Phng php sc k lng ghp u d khi ph

LCMS l phng php c dng trong phn tch vt (ppb, ppm) cc hp cht cn nhn danh chnh xc. Trong nhng iu kin vn hnh nht nh ngoi thi gian lu c trng, cc cht cn c nhn danh bng khi ph ca n.

1. Nguyn ly chung Phng php khi ph (Mass Spectrometry-MS) l phng php nghin cu cc cht bng cch o, phn tch chnh xc khi lng phn t ca cht da trn s chuyn ng ca cc ion nguyn t hay ion phn t trong mt in trng hoc t trng nht nh. T s gia khi lng v in tch (m/z) c nh hng rt ln i vi chuyn ng ny ca ion. Nu bit c in tch ca ion th ta d dng xc nh c khi lng ca ion . Nh vy, trong nghin cu khi ph ca bt k cht no, trc tin n phi c chuyn sang trng thi bay hi, sau c ion ho bng cc phng php thch hp. Cc ion to thnh c a vo nghin cu trong b phn tch khi ca my khi ph. Ty theo loi in tch ca ion nghin cu m ngi ta chn kiu qut ion dng (+) hoc m (-). Kiu qut ion dng thng cho nhiu thng tin hn v ion nghin cu nn c dng ph bin hn. Tuy nhin, s pht trin ca k thut hin nay cng cho php tch hp hai kiu qut ny thnh mt nhm to iu kin thun li nht cho cc nh nghin cu, tuy nhin thng nhy khng cao bng tng kiu qut ring l. Trong rt nhiu nm, cac nha nghin cu ky thut sc ky long ghep khi ph phai i mt vi rt nhiu kho khn trong vic tim cach giai quyt c s tng thich gia h thng sc ky long va u do khi ph. Nguyn nhn la do qua trinh phn tich vi u do MS oi hoi mc chn khng cao, nhit cao, cac cht khao sat phai trang thai khi, vn tc dong chay nho; trong khi h thng LC lai hoat ng ap sut cao vi mt lng dung mi tng i ln, nhit tng i thp, cac cht phn tich th long. khc phuc nhng kho khn trn, cn phai co mt ky thut trung gian gi la giao din. Rt nhiu ky thut giao din (interface technology) nh chum tia hat (FB), bn pha nguyn t nhanh dong lin tuc (CF-FAB), a c nghin cu va ng dung, nhng mai cho n cui thp nhin 80, mi co s t pha tht s vi ky thut ion hoa tai ap sut khi quyn (Atmospheric Pressure Ionization API). u im ni bt ca API l kh nng hnh thnh ion ti p sut kh quyn ngay trong bung ion ha. iu ny khc bit vi cc kiu ion ha s dng trc cho LC/MS nh bn ph nguyn t nhanh vi dng lin tc (continuous flow- fast atom bombardment CF-FAB) hay nh tia nhit (thermospray TS) u i hi p sut thp. Mt thun li na ca API l s ion ha mm (soft ionization), khng ph v cu trc ca hp cht cn phn tch nh thu c khi ph ca ion phn t. Ngoai ra, vi k thut ny, ngi ta c th iu khin c qu trnh ph v ion phn t to ra nhng ion con ty theo yu cu phn tch. C ba kiu hnh thnh ion ng dng cho ngun API trong LC/MS:

Ion ha tia in (electrospray ionization ESI). Ion ha ha hc ti p sut kh quyn (atmospheric pressure chemical ionization APCI). Ion ha bng photon ti p sut kh quyn (Atmospheric Pressure Photoionization APPI).

Trong o, hai k thut APCI v ESI, c bit la ESI c s dng nhiu hn c. 2. Cac loai u d khi ph Hin nay, c bn kiu u d khi ph chnh ang c s dng bao gm:

u d khi ph by ion (Ion Trap, IT) u d khi ph cng hng cyclotron s dung phep bin i Fourier (Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, FTICR hay FT-MS). u d khi ph thi gian bay (Time-of-Flight, TOF) u d khi ph t cc (Quadrupole)

Chng rt khc nhau v thit k v thao tc, vi nhng u va nhc im ring. i vi loi by ion, cc ion trc ht c bt hoc mc by trong mt khong thi gian nht nh ri c phn tch bng MS hoc MS/MS. Loi my ny c s dng kh rng ri (xt theo nhng cng b khoa hc). Tuy nhin, chng c chnh xc khng cao do ch c mt s lng kh hn ch cc ion c th tch ly vo tm im trc khi c tch in trong khng gian, do vy c th phn nh sai lch s phn b v php o. Ngi ta cng tin hnh ci tin k thut trn bng s pht trin cc by ion tuyn tnh hoc hai chiu khi nhng ion c tp hp trong mt th tch hnh ng ln hn by ion ba chiu truyn thng, cho php lm tng nhy, phn gii v chnh xc. FT-MS ban cht cng l mt loi khi ph by ion, cho php bt nhng ion di chn khng su trong mt t trng cao. Do c ci tin v kt hp c hai loi my nn FT-MS c nhy, chnh xc v phn gii cao. Tuy nhin, do vn hnh phc tp v gia thanh qua cao nn chng t c s dng trong phn tich d lng. i vi u do khi ph thi gian bay, tt ca cac ion n in tich nao chiu mt sai bit v th nng V se at mt nng lng chuyn hoa eV (electron volt) nh nhau. Vi vy, nhng ion co khi lng ln hn se co vn tc nho hn nn mt nhiu thi gian hn bay qua cung mt quang ng dai trong mt ng khng co t trng (field-free flight tube). Cac ion, sau khi c tng tc, bay ngang qua vung khng trng, ni y chung se c tach ring nhau ra tuy theo gia tri m/z cua chung, va c tp trung tai b phn thu nhn tin hiu. Do thi gian n ich cua cac ion chi cach nhau rt ngn, 10-7 giy, nn may cn co h thng in t cc nhay co th phn bit c cac ion. u do t cc (mt hoc ba t cc) co nhay cao trong phn tich inh lng mt cht a bit, tao c nhiu phn manh trong ch MS/MS, co th lam c kiu o mt phn t trung hoa (neutral loss), thich hp cho phn tich vi lng cac cht a bit trc cu truc. Tuy nhin, u do t cc kho giai thich c ch phn manh

MS/MS. Ty theo mc ch, cc nh nghin cu s chn cc k thut ng vi cc my khi ph thch hp. Ngoai ra, phat huy nhng u im cung nh khc phuc cac han ch cua tng loai u do, ngi ta con ghep ni kt hp cac ky thut vi nhau, vi du nh h thng kt hp gia by ion va t cc API QTRAP4000 hay QTRAP5500 cua hang Appliedbiosystems, trong o, t cc th ba cua h ba t cc c thay bng by ion. 4. Ngun ion ha trong u d khi ph 4.1. Ion hoa tia in (ESI) ESI l mt k thut ion ha c ng dng cho nhng hp cht khng bn nhit, phn cc, c khi lng phn t ln. ESI c kh nng to thnh nhng ion a in tch (dng hoc m, tuy thuc vao ap cc in th), c xem la ky thut ion hoa m diu hn APCI, thch hp cho phn tch cc hp cht sinh hc nh protein, peptide, nucleotide hoc cc polyme cng nghip nh polyethylen glycol. Trong ESI, cc ion c hnh thnh nh sau:

S tao ion dng bng ngun ESI

c bt ngun t h thng sc ky long, tai u ng dn mao quan, di anh hng cua in th cao va s h tr ca kh mang, mu c phun thnh nhng ht sng nh mang tch in ti b mt. Khi xung quanh cac giot nay tao nhit nng lam bay hi dung mi ra khoi giot sng, khi o, mt in tch ti b mt ht sng gia tng. Mt in tch ny tng n mt im gii hn (gii hn n nh Rayleigh) t ht sng phn chia thnh nhng ht nh hn v lc y lc ny ln hn sc cng b mt. Qu trnh ny c lp li nhiu ln hnh thnh nhng ht rt nh. T nhng ht rt nh mang in tch cao ny, cc ion phn tch c chuyn thnh th kh bi lc y tnh in ri sau i vo b phn tch khi. Trong ky thut ESI, phn t nht thit phai c bin thanh cht in ly, tan trong dung dich dung phun sng. iu nay phu thuc vao: dung mi s dung, pKa cua cht in ly va pH cua dung dich. Cac dung mi phu hp phun sng la: methanol, acetonitrile, ethanol, 4.2. Ion hoa hoa hoc ap sut khi quyn (APCI)

APCI la ky thut ion hoa thng c s dng phn tch nhng hp cht c phn cc trung bnh, c phn t lng nh, d bay hi. Trong APCI, ion c hnh thnh nh sau: mu hp cht cn phn tich, hoa tan trong pha ng, sau khi ra khoi ct sc ky, c cho i ngang qua ng mao quan t nong. Khi ra khoi ng, nh khi N2, dung dich c phun thanh dang sng t u ra ca ngun APCI. Cac giot sng nho c mt dong khi dn n mt ng thach anh un nong, goi la bung dung mi hoa khi. Hp cht i theo lung khi nong ra khoi ng n mt vung co ap sut khi quyn, ni y se xay ra s ion hoa hoa hoc nh vao que phong in corona, tai y co s trao i proton bin thanh ion dng (M + H)+ va trao i electron hoc proton bin thanh ion m (M H)-. Sau o, cac ion se c a vao b phn tich khi.

La chon kiu tao ion

4.3. Ion hoa bng photon tai ap sut khi quyn (APPI) Thng thng, hp cht phn tich thng ion hoa bng ngun ESI hoc APCI, tuy nhin, co mt s cht khng c ion hoa tt bng hai ky thut nay, vi du nh polyaromatic hydrocarbons (PAHs), ngi ta se s dung ngun APPI. Kt hp u im cua gia dong khi phun thng goc vi dong ion, en krypton trong ngun se phat ra cac photon co nng lng cao u ion hoa nhiu hp cht hoa hoc khac nhau. Ngoai ba ngun ion hoa trn, con co ngun ion hoa a phng thc (MMI, Multimode ionization) co th vn hanh, thao tac hai ch ion hoa ESI va APCI. 5. Mt s ky thut ghi ph trong u do khi ph 5.1. Scan Khi thao tc vi ch scan, u d s nhn c tt c cc mnh ion cho khi ph toan ion i vi tt c cc cht trong sut qu trnh phn tch. Thng dng nhn danh hay phn tch khi cht phn tch c nng ln. i vi u do khi ph ba t cc, ch SCAN thng c la chon khao sat ion me.

5.2. Selected Ion Monitoring (SIM) Trong ch SIM, u d MS ch ghi nhn tn hiu mt s mnh ion c trng cho cht cn xc nh. Khi ph SIM ch cho tn hiu ca cc ion c la chn trc , do vy khng th dng nhn danh hay so snh vi cc th vin c sn. i vi u do khi ph ba t cc, ch SIM thng c la chon khao sat nng lng phn manh khi a bit ion me. 5.3. SRM (Selected Reaction Monitoring) va MRM (Multiple Reaction Monitoring) i vi khi ph ba t cc, la may o khi ph hai ln lin tip (MS-MS), 2 ky thut ghi ph co nhay cao thng c s dung la SRM va MRM. SRM: c lp ion cn chn, sau phn mnh ion c lp , trong cc mnh ion sinh ra, c lp 1 mnh ion con cn quan tm v a vo u d pht hin. MRM: trn thc t, do yu cu v mt ky thut i vi phn tich vi lng nn cac ion con cn quan tm thng t 2 tr ln, do vy ky thut ghi ph MRM thng dung hn SRM. u tin, c lp ion cn chon (ion me) t cc th nht, phn manh ion c lp o tai t cc th 2 (thc cht la bung va cham) thu c cac ion con, c lp 2 (hoc nhiu) ion con cn quan tm t cc th 3 va a vao u do phat hin. Ngun: Phng Phn tch sc k (CASE)