28 TCN 186 : 2003 Hm lng cloramphenicol trong sn phm thu sn - Phng php nh lng bng sc k kh

Hm lng cloramphenicol trong sn phm thu sn - Phng php nh lng bng sc k kh Chloramphenicol in fishery products - Method for quantitative analysis by Gas Chromatography
1

Tiu chun ny qui nh phng php xc nh hm lng cloramphenicol (sau y gi tt l CAP) trong thy sn v sn phm thy sn bng h thng sc k kh (sau y gi tt l GC). Gii hn pht hin ca phng php l 0,3 g/kg.
2 Phng php tham chiu

Phm vi p dng

Tiu chun ny c xy dng da theo phng php c cng b trong tp ch ca Hip hi cc nh ho hc phn tch; tp 77, s 3, nm1994 (Journal of AOAC international; Vol. 77, No. 3, 1994: Gas chromatographic determination of chloramphenicol residues in shrimp).
3 Nguyn tc

CAP trong mu thy sn c chit tch bng etyl axetat. Dch chit sau c c cn, cn c x l vi sylon (cht to dn xut trimetylsylyl), to dn xut trimetylsylyl ca CAP. Hm lng dn xut CAP c xc nh trn h thng GC vi u d bt gi in t (sau y gi tt l ECD) theo phng php ni chun.
4 Thit b, dng c, ho cht, dung dch chun v dung dch th

4.1 THIT B, DNG C

4.1.1 H thng GC vi u d ECD v b phn tim mu t ng. 4.1.2 Ct mao qun HP-5, kch thc 25m x 0,2 mm, lp film 5% phenyl metyl silicon dy 0,33 m hoc ct tng ng.

4.1.3 H thng c quay chn khng: Buchi R 110 hoc tng ng. 4.1.4 H thng c N - Evap model 111 hoc tng ng.
4.1.5 Xilanh 10 l.

4.1.6 Bnh qu l dung tch 100 ml.

4.1.7 ng ly tm dung tch 15 ml, 50 ml. 4.1.8 Pipet Pasteur vi qu bp cao su. 4.1.9 My ly tm tc 5000 vng/pht. 4.1.10 My rung trn mu. 4.1.11 My nghin ng th. 4.1.12 B iu nhit 4.2 HO CHT Ho cht phi l loi tinh khit c s dng phn tch, gm: 4.2.1 Etyl axetat. 4.2.2 Nc loi dng cho HPLC. 4.2.3 Hexan. 4.2.4 Toluen. 4.2.5 Natri clorua (NaCl). 4.2.6 Tc nhn to dn xut Trimetyl silyl: Sylon HTP kit. 4.2.7 Chun CAP. 4.2.8 Cht ni chun m-nitro CAP (M-CAP). 4.2.9 Metanol. 4.3 DUNG DCH CHUN V DUNG DCH TH 4.3.1 Dung dch mui 4 %: ho tan 40 g NaCl (4.2.5) trong 1000 ml nc ct (4.2.2).
4.3.2 Dung dch ni chun CAP gc (100 g/ml): cn 10,0 mg CAP (4.2.7) vo bnh nh mc dung tch 100 ml. Ha tan v nh mc n vch 100 ml bng metanol (4.2.9).

4.3.3 Dung dch ni chun CAP lm vic (1000 ng/ml): ht chnh xc 1,00 ml dung dch chun CAP gc (4.3.2) vo bnh nh mc 100 ml, nh mc n vch bng Metanol (4.2.9).
4.3.4 Dung dch chun M-CAP gc (100 g/ml): cn 10,0 mg M-CAP (4.2.8) vo bnh nh mc 100 ml. Ha tan v nh mc n vch 100 ml bng metanol (4.2.9).

4.3.5 Dung dch chun M-CAP lm vic (1000 ng/ml): ht chnh xc 1,00 ml dung dch chun M-CAP gc (4.3.4) vo bnh nh mc 100 ml, nh mc n vch bng metanol (4.2.9).
4.3.6 Dung dch cc chun c to dn xut: ht chnh xc 100 l dung dch t cc chun lm vic (4.3.3) v (4.3.5) vo ng ly tm 15 ml (4.1.7). Lm kh bng dng nit. To dn xut nh c m t ti iu 5.4, nhng th tch cui cng l 1,0 ml.

4.3.7 Dung dch CAP (100 ng/ml): ht chnh xc 10,0 ml dung dch chun CAP lm vic (4.3.3) vo bnh nh mc 100 ml, nh mc n vch bng metanol (4.2.9).

4.3.8 Dung dch M-CAP (100 ng/ml): ht chnh xc 10,0 ml dung dch ni chun M-CAP lm vic (4.3.5) vo bnh nh mc 100 ml, nh mc n vch bng metanol (4.2.9)
5 5.1 Phng php tin hnh CHUN B MU TH

5.1.1 Cn 10,0 g mu thu sn c nghin nhuyn cho vo ng ly tm thy tinh dung tch 50 ml (4.1.7). Thm 100 l dung dch M - CAP lm vic (4.3.5) v 20 ml etyl axetat (4.2.1) vo trong ng ri nghin bng my nghin ng th (4.1.11) trong khong 10 -15 giy. Sau , ly tm trong 5 pht tc 3000 vng/pht. Gn dch trong vo bnh qu l 100 ml (4.1.6). 5.1.2 Cho thm 20 ml etyl axetat (4.2.1) vo phn cn trong ng ly tm ri trn u. Sau , ly tm trong 5 pht tc 3000 vng/pht. Gn dch trong vo bnh qu l trn. 5.1.3 C dch trong bnh qu l trn my c quay chn khng (4.1.3) cho n khi cn khong 2,0 - 4,0 ml dung dch nhit 50oC. Chuyn dch cn li sang ng ly tm 50 ml bng pipet Pasteur. Ra bnh qu l 3 ln, mi ln bng 2,0 ml etyl axetat. Tt c dch ra c cho vo ng ly tm. 5.1.4 C cn dung dch trong ng ly tm bng dng kh nit nhit 50 oC. 5.1.5 Thm 25 ml dung dch mui NaCl 4 % (4.3.1), 2 ml metanol (4.2.9) v 15 ml hexan (4.2.3) vo ng ly tm. y np ng ly tm v lc tht mnh trong 1 pht. Sau , ly tm trong 1 pht tc 800 vng/pht. 5.1.6 Loi b lp hexan trn bng pipet Pasteur (4.1.8). Lp li qu trnh ny thm 2 ln, mi ln 15 ml hexan.

5.1.7 CAP trong pha nc c chuyn sang pha hu c bng cch lc mnh vi 15 ml etyl axetat (4.2.1) trong 30 giy. Ly tm hn hp trong 1 pht tc 800 vng/pht. Dng pipet Pasteur chuyn lp etyl axetat trn vo bnh qu l 100 ml (4.1.6). Lp li qu trnh ny mt ln na vi 15 ml etyl axetat (4.2.1). Dch chit cng c a vo bnh qu l. 5.1.8 C hn hp dch chit (5.1.7) cn li khong 2 - 4 ml trn my quay chn khng nhit 55oC. Chuyn lng dch cn li vo ng ly tm 15 ml. Ra bnh qu l 3 ln, mi ln vi 2 ml etyl axetat. C cn dch thu c bng dng nit nhit 50 oC. Chuyn tip sang bc to dn xut Sylyl theo iu 5.4. 5.2 CHUN B MU TRNG Mu trng c nh ngha l mu thy sn c xc nh khng c cloramphenicol. Tin hnh chun b mu trng ging nh chun b i vi mu th qui nh ti iu 5.1. 5.3 CHUN B MU XC NH THU HI
CHO THM 30,0 L DUNG DCH CHUN CAP 0,1 G/ML (4.3.7) V 30 L DUNG DCH CHUN M-CAP 0,1 G/ML (4.3.8) VO 10,0G MU TRNG RI TIN HNH CHUN B MU GING NH CHUN B I VI MU TH QUI NH TI IU 5.1.

5.4 TO DN XUT SYLYL

Thm 100 l Sylon (4.2.6) vo cn trong ng ly tm, ng np ri lc mnh trn my rung trn mu (4.1.10). t ng ly tm vo trong b iu nhit (4.1.12) nhit 55oC trong 40 pht. Lm bay hi dung dch trong ng cho n gn kh (lu khng kh hon ton) di dng nit ti nhit trong phng. Thm 500 l toluen ri lc mnh cho tan cn hon ton.

5.5 Tin hnh phn tch trn h thng GC 5.5.1 iu kin phn tch t ch lm vic cho h thng GC nh sau: a. Chng trnh nhit ct: - Tng t nhit 1500C ln nhit 2700C vi tc 200C/pht, gi 8,5 pht. - Tng t nhit 2700C ln nhit 2900C vi tc 200C/pht, gi 2,0 pht. b. Nhit u tim: 270oC. c. Nhit detector: 340oC. d. Tc dng kh mang He: 1 ml/pht. . Kh b tr: N2, theo iu kin ca GC. e. Th tch tim: 1 l. 5.5.2 n nh ct sc k trong 3 gi ti ch lm vic. 5.5.3 Tim cc dung dch chun c to dn xut (4.3.6) trc v sau mi 5 dung dch th. Xc nh t s din tch pic (CAP/ M-CAP). 5.5.4 Tim cc dung dch c to dn xut ca mu th, mu trng, mu xc nh thu hi vo h thng GC mi mu 2 ln. Tnh kt qu thu c theo iu 6. 5.6 YU CU V TIN CY CA PHP PHN TCH 5.6.1 lp li ca 2 ln tim lch chun (CVS) tnh theo din tch pic sc k ca 2 ln tim cng mt dung dch chun phi nh hn 0,5 %. 5.6.2 thu hi (R) thu hi c xc nh cho mi ln chy mu. thu hi tnh c phi ln hn 80 %. 5.6.3 Kim tra xc nhn (confirmatory test) i vi cc mu pht hin Cloramphenicol bng phng php GC-ECD ny, phi kim tra xc nhn kt qu bng GC-MS.
6 Tnh kt qu

Hm lng cloramphenicol (C CAP) c tnh theo cng thc: C

CAP

XxCxV = -----------------YxW

Trong : - X l t s trung bnh din tch pic (CAP/M-CAP) cho dung dch mu th. - C l hm lng ca CAP trong dung dch cc chun c dn xut (4.3.6), tnh theo ng/g. - V l th tch cui cng ca dung dch mu th, tnh theo ml.

- Y l t s din tch trung bnh CAP/M-CAP trong dung dch cc chun c dn xut (4.3.6). - W l khi lng mu th, tnh theo g.

Thuc tr su gc phospho hu c trong thu sn v sn phm thu sn - Phng php nh lng bng sc k kh
Organophosphorus pesticides residues in fish and fishery products - Method for quantitative analysis by Gas Chromatography 1 Phm vi p dng Tiu chun ny qui nh phng php xc nh hm lng cc thuc tr su gc phospho hu c (sau y gi tt l OP) gm 5 loi thuc tr su chnh: dimethoate, chlorfenvinphos, chlorpyrifos, methidathion v phosmet trong thu sn v sn phm thu sn bng h thng sc k kh (sau y gi tt l GC). Gii hn pht hin ca phng php t 0,2 n1 mg/kg. 2 Phng php tham chiu Tiu chun ny c xy dng da theo phng php So snh cc k thut lm sch trong cc phng php phn tch chn lc d lng cc thuc tr su OP trong nhuyn th ' ng trong Journal of AOAC international phn 79, s 1, trang 123-131, nm 1996. 3 Nguyn tc Thuc tr su OP trong mu thy sn c chit tch ra bng hn hp axetonitrilaxeton. Dch chit c lm sch trn ct silicagel, sau c chit tch lm 2 phn on. Hm lng cc thuc tr su OP trong cc phn on chit c xc nh trn h thng GC vi 2 u d: u d bt gi in t (sau y gi tt l ECD) v u d ion ho nhit pht hin nit-phospho (sau y gi tt l NPD). 4 Thit b, dng c, ho cht, dung dch th v dung dch chun 4.1 Thit b v dng c 4.1.1 My sc k kh vi u d ECD v NPD. 4.1.2 Ct sc k thy tinh (c van kho), kch thc L x ID l 150 x 5mm v 50 x 20mm. 4.1.3 Ct mao qun (cho my GC): loi HP Ultra 2, kch thc L x ID = 25 m x 0,2 mm, dy lp pha tnh l 0,33 mm hoc tng ng. 4.1.4 My c quay chn khng. 4.1.5 My nghin ng th. 4.1.6 My trn cao tc. 4.1.7 T sy. 4.1.8 L nung. 4.1.9 Cn phn tch c chnh xc 0,0001 g. 4.1.10 Cc dng c thu tinh, bnh nh mc. 4.2 Ho cht Ho cht phi l loi tinh khit c s dng phn tch, gm: 4.2.1 Hexan loi dng cho GC. 4.2.2 Etyl axetat loi dng cho HPLC. 4.2.3 Sunfat natri dng ht, c lm kh bng sy qua m nhit 350 0 C v gi trong chai thu tinh. 4.2.4 Axeton loi dng cho GC. 4.2.5 Axetonitril loi dng cho GC 4.2.6 Kh mang: heli loi dng cho GC. 4.2.7 Bng thu tinh (SUPELCO, 2-0411).

4.2.8 Silicagel c ht 60 mesh: silica-60 (Merck, Darmstadt, Germany). 4.2.9 Silicagel c hot ho: cn mt lng silicagel (4.2.8), ra sch bng iclometan ri cho vo trong chn s v trong t sy nhit 120 0C trong vng 12 gi. Sau , dng giy thic bc kn ming chn s ri li vo t sy nhit 120 0C trc khi s dng. 4.2.10 Xelit 4.2.11 Cc cht chun thuc tr su OP (Promochem, Wesel, Germany): dimethoate, chlorfenvinphos (ng phn E+Z), chlorpyrifos, methidathion v phosmet. 4.3 Dung dch th v dung dch chun 4.3.1 Dung dch ra gii: hn hp gm dung dch axetonitril v axeton theo t l v th tch l 9:1 c pha trc khi s dng. 4.3.2 Chun b cc dung dch chun thuc tr su OP trong axeton t cc cht chun tinh khit (ln hn 99%). Tu theo nng thuc tr su c trong ng chun, dng bnh nh mc v lng axeton thch hp. Ch thch: cc chun phi c bo qun nhit - 200C. 4.3.3 Chun thu hi: pha cc chun ca cc OP trong dung mi axeton c hm lng trong khong 0,005 - 50 mg/ml. 4.3.4 Dung dch chun trung gian: pha long cc chun ban u (4.3.2) trong axeton c cc chun trung gian. 4.3.5 Dung dch chun cng tc: cc chun cng tc c chun b t cc chun trung gian (4.3.4) c pha long trong hexan (4.2.1) bm vo GC. 5 Phng php tin hnh 5.1 Chun b mu th 5.1.1 Cn chnh xc 5 g mu (k hiu m) c nghin bng my nghin ng th (4.1.5) ri cho vo cc thu tinh c m. Thm 1 g xelit (4.2.10), 30 g sunfat natri khan (4.2.3) ri nghin u. Tip tc thm vo 150 ml hn hp dung dch axetonitril-axeton theo t l v th tch l 9:1 (4.3.1) ri trn hn hp trong 3 pht trong my trn cao tc (4.1.6) chit ly cc thuc tr su t mu. Sau , lc dung dch bng giy lc. 5.1.2 Sn phm chit c c c trn my c quay n khong 5 ml, sau c tip cho n kh trn my c quay (4.1.4) trong ch chn khng nhit 40 oC. Thm vo dch thu c 1ml hexan (4.2.1) ri lm sch dch chit theo iu 5.4. 5.2 Chun b mu trng Mu trng c nh ngha l mu thy sn c xc nh khng c cha thuc tr su gc OP. Tin hnh chun b mu trng ging nh chun b i vi mu th theo qui nh ti iu 5.1. 5.3 Chun b mu xc nh thu hi Thm 1ml dung dch chun c hm lng t 0,005 n 50mg/ml vo 5 g mu trng, trn u mu bng my nghin ng th (4.1.5). Tin hnh chun b mu ging nh chun b i vi mu th theo qui nh ti iu 5.1. 5.4 Lm sch dch chit 5.4.1 Chun b ct Gm: bng thu tinh (4.2.7), 1,3 g silicagel c hot ho (4.2.9), 1 g sunfat natri khan (4.2.3). Trc khi tin hnh phn tch, cho vo ct 5 ml etyl axetat (4.2.2) v sau l 5 ml hexan (4.2.1). 5.4.2 Lm sch dch chit 5.4.2.1 Chuyn dch thu c ti cc iu 5.1.2, iu 5.2 v iu 5.3 vo cc ct sc k c chun b (5.4.1).

5.4.2.2 Ra dung dch trong ct vi 5 ml hexan, sau vi 2 ml hn hp hexan-etyl axetat theo t l v th tch l 95:5. Tip theo, ra gii vi 8 ml hn hp hexan-etyl axetat theo t l v th tch l 50:50 ri tch ly phn on 1. Sau , tip tc ra gii vi 5 ml etyl axetat ri tch ly phn on 2. Tc ra gii phi lun iu chnh t 10 git/pht. 5.4.2.3 Thu cc phn on chit vo cc bnh thu tinh y trn ca my c quay chn khng (4.1.4). Tin hnh c trn my c quay cho n kh. Sau , thm chnh xc 1 ml hexan vo tng bnh, lc cho tan u ri ht bm vo my GC theo iu 5.5. 5.5 Tin hnh phn tch trn GC 5.5.1 iu kin phn tch a. My sc k kh s dng u d ECD v NPD, chn ch bm t ng, mi ln mt lng 2ml theo ch tim khng chia. b. Ch nhit ca h thng GC: - Nhit ca bung tim: 2700C - Nhit u d ECD: 300 0C, tc dng N2: 60ml/pht - Nhit u d NPD: 2700 C, tc dng He: 30ml/pht, H2: 3ml/pht, khng kh: 100ml/pht. - Chng trnh nhit ct (m t chi tit trong Biu 1): Duy tr 90 0C trong 1 pht; tng 300C/pht, ln ti 1800C; tng 40C/pht, ln ti 2700C v duy tr 2700C trong 20 pht c. Ct sc k: Ct mao qun HP Ultra 2, cht nhi ct cha 5% metylsilicol, chiu di 25 m, ng knh trong 0,2 mm, dy lp pha tnh 0,33 mm. d. Kh: Kh mang: heli, tc 0,5ml/pht. 5.5.2 Tin hnh phn tch 5.5.2.1 Tim cc dung dch chun cng tc (4.3.5) vo my GC theo th t t nng thp n cao. Mi dung dch tim 2 ln, tnh din tch pic trung bnh. Dng ng chun biu th mi quan h gia cc din tch pic thu c v nng tng loi thuc tr su theo quan h tuyn tnh bc 1 (phng trnh y = ax+b). 5.5.2.2 Tim dung dch mu th, dung dch mu trng, dung dch xc nh thu hi c lm sch (5.4.2.3) vo h thng GC. Mi dung dch mu tim 2 ln. Tnh gi tr trung bnh. 5.6 Yu cu v tin cy ca php phn tch 5.6.1 lp li ca 2 ln tim lch chun (CVS) tnh theo din tch pc sc k ca 2 ln tim cng mt dung dch chun phi nh hn 0,5%. 5.6.2 thu hi (R) thu hi c xc nh bng cch s dng 10 mu trng c b sung mt lng dung dch chun tnh uc hm lng chnh xc. thu hi tnh c phi nm trong khong t 70 n 110% (tu theo hm lng OP c trong mu). 4.6.3 ng chun ca mi cu t phi c tuyn tnh tt, h s tng quan hi quy tuyn tnh (R2) phi ln hn hoc bng 0,99. 6 Tnh kt qu Hm lng tng loi thuc tr su c trong mu c tnh trn c s ng chun thu c ti iu 5.5.2.1. Vi ng chun dng y =ax+b, hm lng tng loi thuc tr su c trong mu c tnh theo cng thc sau:

(Y - b) C (g/kg) = ------------ x F a

Trong : - C l nng tng loi thuc tr su c trong mu, tnh theo mg/kg. - Y l hiu s gia din tch pic ca dch chit v din tch pic c trong mu trng tim vo GC, tnh theo n v din tch. - a, b l cc thng s ca ng chun y = ax + b, c xc nh theo iu 5.5.2.1. - F l h s pha long mu v c gi tr bng t s gia th tch phn on dch chit thu c sau khi lm sch (5.4.2.3) v khi lng mu m (5.1.1) s dng.

28 TCN 177 : 2002 Hm lng thuc khng sinh nhm Tetracyclin trong sn phm thy sn

Hm lng thuc khng sinh nhm tetracyclin trong sn phm thu sn Phng php nh lng bng sc k lng hiu nng cao

Tetracyclines group in fishery products - Method for quantitative analysis by high performance liquid chromatography

1 Phm vi p dng
Tiu chun ny qui nh phng php xc nh hm lng khng sinh nhm tetracyclin trong thy sn v sn phm thy sn bng sc k lng hiu nng cao (sau y gi tt l HPLC). Gii hn pht hin ca phng php l 10 g/kg.

2 Phng php tham chiu

Tiu chun ny c xy dng da theo phng php s 995.09 Chlortetracyclin, Oxytetracycline, and Tetracycline trong phn n c ca ng vt, quyn I, chng 23, trang 19 - 23 (23.1.17), phng php chun ca Hip hi cc nh ho hc phn tch (AOAC) ban hnh nm 1997.

3 Nguyn tc

3.1 Khng sinh nhm tetracyclin bao gm 3 khng sinh chnh sau: tetracyclin (gi tt l TC), oxytetracyclin (gi tt l OTC) v clortetracyclin (gi tt l CTC). 3.2 Cc khng sinh ny trong mu thy sn c chit tch bng dung dch m (pH 4). Dch chit c lm sch bng phng php chit pha rn (SPE) trn ct tch chit pha o Sep Pak Cartrige C18. Hm lng TC, OTC, CTC c trong dch chit c xc nh trn h thng HPLC vi u d UV ti bc sng 350 nm theo phng php ngoi chun.

4 Thit b, dng c, ho cht, dung dch chun v dung dch th 4.1 THIT B, DNG C 4.1.1 H thng HPLC vi u d UV. 4.1.2 Ct sc k pha o gm c: a. Ct sc k pha o C8, kch thc ct L x ID : 250 x 4,6 mm, kch thc ht 5 m.

b. Ct sc k pha o C18, kch thc ct L x ID : 250 x 4,6 mm, kch thc ht 5m.

4.1.3 My nghin ng th tc 10 000 vng/pht. 4.1.4 Cn phn tch c chnh xc 0,0001 g. 4.1.5 My ly tm tc 5 000 vng/pht (s dng ng ly tm dung tch 50 ml). 4.1.6 pH k 4.1.7 ng ly tm thy tinh dung tch 50 ml. 4.1.8 Bnh nh mc dung tch 10 ml, 100 ml v 1 000 ml. 4.1.9 Ct Sep-Pak C18, dung tch 10 ml. 4.1.10 Phu Buch-ner ng knh 5,5 cm. 4.1.11 Pipet t ng iu chnh c t 2 n10 ml.
4.1.12 B siu m.

4.2 HO CHT 4.2.1 inatri photphat khan (Na2HPO4) tinh khit, loi dng cho phn tch. 4.2.2 Nc ct loi dng cho HPLC. 4.2.3 Axit xitric ngm 1 phn t nc, tinh khit, loi dng cho phn tch. 4.2.4 Ethyleninitrilotetraaxtat inatri (sau y vit tt l EDTA) ngm 2 phn t nc tinh khit, loi dng cho phn tch. 4.2.5 Axt oxalic ngm 2 phn t nc, tinh khit, loi dng cho phn tch. 4.2.6 Metanol loi dng cho HPLC. 4.2.7 Axetonitril loi dng cho HPLC. 4.3 DUNG DCH CHUN V DUNG DCH TH 4.3.1 Dung dch m McIlvaine (pH 4,0 +/- 0,05) gm:

a. Dung dch A: ho tan 28,4 g Na2HPO4 khan (4.2.1) bng nc ct (4.2.2) trong bnh nh mc dung tch 1 000 ml (4.1.8). nh mc ti vch.
b. Dung dch B: ho tan 21,0 g axt xitric (4.2.3) bng nc ct (4.2.2) trong bnh nh mc (4.1.8). nh mc ti vch. Cho t t 625 ml dung dch A vo 1 000 ml dung dch B. Chnh pH ti 4,0 (+/- 0,05) bng cch cho tng git dung dch HCl nng 0,1M hoc dung dch NaOH nng 0,1M (s dng pH k xc nh pH). Ch thch: Dung dch bn trong vng 1 tun nhit trong phng.

4.3.2 Dung dch m McIlvaine - EDTA

Ho tan 60,5 g EDTA (4.2.4) vo 1 625 ml dung dch m McIlvaine (4.3.1). Ch thch: Dung dch bn trong vng 1 tun nhit trong phng.

4.3.3 Dung dch axit oxalic-metanol: ho tan 1,26 g axt oxalic (4.2.5) bng metanol (4.2.6) trong bnh nh mc 1 000 ml (4.1.8). nh mc ti vch.
Ch thch: Dung dch chit khng bn, ch pha trc khi s dng.

4.3.4 Pha ng cho HPLC: ho tan 1,26 g axt oxalc (4.2.5) bng nc ct (4.2.2) trong bnh nh mc 1 000 ml (4.1.8). nh mc ti vch. Thm vo dung dch 500 ml axetonitril (4.2.7) v 166 ml metanol (4.2.6). Lc v ui kh.
Ch thch: Dung dch pha ng khng bn, ch pha trc khi s dng.

4.3.5 Dung dch chun gc 1 000 g/ml: cn 108 mg (+/- 0,1) mi loi khng sinh chun (TC, OTC v CTC) loi c giy chng nhn hm lng (lng cn c iu chnh theo hm lng khng sinh ghi trong giy chng nhn) vo 3 bnh nh mc dung tch 100 ml (4.1.8) ring bit. Ha tan v nh mc ti vch 100 ml bng metanol (4.2.6). Ch thch: Bo qun dung dch ti nhit -20 0C. Dung dch bn trong vng 3 thng. 4.3.6 Dung dch chun hn hp 100 g/ml: ht chnh xc 10 ml cc dung dch chun gc (4.3.5) vo bnh nh mc 100 ml (4.1.8). nh mc ti vch bng metanol (4.2.6). 4.3.7 Dung dch chun hn hp 25 g/ml: ht chnh xc 2,5 ml dung dch chun hn hp 100 g/ml (4.3.6) vo bnh nh mc 10 ml (4.1.8). nh mc ti vch bng metanol (4.2.6). Ch thch: Bo qun dung dch trong t lnh nhit t 0 n 5oC. Dung dch bn trong vng 1 tun. 4.3.8 Dung dch chun hn hp 0,05; 0,10; 0,25; 0,50 v 1,00g/ml: ht chnh xc 20, 40, 100, 200, 400 l dung dch chun hn hp 25 g/ml (4.3.7) vo cc bnh nh mc 10 ml (4.1.8). Thm 6 ml

dung dch axt oxalic-metanol (4.3.3) vo mi bnh. nh mc ti vch bng nc ct (4.2.2). Ch thch: Bo qun dung dch trong t lnh nhit t 0 n 5oC. Dung dch bn trong vng 1 tun. 5 Phng php tin hnh 5.1 CHUN B MU TH 5.1.1 Cn 5,00 g (+/- 0,05) mu (m) c bm nhuyn cho vo ng ly tm thy tinh (4.1.7) th 1. Thm 20 ml dung dch m McIlvaine - EDTA (4.3.2) vo ng ri nghin trong 30 giy bng my nghin ng th (4.1.3). Sau , ly tm ng bng my ly tm (4.1.5) trong 10 pht tc 3 000 vng/pht. 5.1.2 Gn dch trong ca ng th 1 vo ng ly tm thy tinh (4.1.7) th 2. Cho thm 20 ml dung dch m McIlvaine - EDTA vo ng ly tm thu tinh th 1. Trn u, ly tm ng trong 10 pht tc 3 000 vng/pht ri li gn dch trong vo ng ly tm thy tinh th 2. 5.1.3 Cho thm 10 ml dung dch m McIlvaine - EDTA vo ng ly tm thu tinh th 1. Trn u ri ly tm ng trong 10 pht tc 3 000 vng/pht. Tip tc gn dch trong vo ng ly tm thy tinh th 2. 5.1.4 Ly tm ng thy tinh th 2 trong 20 pht tc 3 000 vng/pht. Sau , lc dch trong qua giy lc trn phu Buch-ner. Ra ng ly tm 2 ln, mi ln ra s dng 2 ml dung dch m McIlvaine - EDTA. 5.2 CHUN B MU TRNG
Mu trng c nh ngha l mu thy sn c xc nh khng c cc khng sinh thuc nhm TC. Tin hnh chun b mu trng ging nh chun b vi mu th theo qui nh ti iu 5.1.

5.3 CHUN B MU XC NH THU HI

Thm 100 l dung dch chun hn hp 25 g/ml (4.3.7) vo 5,00 g mu trng. ng nht mu bng my nghin ng th (4.1.3).Tin hnh chun b mu ging nh chun b vi mu th theo qui nh ti iu 5.1.

5.4 LM SCH DCH CHIT 5.4.1 Chun b ct Ni ct Sep - Pak C18 vo u ra ca mt xi lanh thy tinh dung tch 100 ml. Thm ln lt 20 ml metanol (4.2.6), 20 ml nc ct (4.2.2) vo xi lanh thy tinh. Loi b dung dch chy qua ct. 5.4.2 Lm sch dch chit

5.4.2.1 Cho ln lt cc dch chit trong ng ly tm thu c ti cc iu 5.1.4, 5.2 v 5.3 vo cc ct Sep - Pak c chun b iu 5.4.1. Trng ra bnh cha bng 2,0 ml dung dch m McIlvaine - EDTA (4.3.2). Trong giai on ny, cc khng sinh nhm tetracyclin s c hp ph ln b mt cc ht rn cha trong ct. Ch thch: Khng c cho ct Sep - Pak kh gia hai giai on chun b ct v lm sch dch chit. iu chnh cho dch chy ra khi ct tng git. 5.4.2.2 Cho 20 ml nc ct (4.2.2) vo xi lanh thy tinh v cho chy qua ct. Loi b dch chy ra khi ct. Lm kh ct bng dng khng kh sch trong 2 pht.

5.4.2.3 Gii hp cc khng sinh nhm tetracyclin bng cch cho 6,0 ml dung dch axt oxalic-metanol (4.3.3) chy qua ct vi tc 1,5 ml/pht. Thu dch ra khi ct vo bnh nh mc 10 ml. nh mc ti vch (th tch V) bng nc ct (4.2.2). Tin hnh phn tch dch thu c trn HPLC theo qui nh ti iu 5.5. 5.5 TIN HNH PHN TCH TRN HPLC 5.5.1 iu kin phn tch
a. Ct sc k : b. Nhit ct: c. Pha ng: d. Tc dng: Ct pha o C8, L x ID : 250 x 4,6 mm, kch thc ht 5 m. Nhit trong phng. Hn hp dung dch axit oxalic-metanol-axetonitrile (4.3.4). 1,2 ml/pht.

. Bc sng ci t cho u d UV l 350 nm. e. Th tch tim: 60 l.

5.5.2 n nh ct sc k trong 30 pht bng pha ng (4.3.4). 5.5.3 Tim cc dung dch chun (4.3.8) vo my HPLC theo th t nng t thp n cao. Mi dung dch tim 2 ln, tnh chiu cao pic trung bnh. Dng ng chun biu th mi quan h gia cc chiu cao pic thu c v nng (g/ml) tng loi khng sinh theo quan h tuyn tnh bc 1 (phng trnh y = ax + b). 5.5.4 Tim dung dch mu th, dung dch mu trng, dung dch xc nh thu hi theo qui nh ti iu 5.4.2.3 vo h thng HPLC. Mi dung dch mu tim 2 ln. Tnh gi tr trung bnh.
Ch thch: Sau khi phn tch xong, lm sch h thng HPLC (bao gm c ct sc k) bng hn hp: nc ct (4.2.2) - metanol (4.2.6) - axetonitrile (4.2.7) theo t l v th tch l 7-1-2 trong 30 pht.

5.6 YU CU V TIN CY CA PHP PHN TCH 5.6.1 lp li ca 2 ln tim

 lch chun (CVS) tnh theo chiu cao pic sc k ca 2 ln tim cng mt dung dch chun phi nh hn 0,5 %.

5.6.2 thu hi (R)

thu hi c xc nh cho mi ln chy mu phi ln hn 80 %.

5.6.3 ng chun i vi mi khng sinh phi c tuyn tnh tt, h s tng quan quy hi tuyn tnh (R2) phi ln hn hoc bng 0,995.

5.6.4 i vi cc mu cha t hn 0,5 g/g khng sinh nhm TC, phi kim tra xc nhn kt qu bng cch tim li dch chit mu v dung dch chun trn ct C18 (4.1.2.b) chy cng ch nh ct C8 (4.1.2.a) v so snh thi gian lu ca pc mu v pc chun. 6 Tnh kt qu
Hm lng cc khng sinh c trong mu c tnh trn c s ng chun thu c (5.5.3). Vi ng chun dng y = ax + b, hm lng cc khng sinh c trong mu c tnh theo cng thc sau: (Y - b) C (g/kg) = ------------ x F x 1 000 a Trong : - C l nng cc khng sinh c trong mu, tnh theo g/kg. - Y l hiu s gia chiu cao pic ca dch chit v chiu cao pic c trong mu trng tim vo HPLC, tnh theo n v di. - a, b l cc thng s ca ng chun y = ax + b, c xc nh theo iu 5.5.3. - F l h s pha long mu v c gi tr bng t s gia th tch dch chit thu c sau khi lm sch V ( 5.4.2.3) v khi lng mu m ( 5.1.1) s dng.