nh lng Sc k lng. Phn tch Melamine trong sn phm sa.

S dng Agilent 1120 Compact LC v1200 Rapid Resolution LC.

Tm tt
+Trong ng dng ny, ba phng php LC khc nhau c pht trin xc nh melamine trong cc sn phm sa. +u tin l mt sa i phng php ca Hoa K FDA [1]. Mt h thng Agilent LC (1120 hoc 1200) c s dng vi mt ct ZORBAX LC SBC8 chy trong ch cp ion pha o ngc l phng php thng xuyn nh lng melamine. +Phng php th hai nhm mc tiu cho nng sut cao bng cch s dng mt h thng Agilent Rapid Resolution LC (RRLC) (1200SL) tng tc phn tch melamine hn ba ln vi mt ct Rapid Resolution High Throughput (RRHT). +Th ba l phng php LC trao i ion thay th, trong ct ZORBAX 300SCX c s dng gi melamine li bng cch s dng mt giai on n gin, pha ng gm dung dch m / acetonitrile m khng c s hin din ca cp ion tinh khit. Do s phc tp ca cc yu t nhiu gy ra bi nn sn phm sa, bc lm sch s dng chit pha rn (SPE) l cn thit cho cc phng php trn. +Agilent SampliQ SCX, phng php SPE s dng cht hp ph dng hn hp polymer kt hp vi pha o ngc. Cc cation mnh lm thay i tnh cht, v c s dng loi b cc yu t nhiu bi nn . Gii thiu +Thng 3 nm 2007, Hoa K nhp khu nguyn liu thc n vt nui b nhim vi melamine, gy ra suy thn ch, mo. Mt ln na, hp cht ny

b cm trong cc sn phm sa nhp khu t Trung Quc v l mi quan tm ca ton cu, cc xt nghim melamine c tin hnh mt cch rng ri. Cc phng php phn tch ph bin bao gm LC, LC / MS, v GC / MS. Cc phng php LC ang c s dng cho phn tch nh lng melamine. Phng php tch cc hp cht cc nh c th t c bng sc k lng cp ion pha o ngc. FDA Hoa K pht trin phng php ny trong thc n cho vt nui c cha melamine trong nm 2007. Vi mt cht sa i t l cc cht trong pha ng, phng php ny c th p dng thnh cng tch melamine trong cc loi sn phm sa. +Nhng bt li thng gp ca HPLC l thi gian v dung mi dng phn tch. Cc h thng Agilent 1200 Series Rapid Resolusion LC c thit k cho nng sut cao nht, hoc tt hn khi kt hp vi ct Agilent ZORBAX RRHT. Lu trong ng dng ny, phng php HPLC thng c chuyn t ct ZORBAX SB-C8 4,6 mm 250 mm, 5 micromet thnh ct RRHT ZORBAX SB-C8 4,6 mm 50 mm, 1,8 micron vi kt qu tng ng, v thi gian chy LC c rt ngn t 20 pht cn li l 6 pht. +Phng php tip cn mi thay th cho phng php tch sc k lng l phng php sc k trao i ion. +Agilent ZORBAX 300SCX l mt ct c pha gn ionic dng trao i cation trong sc k lng hiu sut cao. Ct ny c p dng thnh cng gi li melamine bng cch s dng mt pha ng gm dung dch m / acetonitrile m khng c s hin din ca cp ion tinh khit. +i vi nn sn phm sa phc tp, n l cn thit loi b nhiu chng hn nh protein, ng v cht bo trc khi phun mu. Chit pha rn (SPE) l mt cch n gin lm sch cc nn phc tp. SampliQ l mt phng php mi trong phng php SPE s dng cht hp ph ion t Agilent . Trong s cc phng php ny, cc cation trao i l mt polymer ca acid sulfonic v divinyl benzen. iu ny lm cho phng php SampliQ SCX rt hiu qu lm sch sau khi tch chit dung mi. Th nghim Chun b cht chun Mt dung dch cha melamine c nng l 1.000 mg / mL c chun b trong methanol. Pha long trong pha ng c thc hin nng 0.05, 0.1, 0.5, 1.0, 5.0, 10,0, 50,0, v 100,0 mg / mL

Chun b mu Qu trnh chun b mu c sa i theo tiu chun ca Trung Quc Th tc chit mu i vi mu sa nc, sa bt, sa chua, kem, v ko sa: Cn 2 0,01 g mu th v thm vo mt ng ly tm 50 ml, thm 15 ml dung dch axit tricloaxetic 5% trong nc v 5 ml dung dch acetonitrile, sau y np. Khy mu trong 10 pht v sau t mu trn theo chiu dc ca my rung trong10 pht. Ly tm trong 10 pht 4000 rpm. Lm t giy lc bng dung dch axit tricloaxetic 5%, sau lc cc cht ni trn b mt vo mt bnh c th tch 25,0 mL bnh, sau thm mt lng 5% axit tricloaxetic vo bnh cho n vch 25,0ml. Ly 5.0 ml sn phm chit xut vo mt ng thy tinh, v sau thm 5,0 mL tinh khit nc, sau dng my xoy trn. i vi mu pho mt, kem v s c la: Cn 2 0,01 g mu th cho vo ci xay vi 8 ~ 12 g ct bin, v sau chuyn vo mt ng ly tm 50 ml. Ra sch cc va vi 5 mL tricloaxetic 5% axit trong nc ba ln, chuyn sn phm vo ng li tm 50 ml, v sau thm 5 ml dung dch acetonitrile. Tip tc, khy mu v cc bc khc nh m t cch tin hnh trn .. Nu mu nhiu cht bo, ly cht bo khi cht chit xut bng cch s dng chit cht bo vi hexane bo ha vi 5% tricloaxetic axit trong nc trc khi c lm sch bi SPE. Tin trnh x l mu Mt hp cht hp ph (cation trao i) SampliQ SCX SPE (p/n 5982-3236, 3 mL, 60 mg, hoc p/n 5982-3267, 6 mL, 150 mg) c th c s dng lm sch mu chit, lu ng dng ny c s dng sau ny. Tt c cc bc ra gii bng SPE, bao gm : cc bc chun b ban u (iu kin), ti mu, ra v ra gii cui cng : tc dng chy phi nh hn 1ml/pht, ngoi tr vic lm kh cht hp th bng chn khng. iu kin hp cht hp ph (cation trao i) vi 5ml methanol v tip theo l 6ml nc. Ti cc mu chit sut trn ln cht hp ph c iu kin.Ra sch cht hp th vi 5 ml nc,sau ra tip bng 5 ml methanol.

 Lm kh cht hp ph bng chn khng, v sau ra gii vi 5 ml dung dch amoni hydroxit 5% trong methanol. Bay hi nc gii hp bng cch lm kh di mt dng nit khong 50 C Khi phc li cc chit sut kh trong 1,0 ml dung mi pha ng, xoy bng sng m trong 1 pht v lc qua mt mng lc cellulose ti sinh (p / n 5064-8222) vo mt l thy tinh Thit b v iu kin Phng php HPLC thng thng s dng 1120 Compact LC hoc 1200 LC: H thng Agilent 1120 Compact LC vi bm gradient (kh kh bn trong), mu t ng, ct ngn,pht hin v thay i bc sng (VWD) hoc 1200 thnh phn tng ng. S dng phn mm EZChrom Elite Compact hoc ChemStation (Ver. B.04.01 or later) Ct ZORBAX SB-C8 (cn c gi l ZORBAXRx-C8), 4.6 mm 250 mm, 5 micromet(p / n 880975-906) m 10 mM citric acid, 10 mM sodium c ch s octansulfonate, iu chnh pH 3,0 Pha ng 92:8 m: acetonitrile Tc dng chy 1,5 ml / pht Khi lng tim 20 mlCt nhit 30 C Pht hin bc sng 240 nm Thi gian chy 20 pht Phng php cng sut cao s dng 1200SL RRLC: Agilent 1200SL Dng nh phn bm, kh kh, a mng ly mu, ngn ct iu nhit t ng v diode pht hin mng (DAD) Phn mm ChemStation (Ver. B.04.01 hoc sau ) Ct ZORBAX SB-C8 RRHT, 4.6 mm 50 mm,1,8 micron (p / n 827975-906) m 10 mM citric acid, 10 mM sodium c ch s octansulfonate, iu chnh pH 3,0 Pha ng 92:8 m: acetonitrile Tc dng chy 1,5 ml / pht Th tch phun 8 ml Ct nhit 30 C Pht hin bc sng 240 nm Chy thi gian 6 pht Phng php sc k trao i ion vi 1120 Compact LC hoc 1200 LC:

Mt lot bm nh phn Agilent 1200, kh kh, a mng ly mu, ngn ct iu nhit t ng v bc sng detector thay i (VWD) hay 1200 thnh phn tng ng. Phn mm EZChrom Elite Compact hoc ChemStation (Ver. B.04.01 hoc sau ) Ct ZORBAX 300SCX, 4.6 mm 150 mm, 5 micromet(p / n 883952-704) m 50 mM formate amoni, iu chnh n pH 3,0 vi axit formic Pha ng 15:85 m: acetonitrile Lu lng 1.0 mL /pht Th tch phun 10 ml Ct nhit 30 C Pht hin bc sng 240 nm Thi gian chy 5,5pht Kt qu v tho lun Tch Melamine trong sn phm sa bng cch s dng phng php LC cp ion pha o ngc Melamine khng c gi li bi LC cp ion pha o ngc v do c tch ra vi dung mi v khng tip tc b nhiu do cht nn.Tuy nhin, bng cch s dng mt thuc th ion-ghp ni vi sc k pha o ngc, melamine c th c gi li v c tch khi nhiu. Hnh 1 (a) l sc k ca dung dch melamine chun bng LC cp ion pha o ngc Hnh 1 (b)l sc k ca mt mu sa chua tch cc sau khi c lm sch mu bng cht hp ph (cation trao i) SCX SPE SampliQ Agilent.

Hnh1 (a) Tch 20 ug / mL melamine tiu chun, v (b) mu sa chua sau khi lm sch bi SampliQ SCX SPE. Thi gian lu ca melamine l 14,2 pht.

Phn tch vi nng sut cao: tin hnh bi Agilent 1200SL RRLC v Ct RRHT. Vi h thng Agilent Dng RRLC s 1200, nng sut cao nht c th t khi kt hp ZORBAX Agilent vi ct RRHT, sc k c th t c phn gii cao nht vi thi gian chy ngn hn nhiu so vi quy c LC h thng. Phng php RRLC c pht trin ng k thng qua mu xc nh melamine trong cc sn phm sa. Hnh 2 (a) l sc ca mt mu melamine tiu chun theo phng php RRLC vi thi gian lu ca melamine l 2,8 pht. Hnh 2 (b) l sc ca cng mu sa chua trong hnh 1 (b). chc chn rng ct sch cho vic phun tip theo, tng thi gian chy c ko di thm 6 pht. Phng php RRLC nng sut cao c p dng cho vic phn tch cc sn phm t sa bao gm sa chua lng v sa bt chng minh rng phng php ny t cng phn gii nh vi phng php HPLC thng thng. Cc ng chun ca phng php RRLC c th hin trong hnh 3. Cc im chun bao gm 0.8, 2.0, 20,0, 40,0 v 80,0 ug / mL. +cc cng c LOD (gii hn pht hin) ca phng php RRLC l 0,03 mg / mL.

Hnh 2(a) Tch 0,8 ug / mL melamine tiu chun, v (b) mu sa chua sau khi lm sch
bi SampliQ SCX SPE. Thi gian lu ca melamine l 2,8 pht.

Hnh 3: ng chun ca phng php RRLC.

Phng php sc k trao i ion Cc Agilent ZORBAX 300SCX c s dng trao i cation trong phng php sc k lng hiu sut cao (HPLC). Ct ny c s dng tch ring bit melamine trong sn phm sa, vi s lu gi tch chng ring r. i vi melamine ta chn phng php sc k trao

i ion (IEC). Hnh 4 th hin vic tch ring bit melamine t mu sa m khng c bc lm sch SPE. Ni chung, tng thi gian chy ca sc k trao i ion ch khong 5,5 pht, vi mt LOD( gii hn pht hin): 0,05 ug / mL, nh th hin trong hnh 5. Cc im chun cho cc phng php IEC th hin trong hnh 6. Cc im chun bao gm 0.5, 1.0, 5.0, 10, 50, v 100 ug / mL. Mc d s tch bit c tin hnh vi nguyn liu sa v sa nc khng c bt k ph gia no, n vn c khuyn co phi tin hnh bc lm sch m bo vng chc phng php c th chy trn nhiu mu v cc mu khc nhau.

Hnh 4: Tch melamine trong sn phm sa bt bng phng php IEC khng qua bc lm sch bi SPE.

Hnh 5: Gii hn ca s pht hin( LOD) melamine nng 0,05 ug/ml.

Hnh 6: ng chun ca phng php IEC.

Kt lun Ba cch tip cn c m t trong lu ng dng; u tin l phng php LC cp ion-pha o ngc s dng Agilent 1120 Compact LC hoc 1200 HPLC vi mt ct SB-C8. Th hai l mt phng php hiu nng cao, lm gim thi gian chy LC t 20 pht cn 6 pht, bng cch s dng Agilent 1200 RRLC vi ct ZORBAX SB-C8 RRHT. Cui cng l phng php IEC s dng Agilent ZORBAX 300SCX ct. Cc phng php thnh cng trong vic loi b nhiu do nn sn phm sa, cung cp cch xc nh bng thi gian lu gi v kt qu nh lng. Kt qu ca nghin cu ny, bao gm lm sch mu vi phng php s dng cht hp ph (cation trao i) SampliQ SCX SPE v ba phng php tch, cung cp mt gii php Agilent hon chnh cho vic xc nh melamine trong cc sn phm sa. Phng php cp ion-pha o ngc da trn cc tiu chun ca FDA v Trung Quc. Tuy nhin ,IEC l phng php n gin, nhanh chng, nhy cm v mnh. Vi iu ny, melamine c th c gi li thnh cng bng cch s dng mt pha ng n gin m khng c s hin din ca mt thuc th ion cp.

Tham kho 1. Updated FCC Developmental Melamine Quantitation (HPLC-UV), April 2, 2007

2. GB/T 22388-2008 Determination of melamine in raw milk and dairy products, October 7, 2008 For More Information For more information on our products and services, visit Web site at www.agilent.com/chem.