phi
thay i phng php loi tr cc nh hng.
- Phn tch mu th hoc mu trng thm chun hm lng gn
LOQ, lp li ti thiu 6 ln. So snh kt qu vi mu trng, phi cho
tn hiu cht cn phn tch.
- S dng phng php thm chun sau chun b mu (co-
chromatography), cch ny thng p dng i vi cc phng php
sc k. Sau khi chun b mu (mu trng hoc mu thc) v phn
tch mu trn thit b sc k thu c cc pic sc k, ta thm chun
vo mu chit xut v phn tch mu ny. So snh sc k ca
hai mu nh gi tnh c hiu/chn lc.
- Phn tch mu khng c cht phn tch nhng c cht cu trc tng
t cht phn tch (nu c): Phi cho kt qu m tnh (i vi phng
php nh tnh) v khng c nh hng n kt qu nh lng
ca cht phn tch (i vi phng php nh lng).
Trong trng hp nhng ch tiu phn tch khng th c mu trng
(sample blank) xc nh tnh chn lc/c hiu, c th thc hin cc th
nghim trn cc mu trng thuc th (reagent blank), tc l thc hin phn
tch cc bc tng t nh khi phn tch mu nhng khng c mu th.
1.2.2. Cc trng hp c bit
Sc k lng s dng detector DAD (mng diod)
Tnh chn lc trong sc k lng c th t c thng qua vic la
chn ct ti u, iu kin sc k ti u, nhit ct v bc sng. Ngoi
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 21
vic thay i iu kin sc k, th giai on x l mu cng phi ti u
t c tnh chn lc cao nht. Trong sc k, cn xc nh rng pc sc k
c phi l mt pc n hay c ln cc tp cht khc. i vi cc h thng
sc k c detector mng diod (DAD) c th xc nh c tnh chn lc
thng qua xc nh tinh khit (peak purity) ca pc, hay so snh ph ca
pc vi th vin ph sn c.

Hnh 1: Xc nh tinh khit ca pic trong sc k lng HPLC-DAD
tinh khit ca pc c xc nh bng cch so snh ph ti cc
im khc nhau trn pic sc k. Cch ph bin nht l chn ti ba im:
nh pc (apex), v hai im v hai bn sn ca pc (upslope v
downslope). Thng thng chn hai im ti 2/3 rng ca pc v hai
pha. Ngoi ra hin nay a s cc thit b u c phn mm tnh ton tinh
khit ca pc, c th da vo 3 im, 5 im, 7 im hay tt c cc im to
thnh pc. Cc thit b hin i c th tnh ton tinh khit trc tip thng
qua phn mm iu khin.
22 CHNG II: THM NH PHNG PHP PHN TCH HA HC


Hnh 2: Cc cch xc nh tinh khit ca pic sc k HPLC-DAD
So snh ph ca pic vi ph chun cng l mt cch ph bin xc
nh s tinh khit ca pc. Mt pc c xem l khng tinh khit khi gi tr
ph hp (h s match) khng t xp x 100%. Tuy nhin, nu gi tr ny c
gn 100% cng khng th khng nh c chc chn s tinh khit ca pc,
c th do mt trong s cc nguyn nhn sau:
- Tp cht tn ti vi lng thp hn nhiu so vi cht phn tch do
ph UV-Vis khng nh hng nhiu n ph ca cht phn tch.
- Ph ca tp cht v cht phn tch tng t nhau.
Sc k khi ph
Cc thit b sc k kh c gn detector MS, thng so snh ph ca
cht phn tch vi ph chun c sn trong th vin i km hoc ph ca cht
chun tng ng.
S dng cc phng php xc nhn (confirmation method) l mt
cch rt tt m bo tnh c hiu ca phng php. Hi ng chu u
quy nh cch tnh im IP (im nhn dng identification point) i vi
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 23
cc phng php khc nhau khng nh chc chn s c mt ca mt
cht. Cch tnh im IP i vi cc k thut khi ph khc nhau c quy
nh nh sau:
Bng 3: Quan h gia cc k thut khi ph v s im IP t c
K thut khi ph S im IP t c vi 1 ion
MS phn gii thp (LR-MS) 1,0
LR-MS (ion m) 1,0
LR-MS (ion con) 1,5
MS phn gii cao (HR-MS) 2,0
HR-MS (ion m) 2,0
HR-MS (ion con) 2,5
Bng 4: V d v s im IP t c i vi cc k thut khi ph khc nhau
K thut S ion S im IP
GCMS (EI hoc CI) n n
GCMS (EI v CI) 2 (EI) + 2 (CI) 4
GCMS (EI hoc CI) hai dn
xut
2 (dc A) + 2 (dc B) 4
LC-MS n n
GC-MS-MS 1 ion m, 2 ion con 4
LC-MS-MS 1 ion m, 2 ion con 4
GC-MS-MS 2 ion m, mi ion m c 1 ion con 5
LC-MS-MS 2 ion m, mi ion m c 1 ion con 5
LC-MS-MS-MS 1 ion m, 1 ion con v 2 ion chu 5,5
24 CHNG II: THM NH PHNG PHP PHN TCH HA HC

HRMS n 2n
GC-MS v LC-MS 2+2 4
GC-MS v HRMS 2+1 4
Cc thit b sc k kh c gn detector MS, thng so snh ph ca
cht phn tch vi ph chun c sn trong th vin i km hoc ph ca cht
chun tng ng.

V d: Phng php phn tch chloramphenicol bng k thut sc k lng
khi ph hai ln, ngi ta thc hin bn ph ion m m/z 321 v nh lng
theo ion con to thnh l 152 v 194. Theo cch tnh im IP th vi 1 ion
m v 2 ion con thu c 4 im IP, nh vy phng php c tnh c
hiu p ng c yu cu.
Cn thc hin thm cc php th trn mu trng (n 6) v mu c nng
gn LOQ (n 6) xc nh chc chn phng php c tnh c hiu
cao.
1.3. Tnh c hiu/chn lc i vi phng php chun
i vi cc phng php chun, nh nu trong bng 2, thng
thng khng cn xc nh tnh c hiu/chn lc. Tuy nhin, cn cn nhc
thc hin vic xc nh c hiu trong cc trng hp sau:
- Nn mu phn tch ti phng th nghim khc vi nn mu vi mu
nu trong phng php tiu chun. Trong trng hp ny cn thc
hin y nh khi thm nh phng php ni b.
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 25
- C s khc nhau v thit b phn tch m s khc nhau ny c th
nh hng n tnh chn lc. C th thc hin y hoc xc nh
nh hng nu c thng qua xc nh hiu nng ca thit b.
2. Khong tuyn tnh v ng chun
2.1. nh ngha
Khong tuyn tnh ca mt phng php phn tch l khong nng
c s ph thuc tuyn tnh gia i lng o c v nng cht
phn tch.
Khong lm vic ca mt phng php phn tch l khong nng
gia gii hn trn v gii hn di ca cht phn tch (bao gm c cc gii
hn ny), ti c chng minh l c th xc nh c bi phng php
nht nh vi ng, chnh xc v tuyn tnh nh nu.
n gin hn, hai khi nim ny c m t trong hnh 3 di y:

Hnh 3: Khong tuyn tnh (linear range) v khong lm vic
(working range)
26 CHNG II: THM NH PHNG PHP PHN TCH HA HC

2.2. Cch xc nh khong tuyn tnh
i vi hu ht cc phng php nh lng, cn phi thc hin vic
xc nh khong tuyn tnh. Vic xc nh khong tuyn tnh thng c
kho st bt u t gii hn nh lng (im thp nht) v kt thc l gii
hn tuyn tnh (im cao nht). Ni chung, xc nh khong tuyn tnh
cn khong 10 (ti thiu l 6) nng khc nhau.
xc nh khong tuyn tnh cn thc hin o cc dung dch chun
c nng thay i v kho st s ph thuc ca tn hiu vo nng . V
ng cong ph thuc gia tn hiu o v nng , sau quan st s ph
thuc cho n khi khng cn tuyn tnh. Khong tuyn tnh di hay ngn
ph thuc vo nhiu yu t, trong quan trng nht l bn cht ca cht
phn tch v k thut s dng. Cc cht khc nhau c khong tuyn tnh
khc nhau do s khc nhau v tnh cht l ha. Trong khi cc k thut s
dng khc nhau nh hng ln n di ngn ca khong tuyn tnh. V
d, k thut HPLC nu s dng detector UV-Vis hoc DAD c th cho
khong tuyn tnh n 10
6
thm ch n 10
7
, trong khi nu s dng detector
hunh quang hay ch s khc x th ch cho khong tuyn tnh khong 10
4

10
5
.
2.3. Xy dng ng chun
Sau khi xc nh khong tuyn tnh cn xy dng ng chun v xc
nh h s hi quy tng quan. Trong phn tch thc t, c th xy dng cc
ng chun ngn, trm ln vng nng trong mu, khng nht thit phi
lp ng chun ton b khong tuyn tnh. Nng trong mu khng c
vt ra ngoi gii hn cao nht v thp nht ca ng chun v tt nht
phi nm vng gia ng chun.
C nhiu loi ng chun khc nhau ty thuc vo cc phng php
v k thut khc nhau, sau y l cc loi ng chun ch yu:
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 27
2.3.1. ng chun vi chun tinh khit
Chun b dy nng chun (ti thiu 6 nng ). Xc nh cc gi tr
o c y theo nng x (lp li 2 ln ly gi tr trung bnh). Nu s ph
thuc tuyn tnh, ta c khong kho st ng biu din l mt phng
trnh:
y = ax + b
Trong : a: gi tr dc slope;
b: gi tr h s chn intercept
v h s tng quan:

) Y y ( ) X x (
) Y y )( X x (
R
i
2
i
i i

Nu 0,995 < R 1: C tng quan tuyn tnh r rt.
C th kho st tuyn tnh da vo tnh h s p ng ( th
di). Khong tuyn tnh nm trong khong th nm ngang.

Hnh 4: Cc cch lp ng chun tuyn tnh
28 CHNG II: THM NH PHNG PHP PHN TCH HA HC

2.3.2. ng chun trn mu trng
Phn tch cc mu trng thm chun vi nng khc nhau (t nht 6
nng ), trong khong tuyn tnh c lng trn, mi nng lm 3 ln.
V ng cong ph thuc gia tn hiu o (trc tung y) ph thuc vo nng
(trc honh x). Tnh cc h s hi quy (a, b trong phng trnh hi quy y
= ax + b) v h s tng quan (R) tng t nh trn.
ng chun xy dng trn nn mu trng thng cho tin cy cao
hn khi xy dng vi chun tinh khit, do c th loi tr phn no cc nh
hng ca nn mu. Tuy nhin, trong nhiu trng hp kh tm c mu
trng ph hp v khng c c cht phn tch. Do , c th s dng
phng php lp ng chun trn nn mu thc nh sau.
2.3.3. ng chun trn mu thc
Phn tch mu thc c cho thm cc nng chun khc nhau tng
t nh trong phn lm vi mu trng. V ng cong tn hiu o (trc tung
y) ph thuc vo nng chun thm (trc honh x). Dng ng chun
trn nn mu thc thng c dng nh hnh 5:

Hnh 5: ng chun trn nn mu thc
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 29
Khi s dng ng chun trn nn mu thc c th loi tr c cc
nh hng ca nn mu n kt qu phn tch. Sau khi lp c phng
trnh ng chun y = ax + b, c th d dng tnh c nng : X = b/a
2.3.4. ng chun c s dng ni chun
Mt phng php rt hu ch trong phn tch, c bit trong phn tch
hin i l s dng ni chun. Ni chun c thm vo dung dch chun
o my, vi nng ph hp v ging nhau (C
IS
). V ng cong ph
thuc gia t l tn hiu cht ngoi chun chia cho ni chun (trc tung y)
ph thuc vo nng ngoi chun (trc honh x). Tnh cc h s hi quy
(a, b trong phng trnh hi quy y = ax + b) v h s tng quan (R) tng
t nh trn.

Hnh 6: V d ng chun s dng cht ni chun
Khi phn tch mu, ni chun cng phi c thm vo (tt nht l t
u, sau khi cn, ong) sao cho to c nng cui cng bng nng
ni chun trong cc dung dch chun. Vi cch tin hnh nh th ny c
th hn ch c hu ht cc nh hng trong qu trnh phn tch (bao gm
30 CHNG II: THM NH PHNG PHP PHN TCH HA HC

t cn mu, chun b mu n phn tch trn thit b) n kt qu phn tch.
i vi cc k thut phn tch hin i nh khi ph, c bit l sc k lng
khi ph, vic s dng ni chun l mt yu cu tin quyt, nu khng
mun ni l bt buc. Thng thng trong sc k lng khi ph, cc cht
ni chun c u tin la chn l cc cht ng v, thng thng nh
2
H
(D),
13
C,
15
N... V d, khi phn tch chloramphenicol th ngoi cht ngoi
chun l chloramphenicol, s dng thm cht ni chun l chloramphenicol-
d5; clenbuterol th s dng clenbuterol-d9, melamin th s dng melamin-
13
C
15
N... Cc cht ng v ny c u im ni bt l tnh cht ha l gn
nh tng t cht phn tch do i din tt cho cht phn tch trong c
qu trnh. Ngoi ra, trong mt s trng hp c th chn cc cht ni chun
khc, vi iu kin l cc cht ny phi c mt s tnh cht c bn ging
cht phn tch, v c th phn tch c bng phng php ang thc hin.
2.4. Cc lu khi xy dng ng chun
- Cn m bo nng chun chnh xc: ng chun l yu t sng
cn, quyt nh s ng n ca kt qu phn tch, do nu trong qu trnh
xy dng ng chun mc nhng sai s ln s dn n s mt chnh xc
ca kt qu. iu u tin kim sot c s chnh xc ca cc nng
chun khi xy dng ng chun l cn m bo chnh xc ca cht
chun (cht chun mua t nh sn xut) v hm lng, tinh khit. Theo
yu cu ca ISO 17025, cc cht chun khi s dng cn c chng nhn ca
nh sn xut v vn cn hn s dng. Trong mt s trng hp, c th b tr
cc th nghim nh gi cht lng cc cht chun trc khi a vo s
dng.
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 31
- Tn hiu cc ln o ca mi nng phi c lp li t yu cu:
Khi thm nh phng php, mi nng cn c o vi ln (ba ln)
kim tra lp ca cc nng chun.
- Loi tr sai s th nu cn thit: Mt s trng hp, c th gp cc
sai s th (ngu nhin) xut hin dn n vic ng chun khng p ng
yu cu. Trong trng hp ny, c th cn nhc loi tr cc im ny
m bo s chnh xc.
2.5. Gii hn chp nhn ca ng chun
- H s hi quy tuyn tnh (R): Ch tiu u tin ca mt ng chun
t yu cu l h s tng quan hi quy (Coefficient of correlation). R phi
t theo yu cu sau:
0,995 R 1
Hay 0,99 R
2
1
- chch cc im nng dng xy dng ng chun. Sau khi
lp ng chun xong cn kim tra bng phng php tnh ngc li nng
ca cc im chun s dng xy dng ng chun, t tnh cc gi
tr chch theo cng thc sau:
100
C
C C
c
c t
i


Trong :
i
: chch ca tng im chun dng xy dng ng chun

t
C : Nng tnh ngc theo ng chun ca cc im chun

c
C : Nng ca cc im chun
Theo quy nh ca nhiu t chc ca M, Canada, chu u, gi tr
khng c vt qu t 15% cho tt c cc nng , ring nng LOQ
c th chp nhn gii hn t 20%.
32 CHNG II: THM NH PHNG PHP PHN TCH HA HC

3. Gii hn pht hin
3.1. nh ngha
Gii hn pht hin l nng m ti gi tr xc nh c ln hn
khng m bo o ca phng php. y l nng thp nht ca cht
phn tch trong mu c th pht hin c nhng cha th nh lng c
(i vi phng php nh lng).
3.2. Cch xc nh
3.2.1. LOD ca phng php nh tnh
Cn xc nh c nng no m ti s xc nh chc chn s c
mt ca cht phn tch.
Phn tch cc mu trng thm chun cc nng nh khc nhau,
mi nng phn tch lp li 10 ln. Xc nh t l phn trm s ln pht
hin (dng tnh) hoc khng pht hin (m tnh).
V d:
Bng 5: V d cch tnh LOD ca phng php nh tnh
Nng
(ppm)
S ln
th
T l dng tnh/m tnh T l %
200 10 10/0 100
100 10 10/0 100
75 10 5/5 50
50 10 1/9 10
25 10 0/10 0

THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 33
Trong v d trn, vi nng di 100 ppm kt lun dng tnh
khng cn chc chn 100%, gii hn pht hin trong trng hp ny l 100
ppm.
3.2.2. LOD ca phng php nh lng
C nhiu cch xc nh LOD khc nhau ty thuc vo phng php
p dng l phng php cng c hay khng cng c. Cc cch tip cn c
th chp nhn c bao gm:
Da trn lch chun
Cch 1. Lm trn mu trng (mu trng c thnh phn nh mu th nhng
khng c cht phn tch).
Phn tch mu 10 ln song song, tnh lch chun. lch chun
ny phi khc 0.
Tnh LOD: LOD =
0 0
SD 3 x +


Vi SD
0
=
1 n
) x x (
2
0
i 0


Trong :

0
x = trung bnh ca mu trng
SD
0
= lch chun ca mu trng

Cch 2. Lm trn mu th: Lm 10 ln song song. Nn chn mu th c
nng thp (v d, trong khong 5 n 7 ln LOD c lng).
Tnh LOD: Tnh gi tr trung bnh

x , v lch chun SD
34 CHNG II: THM NH PHNG PHP PHN TCH HA HC

LOD = 3 x SD
1 n
) x x (
SD
2
i



nh gi LOD tnh c: tnh R =

x / LOD
Nu 4 < R < 10 th nng dung dch th l ph hp v
LOD tnh c l ng tin cy
Nu R < 4 th phi dng dung dch th m c hn, hoc
thm mt t cht chun vo dung dch th dng v lm
li th nghim v tnh li R
Nu R > 10 th phi dng dung dch th long hn, hoc
pha long dung th dng v lm li th nghim v tnh li
R
Da trn t l tn hiu trn nhiu (S/N)
Cch ny ch p dng i vi cc quy trnh phn tch s dng cc cng
c c nhiu ng nn. Thng thng cch tnh ny p dng ph bin cho
cc phng php sc k, in di.
Phn tch mu (mu thc, mu thm chun hoc mu chun) nng
thp cn c th xut hin tn hiu ca cht phn tch. S ln phn tch lp
li 4 ln. Xc nh t l tn hiu chia cho nhiu (S/N = Signal to noise
ratio),
trong S l chiu cao tn hiu ca cht phn tch,
N l nhiu ng nn
Nhiu ng nn c tnh v hai pha ca ng nn v tt nht l
tnh nhiu ln cn hai bn ca pc, b rng mi bn ti thiu gp 10 ln
chiu rng ca pc ti na chiu cao.
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 35
LOD c chp nhn ti nng m ti tn hiu ln gp 2-3 ln
nhiu ng nn, thng thng thng ly S/N =3

Hnh 7: Xc nh LOD bng cch tnh S/N
Da trn ng chun (tham kho)
Ch p dng c cho cc phng php c xy dng ng chun.
LOD c th c xc nh da vo dc ca ng chun v
lch chun ca tn hiu o.
a
SD 3 , 3
LOD


Trong : SD: lch chun ca tn hiu
a: dc ca ng chun
Gi tr a c th d dng tnh c t ng chun, gi tr SD c th
c tnh theo nhiu cch khc nhau, bao gm:
- Da trn lch chun ca mu trng: Phn tch mu trng lp li
10 ln v tnh SD tng ng;
- Da trn lch chun ca mu thm chun nng nh gn
LOD, lp li 10 ln (xem phn kim tra pha trn) v tnh SD;
- Da trn h s chn ca ng chun, lm nhiu ln tnh SD ca
gi tr b (intercept);
36 CHNG II: THM NH PHNG PHP PHN TCH HA HC

- Da trn lch chun ca khong cch cc gi tr o thc vi
ng chun.
4. Gii hn nh lng
4.1. nh ngha
LOQ l nng ti thiu ca mt cht c trong mu th m ta c th
nh lng bng phng php kho st v cho kt qu c chm mong
mun.
LOQ ch p dng cho cc phng php nh lng.
Ging nh LOD, c nhiu cch khc nhau xc nh LOQ, ph
thuc vo tng phng php c th m la chn cho ph hp.
Vic xc nh LOQ cn tnh n cc yu t nh hng trong mu phn
tch, do cn thc hin trn nn mu tht.
LOQ trong nhiu trng hp c th l im thp nht ca khong
tuyn tnh. Hnh 8 m t mi quan h gia LOD, LOQ v khong tuyn
tnh.

Hnh 8: Mi quan h gia LOD, LOQ v khong tuyn tnh
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 37
4.2. Cch xc nh
Vic b tr th nghim xc nh LOQ thng kt hp vi tnh LOD.
C nhiu cch khc nhau tnh LOQ nh sau:
Da trn lch chun:
C hai trng hp nh trong phn tnh LOD l thc hin trn mu
trng v thc hin trn mu th. Cc cng thc tnh ton nh sau:
Tnh trn mu trng: LOQ =
0 0
SD 10 x +


Tnh trn mu th: LOQ = 10 SD

V d: xc nh gii hn pht hin v gii hn nh lng ca phng
php phn tch metanol trong ru, thc hin phn tch mu trng (mu c
hm lng metanol rt thp, gn vi gii hn di ca ng chun),
thc hin phn tch 10 ln lp li v tnh gi tr trung bnh v lch chun,
thu c cc kt qu nh sau:
Phng trnh ng chun: y = 13,227 x + 0,004
Ln
Abs ti
575nm
HL metanol
(mg/l)
Trung bnh
(mg/l)
SD (mg/l)
1 0,021 12,85
2 0,024 15,12
3 0,022 13,61
4 0,024 15,12
13,61 1,75
38 CHNG II: THM NH PHNG PHP PHN TCH HA HC

5 0,019 11,34
6 0,025 15,88
7 0,018 10,58
8 0,024 15,12
9 0,022 13,61
10 0,021 12,85
Nh vy, LOD = 13,61 + 3x1,75 = 18,86 mg/l
LOQ = 13,61 + 10x1,75 = 31,11 mg/l
Da trn t l tn hiu trn nhiu:
Cch ny ch p dng i vi cc quy trnh phn tch s dng cc cng
c c nhiu ng nn. Cch tnh ton hon ton tng t nh trong phn
tnh LOD.
LOQ c chp nhn ti nng m ti tn hiu ln gp 10-20 ln
nhiu ng nn, thng thng thng ly S/N = 10.


V d: Xc nh LOD ca phng php phn tch axit benzoic trong nc
gii kht bng HPLC, ngi ta thc hin phn tch cc mu trng c thm
chun axit benzoic cc nng thp di gii hn thp nht ca ng
chun. Tnh chiu cao ca pic sc k (H l tn hiu S) v chiu cao ca
nhiu ng nn (h = 2N) v hai pha ca pc. Thu c cc kt qu nh
sau:
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 39
Nng
(mg/kg)
Ln
Chiu cao pic
(H = S)
Chiu cao nhiu
(h = 2N)
S/N
1 1765 1231 2,87
2 1723 1429 2,41
3 1598 1364 2,34
0,01
4 1628 1151 2,83
1 3021 1351 4,47
2 3109 1491 4,17
3 2919 1502 3,89
0,02
4 2934 1385 4,24
1 6531 1421 9,19
2 6691 1323 10,1
3 6567 1299 10,1
0,05
4 6519 1313 9,93
Nh vy LOD = 0,02 mg/kg
LOQ = 0,05 mg/kg
Da trn ng chun:
Cch tnh tng t nh trong phn LOD nhng theo cng thc sau:

a
SD 10
LOQ


Lu , ngoi vic tnh ton nh trn cn quan tm n lp li ti
LOQ, ty theo mi phng php, gi tr RSD% cn phi t c theo mt
mc yu cu no . Xem thm phn chm v yu cu RSD% cho tng
nng c th.
5. chnh xc ( ng v chm)
Hin nay c nhiu cch hiu khc nhau v thut ng chnh xc.
Trc y v n by gi nhiu ti liu c ni v ng v chnh xc
40 CHNG II: THM NH PHNG PHP PHN TCH HA HC

nh l hai khi nim khc nhau. Trong ti liu ny chng ti s dng hai
thut ng ng v chm din t chnh xc ca mt phng php
phn tch theo quan im mi nht ca tiu chun quc t (ISO 5725 1-
6:1994) v tiu chun quc gia (TCVN 6910 1-6:2005). ng ch mc
gn nhau gia gi tr trung bnh ca kt qu th nghim v gi tr thc
hoc gi tr c chp nhn l ng . chm ch mc mc dao
ng ca cc kt qu th nghim c lp quanh tr gi trung bnh. Hnh 9
m t s mi quan h gia chm, ng v chnh xc.
chnh xc (accuracy) = chm (precision) + ng (trueness)

Hnh 9: Minh ha khi nim chnh xc ( chm v ng)
5.1. chm
5.1.1. nh ngha
Trong nhiu trng hp cc php th nghim trn nhng i tng v
vi nhng iu kin khc nhau thng khng cho kt qu ging nhau. iu
ny do cc sai s ngu nhin ca mi quy trnh gy ra, ta khng th kim
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 41
sot c hon ton tt c cc yu t nh hng n kt qu th nghim. Do
, kim sot c cc sai s ny, phi dng n khi nim chm.
chm ch ph thuc vo sai s ngu nhin v khng lin quan n gi tr
thc. chm l mt khi nim nh tnh v c biu th nh lng bng
lch chun hay h s bin thin. chm cng thp th lch chun
hay h s bin thin cng ln.
chm c th c phn ra thnh ba trng hp sau:
- lp li (repeatability)
- chm trung gian (intermediate precision)
- ti lp (reproducibility)
S khc nhau gia cc khi nim lp li, chm trung gian v
ti lp c tm tt trong bng 6.
Bng 6: S khc nhau gia lp li, chm trung gian v ti lp
iu kin lp li chm trung
gian
ti lp
Nn mu Khc Khc Khc
Nng Khc Khc Khc
Thit b Ging Khc Khc
Ngi phn tch Ging Khc Khc
Dng c, ha cht Ging Khc Khc
iu kin mi
trng
Ging Khc Khc
Phng th nghim Ging Ging Khc
42 CHNG II: THM NH PHNG PHP PHN TCH HA HC

5.1.2. Cch xc nh
Cch 1. B tr th nghim
Tin hnh lm th nghim lp li 10 ln (t nht 6 ln) trn cng mt
mu (mi ln bt u t cn hay ong mu). Mu phn tch c th l mu
chun, hoc mu trng c thm chun, tt nht l lm trn mu th hay mu
th thm chun.
Tng phng th nghim, c th b tr th nghim tnh lp li
hoc chm trung gian. Trong mt s trng hp tham gia so snh vi cc
phng th nghim khc (v d trong chng trnh th nghim thnh tho, so
snh lin phng).
Nn tin hnh nng khc nhau (trung bnh, thp, cao) trong
khong lm vic, mi nng lm lp li 10 ln (t nht 6 ln). Tnh lch
chun SD v lch chun tng i RSD hay h s bin thin CV theo cc
cng thc sau:
( )
1 n
x x
SD
2
i



100
x
SD
% CV % RSD
Trong :
SD: lch chun
n: S ln th nghim
x
i
: Gi tr tnh c ca ln th nghim th i
x : Gi tr trung bnh ca cc ln th nghim
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 43
RSD%: lch chun tng i
CV%: H s bin thin
Cch 2. Tnh ton trn cc kt qu phn tch mu thc lm.
Trong mt s trng hp vic c lng lp li c th thng qua
tnh ton da trn kt qu phn tch cc mu thc. Do vic lu gi cc
kt qu phn tch c vai tr rt quan trng.
Da trn kt qu phn tch lm trn mu thc trong nhiu tun, t nht
l 10 mu, c th l cc nn mu khc nhau, nng khc nhau nhng phi
c kt qu lm lp 2 ln .
Trng hp cc mu c nng , hm lng gn nh nhau. Tnh
lch gia hai kt qu lp ca mi mu d
i
ri tnh lch trung bnh d
tb
, sau
tnh lch chun s:
2 i 1 i i
x x d
n
d
d
i
tb


2
x x
x
2 i 1 i
i
+

n
x
X
i

118 , 1
d
s
tb

100
X
s
% RSD
44 CHNG II: THM NH PHNG PHP PHN TCH HA HC

Nu cc mu c nng x
i
khc nhau nhiu th thay cho lch d
i
,
tnh lch tng i D
i
, v lch tng i trung bnh D
tb
v sau tnh
lch chun tng i:

i
i
i
x
d
D
n
D
D
i
tb


100
118 , 1
D
% RSD
tb

5.1.3. Tiu ch nh gi
i chiu gi tr tnh c vi gi tr mong mun hay gi tr yu cu
hoc so vi RSD% lp li cho trong bng 7 (RSD% tnh c khng c
ln hn gi tr trong bng hm lng cht tng ng). chm thay i
theo nng cht phn tch. Nng cht cng thp th kt qu cng dao
ng nhiu (khng chm) ngha l RSD cng ln.
Bng 7: lp li ti a chp nhn ti cc nng khc nhau (theo AOAC)
TT Hm lng % T l cht n v RSD (%)
1. 100 1 100% 1,3
2. 10 10
-1
10% 1,8
3. 1 10
-2
1% 2,7
4. 0,1 10
-3
0,1 % 3,7
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 45
5. 0,01 10
-4
100 ppm 5,3
6. 0,001 10
-5
10 ppm 7,3
7. 0,0001 10
-6
1 ppm 11
8. 0,00001 10
-7
100 ppb 15
9. 0,000001 10
-8
10 ppb 21
10. 0,0000001 10
-9
1 ppb 30



V d: Kt qu cc ln phn tch lp li ch tiu lipit tng s trong sa bt:
Ln 1 2 3 4 5 6 7 8 9 10
1 i
x

21,3 26,5 24,1 25,7 24,6 27,8 26,6 25,4 20,1 18,5
2 i
x

21,5 25,9 24,1 25,3 24,5 27,6 26,5 25,1 20,5 18,3
x

21,4 26,2 24,1 25,5 24,55 27,7 26,55 25,25 20,3 18,4
d
i
0,2 0,6 0,0 0,4 0,1 0,2 0,1 0,3 0,4 0,2
D
i
0,0093 0,0229 0,0000 0,0157 0,0041 0,0072 0,0038 0,0119 0,0197 0,0109

46 CHNG II: THM NH PHNG PHP PHN TCH HA HC

Ln 11 12 13 14 15 16 17 18 19 20
1 i
x

27,1 25,3 24,6 25,1 27,6 22,4 26,3 19,8 22,6 24,5
2 i
x

27,4 25,2 25 25,8 26,9 22,4 26,1 20,4 23,1 24,9
x

27,25 25,25 24,8 25,45 27,25 22,4 26,2 20,1 22,85 24,7
d
i
0,3 0,1 0,4 0,7 0,7 0,0 0,2 0,6 0,5 0,4
D
i
0,0110 0,0040 0,0161 0,0275 0,0257 0,0000 0,0076 0,0299 0,0219 0,0162
0133 , 0
20
265 , 0
n
D
D
i
tb


;

tb
D 0,0133
RSD% 100 100 1,19
1,118 1,118
%
khong nng ny, theo AOAC RSD% ti a chp nhn c l 1,8%,
nh vy phng php p dng c chm t yu cu.
Trong ni b mi phng th nghim ngoi vic tnh ton lp li,
cn phi b tr th nghim tnh c chm trung gian (mt s tc gi
gi l ti lp ni b phng th nghim) theo mt trong cc cch sau y:
- S dng cc nhn vin th nghim khc nhau;
- S dng cc thit b vi mt s c tnh khc nhau, v d cc h
thng HPLC khc nhau ca cc hng khc nhau, hoc ca cng mt
hng nhng vi cc model khc nhau;
- S dng cc dung mi ha cht, thuc th c cht lng khc nhau;
- Khc nhau v nhit v m ca phng th nghim;
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 47
- Khc nhau cc iu kin c th ca thit b, v d thnh phn dung
mi pha ng, tc dng, pH ca pha ng...
Vic tham gia th nghim thnh tho, so snh lin phng th nghim
l iu kin rt quan trng trong nh gi phng php. Cc phng th
nghim tham gia phi c kt qu th nghim lin phng trc khi c
cng nhn t ISO 17025 v duy tr cng nhn ISO 17025.
5.2. ng (trueness)
5.2.1. nh ngha
ng ca phng php l khi nim ch mc gn nhau gia gi
tr trung bnh ca kt qu th nghim v gi tr thc hoc gi tr c chp
nhn l ng ().
i vi a s mu phn tch, gi tr thc khng th bit mt cch
chnh xc, tuy nhin n c th c mt gi tr quy chiu c chp nhn l
ng (gi chung l gi tr ng).
Ging nh chm, ng l mt khi nim nh tnh. ng
thng c din t bng chch (bias).
100
X
tb



Trong : : chch (bias), %
X
tb
: Gi tr trung bnh ca kt qu th nghim
: Gi tr thc hoc gi tr c chp nhn l ng
5.2.2. Cch xc nh ng
Mun xc nh ng cn phi tm c gi tr ng, c nhiu cch
khc nhau xc nh ng, bao gm vic so snh kt qu thc nghim
vi kt qu thc hin bi mt phng php i chiu hoc s dng mu
48 CHNG II: THM NH PHNG PHP PHN TCH HA HC

bit nng (mu kim tra hoc mu chun c chng nhn) v phng
php xc nh thu hi ( tm li).
Cch 1: So snh vi phng php chun/i chiu
Phn tch mu chun hoc mu th, thc hin 10 ln bng phng
php kho st v bng mt phng php i chiu. Phng php i chiu
tt nht l phng php tiu chun ca cc t chc c uy tn, nu khng
phng php i chiu l phng php qua thm nh cho kt qu tin cy
trong di o ang thc hin. Tnh ton cc kt qu trung bnh v lp li
(h s bin thin) ca hai phng php.
nh gi tng ng v chm ca hai phng php bng cch
so snh phng sai s
2
ca hai phng php , dng tiu chun F (Fisher)
v so snh hai tr gi trung bnh bng tiu chun t (Student). Vic b tr cc
th nghim phi c thc hin theo phng php tham chiu mt cch
nghim ngt v cc php o phi c tin hnh di iu kin lp li.
- So snh hai phng sai (chun F Fisher)
Chun F dng so snh lp li ca hai tp s liu hoc hai
phng php khc nhau. Vi tp s liu nh, tnh ton gi tr F
tn
(F thc
nghim) theo cng thc sau y v so snh vi gi tr F
c
(F tra bng).
1
S
S
F
2
2
2
1
tn
>
Trong : F
tn
: Gi tr F thc nghim
2
2
2
1
S , S : Cc phng sai ca hai phng php vi
quy c
2
2
2
1
S S >
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 49
Nu: F
tn
F
c
(, k
1
, k
2
): Hai phng php c lp li ( chm)
ging nhau.
Nu: F
tn
> F
c
(, k
1
, k
2
): Hai phng php c lp li khc nhau,
trong trng hp ny nu lp li ca phng php th nghim khc vi
phng php chun th cn xem xt thm v lp li nh m t trong
phn trn.
Trong : F
c
(, k
1
, k
2
): Gi tr F tra bng (xem ph lc 2) vi:
k
1,
k
2
: Bc t do (k
1
= n
1
- 1; k
2
= n
2
- 1)
n
1
, n
2
: S ln lm thc nghim ca hai phng php
: Mc ngha (significance level), thng ly =
0,05 (tng ng vi tin cy (confidence level) 95%)
- So snh hai gi tr trung bnh (chun t Student)
Chun t c dng so snh xem c s khc nhau gia gi tr thc
nghim v gi tr thc hay khng; phng php ny c ng dng hoc
so snh kt qu thc nghim vi gi tr chun trong mu kim tra (xem thm
cch 2) hoc so snh kt qu ca phng php phn tch vi phng
php i chiu.
Trc khi so snh hai gi tr trung bnh cn so snh hai phng sai.
Vi s ln phn tch nh hn 30, khi hai phng sai c s ng nht, tnh
lch chun chung v gi tr t
tn
(t thc nghim) theo cng thc sau y v
so snh vi gi tr t
c
(t tra bng):

( ) ( )
2 n n
S 1 n S 1 n
S
2 1
2
2 2
2
1 1
2
c
+
+

50 CHNG II: THM NH PHNG PHP PHN TCH HA HC

,
_

2 1
2
c
2 1
tn
n
1
n
1
S
x x
t
k = n
1
+ n
2
- 2
Trong : t
tn
: Gi tr t thc nghim
t
c
(, k): Gi tr t tra bng mc ngha , bc t do k
(xem ph lc 1)
n
1
, n
2
: S ln th nghim ln lt ca phng php
th nghim v phng php i chiu
2
2
2
1
S , S : Phng sai ln lt ca phng php th
nghim v phng php i chiu
2 1 x , x : Gi tr trung bnh ln lt ca phng php
th nghim v phng php i chiu
Nu t
tn
t
c
(, k) : Khng c s khc nhau v kt qu ca hai phng
php.
Nu t
tn
> t
c
(, k) : C s khc nhau v kt qu ca hai phng php,
phng php th nghim mc sai s h thng.
Trong trng hp hai phng sai khng ng nht (khc nhau c
ngha), tnh gi tr t
tn
v bc t do k theo cc cng thc sau v so snh nh
trn.
2
2
2
1
2
1
2 1
tn
n
S
n
S
x x
t
+


THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 51
2
1 n
n
S
1 n
n
S
n
S
n
S
k
2
2
2
2
2
1
2
1
2
1
2
2
2
2
1
2
1

1
1
1
1
1
1
1
1
]
1

,
_

+
+

,
_

,
_

+

V d: nh gi phng php mi xut xc nh hm lng nit
trong mu thc phm, ngi ta so snh phng php ny vi phng
php tiu chun (phng php Kjeldahl) qua th nghim sau:
Phng php tiu chun: n
1
= 10; 1 x = 10,5; s
1
= 0,65
Phng php nghin cu: n
2
= 10; 2 x = 10,9; s
2
= 0,76
Hy nh gi ng ca phng php xut?
Gii:
- So snh phng sai:
2
2
tn 2
1
S
F 1,3671
S

Tra bng ta c: F (0,05; 9; 9) = 3,18 > F
tn
: Hai phng sai khng c
s khc nhau c ngha, hai phng php c lp li tng ng..
- So snh hai gi tr trung bnh:
( ) ( ) +
+

+
2 2
2
c
10 1 0, 65 10 1 0,76
3,8025 5,1984
S 0,50
10 10 2 18


_
+

,
tn
10,5 10,9
t 1,265
1 1
0,50
10 10

Tra bng c t(0,05; 18) = 2,101 > t
tn
, do khng c s khc nhau
v kt qu ca hai phng php.
52 CHNG II: THM NH PHNG PHP PHN TCH HA HC

Cch 2: S dng vt liu chun (Reference material)
Vt liu chun (cn gi l mu chun) l mu phn tch c hm lng
cht phn tch c xc nh trc v l ng. C nhiu cp vt liu
chun khc nhau, trong cao nht l CRM (certified reference material
vt liu chun c chng nhn) c cung cp bi cc t chc c uy tn
trn th gii.
Cc mu CRM lun c kt qu km theo khong dao ng, do khi
phn tch mu CRM c th nh gi c ng da vo khong dao
ng cho php (v d: Mu tht, ch tiu clenbuterol l 1 ng/g 0,05 ng/g;
nu kt qu phn tch c trong khong t 0,95-1,05 th t).
Nu khng c cc mu CRM c th s dng cc mu kim tra (QC-
Quality Control) bit nng . Phng th nghim c th t chun b cc
loi mu ny, hoc s dng cc mu thc c hm lng bit hoc s
dng cc mu lu t chng trnh so snh lin phng th nghim. Trong
trng hp khc phng th nghim c th s dng cc mu thm chun
nh gi ng, ni dung ny s c m t c th trong cch 3 di y.
Nhiu t chc c uy tn nh USFDA, EURACHEM, ICH... quy nh
tnh chch (bias) xc nh ng nh sau:
100
x



Trong : : chch (bias), %
x : Gi tr trung bnh ca kt qu th nghim
: Gi tr thc hoc gi tr c chp nhn l ng
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 53
USFDA quy nh chch ca cc phng php xc nh d lng
phi khng c ln hn 15% v khng ln hn 20% ti LOQ.
C th s dng chun t nh gi kt qu nh sau:
Phn tch mu chun lp li 10 ln (ti thiu 6 ln), tnh gi tr trung
bnh v lch chun, t tnh gi tr t
tn
theo cng thc sau y v so snh
vi t
c
(p) :
n
S
x
t
2
tn


Trong : t
tn
: Gi tr t thc nghim
t
c
(, k): Gi tr t tra bng vi mc ngha 0,05 (xem
ph lc 1) v bc t do k = n - 1.
: Gi tr thc hoc gi tr c chp nhn (tham
chiu)
x : Gi tr trung bnh ca phng php th nghim
2
S : Phng sai ca phng php th nghim
n: S ln th nghim
Nu t
tn
t
c
: Khng c s khc nhau v kt qu ca gi tr trung bnh
so vi gi tr tham chiu mc ngha , tc l phng php c ng
t yu cu.
Nu t
tn
> t
c
: C s khc nhau v kt qu ca phng php th nghim
so vi kt qu tham chiu mc ngha , phng php th nghim mc
sai s h thng.
54 CHNG II: THM NH PHNG PHP PHN TCH HA HC


V d: Phn tch mu QC bng phng php ang nghin cu cho kt qu
nh sau:
n = 10; x = 46,5 ng/g; SD = 4,5 ng/g
Cho bit mu QC c kt qu ng l: 50 ng/g.
nh gi ng ca phng php ang nghin cu?
Gii:
Tnh gi tr t
tn
:


tn
2
50 46,5
t 2, 46
4,5
10

Tra bng: t (0,95; 9) = 2,262 < t
tn
, do kt qu mu QC khng t yu
cu.
Cch 3: Xc nh thu hi
Cc phng php tnh ng theo cch 1 hay cch 2 u gp nhng
kh khn nht nh. Trong nhiu trng hp khng th tm hoc p dng
mt phng php tiu chun so snh kt qu, cng nh khng th d
dng c c cc mu chun hoc mu chun c chng nhn ph hp vi
phng php. Vic xc nh ng do c th thc hin thng qua xc
nh thu hi (cn gi l tm li) ca phng php.
Thm mt lng cht chun xc nh vo mu th hoc mu trng,
phn tch cc mu thm chun , lm lp li ti thiu bn ln bng phng
php kho st, tnh thu hi theo cng thc sau y:
- i vi mu th:
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 55
100
C
C C
% R
c
m c m

+

- i vi mu trng:
100
C
C
% R
c
tt

Trong : R%: thu hi, %
C
m+c
: Nng cht phn tch trong mu thm chun
C
m
: Nng cht phn tch trong mu th
C
c
: Nng chun thm (l thuyt)
C
tt
: Nng cht phn tch trong mu trng thm chun
Sau tnh thu hi chung l trung bnh ca thu hi cc ln lm
lp li.
Thm cht chun ba mc nng l mc thp, trung bnh v cao
trong khong nng lm vic. Theo quy nh ca hi ng chu u i
vi cc ch tiu an ton (cc ch tiu thuc nhm c, c quy nh gii hn
cho php, v d tn d hormon, khng sinh, ha cht bo v thc vt...)
thm chun vo mu trng ba mc nng ti 0,5 ln, 1 ln v 2 ln gii
hn cho php (MRL).
Hi ng chu u cng quy nh i vi cc mu phn tch hng ngy
(routine) cc ch tiu thuc cng nhm (v d: ha cht bo v thc vt
nhm clo hu c) cn kim sot cht lng bng cch phn tch mu thm
chun ti thiu 10% s lng cht, cc cht khc cn thay phin kim tra
vi tn sut ti a 1 nm/ln cho tng cht.
56 CHNG II: THM NH PHNG PHP PHN TCH HA HC


V d: xc nh thu hi ca phng php phn tch hm lng SO
2

trong mu ru vang, thc hin phn tch cc mu ru vang v ru
vang c cho thm chun ba mc nng , mi nng thc hin phn
tch bn ln lp li, thu c cc kt qu nh sau:
TT
Mu
(ml)
Chun thm (mg/100
ml)
HL SO
2

(mg/100ml)
TB
R
(%)
1 20 0 7,72
2 20 0 7,69
3 20 0 7,76
4 20 0 7,73
7,73 0
5 20 5 12,4
6 20 5 12,5
7 20 5 12,6
8 20 5 12,9
12,6 97,5
9 20 10 17,6
10 20 10 17,8
11 20 10 17,8
12 20 10 17,7
17,7 100
13 20 25 32,4
14 20 25 32,1
15 20 25 32,9
16 20 25 31,9
32,3 98,4
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 57
- nng thm 5 mg/100 ml: R % =


12,6 7,73
100 97,5%
5

- nng thm 10 mg/100 ml: R % =


17,7 7,73
100 100%
10

- nng thm 25 mg/100 ml: R % =


32,3 7,73
100 98, 4%
25

5.2.3. Tiu ch nh gi
Sau khi nh gi thu hi, so snh kt qu vi cc gi tr cho trong
bng 8. thu hi cc nng khc nhau c k vng khc nhau. Trong
trng hp phn tch cc cht hm lng vt c th tham kho tiu chun
ca hi ng chu u (bng 9).
Bng 8: thu hi chp nhn cc nng khc nhau (theo AOAC)
TT Hm lng
[%]
T l cht n v thu hi [%]
1. 100 1 100% 98-102
2. 10 10
-1
10% 98-102
3. 1 10
-2
1% 97-103
4. 0,1 10
-3
0,1 % 95-105
5. 0,01 10
-4
100 ppm 90-107
6. 0,001 10
-5
10 ppm 80-110
7. 0,0001 10
-6
1 ppm 80-110
8. 0,00001 10
-7
100 ppb 80-110
9. 0,000001 10
-8
10 ppb 60-115
10. 0,0000001 10
-9
1 ppb 40-120
58 CHNG II: THM NH PHNG PHP PHN TCH HA HC

Bng 9: Quy nh v thu hi ca hi ng chu u
TT Hm lng cht (g/kg) n v thu hi [%]
1. 1 1 ppb 50-120%
2. > 1 n < 10 1-10 ppb 70-110%
3. 10 10ppb 80-110%
6. n nh (hay vng/ chc chn) ca phng php
6.1. nh ngha
n nh ca phng php l kh nng cung cp cc kt qu c
chnh xc ( ng v chm) chp nhn c di nhng iu kin c
s thay i v mt s iu kin thc hin phng php.
Ngi ta phn bit hai khi nim vng (robustness) v chc
chn (rugedness) nh sau:
- vng: L mc lp li cc kt qu phn tch khi c s thay i
nh mt s thng s ca phng php (method parameters). V d
i vi phng php sc k lng c th k ra cc yu t ny bao
gm pH pha ng, tc dng, nhit ct, th tch tim mu,
thnh phn pha ng, bc sng pht hin...
- chc chn: L mc lp li cc kt qu phn tch khi c s thay
i mt s yu t thuc v iu kin phn tch (test conditions). Cc
iu kin ny c th l ngi phn tch, thit b phn tch, ct sc k
do cc nh cung cp khc nhau, l ha cht thuc th, thi im
thc hin th nghim... Ni chung, chc chn thng lin quan
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 59
n vic tnh chm trung gian hay ti lp ni b phng th
nghim.
Trong ti liu ny chng ti ni n n nh bao gm c hai yu t
vng v chc chn ca phng php.
6.2. Cch xc nh
Cho d c s khc nhau v iu kin phn tch hay khc nhau v
thng s phng php, th cng cn phi b tr cc th nghim so snh
cc kt qu t c trong cc iu kin thay i ny bng cch so snh hai
phng sai (chun F) v so snh hai gi tr trung bnh (chun t) ca hai
nhm kt qu A v B. V d, gia ngi A vi ngi B, gia my A v my
B, gia iu kin A v iu kin B... Cch tin hnh so snh ging nh
trong phn xc nh ng khi so snh hai phng php nu trn.
Trong trng hp c mu CRM c th b tr cc th nghim (c s
thay i iu kin phn tch hay cc thng s ca phng php) tnh kt
qu mu CRM v so snh vi kt qu chun nh gi n nh ca
phng php.
60 CHNG III: THM NH PHNG PHP PHN TCH VI SINH VT


Chng III: THM NH PHNG PHP
PHN TCH VI SINH VT
Phng php vi sinh vt c s khc bit r rt vi cc phng php
ha hc. Do vic thm nh phng php phn tch vi sinh vt cng c
nhng yu cu v k thut ring. Chng ny s gii thiu mt cch s lc
cc khi nim v cch tin hnh thm nh cc thng s c bn nht ca
phng php phn tch vi sinh vt thc hin theo k thut nui cy truyn
thng.
1. Cc yu cu chung
1.1. Chun b thm nh
Trc khi thm nh bt k mt phng php no, phng th nghim
phi c y cc trang thit b, dng c v my mc thc hin phng
php cng nh cc yu cu nghim ngt v cht lng cc ch s k
thut ca trang thit b v dng c (phi c hiu chun nu cn, bi mt
n v/phng hiu chun c iu kin, thch hp so vi yu cu). C th
thc hin kim tra cht lng mi trng nui cy, thuc th theo hng
dn ti tiu chun ISO 11133 phn I v II.
Thm nh mt phng php cn c thc hin bi cc kim nghim
vin nm vng kin thc chuyn mn, k thut thao tc tt v c kinh
nghim trong phn tch vi sinh vt.
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 61
Chun b mu cho qu trnh thm nh: i vi phng php vi
sinh, vic chun b mu thm nh rt quan trng do cc mu vi sinh thng
c n nh khng cao. Qu trnh la chn mt dng thc phm lm
tng t l mu b nhim t nhin cn c tnh ton ht sc cn thn.
thm nh trn i tng mu thc phm, cn la chn nm loi
thc phm khc nhau thuc nm nhm. Chi tit vic la chn loi thc
phm thm nh c m t trong ph lc 3.
- Cc mu t nhin c u tin nht. l cc sn phm c phn
tch c cha/nhim vi sinh vt ch. Qu trnh bo qun mu cn hn
ch ti a s thay i ca vi sinh vt v trng thi ca chng. Cn
phi kim tra mc n nh hm lng vi sinh nhim ngay trc
thi im s dng mu lm thm nh.
- Mu t nhim: Cc dng mu lng hoc st c th c gy nhim
bng cch trn vi cc sn phm tng t c nhim vi sinh vt t
nhin. Cn phi m bo tnh ng nht ca mu sau khi trn.
- Mu thm: S dng cc vi sinh vt ch gy nhim vo mu
trng. Hm lng vi sinh vt gy nhim cn tng ng vi lng
thng c trong cc sn phm t nhin.
- Mu chun: Mu chun, c bit l cc mu chun c chng
nhn, c cha mt lng vi sinh vt bit v c n nh cao.
Mu chun c th c s dng lm mu thm chun khi thm
nh cc phng php nh lng. i vi cc phng php nh
tnh, vic s dng thng khng cn thit.
62 CHNG III: THM NH PHNG PHP PHN TCH VI SINH VT

1.2. La chn thng s thm nh
Cc phng php vi sinh, ging nh cc phng php ha hc cng
c th phn loi thnh hai nhm phng php l cc phng php tiu
chun (reference method) v cc phng php khng tiu chun hay cc
phng php thay th (alternative method). Hin nay, cc phng th nghim
Vit Nam a s u s dng cc phng php tiu chun thc hin cc
ch tiu vi sinh. Do , trong phm vi ca ti liu ny, chng ti ch xin gii
thiu vic thm nh i vi phng php tiu chun, cc thng s cn thm
nh nh sau:
Bng 10: La chn thng s thm nh phng php phn tch vi sinh vt
Phng php nh tnh Phng php nh lng
Gii hn pht hin
c hiu, nhy
lch dng, lch m
chnh xc ( ng)
Gii hn pht hin
Gii hn nh lng
lp li
ti lp
Cc khi nim v thng s thm nh vi sinh cng c nhng im
khc so vi thm nh ha hc, cc khi nim ny s c lm r trong cc
phn sau.
i vi cc phng php khng tiu chun, qu trnh thm nh phc
tp hn v thng thng cn tri qua hai giai on, giai on I l thc hin
so snh vi mt phng php chun v giai on II l so snh lin phng
th nghim. tm hiu k hn v thm nh phng php tiu chun, c
th tham kho thm ISO 16140:2003.
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 63
2. Thm nh phng php tiu chun (method verification)
2.1. Phng php nh tnh
2.1.1. Gii hn pht hin
a. nh ngha
Gii hn pht hin ca mt phng php l nng vi sinh vt thp
nht trong mu c th xc nh c bng phng php .
i vi phng php nh tnh gii hn pht hin c tnh nng
c ti thiu 90% mu c kt qu dng tnh.
b. Cch xc nh
Thc hin theo cc bc sau:
- Chn loi i tng mu cn thc hin thm nh.
- Th nm mt vi khun cho mi loi vi sinh vt ch cho mt loi
mu, gm c chng m. Cc mc c th nh sau:
Mc 1 (L
o
) l chng m, ch cn thc hin 1 ln.
Mc 2 l mc pht hin theo l thuyt t [1 - 9] CFU/ 25g
v mi mc gy nhim k hiu L
1a
, L
1b
, L
1c
vi (1 L
1a
<
L
1b
< L
1c
9) CFU/ 25g. Mc 2 c thc hin 5 ln cho mi
mu nhim.
Mc 3 (L
2
) l mc trn mc pht hin theo l thuyt [10-50]
CFU/ 25g, mc ny ch cn thc hin 1 ln.
c. Tnh kt qu
LOD ca mi loi thc phm c tnh nh sau:
64 CHNG III: THM NH PHNG PHP PHN TCH VI SINH VT

LOD = nng vi khun nhim vo mu m c t nht 90%
mu phn tch c kt qu dng tnh.
d. nh gi kt qu
LOD < 10 l t yu cu.
2.1.2. Xc nh chnh xc (accuracy:AC), c hiu
(specificity:SP), nhy (sensitivity:SE), lch dng
(positive deviation:PD) v lch m (negative deviation:ND)
a. nh ngha
- chnh xc (hay ng) l mc ging nhau gia kt qu
phn tch c vi gi tr tham chiu.
- c hiu l kh nng phn bit vi sinh vt ch vi cc vi sinh
vt khc.
- nhy l t l ca cc vi sinh vt ch c th c pht hin.
b. Cch xc nh
Vi mi loi i tng, chun b cc mu khc nhau, chun b cc
mu ny thnh hai nhm, trong c mt nhm mu m tnh v mt nhm
mu c gy nhim mc nng khong bng 10 ln LOD (khng thng
bo cho kim nghim vin). B tr cc kim nghim vin phn tch c lp
theo cng mt phng php ang nghin cu.
c. Tnh kt qu
Tnh kt qu theo cc cng thc sau:

THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 65
Kt qu Mu chng (+) Mu chng (-)
Mu c kt qu dng tnh (+) TP FP
Mu c kt qu m tnh (-) FN TN
Trong : TP (True Positive): Dng tnh ng (Mu chng c kt qu
dng tnh, kt qu phn tch cho kt qu dng tnh)
FP (False Positive): Dng tnh gi (Mu chng c kt qu
m tnh, kt qu phn tch cho kt qu dng tnh)
FN (False Negative): m tnh gi (Mu chng c kt qu
dng tnh, kt qu phn tch cho kt qu m tnh)
TN (True Negative): m tnh ng (Mu chng c kt qu
m tnh, kt qu phn tch cho kt qu m tnh)
n = TP + FP + FN + TN: Tng s cc kt qu
chnh xc, c hiu, nhy, lch dng, lch m c
tnh ton theo cc cng thc sau y:
chnh xc 100
N
TN TP
AC
+

c hiu 100
FP TN
TN
SP
+

nhy 100
FN TP
TP
SE
+

lch dng 100
TP FP
FP
PD
+

66 CHNG III: THM NH PHNG PHP PHN TCH VI SINH VT

lch m 100
TN FN
FN
ND
+

d. nh gi kt qu
Cc thng s cn c nh gi theo cc chun mc sau:
chnh xc (AC) 90 %
c hiu (SP) 90 %
nhy (SE) 90 %
lch dng (PD) 10 %
lch m (ND) 10 %

V d: B tr cc th nghim xc nh chnh xc, c hiu v
nhy ca mt phng php vi sinh, thu c cc kt qu nh sau:
- S mu dng tnh pht hin dng tnh: 37 (TP)
- S mu m tnh pht hin dng tnh: 3 (FP)
- S mu dng tnh pht hin m tnh: 2 (FN)
- S mu m tnh pht hin m tnh: 38 (TN)
Ta c cc kt qu:
- Tng cc kt qu: n = 80
- nhy: SE =
+ +
TP 37
100 100 95
TP FN 37 2
%
- c hiu: SP =
+ +
TN 38
100 100 93
TN FP 38 3
%
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 67
- lch dng:
+ +
FP 3
PD 100 100 7,5
FP TP 3 37
%
- lch m:
+ +
FN 2
ND 100 100 5
FN TN 2 38
%
- chnh xc:
+ +

TP TN 37 38
AC 100 100 94%
n 80

2.2. Phng php nh lng
2.2.1. Gii hn pht hin v gii hn nh lng
a. nh ngha
- i vi phng php nh lng, gii hn pht hin l nng vi
sinh vt thp nht m phng php c th xc nh c nhng
cha nh lng c.
- Gii hn nh lng ca phng php l nng vi sinh vt thp
nht m phng php c th nh lng c vi mt chm
mong mun.
b. Quy trnh thc hin
Thc hin kim nghim nm mu nng ti thiu - ti mc pht
hin l trn 0 (a minimum non zero level) hoc gn 0 (near zero level) -
chc chn rng s lng mu dng tnh c t l < 50%.
Chn 3-5 nng trong khong nng vi sinh vt d kin phn
tch (v d 10, 20, 50 CFU/25g). Mi nng thc hin lp li 6 ln. c
tnh lng vi sinh vt thp nht trong mu c th pht hin 50% kt qu
dng tnh.
68 CHNG III: THM NH PHNG PHP PHN TCH VI SINH VT

c. Tnh kt qu
Tnh gii hn pht hin v gii hn nh lng theo cng thc sau:

65 , 1
LC
S
0


0
S 3 , 3 LOD

0
S 10 LOQ
Trong : LC (Critical Level): Lng vi sinh vt thp nht trong
mu c th pht hin 50% kt qu dng tnh.
S
0
: Tn hiu nn (baseline spread)

V d: Cc kt qu dng tnh: 0/6 ti nng 10, 2/6 ti nng 20 v
5/6 ti nng 50
c tnh mc dng tnh xp x pht hin 50% l LC 20 CFU/25g
Do , S
0
= 20/1,65 12
Gii hn pht hin: LOD = 3,3 x 12 40 (CFU/25g)
Gii hn nh lng: LOQ = 10 x 12 = 120 (CFU/25g)
2.2.2. Xc nh chm ( lp li v ti lp ni b)
a. nh ngha
Nh nu trong chng 2, thm nh phng php ha hc, khi
nim chm c s dng ch mc gn nhau gia cc kt qu phn
tch.
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 69
lp li ch chm c thc hin trong nhng iu kin ging
nhau, bi cng mt kim nghim vin v trong mt khong thi gian tng
i ngn.
ti lp ch chm c thc hin bi cc kim nghim vin khc
nhau ( ti lp ni b phng th nghim hay cn gi l chm trung
gian), hoc cc phng th nghim khc nhau ( ti lp lin phng th
nghim).
b. Quy trnh thc hin
Chn khong 3-5 loi nn mu thc phm, mi loi chn 3-5 sn
phm (mu t nhin hoc mu t nhim). i vi mu t nhim, cy vi sinh
vt vi nng khong 10
2
-10
3
CFU/n v.
Xc nh lp li: Mt kim nghim vin thc hin kim
nghim cc mu ni trn, mi mu lp li hai ln, trong mt
khong thi gian ngn.
Xc nh ti lp ni b phng th nghim ( chm trung
gian): Hai kim nghim vin, kim nghim cc mu ni trn, lp
li hai ln song song; c th thc hin trong khong thi gian
tng i di.
Cc d liu xc nh ti lp c th thu c t qu trnh kim
nghim mu thc t, lu li cc s liu song song tnh ton.
c. Tnh kt qu
v lp li: Tnh lch chun tng i (RSD) theo cng thc:
2
b log a log
x
i i
i
+

70 CHNG III: THM NH PHNG PHP PHN TCH VI SINH VT

( ) [ ]
n 2
x b log a log
RSD
n
1 i
2
i i i
r


Trong : a
i
, b
i
: Cc kt qu phn tch song song ca kim
nghim vin.
i
x : Kt qu trung bnh ca hai ln th nghim song
song ().
n: S kt qu phn tch song song ca kim nghim
vin.
v ti lp ni b phng th nghim: C nhiu cp kt qu lp li
(song song) ca hai kim nghim vin A v B. Tnh lch chun
tng i (RSD) theo cng thc sau:
2
b log a log
x
i i
i
+

( ) [ ]
n 2
x b log a log
RSD
n
1 i
2
i i i
R


Trong : a
i
, b
i
: Cc kt qu phn tch song song ca c hai kim
nghim vin A v B.

i
x : Kt qu trung bnh ca hai ln phn tch song song.
n: S kt qu phn tch song song ca c hai kim nghim
vin A v B.
Trng hp c nhiu kim nghim vin cng thc hin cng c th p
dng cng thc trn tnh ti lp.
d. nh gi kt qu
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 71
Khi trong phng php chun c cung cp cc thng s v chm
th kt qu xc nh lp li SD
r
v ti lp SD
R
cn phi nh hn cc
gi tr tng ng nu trong phng php chun.
GHI CH: Nu trong phng php chun c nu gii hn lp li r
hay gii hn ti lp R th cch thm nh/kim tra ti thiu l: hiu hay
thng (ty theo quy nh trong phng php tiu chun) gia hai kt qu
phn tch song song phi nh hn r (nu l hai kt qu lp li) hay nh hn
R (nu l hai kt qu ti lp).

V d: Kt qu xc nh lp li v ti lp ca hai kim nghim vin A
v B khi nh lng mt loi vi khun trn mu thc phm v cc tnh ton
lp li v ti lp nh sau:
Kt qu
Mu KNV
Ln
1 (a
i
)
Ln
2 (b
i
)

ai
x
log(a
i
)

bi
x
log(b
i
)
Hiu

ai bi
x x
TB x
i
=

+
ai bi
x x
2

Hiu/
Trung
bnh

(Hiu/
Trung
bnh)
2
1 A 93 86 1,968 1,934 0,034 1,951 0,017415 0,000303
2 B 36 28 1,556 1,447 0,109 1,502 0,072679 0,005282
3 A 34 30 1,531 1,477 0,054 1,504 0,036135 0,001306
4 B 70 64 1,845 1,806 0,039 1,826 0,021318 0,000454
5 A 98 73 1,991 1,863 0,128 1,927 0,066365 0,004404
6 B 262 242 2,418 2,384 0,034 2,401 0,014363 0,000206
72 CHNG III: THM NH PHNG PHP PHN TCH VI SINH VT

7 A 89 83 1,949 1,919 0,030 1,934 0,015671 0,000246
8 B 136 105 2,134 2,021 0,112 2,077 0,054083 0,002925
9 A 116 105 2,064 2,021 0,043 2,043 0,021181 0,000449
10 B 54 49 1,732 1,690 0,042 1,711 0,024658 0,000608
11 A 168 156 2,225 2,193 0,032 2,209 0,014568 0,000212
12 B 86 68 1,934 1,833 0,102 1,884 0,054149 0,002932
13 A 62 56 1,792 1,748 0,044 1,770 0,024970 0,000623
14 B 35 28 1,544 1,447 0,097 1,496 0,064796 0,004199
15 A 38 28 1,580 1,447 0,133 1,513 0,087630 0,007679
16 B 71 64 1,851 1,806 0,045 1,829 0,024650 0,000608
Tng KNV A 0,015222
Tng KNV B 0,017214
Tng A+B 0,032436
lp li ca KNV A: RSD
r(A)
=
0,015222
0,0308
2 * 8
;
lp li ca KNV B: RSD
r(B)
=
0,017214
0,0328
2 * 8
;
ti lp ni b: RSD
R
=

0,032436
0,045
2 16

THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 73
74 CHNG IV: C LNG KHNG M BO O

Chng IV: C LNG
KHNG M BO O
Theo yu cu ca ISO 17025, khng m bo o l mt yu cu
cn thc hin cho tt c cc phng php th c p dng. Vic c lng
khng m bo o l mt vic kh v rt phc tp, v khng phi mt
phng th nghim no cng c th thc hin c. Tuy nhin, c th dn
dn nng cao cht lng h thng phng th nghim phn tch ha hc v vi
sinh vt th cn phi thc hin cng vic ny. Cc phng th nghim c th
phi hp xc nh khng m bo o nh l mt phn ca qu trnh
thm nh phng php phn tch. Ni dung ca chng ny s mang n
nhng khi nim c bn nht v cc cch thc hin cc php nh gi
khng m bo o.
1. Khi nim v khng m bo o
khng m bo o ca php o l thng s gn vi kt qu ca
php o, thng s ny c trng cho mc phn tn ca cc gi tr c th
chp nhn c quy cho i lng o ca php o.
- khng m bo o ni ln tin cy ca php o.
- Kin thc v khng m bo o rt hu ch cho vic pht trin
phng php, thm nh phng php, hay thay i b sung iu
chnh phng php tt hn.
2. Cc ngun gy ra khng m bo o
Trong mt php th c rt nhiu ngun gy ra khng m bo o:
- Mu th: bn cht ca mu th khng ng nht, trng thi vt l,
n nh ca mu th, nh hng ca cc yu t nhit v mi
trng...
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 75
- Ly mu: Ly mu khng i din, khng ng nht...
- iu kin bo qun: Qu trnh vn chuyn, bo qun v thi gian
bo qun mu c th nh hng ln n kt qu phn tch.
- Chun b mu: Qu trnh ng nht, cn mu, chit tch...
- Dung mi v thuc th: tinh khit, tp cht...
- Thit b: Thit b c nhng sai s trong qu trnh hiu chun hoc
cha c hiu chun, sai s cc khong o khc nhau...
- iu kin mi trng: Cc nh hng ca nhit , m...
- Con ngi: K nng, trnh , sai s tnh ton...
- Ngun ngu nhin khc.
3. Cc cch nh gi khng m bo o
Ti liu ny gii thiu hai cch nh gi khng m bo o hin
nay ang c p dng, u tin l c lng khng m bo o t tt
c cc yu t cu thnh n (theo hng dn ca EURACHEM v cch th
hai l c lng khng m bo o tng t vic b tr th nghim v xc
nh theo phng php thng k. Trong phm vi cc phng th nghim hin
nay Vit Nam th cch tip cn th hai thun li hn v c tnh kh thi
cao.
3.1. Cch 1: c lng khng m bo o theo hng dn ca
EURACHEM
3.1.1. Bc 1: Xc nh cc i lng o
Kt qu o thu c cui cng l gi tr s hc ca i lng o, gi
tr ny ph thuc vo gi tr cc i lng u vo v gi tr cc i lng
trung gian.
Lit k cc i lng o c nh hng n kt qu o cui cng,
thng thng da vo quy trnh thao tc chun (SOP) v cng thc tnh ton
xc nh cc i lng ny.
76 CHNG IV: C LNG KHNG M BO O

3.1.2. Bc 2: Xc nh cc ngun gy ra khng m bo o
T cc i lng o cn xc nh ngun gc c th gy ra khng
m bo o ca cc i lng . Cc ngun gy ra khng m bo o
c lit k trong mc 2. Cch n gin l phn thnh cc nhm chung
v lp s xng c xc nh ngun gy ra khng m bo o.


V d: Xc nh ngun gy ra khng m bo o ca php cn 0,5g
NaOH
Cc i lng o:
- Khi lng mu (i lng cui)
- Khi lng b (i lng trung gian)
- Khi lng b + mu (i lng trung gian)


THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 77
3.1.3. Bc 3: Tnh cc thnh phn khng m bo o
Sau khi xc nh cc ngun gy ra khng m bo o, bc tip
theo l tnh khng m bo o t cc ngun ny sau kt ni cc gi tr
ny li vi nhau thu c khng m bo o tng.
Khng phi tt c cc ngun gy ra khng m bo o u nh
hng trc tip c ngha n khng m bo o tng hp m thc t
ch c mt s lng t l c nh hng trc tip. Do u tin cn nh
gi s b s ng gp ca tng ngun thnh phn n khng m bo o
tng. Thc t, nn nhm cc thnh phn n l thnh cc nhm ring bit
n gin ha vic tnh ton.
Cc phng php c bn tnh tng thnh phn khng m bo
o l:
- Tnh lch chun ca cc gi tr bng cch tin hnh cc php o
lp li: khng m bo o chun xut pht t cc sai s ngu
nhin v c tnh ton t lch chun ca php o lp li. Yu
cu s ln lp li ti thiu phi t 6 ln, thng thng 10 ln. Cc
d liu thu c trong qu trnh thm nh phng php rt c ch
tnh khng m bo o.
- Tin hnh cc php o trn cht chun: Khi nim cht chun c
ngha l mt c tnh no ca cht c xc nh chnh xc, s
dng hiu chun thit b hay thm nh phng php. Cht
chun phi c chng nhn c cung cp bi cc t chc uy tn c
nng lc thc hin.
- S dng d liu v kt qu ca cc php o trc , c bit hu
ch l cc kt qu t cc chng trnh th nghim lin phng.
78 CHNG IV: C LNG KHNG M BO O

- T s suy lun da vo kinh nghim ca ngi phn tch: C nhiu
trng hp khng th tin hnh cc php th nghim lp li hoc
khng c cc thng tin cn thit tnh khng m bo o ca
mt thnh phn no , trong nhng trng hp ny th kinh nghim
v kin thc ca ngi lm phn tch cng l mt yu t quan trng
c bit trong trng hp tnh khng m bo o bng cc
phng php khng phi phn tch thng k ca mt dy cc gi tr
quan st (loi B).
a. Tnh khng m bo o chun theo loi A
Phng php ny nh gi khng m bo o bng cch tin hnh
phn tch thng k dy gi tr, vi s ln lp li 6 ln (phn phi chun).
khng m bo o chun c th c biu th bng lch chun hoc
lch chun tng i:

( )
1 n
x x
SD u
2
i





x
SD
% RSD % u
Trong : u: khng m bo o
SD: lch chun
x
i
: Gi tr th i
x : Gi tr trung bnh
n: S ln lm lp li
RSD: lch chun tng i
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 79
Trong trng hp khng ch r dng phn b, khng m bo o
c chuyn i t khong tin cy x :
- i vi mu nh (n < 10):
2
u

vi mc ngha 0,05 ( tin cy 95%)
3
u

vi mc ngha 0,003 ( tin cy
99,7%)
- i vi mu ln (n 10):
Z
u


trong Z c gi tr: 1,96 vi mc ngha 0,05 ( tin cy 95%)
2,575 vi mc ngha 0,01 ( tin cy 99%)

V d: Chng ch hiu chun cn cho bit ti lng cn 100 mg sai s l
0,2 mg vi mc ngha 0,05. khng m bo o l u = 0,4/1,96 =
0,204 (mg)
b. Tnh khng m bo o chun theo loi B: nh gi khng
m bo o khng bng phng php thng k. Ty thuc vo xc
sut xut hin cc gi tr m chn hm phn b ph hp tnh ton:
- Phn b hnh ch nht: Khi cc gi tr gy ra khng m bo
o c phn b ri rc trong mt vng m ti kh nng xut
hin ca cc s liu cc v tr l nh nhau. Trong trng hp
80 CHNG IV: C LNG KHNG M BO O

ny cn xc nh khong dao ng ( rng 2a, dao ng a) v
tnh khng m bo o theo cng thc sau:

3
u


Trong l gii hn thng c ghi trong chng nhn tiu
chun.

V d: Sai s bnh nh mc 10 ml loi A ca nh sn xut cng b l 0,2 ml
nhit 20
o
C nhng khng cng b tin cy bao nhiu. Theo phn
phi hnh ch nht, khng m bo o c tnh:
0, 2
u
3
= 0,12 ml
- Phn b hnh tam gic: Khi hu ht cc gi tr gy ra khng
m bo o c phn b gn trung tm. Trong trng hp
ny cn xc nh khong dao ng ( rng 2a, dao ng a) v
tnh khng m bo o theo cng thc sau:

6
u


Trong l gii hn thng c ghi trong chng nhn tiu
chun.

V d: Sai s bnh nh mc 10 ml loi A ca nh sn xut cng b l 0,2
ml nhit 20
o
C nhng khng cng b tin cy bao nhiu. Nhng
cc kt qu kim tra phng th nghim cho thy gi tr bt thng khng
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 81
bao gi c, cc gi tr u tp trung gn gi tr trung bnh. Theo phn phi
hnh tam gic, khng m bo o c tnh:
0, 2
u
6
= 0,08 ml
3.1.4. Bc 4: Tnh khng m bo o tng hp v m rng
a. i vi m hnh lin quan n php tnh tng hay hiu s: y = a
+ b c...
khng m bo o tng hp c tnh:
... u u u U
2
c
2
b
2
a TH
+ + +

c b a
u , u , u : khng m bo o ca cc u vo a, b, c

V d: Cn chnh xc khong 0,25 g cht chun A trn cn phn tch 4 s.
Cc s liu nh sau:
- Chng ch hiu chun cn cng b khng m bo o l
0,0005 g mc ngha 0,05.
- Qu trnh cn lp li cho lch chun l 0,000075
Cch tnh khng m bo o:
- khng m bo qu trnh hiu chun: u = 0,0005/1,96 =
0,000255 g = 0,255 mg
- khng m bo o qu trnh lp li: u = SD = 0,000075 g =
0,075 mg
- khng m bo o tng: U = ( ) ( )
2 2
075 , 0 255 , 0 + = 0,266 mg
82 CHNG IV: C LNG KHNG M BO O

b. i vi m hnh lin quan n php tnh tch hay thng s:
Vi dng y = a.b.c hoc dng y = (a.b)/c
2
c
2
b
2
a TH
c
u
b
u
a
u
y
U

,
_

+
,
_

+
,
_



V d: Cn chnh xc khong 0,25 g cht chun A, pha trong nc ct v
nh mc n 100ml bng nc ct trong bnh nh mc. tinh khit ca
chun A l P = 99,9 0,1%. khng m bo o ca bnh nh mc l
0,1 ml. Tnh U ca nng mc ngha 0,05.
Nng dung dch c tnh theo cng thc sau:


m P
C 1000
V
=
2,498 mg/l
C ba ngun gy ra khng m bo o ca nng :
- Qu trnh cn: u
a
= 0,266 mg (theo tnh ton v d trc)
- tinh khit mu th: u
b
=
0,001
3
=0,00058
- Th tch: u
c
= 0,1 ml
Vy khng m bo o tng ca nng c tnh theo dng y =
(a.b)/c:

_ _ _
+ +

, , ,
2 2 2
TH
0, 266 0,00058 0,1
U 2, 4975 0,004
250 0,999 100
mg/l
- khng m bo o m rng: Vic bo co khng m bo
o cui cng thng cn quy v khng m bo o m rng. Tnh
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 83
khng m bo o m rng theo cng thc sau:

TH
U k U
k l h s ph
Trong a s trng hp, k c cho gi tr bng 2 tng ng vi
tin cy 95%. Tuy nhin trong mt s trng hp c th k cn ph thuc vo
tin cy mong mun v s bc t do, khi k c tnh bng cch tra
bng t-student (theo ph lc 1)

Nh v d trn y, khng m bo o m rng U = 2 x 0,004 = 0,008 g/l
Vy, cch ghi l 2,4980,008 mg/l tin cy 95%.
3.2. Cch 2: c lng khng m bo o t d liu phn tch mu
thc
Trong a s trng hp, vic tnh ton khng m bo o theo
tng bc nh trn rt tn km thi gian v cng sc. Vi mt phng php
thm nh, th vic ch b tr cc php th nghim v tnh khng m
bo o theo phng php thng k l tnh khoa hc v tit kim.
3.2.1. Xc nh khng m bo o trn mu cng nng
Thc hin theo cc bc sau:
- Phn tch mu chun bit hm lng, hoc phn tch mt mu
ng nht c hm lng xc nh;
- Phn tch mu lp li ti thiu 20 ln, tnh kt qu cc ln phn tch
(cc ln phn tch nn c b tr cc ngy khc nhau);
84 CHNG IV: C LNG KHNG M BO O

- Tnh gi tr trung bnh, lch chun v h s bin thin ca kt qu
phn tch;
- khng m bo o c tnh theo cng thc sau:
(%) CV t (%) U
k ,



Trong : U : khng m bo o tng (%)
CV : H s bin thin ca kt qu o (%)

k ,
t

: Gi tr t tra bng vi mc ngha = 0,05; bc t do k = n - 1


n : S ln phn tch lp li

V d: nh gi khng m bo o, ngi ta thc hin trn mu
chun o lp li 20 ln v thu c cc gi tr trung bnh x 39,5 mg/ kg
v lch chun SD=2,417 mg/kg. Tnh khng m bo o nh sau:
CV% =
SD
100
x
=6,12


,k
U(%) t CV(%) =1,73 x 6,12 = 10,6 %
U = 10,6% x 39,5 = 4,19 mg/kg
Cng b U nh sau: 39,5 mg/kg 10,6% hoc 39,5 4,19 mg/kg
3.2.2. Xc nh khng m bo o trn cc mu nng khc nhau
- Thc hin phn tch trn mu thm chun (vi cc nng khc
nhau).
- Phn tch mu lp li ti thiu 20 ln, tnh kt qu cc ln phn tch
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 85
(cc ln phn tch nn c b tr cc ngy khc nhau) t xc nh thu
hi ca cc ln phn tch.
- Tnh gi tr trung bnh, lch chun v h s bin thin ca thu
hi phn tch.
- khng m bo o c tnh theo cng thc sau:
(%) CV t (%) u
R k , R



Trong : u
R
: khng m bo o tng theo thu hi (%)
CV
R
: H s bin thin ca thu hi (%)

k ,
t

: gi tr t-student tra bng vi mc ngha = 0,05


k : Bc t do (k = n - 1)
n : S ln th nghim

V d: nh gi khng m bo o, ngi ta thc hin trn cc mu
trng thm chun trong khong nng t 10 n 100 mg/kg, tnh thu
hi v thc hin lp li 20 ln v thu c gi tr thu hi trung bnh l
97% v lch chun ca thu hi l 7%.
Tnh khng m bo o nh sau: CV% =
SD
100
x
= 6,87



,n 1
U(%) t CV(%) = 1,73 x 6,87 = 11,7 %
Cng b U nh sau: t 10-100 mg/kg 11,7%

4. Cng b khng m bo o
Phi thng bo cc thng tin sau y khi trnh by kt qu trong
cc bo co thm nh hoc khi c yu cu.
86 CHNG IV: C LNG KHNG M BO O

a. Kt qu o.
b. khng m bo o.
c. Mc tin cy c s dng trong vic xc nh khong ca
khng m bo o m rng.
4.1. Cch vit khng m bo o chun tng hp
Khi s dng h s ph k = 1 th khng m bo o chnh l
khng m bo o chun v c vit nh sau:
Kt qu (n v) vi khng m bo o chun U (n v)
Ch : Khng s dng du khi bo co khng m bo o chun v
du ny thng gn vi tin cy cao nh 95% v 99%.
4.2. Cch vit khng m bo o m rng
Thng thng, khng m bo o m rng vi h s ph k = 2 c vit
nh sau:
Kt qu (n v) U (n v)
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 87


88 CHNG V: M BO CHT LNG KT QU TH NGHIM

Chng V: M BO CHT LNG
KT QU TH NGHIM
Thm nh phng php phn tch khng phi l tt c m bo
cht lng kt qu th nghim. Theo yu cu ca ISO 17025, m bo cht
lng l mt qu trnh v cn thng xuyn thc hin. Phng th nghim
c cng nhn v duy tr ISO 17025 chnh l qu trnh m bo cht lng
lin tc, ngc li cc chng trnh m bo cht lng l mt yu t cc
k quan trng gip duy tr c ISO 17025. Trong khun kh ti liu ny
chng ti nu ln cc chng trnh v cch t chc vic m bo cht lng
ti phng th nghim.
C nhiu cch cc phng th nghim t chc cc chng trnh m
bo cht lng. Theo quy nh ca ISO 17025 (mc 5.9), phng th nghim
phi c cc th tc kim sot cht lng kim tra tnh hiu lc ca php
th nghim thc hin. D liu kt qu phi c ghi chp sao cho c th
nhn bit cc khuynh hng din bin ca cc kt qu v nu c th cn
phi p dng k thut thng k xem xt cc kt qu. Vic kim tra ny
phi c ln k hoch, sot xt li. Cc d liu v kim sot cht lng
phi c phn tch v khi nhng d liu ny nm ngoi chun mc nh
th phi c hnh ng khc phc iu ny v ngn nga kt qu sai c
thng bo.
Chng ny gii thiu cc k thut v cch t chc cc chng trnh
m bo cht lng phng th nghim.
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 89
1. Php th nghim lp li
Cho cng mt nhn vin thc hin vic phn tch mt ch tiu no
xc nh trn cng mt mu th ng nht, trn cng mt thit b, cng
phng php trong mt khong thi gian tng i ngn v nh gi mc
lp li cc kt qu kim tra.
Trong phn tch hng ngy, cn thc hin song song ti thiu hai ln
nhm trnh c cc sai s ngu nhin gp phi. nh gi mc chnh
lch gia hai ln lm vi gi tr trung bnh, s chnh lch ny phi tha mn
theo yu cu ca tng phng php.
2. Php th nghim ti lp
Cc phng th nghim nn b tr cho nhn vin thc hin cc php
th nghim ti lp, bng cch cho thc hin trn cc thit b khc nhau hoc
cho cc nhn vin khc nhau thc hin cng mt php th nghim (i tay
nhn vin).
3. Php th nghim trn mu lu
C th b tr nhn vin thc hin cc php th nghim trn mu
lu, tuy nhin nh gi c kt qu cn m bo n nh ca mu
theo thi gian v trong iu kin bo qun ca phng; ng thi ch nn p
dng i vi cc cht c bit l tng i bn vng trong iu kin nht
nh no .
4. Php th nghim trn mu trng
Mu trng l mu tng t nh mu th nhng khng c cht phn
tch. Vic thc hin php th trn mu trng l yu cu cn thit i vi a
90 CHNG V: M BO CHT LNG KT QU TH NGHIM
s phng php. Trong mt s trng hp khi khng th c c mu trng
th c th thc hin mu trng thuc th, tc l thc hin tng t nh mu
nhng khng c giai on cn mu.
i vi cc phng php phn tch vi lng v phn tch hm lng
vt, vic s dng cc mu trng ng vai tr rt quan trng loi tr
nhim cho trong qu trnh phn tch. Trong mt l mu phn tch (chy
my) cn chy mu trng sau mt khong nht nh (v d sau mi 10 mu)
kim tra lng qu mu cn st li trong h thng. S lng cc mu
trng cn phi chim t nht 5% tng s mu phn tch.
5. Php th nghim trn mu chun
Mu chun c chng nhn (CRM) hoc mu chun th cp l bng
chng quan trng khng nh kt qu phn tch. Vic c c cc mu
chun c chng nhn thng rt kh khn do kinh ph, khi c th cn
nhc s dng cc mu chun th cp nh l mu kim tra. Cc mu ny c
th l mu thu c t cc chng trnh so snh lin phng th nghim,
mu c chun li t cc mu CRM hoc cc mu chun do phng th
nghim t to.
6. Php th nghim trn mu thm
Trong trng hp khng c cc mu chun, th s dng mu thm
chun l mt cch ph bin v cn thit. Bi v chun thm vo mu v bn
thn cht phn tch c s tng tc khc nhau vi nn mu nn mu thm
cn c thc hin sao cho s khc bit ny l nh nht. Chun cn c
thm ngay t u, sau giai on cn ong v m bo thi gian ng nht
vi nn mu. V d nh, i vi cc php phn tch d lng trn cc nn
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 91
mu sinh hc thng thm chun vo ngy hm trc khi phn tch, c th
qua m ng nht.
7. S dng cc phng php khc nhau
Phng th nghim c th s dng nhiu phng php khc nhau
kim tra mt ch tiu no . Tt nht l so snh kt qu ca phng php
thc hin vi mt phng php tiu chun.
8. nh gi s ph hp ca h thng
Thng thng cc phng php phn tch sc k hay quang ph cn
c s nh gi m bo h thng ph hp phn tch. nh gi s ph
hp ca h thng l cc php th chng minh rng h thng hot ng
ng theo mc ch s dng. Cn nh gi hng ngy ty thuc vo n
nh ca h thng hoc s lng mu phn tch hng ngy.
Php th nh gi s ph hp ca h thng ca cc phng php sc
k c quy nh theo nhiu t chc (USP, USFDA) nh sau:
- chm ca thi gian lu, din tch pc sc k: Tim trc tip dung
dch chun nhiu ln lp li vo h thng sc k v xc nh lch
chun tng i (RSD). S ln bm ti thiu 5 ln phi cho RSD
nh hn 2%. Nu RSD ln hn 2% cn s dng 6 im;
- phn gii gia hai cht;
- H s ko ui;
- Cc thng s khc: nh tri ng nn, nhiu ng nn (khi
gii hn pht hin ng vai tr quan trng trong phng php).
92 CHNG V: M BO CHT LNG KT QU TH NGHIM
9. Tham gia cc chng trnh th nghim lin phng
Tham gia cc chng trnh th nghim lin phng l mt yu cu bt
buc nu phng th nghim mun c cng nhn ISO 17025. Hng nm,
phng th nghim cn lp k hoch tham gia cc chng trnh th nghim
lin phng, xem y l cch tt nht nh gi nng lc bn thn phng
th nghim ca mnh. Hin nay c nhiu t chc Vit Nam v trn th
gii cung cp cc chng trnh th nghim lin phng, cc phng th
nghim cn tm kim cc chng trnh ph hp vi phm vi ca phng
mnh tham gia.

Hnh 10: So snh lin phng th nghim kiu mu
Tr mt s trng hp c bit, thng thng da vo cc kt qu thu
c t cc chng trnh th nghim lin phng, phng th nghim cn c
hng x l v khc phc:
- Nu |Z| 3: C s lc. Phng th nghim cn tm hiu nguyn nhn
gy sai lch v c hnh ng khc phc ph hp, thng thng kt qu khc
phc cn c gi cho n v t chc.
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 93
- Nu 2 < |Z |< 3: Nghi ng. Phng th nghim khng bt buc phi
thc hin hnh ng khc phc, tuy nhin phng cn tm hiu cc nguyn
nhn v c hnh ng phng nga.
- Nu |Z| 2: Khng c s lc.
10. S dng biu kim sot
Biu kim sot (Control Chart) l mt trong nhng cng c thng
k rt hu ch gip cho qu trnh thc hin cc chng trnh m bo cht
lng. C nhiu dng biu kim sot khc nhau, trong dng thng
c s dng trong phng th nghim l biu trung bnh. Biu kim
sot loi ny c xy dng t gi tr trung bnh v lch chun ca mu
kim tra (QC), bao gm gi tr trung tm v cc gi tr gii hn cnh bo
trn (UWL Upper Warning Limit), gii hn cnh bo di (LWL Lower
Warning Limit), gii hn hnh ng trn (UAL Upper Action Limit) v
gii hn hnh ng di (LAL Lower Action Limit). Thng thng cc
gii hn ny tng ng vi quy tc 2SD v 3SD (SD l lch chun).

Hnh 11: M hnh biu kim sot dng trung bnh
94 CHNG V: M BO CHT LNG KT QU TH NGHIM
Trong qu trnh phn tch, ty theo gi tr v xu hng ca mu QC
m c bin php khc phc. Cn ngng thc hin phn tch, kim tra cc kt
qu ca cc mu thc phn tch km theo v c hnh ng ph hp nu xy
ra mt trong cc trng hp sau:
- C bt k mt mu QC vt ra ngoi khong hnh ng (LAL
UAL)
- C nhiu hn hai trong ba mu lin tc vt ra ngoi khong cnh
bo (LWL UWL)
- Cc gi tr mu QC c xu hng nm v mt pha ca gi tr thc
hoc gi tr c chp nhn l ng (trong trng hp bit nng mu
QC): Theo ISO 8258, nu c ln hn 9 im nm v mt pha th khng
chp nhn.
- Hm lng mu QC c xu hng tng hoc gim theo thi gian phn
tch: Theo ISO 8285, nu c 6 im lin tc ca mu QC tng hoc gim
(vn nm trong gii hn) th cn dng li v kim tra li kt qu.
Tt c cc hot ng ni trn u l cc chng trnh nht nh nhm
m bo cht lng kt qu th nghim. hng ti m bo cht lng
ton din, th p dng v thc hin ng theo cc yu cu ca ISO 17025 l
cch tt nht.
THM NH PHNG PHP TRONG PHN TCH HA HC V VI SINH VT 95
Ph lc 1: Bng phn phi chun Student
vi cc mc ngha t 0,10 n 0,001

Bc t do
0,10 0,05 0,01 0,005 0,001
1 6,314 12,71 31,82 63,66 318,3
2 2,920 4,303 6,965 9,925 22,33
3 2,353 3,182 4,541 5,841 10,21
4 2,132 2,776 3,747 4,604 7,173
5 2,015 2,571 3,365 4,032 5,893
6 1,943 2,447 3,143 3,707 5,208
7 1,895 2,365 2,998 3,499 4,785
8 1,860 2,306 2,896 3,355 4,501
9 1,833 2,262 2,821 3,250 4,297
10 1,812 2,228 2,764 3,169 4,144
11 1,796 2,201 2,718 3,106 4,025
12 1,782 2,179 2,681 3,055 3,930
13 1,771 2,160 2,650 3,012 3,852
14 1,761 2,145 2,624 2,977 3,787
15 1,753 2,131 2,602 2,947 3,733
16 1,746 2,120 2,583 2,921 3,686
17 1,740 2,110 2,567 2,898 3,646
18 1,734 2,101 2,552 2,878 3,610
19 1,729 2,093 2,539 2,861 3,579
20 1,725 2,086 2,528 2,845 3,552
21 1,721 2,080 2,518 2,831 3,527
22 1,717 2,074 2,508 2,819 3,505
23 1,714 2,069 2,500 2,807 3,485
24 1,711 2,064 2,492 2,797 3,467
25 1,708 2,060 2,485 2,787 3,450
26 1,706 2,056 2,479 2,779 3,435
27 1,703 2,052 2,473 2,771 3,421
28 1,701 2,048 2,467 2,763 3,408
29 1,699 2,045 2,462 2,756 3,396
30 1,697 2,042 2,457 2,750 3,385

1,645 1,960 2,326 2,576 3,090

Ph lc 2: Bng phn phi chun Fisher vi k1, k2 l cc bc t d o, l mc ngha

k
2

k
1

97
Ph lc 2 (tip theo)

k
2
k
1

Ph lc 3: La chn i tng mu thm nh phng php vi sinh
Sn
phm
Yersi-
nia
spp.
Cl.
Perfr-
ingens
L.
monocy-
togenes
E. coli
O157
&VTEC
Staph.
aureus
S.aureus
entero-
toxins
Campy-
lobacter
spp.
Salmo-
nella
spp.
Bacillus
cereus
Men
mc
VK
Lactic
Tng
khun
lc
Coliforms E.
coli
Sn phm tht
Ti
sng
X X X X X X X X X X
Ch
bin
X X X X X X
ng
lnh
X X X X
Ln
men
X X X X
p X X X X X
Khc Mn
n,
nc
st
Pat X X
Sn phm tht gia cm
Ti
sng
X X X X X X X
Ch
bin
X X X
ng
lnh
X X
Khc
C v thy sn
Ti
sng
X X X X X X X X
Ch
bin
X X X
ng X X

99
lnh
Hun
khi
X X X X X X
Khc
Sn phm t rau qu
Ti
sng
X X X X X X X X
Ch
bin
X X
ng
lnh
X X
Kh X X X X
Ln
men
X
p X
Nc
p
X X
m
thp
X X X
Khc

Ph lc 3 (tip)

Sn phm Yersi-
nia
spp.
Cl.
Perfr-
ingens
L.
monocy-
togenes
E. coli
O157
&VTEC
Staph.
aureus
S.aureus
entero-
toxins
Campy-
lobacter
spp.
Salmo-
nella
spp.
Bacillus
cereus
Men
mc
VK
Lactic
Tng
khun
lc
Coliforms E.
coli
Ng cc v sn phm
Ti sng X X X X X X X X X X X
Ch bin X X X
ng lnh X X X X X X X X
Ln men X X X X X X X
Kh X X X X X X X
Khc
Sn phm chocola v bnh m
m thp X X X X
Kh X X X
Khc Trng
sa

Sn phm khc
Bia X
Nc st X X
Gia v X X
Mayonnaise X X X
M
Trng X
Go
Thc n
chn nui
X X X

101
TI LIU THAM KHO
1. Ludwig Huber (2007), Validation and Qualification in Analytical
Laboratories, Informa Healthcare, London.
2. TCVN ISO/IEC 17025:2005, Yu cu chung v nng lc ca phng th
nghim v hiu chun.
3. AOAC International (2007), How to meet ISO 17025 requirements for
method verification, USA.
4. Vn phng cng nhn cht lng (2010), Yu cu b sung cng nhn
cc phng th nghim lnh vc Ha (AGL3).
5. Hewlett Packard (HP) (1993), Peak purity analysis in HPLC and CE
using diode-array technology, Germany.
6. ICH (1996), Validation of Analytical Procedures: Text and
methodology, ICH Hamonised Tripartite Guideline.
7. Eurachem (1998), The fitness for purpose of Analytical methods A
laboratory guide for method validation and related topics, Eurachem
guide.
8. Centre dexpertise en analyse environnementale du Qubec (2009),
Protocole pour la validation dune mthode danalyse en chimie (DR
12 VMC), Canada.
9. Elison S. L. R. & al. (2000), Quantifying Uncertainty in Analytical
Measurement, 2 edition, Eurachem/Citac Guide.

10. Standards Council of Canada (2009), PALCAN Interpretation and
Guidance on the Estimation of Uncertainty of Measurement in Testing
(APLAC TC 005), Canada.
11. T Th Tho, Thng k trong ha phn tch, Gio trnh Trng i hc
Khoa hc T nhin.
12. National Public Health Service for Wales (2005), Uncertainty of
Measurement in Testing.
13. European Communities (2002), The performance of Analytical Methods
and interpretation of results, (2002-657-EC)
14. SANCO (2007), 3131, Method validation and quality control procedure
for pesticide residues analysis in food and feed, SANCO 2007/3131.
15. Singapore Accreditation Council (2002), Method Validation for
Microbiological methods, Guidance Note.
16. ISO 16140 (2003), Microbiology of food and animal feeding stuffs
Protocol for the validation of alternative methods.

103
VIN KIM NGHIM AN TON V SINH THC PHM QUC GIA



THM NH PHNG PHP
TRONG PHN TCH
HA HC V VI SINH VT


Chu trch nhim xut bn: TS. Phm Vn Din
Bin tp: Nguyn Kim Dung
Trnh by ba: Trnh Th Thy Dng






NH XUT BN KHOA HC V K THUT
70-TRN HNG O - H NI

In 1000 cun kh 16x24cm ti
S ng k KHXB s 215-2010/CXB/54.1-17/KHKT, ngy 05/3/2010.
Quyt nh XB s 182/QXB-NXBKHKTngy 13 thng 8 nm 2010.
In xong v np lu chiu thng 8 nm 2010.

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