B Y T TRNG I HC DC H NI PHM KIM ANH NGHIN CU BN NH LNG MT S VITAMIN TRONG HN HP BNG PHNG PHP HPTLC KHA LUN TT NGHIP DC S H NI - 2014 B Y T TRNG I HC DC H NI PHM KIM ANH NGHIN CU BN NH LNG MT S VITAMIN TRONG HN HP BNG PHNG PHP HPTLC KHA LUN TT NGHIP DC SNgi hng dn:TS. Nguyn Th Thun Ni thc hin:B mn Ha Dc H NI - 2014 LI CM N t c kt qu nh ngy hm nay, xin cho php ti gi li cm n ti nhng ngi hng dn, gip v lun bn ti trong sut thi gian thc hin kha lun. Vilngbitnsusc,tixincgilicmnutintiTS. NguynThThun,gingvinbmnHaDctrngihcDc H Ni, ngi thy trc tip hng dn cho ti thc hin ti ny. Cng vi tri thc v tm huyt ca mnh, c truyn t cho ti vn kin thc qu bu,lng saymcng vic, tcphong lmvic nghimtc,khoahc...Em xin cm n c! Ti cng xin chn thnh cm n ton th cc thy c v anh ch k thut vin trong b mn Ha Dc trng i hc Dc H Ni, cc anh ch trong khoa Vt l o lng Vin Kim nghim thuc Trung ng to mi iu kin thun li cho ti hon thnh c kha lun. Cui cng, ti xin cm n gia nh, bn b l nhng ngun c v tinh thn ln lao, lun bn cnh ng vin, khch l ti trong sut thi gian qua. H Ni, 5/2014 Sinh vin Phm Kim Anh MC LC CC CH V K HIU VIT TT DANH MC CC BNG DANH MC CC HNH T VN Trang Chng 1. TNG QUAN ................................................................................. 2 1.1. Tng quan v i tng nghin cu. ......................................................... 2 1.1.1. Vi nt v cc vitamin B1, B6, B12 ....................................................... 2 1.1.1.1. Vitamin B1 (Thiamin) .................................................................... 2 1.1.1.2. Vitamin B6 (Pyridoxin) .................................................................. 2 1.1.1.3. Vitamin B12 (Cobalamin) ............................................................... 3 1.1.2. Mt s ch phm cha vitamin B1, B6, B12 trn th trng ................. 4 1.1.3. Mt s phng php nh lng hn hp vitamin 3B ang c s dng hin nay ................................................................................................. 5 1.2. C s l thuyt chung ca phng php sc k lp mng (TLC) ............. 6 1.2.1. Nguyn l chung ca TLC ................................................................... 6 1.2.2. Pha tnh trong TLC .............................................................................. 7 1.2.3. Pha ng trong TLC. ............................................................................ 7 1.2.4. Cc bc tin hnh nh lng bng TLC .......................................... 8 1.2.4.1. Chun b bn mng ........................................................................ 8 1.2.4.2. a mu phn tch ln bn mng .................................................. 8 1.2.4.3. Khai trin sc k vi pha ng ph hp ........................................ 8 1.2.4.4. Pht hin cc vt trn sc k ..................................................... 9 1.2.5. ng dng ca TLC ............................................................................ 10 1.3. Sc k lp mng hiu nng cao (HPTLC) ............................................... 10 1.4. Vi nt v bn nh lng (Semiquantitative) ......................................... 12 1.4.1. Khi nim bn nh lng ................................................................. 12 1.4.2. Bn nh lng bng HPTLC vi phn mm VideoScan ................. 12 Chng 2: I TNG V PHNG PHP NGHIN CU .................. 14 2.1. i tng nghin cu, dung mi ha cht v dng c. ........................... 14 2.1.1. i tng nghin cu. ....................................................................... 14 2.1.2. Dung mi, ha cht, thuc th. .......................................................... 15 2.1.3. Dng c, trang thit b. ...................................................................... 16 2.2. Ni dung nghin cu ................................................................................ 16 2.2.1. Kho st iu kin sc k v iu kin pht hin .............................. 16 2.2.2. nh gi phng php ....................................................................... 17 2.2.2.1. Tnh chn lc c hiu .............................................................. 17 2.2.2.2. ng chun v khong tuyn tnh ............................................. 17 2.2.2.3. lp li ...................................................................................... 18 2.2.2.4. ng ........................................................................................ 18 2.2.3. ng dng phng php trn mt s ch phm thuc lu hnh trn th trng ........................................................................................................... 19 2.3. Phng php nghin cu. ......................................................................... 19 Chng 3. THC NGHIM, KT QU V BN LUN. .......................... 21 3.1. Chun b mu trng, dung dch chun v th. ......................................... 21 3.1.1. Pha mu trng v dung dch chun gc ............................................. 21 3.1.2. Pha dung dch chun .......................................................................... 21 3.1.3. Pha dung dch th .............................................................................. 22 3.2. Kho st iu kin sc k ......................................................................... 22 3.2.1. Kho st thnh phn pha ng ........................................................... 22 3.2.2. La chn bc sng pht hin ........................................................... 24 3.2.3. Kho st th tch dung dch mu a ln bn mng v cc iu kin sc k khc. .................................................................................................. 24 3.3. nh gi phng php phn tch ............................................................. 26 3.3.1. Bn nh lng B1 v B6 .................................................................... 26 3.3.1.1. c hiu chn lc ................................................................. 26 3.3.1.2. ng chun khong tuyn tnh. .............................................. 27 3.3.1.3. lp li v ng. .................................................................. 29 3.3.2. Bn nh lng B12 ............................................................................ 32 3.3.2.1. c hiu chn lc ................................................................. 32 3.3.2.2. ng chun khong tuyn tnh ............................................... 33 3.3.2.3. lp li v ng ................................................................... 35 3.4. ng dng bn nh lng vitamin trong mt s ch phm thuc ........... 37 KT LUN V XUT........................................................................... 40 TI LIU THAM KHO CC CH V K HIU VIT TT AOAC : Hip hi cc nh ho phn tch chnh thng (Association ofOfficial Analytical Chemists) CT CPDP : Cng ty c phn dc phm CT TNHH: : Cng ty trch nhim hu hn EtOH : Ethanol HPLC : Sc k lng hiu nng cao (High Performance Liquid Chromatography) HPTLC :Scklpmnghiunngcao(HighPerformanceThin Layer Chromatography) Pyridoxin .HCl: Pyridoxin hydroclorid r : H s tng quan Rf: H s lu gi RSD: lch chun tng i (Relative Standard Deviation). Thiamin .HCl: Thiamin hydroclorid TLC: Sc k lp mng (Thin Layer Chromatography). USP: Dc in M (United States Pharmacopoea). UV: T ngoi (Ultra Violet). VIS: Kh kin (Visible). DANH MC CC BNG Bng Trang Bng 1.1: Cc phng php nh lng hn hp vitamin 3B thng dng v u nhc im............................................................................. 5 Bng 1.2: So snh mt s tham s ca TLC v HPTLC...........................11 Bng 2.1: Cc ch phm c nghin cu trong kha lun......................14 Bng 2.2: Cc ch phm c p dng phng php phn tch...............15 Bng 3.1: Kt qu phn tch vitamin B1, B6 trn dy dung dch chun.....28 Bng 3.2: Kt qu kho st lp li B1, B6..........................................30 Bng 3.3: Kt qu kho st ng B1, B6............................................31 Bng 3.4: Kt qu phn tch vitamin B12 trn dy dung dch chun..........34 Bng 3.5: Kt qu kho st lp li B12...............................................36 Bng 3.6: Kt qu kho st ng B12................................................37 Bng 3.7: Phn trm hm lng cc vitamin trong cc ch phm so vi nhn............................................................................................................ 38 DANH MC CC HNH HnhTrang Hnh 1.1: Mt h thng my mc HPTLC................................................11 Hnh 3.1: Sc k khi trin khai bng pha ng 1.................................23 Hnh 3.2: Sc k khi trin khai bng pha ng 2.................................23 Hnh 3.3: V tr chm mu trn bn mng HPTLC silica gel 60 F254 kch thc 20 x 10 cm............................................................................... 25 Hnh 3.4: Sc k th c hiu B1, B6 khi soi di n UV bc sng 366 nm...................................................................................... 26 Hnh 3.5: Sc k mt vt ca ng chun B1 v B6 thu c vi phn mm VideoScan................................................................................ 27 Hnh 3.6: ng chun vitamin B1...........................................................28 Hnh 3.7: ng chun vitamin B6...........................................................29 Hnh 3.8: Sc k nh gi lp li v ng ca B1 v B6 bc sng 366 nm............................................................................................... 30 Hnh 3.9: Sc k th c hiu B12 khi quan st di nh sng kh kin...................................................................................................... 32 Hnh 3.10: Sc k mt vt ca ng chun B12 thu c vi phn mm VideoScan......................................................................................... 33 Hnh 3.11: ng chun vitamin B12.......................................................34 Hnh 3.12: Sc k nh gi lp li v ng ca B12 nh sng kh kin...................................................................................................... 35 1 T VN Hinnay,trnthtrngcrtnhiuloithucchahnhpcc vitamin, hay gp l dng kt hp cc vitamin nhm B. Theo thng k t danh mc thuc c cp s ng k ca Cc Qun l Dc Vit Nam, t thng 7/2009nthng1/2014cgn500chphmthucmultivitaminc cp s ng k lu hnh trn th trng, trong khong 150 thuc c thnh phnkthp3loivitamin:B1,B6,B12[2].Vitaminlnhmchtrtdb phnhybinhiutcnhnlha(nhnhit,nhsng,ccionkim loi...) lm gim hm lng trong thuc theo thi gian v iu kin bo qun. V vy, cng tc kim tra cht lng ca cc thuc cha vitamin ni ring v cc thuc ni chung rt cn c quan tm. Cc phng php hay c dng nh lng hn hp vitamin hin nay l HPLC [3], quang ph UV VIS [4], [13]. Phng php TLC mi ch c s dng ch yu vi mc ch nh tnh, th tinh khit. Tuy nhin, t khi xut hin HPTLC, mt k thut ci tin ca TLC th vic s dng HPTLC nh lng/bnnhlnghotchttrnnphbinhn.pdngkthut HPTLCnhlng/bnnhlngccvitamintronghnhpcmts u im nh: tnh c hiu cao, n gin, d thc hin, nh lng c ng thinhiuvitamintronghnhp,titkimthigian,hacht.Vvy, nggpthmmtphngphpkimtrasbchtlngthuchnhp vitaminchoccnhsnxut,kinhdoanhthuc,chngtithchinti Nghin cu bn nh lng mt s vitamin trong hn hp bng phng php HPTLC vi cc mc tiu:1.XydngphngphpbnnhlngccvitaminB1,B6,B12trong hn hp bng HPTLC. 2. nh gi phng php va xy dng. 2 Chng 1. TNG QUAN 1.1. Tng quan v i tng nghin cu. Mc d trn th trng c rt nhiu cc ch phm multivitamin m thnh phnlhnhpnhiuvitamin,thmchcchphmchati910loi vitaminkhcnhau,nhngtrongkhunkhchncakhalun,chngti chnghincubnnhlngccvitaminB1,B6,B12trongccchphm thuc cha 3 loi vitamin ny.1.1.1. Vi nt v cc vitamin B1, B6, B12 [6], [13]. 1.1.1.1. Vitamin B1 (Thiamin) -Trnthtrnghinnay,thiaminthngcdngdidngmui hydroclorid v nitrat, 2 dng mui ny c tnh cht tng t nhau. Cng thc: NN H3C NH2NSCH2CH2OHCH3.Cl-.HCl (NO3-) - Tnh cht: Thiamin hydroclorid l tinh th khng mu hoc bt kt tinh trng, v ng, d tan trong nc (1g/1ml), kh tan trong EtOH (1g/315ml cn tuyt i), khng tan trong ether, benzen v chloroform. n nh: thiamin hydroclorid n nh trong dung dch nc (c pH t 2 3,5). Do cha d vng thm nn thiamin hp th mnh bc x UV. - Tc dng: Phng v iu tr bnh beri beri, phi hp vi B6, B12 iu tr cc trng hp au nhc dy thn kinh lng, hng, dy thn kinh sinh ba, dng trong trng hp mt mi, km n, suy dinh dng. 1.1.1.2. Vitamin B6 (Pyridoxin) -VitaminB6 ltnchungca3chtccutrchahckhcnhauvtr nhmchcs4l:pyridoxol,piridoxal,pyridoxamin(1).Chphmdc 3 dngldngmuipyridoxolhydroclorid,thnggilpyridoxin hydroclorid (2). N CH3OHRHOH2CR= CH2OH: PyridoxolR=CH2NH2: PyridoxaminR= CHO: Pyridoxal

N CH3OHCH2OHHOH2C . HCl (1)(2) - Tnh cht: Pyridoxin hydroclorid l nhng tinh th khng mu hoc bt kt tinh trng, d tan trong nc (1g/4,5ml), tan trong EtOH (1g/90ml), khng tan trong chloroform, ether. Pyridoxin hydroclorid hp th mnh bc x UV, n nh trong mi trng nc vi pH t 2 4,5.- Tc dng: Phng v iu tr bnh thiu vitamin B6, biu hin: tn thng mt, mi, ming, gy co git, sng khp c tay, thiu mu, phng chng vim dy thn kinh ngoi vi do dng isoniazid, iu tr qu liu isoniazid. 1.1.1.3. Vitamin B12 (Cobalamin) -DngdcdngthngcdnghinnayVitNamcavitamin B12 l cyanocobalamin, cng thc: -Tnhcht:Cyanocobalaminlnhngtinhthmutihocbtv nh hnh, tan c trong nc (1g/1,3ml), EtOH (1g/1,3ml), khng tan trong chloroform v ether. Cyanocobalamin hp th mnh c bc x vng t ngoi 4 v kh kin, d b phn hy bi cctc nhn nh oxi, nh sngmt tri, m,ccionkimloi,khitrongdungdchnccmtccchtkhcnh vitamin B1, C, PP... -Tcdng:iutrccbnhthiumuuschngcutodothiu vitamin B12, phng thiu mu khi phu thut ct d dy hoc hi trng, vim au dy thn kinh... 1.1.2. Mt s ch phm cha vitamin B1, B6, B12 trn th trng [2]. Trn th trng hin nay c rt nhiu loi ch phm cha B1, B6, B12 vi cngdngchungl:iutrccbnhlinquannthiuvitaminnhmB, suynhccth,chnn,thiumu,ngimimdy,aunhcdo nguyn nhn thp khp hay thn kinh, ri lon thn kinh do nghin ru mn tnh. Cc dng thuc thng gp l: - Dng phi hp B1 B6 B12 vi t l cc thnh phn rt khc nhau: Vinnn,thngchmlngB1 B6B12l125mg125mg 125g(CaditrivitUSPharma,Vitamin3BCTCPDPQungBnh,...), hoc100mg200mg200g(ScanneuronCTTNHHSTADAVit Nam, Betex CT TNHH The United Drug Thi Lan...)... Dungdchtim(NeurobionH5000Merck,Enpovit3BCTCP SPM...). Ngoi ra cn c dng vin nang mm, dng cm. -DngkthpB1,B6,B12liunhhndngthuc3Bnitrnhng chc ln vi cc loi vitamin v acid amin khc: vin nang (Moriamin Forte CT Roussel Vit Nam, Vitacap CT TNHH Mega Lifesciences Thi Lan...), vinsi(SunlifeMultivitamincac,HaasMultivitamincaHungari...), dng siro ung (Pediakids Php, TGB Kidbaomin Plus Vit Nam...)... 5 1.1.3. Mt s phng php nh lng hn hp vitamin 3B ang c s dng hin nay (bng 1.1). Phng php nh lng u im chnhNhc im chnh Quang ph UV-VIS [3]. - Tin hnh nhanh. - Choktquc chnh xc cao. - Chnhlngchnhp cha2-3thnhphn,khng nhlngcngthi nhiu hot cht. - Khng c hiu. Sc k lng hiu nng cao (HPLC) [4], [13]. - c hiu. - lpliv chnh xc cao. - nhlngc hnhpnhiu thnh phn. -Dungmi,hachtttin (dungmiyuculcloikh hocdngloichuyndng, tn nhiu dung mi).- Thi gian phn tch lu. -Dtcct,khbodng thit b. nhlngring tngvitaminsdng kt hp nhiu phng php:quangphhp th UV, phng php ha hc [12]. - C th p dng cho hnhpchac vitamintantrong duvvitamintan trong nc. -Cnthchinnhiuthaotc, tn cng sc v thi gian. Bng 1.1: Cc phng php nh lng hn hp vitamin 3B thng dng v u nhc im. 6 Ngoira,hinnayphngphpindimaoquncngcnghin cu nh lng hn hp cc vitamin tan trong nc [5]. KthutHPTLCkhcphccccnhcimcaccphng php nh lng trn, l thao tc phc tp, thi gian tin hnhlu (HPLC, phng php nh lng ring tng thnh phn) hoc khng nh lng c mucthnhphnphctp(UVVIS).LdolHPTLCcthaotcn gin,chophpphntchcngthimtslnglnmuthcha nhiu hot cht nn tit kim thi gian. Ngoi ra n cn mt s u im khc nh c chn lc cao, s dng lng nh dung mi cho pha ng nn tit kim chi ph, trnh nhim mi trng... [14], [15]. 1.2. C s l thuyt chung ca phng php sc k lp mng (TLC) [1], [14], [19]. 1.2.1. Nguyn l chung ca TLC -Scklpmnglmttrongcckthuttchsck,trongdung dchchtphntchcdichuyntrnmtlpmngchthpphmn. Trongqutrnhdichuyn,ccthnhphntronghnhpmuthc chuyndchtrnlpmng,theohngphang,vinhngtckhc nhau ty vo bn cht ca chng. Kt qu thu c l mt sc k trn lp mng. C ch ca s tch c th l c ch hp ph, phn b, trao i ion, sng lc phn t hay s phi hp ng thi ca nhiu c ch ty thuc vo tnh cht ca cht lm pha tnh v dung mi lm pha ng. - i lng c trng cho mc di chuyn ca cht phn tch l h s lugiRf ctnhbngtlgiakhongdchchuyncachtthv khong dch chuyn ca dung mi: bafR7 Trong :a l khong dch chuyn ca cht phn tch; b l khong dch chuyn ca dung mi tnh t im chm mu. Rf: Ch c gi tr t 0 n 1. - Tr s Rf nm trong khong 0,2 0,8 s t phn gii cc tr. 1.2.2. Pha tnh trong TLC -Phatnhlmtlpmngccchtdnghtnhckchthcng nht,ctrithnhlpmngvccnhtrnmtmtphng(thy tinh, nhm hoc cht do). Cc pha tnh c s dng da trn nhiu c ch phn tchkhc nhau:phnb,hp thu,ryphnt,traoiion, indi,i lc... Mt s cht thng dng lm pha tnh cho TLC: ph bin nht l silica (SiO2)valumina(Al2O3),ngoiracncdnchtsiloxan,cellulose, cellulosetraoiion,ctbin,gelsephadexNhiukhingitacna thm cc cht pht hunh quang khng tan vo pha tnh d pht hin cht cn phn tch. - Khi phn tch vitaminthng dng pha tnh silica, khi qu trnh sc k s da trn c ch hp ph.1.2.3. Pha ng trong TLC -Phanglmthdungminhocathnhphnctrnvi nhau theo t l khc nhau, ty thuc vo c ch sc k. Tiu chun quan trng nhtchnhdungmiphanglphnccvpHcan.Cchtt nht l la chn pha ng bng thc nghim da trn c s:- V phn cc: nu cht phn tch c phn cc km, chn cht hp ph phn cc mnh (silica gel, Al2O3), h dung mi khai trin c phn cc 8 km(hexan,toluen,benzen...).Ngclichtphntchdngionhayphn cc s c ra gii tt bng dung mi phn cc (methanol, nc...). -VpHcaphang:Nuthmmtlngnhacidacetichoc amoniac vo dung mi cha nc s lm tng ng k tan ca base hoc acid tng ng, cht phn tch s di chuyn nhanh hn v vt gn hn. 1.2.4. Cc bc tin hnh nh lng bng TLC 1.2.4.1. Chun b bn mng i vi bn mng silica gel, khi bo qun trong iu kin thng, cc n vsilanolchanhmhydroxylcasilicagelddnghpphnctrong khngkh,lmgimkhnnghpphchtphntchcan.Dovy,bn mng silicagel thng c sy 100 120oC trong 1 2h loi nc hp ph, m khng lm mt i nhm hydroxyl ca n v silica gel. Kh nng hp ph ca silica gel cao nht khi c hot ha 105oC trong 2h. Ngoi ra, bn mng nn c ra sch trc khi khai trin loi b bi bntrnbmtnuboqunkhngtt.Dungmiracthlmethanol, methanol dicloromethan (1:1) hoc isopropanol. Cho dung mi chy ht bn mng, sau hot ha li bn mng ra trong t sy [10]. 1.2.4.2. a mu phn tch ln bn mng Lng mu a ln bn mng nh hng ln ti tr s Rf v hiu qu tch sc k. Nu lng cht qu ln, vt sc k s ln v b ko di, khi cc vt ctrsRfgnnhauschnglnnhau.Nulngchtqunhskhng pht hin c vt. V tr a mu ln bn mng cn m bo khng qu gn b mt pha ng, cc vt cch ra bn mng v cch nhau xa khng b nh hng bi hiu ng b v cc vt khc khi khai trin. 1.2.4.3. Khai trin sc k vi pha ng ph hp 9 Khai trin sc k trong bnh thy tinh c np y kn. tng bo ha dung mi trc khi khai trin, thng t mt t giy lc p st thnh bnh.Sau khi chmmu, bay hi ht dung mi pha mu trn bn mng ri tvobnhsckbohaphang.Mpdibnmngcnhng vo v cch b mt pha ng khong 1 cm. 1.2.4.4. Pht hin cc vt trn sc k Davo tnhcht lha khcnhaucacht phntch la chncch pht hin thch hp. Mt s k thut thng gp: - Phun thuc th hin mu. Thuc th chung nhn bit nhiu cht hu clacidphosphomolypdic,acidsulfuricc,hiiod.Ngoiracncc thuc th c hiu vi tng cht nh ninhydrin nhn bit acid amin v cc nhm chc amin, rhodamin B nhn bit cc cht lipid... -SoidinUV:Nhiuvtchthuctrnsckstiihoc pht quang khi soi di n UV bc sng 254 hoc 366 nm. -Dngdensitometer:Thitbnysocng tiaphnx tbmt bn mng khi soi di n UV VIS. Cht phn tch hp th bc x c ghi li thnh pic sc k. 1.2.4.5. nh lng cc cht trong vt sc k C hai phng php nh lng cc cht trong vt sc k: - Tch chit cht phn tch trong vt sc k bng dung mi thch hp, lm sch dch chit ri nh lng cht phn tch bng phng php thch hp. - nh lng trc tip trn bn mng bng cch o din tch hay cng mu ca vt sc k, s dng 2 k thut:10 Densitometer:chiuchmtiavovtsckvocnghpth hoc hunh quang. Xlnhvicamerakthuts:qutbnmngvihthngphn tch hnh nh, v d camera k thut s c phn gii cao thu nhn hnh nh ca vt sc k, sau x l d liu nh bng my tnh. 1.2.5. ng dng ca TLC TLC c ng dng trong nh tnh, th tinh khit, nh lng, bn nh lng cc cht trong hn hp nhiu thnh phn. 1.3. Sc k lp mng hiu nng cao (HPTLC) V nguyn tc c bn, HPTLC khng khc nhiu so vi sc k lp mng thng thng. im khc l k thut ny s dng bn mng hiu nng cao c lp pha tnh mng hn, vi bt mn c kch thc ht nh hn, ng u cao hn [1]. Hin nay, qu trnh chm sc k c iu khin v thc hin bng my, s vt, lng dch chit s chm trn bn mng c xc lp trn my vi tnh. Qu trnh chm dch chit thc hin vi s h tr ca my nn kh hoc bnh khnn.Saukhitrinkhaitrongbnhtrinkhaiscktng,sck cchpnhquabungchpvktqucnhgibngphnmm chuyn dng (Hnh 1.1). 11 Bng 1.2 sau y s gip so snh mt s tham s ca TLC v HPTLC: Bng 1.2. So snh mt s tham s ca TLC v HPTLC

Tham sHPTLCTLC Cng nghBn t ngTh cng. Kch thc ht5 6 m10 12 m dy lp pha tnh100 m250 m Chiu cao a l thuyt12 m30 m Hiu lc tchCaoThp hn. Gii hn pht hin (s dng my o hp th UV VIS). 100 500 pg1 5 ng Giihnphthin(sdngmy qut hunh quang). 5 10 pg50 100 ng My tnh c phn mm VideoScan My chm mu t ng Bnh trin khai sc k Bung chp Hnh 1.1: Mt h thng my mc HPTLC. 12 So vi TLC, HPTLC k tha c nhng li th vn c v mang mt suimtrihn:chovtsckgnhn,khnngtchtthn.ng thi, cc bc ca qu trnh phn tch bao gm phun mu, khai trin sc k, nhn din vt c tin hnh bn t ng, cc thng s c kim sot mt cch cht ch gip gim thiu ti a cc sai s ngu nhin gp phi, nht l khuamulnbnmng,choktquclplivngcaohn TLC [14]. 1.4. Vi nt v bn nh lng (Semiquantitative) 1.4.1. Khi nim bn nh lng - Bn nh lng l xc nh mt cch gn ng hm lng ca mt cht, ktqubnnhlngrivokhonggiacamtktqunhlngv mt kt qu nh tnh [8]. -Phntchbnnhlngtngtnhphntchnhtnhtrongvic pht hin s c mt hay vng mt ca mt cht phn tch, nhng sau i vo cung cp mt con s i din ca hm lng cht phn tch trong mu vt c lin quan n cc ngng bnh thng/khng bnh thng. - WHO thng khng a ra cc ti liu tham kho i vi kt qu phn tchbnnhlng,vphmvingdngcabnnhlngcthkhc nhau ng k gia cc phng th nghim [20]. 1.4.2. Bn nh lng bng HPTLC vi phn mm VideoScan -KhinhlngccchtbngHPTLC,chnhxccaktquph thucnhiuvokthutxcnhvlnggivttrnsck.Nus dng k thut x l nh sc k vi camera k thut s, th chnh xc ca kt qu phn ln ph thuc vo phn mm x l d liu nh. 13 -PhnmmxlnhVideoScanlmtphnmmchnchv chnh xc khi c kt qu d liu nh. V vy, khi s dng phn mm ny xlnhsckthchcthxcnhcsbhmlngcccht trong mu phn tch (bn nh lng). 14 Chng 2: I TNG V PHNG PHP NGHIN CU 2.1. i tng nghin cu, dung mi ha cht v dng c. 2.1.1. i tng nghin cu. Mu th l mt ch phm cha vitamin B1, B6, B12 trn th trng v mt ch phm khng cha cc vitamin ny c dng lm mu trng (bng 2.1): Tn bit dc Dng bo ch Thnh phn, hm lng hot cht Nh sn xutS l, ngy sn xut, hn dng Betex Vin nn bao phim Thiamin .HCl: 100 mg Pyridoxin .HCl: 200 mg Cyanocobalamin: 200 g Cng ty TNHH The United Drug (1996), Thi Lan. S l: 413028 NSX:29/03/2013 HSD: 28/03/2016 Ko ginszhi (Mu trng) Vin nang mm Cao Linh Chi: 13 mg Cao sm: 13 mg Magnesium sulphate: 5mg Vitamin A: 5 mg Vitamin B2: 3 mg Ferrous Fumarate: 5 mg Cng ty TNHH TC PHARMA, Vit Nam S l: 151114 NSX: 12/10/2013 HSD: 12/10/2016 p dng phng php phn tch mt s ch phm khc trn th trng (bng 2.2): Bng 2.1: Cc ch phm c nghin cu trong kha lun. 15 Bng 2.2. Cc ch phm c p dng phng php phn tch Tn bit dc Dng bo ch Thnh phn, hm lng hot cht Nh sn xutS l, ngy sn xut, hn dng Nebamin Vin nn bao ng Thiamin .HCl: 50 mg Pyridoxin .HCl: 250 mg Cyanocobalamin: 250 g Cng Ty CPDP Trung ng 25 (UPHACE), Vit Nam. S l: 312561 NSX:07/06/2013 HSD: 27/06/2015 Tervit H Vin nn bao phim Thiamin .HCl: 250 mg Pyridoxin .HCl: 250 mg Cyanocobalamin: 250 g Cng Ty CPDP AMPHARCOVitNam. S l: L2007 NSX:29/11/2011 HSD: 20/12/2014 Neurobion Thuc tim (ng 3ml) Thiamin .HCl: 100 mg Pyridoxin .HCl: 100 mg Cyanocobalamin: 5 mg MERCK, Indonesia S l:T 1177904 HSD: 10/09/2015 2.1.2. Dung mi, ha cht, thuc th. - Nc ct, ethanol, chloroform, acid acetic, amoniac t tinh khit phn tch. -ChtchuncaVinKimnghimthucTrungng,ttiuchun Dc in Vit Nam IV: thiamin hydroclorid hm lng 99,87%, pyridoxin hydroclorid hm lng 99,97%, cyanocobalamin 99,67%.16 2.1.3. Dng c, trang thit b. - My siu m Bandelin Sonorex Super RK 106 - My ly tm KUBOTA 6500 - My quang ph UV-VIS Cary 60 - H thng my HPTLC CAMAGgm:My sy bn mng My chm sc k LINOMAT 5 My khai trin sc k ADC2 Bung chp nh sc k TLC VISUALIZED Phn mm x l kt qu: VideoScan - Cn phn tch AUW 220 - Bn mng HPTLC silica gel 60 F254 (MERCK, c) - Cc dng c thy tinh: pipet, bnh nh mc, cc c m, a thy tinh, bnh chit, bnh nn, ng nghim, phu lc, giy lc. 2.2. Ni dung nghin cu 2.2.1. Kho st iu kin sc k v iu kin pht hin - Bn mng: bn mng silica gel c thm cht pht hunh quang bc sng 254 nm ca MERCK. - Chn pha ng: sau khi kho st mt s ti liu tham kho [16], [17] vi bn cht bn mng la chn, chng ti tin hnh kho st hai pha ng (1) v (2) la chn iu kin ti u: (1). EtOH chloroform aceton amoniac, 2:2:2:1. (2). EtOH chloroform acid acetic amoniac nc, 6:5:5:1:1. - Kho st iu kin pht hin: hin vt sc k di n UV VIS, chn bc sng thch hp phn tch cc thnh phn trong hn hp vitamin. 17 - La chn th tch dung dch mu a ln bn mng v tc chm mu, chiu rng vt mu sau khi trin khai sc k thu c sc k c vt r, gn p, cc vt tch nhau hon ton. 2.2.2. nh gi phng php Phng php phn tch c nh gi qua cc ch tiu: tnh chn lc, c hiu, ng chun v khong tuyn tnh, lp li, ng. Hin nay, cha c ti liu chnh thc no a ra cc yu cu v cc gii hn thng s nh gikthutbnnhlngbngHPTLC,nnkhalunchxcnhcc thngstrnvaracngviccgiihnchpnhnkhithmnh1 phngphpnhlngtheoAOACvmtstiliukhc[7],[9],[11], [13], [18], [21]. 2.2.2.1. Tnh chn lc c hiu - X l mu trng v mu chun theo quy trnh c xy dng, tin hnh sc k ng thi cc mu sau theo iu kin chn: mu trng, mu trng c thmchunvitaminB1,mutrngcthmchunvitaminB6,mutrngc thmchunvitaminB12,mutrngcthmcB1,B6,B12.Hinsck di n UV VIS cc bc sng 254 nm, 366 nm v nh sng kh kin. Phng php c coi l c tnh chn lc khi:CcvtB1,B6,B12 tchhontonkhinhauvtchkhiccvttp ca mu trng. TrnsckcamutrngkhngxuthinccvtvtrcRf trng vi Rf ca B1, B6, B12. 2.2.2.2. ng chun v khong tuyn tnh- Qua thc t kho st nhiu ln, chng ti thy rng: khi tin hnh sc k dung dch cha cc vitamin B1, B6, B12 trong iu kin c xy dng ca 18 khalun,vicngmtlngchtcalnbnmng,thvicchm mtthtchnhccdungdchcvchmthtchlnhnccdungdch longuchoccktqutngtnhau.Chnhvvy,thayvvicphi chun b mt dy 5 dung dch chun c nng khc nhau ca cc cht cn phntch,chcnalnbnmngmtdy5vtchaccthtchkhc nhaucadungdchchuntrongkhong20%thtchdungdchthcn nh lng theo l thuyt. - Tin hnh: chun b dung dch chun hn hp c nng B1 khong 0,5 mg/ml, B6 khong1 mg/ml, B12 khong 0,05 mg/ml. Tin hnh sc k 5 vt dung dch chun c th tch: 3, 4, 5, 6, 7 l trn bn mng. Xy dng phng trnhhiquybiudinmitngquangiadintchpicvnngcht chun,xcnhhstngquanr.TheoAOAC:nur0,99thng chun c tuyn tnh cao trong khong nng kho st [9]. 2.2.2.3. lp li - Phn tch 6 mu th: T1 T6, tnh lch chun tng i RSD ca kt qu gia cc ln phn tch.-Giihnlpliivi1phngphpnhlngtheoAOACl RSD 2% nng cht phn tch 1% v RSD 3% nng cht phn tch 0,1% [9]. Theo USP 28 v mt s ti liu khc, i vi k thut sc k, RSD 3% c coi l rt chnh xc, RSD trong khong 3 10% c coi l c chnh xc thng thng [13], [18]. 2.2.2.4. ng -ngcnhgiquathuhi,sdngphngphpthm chun. Tin hnh: thm cng mt lng chun vo 6 dung dch th T1 T6 (vi t l 1:1 v th tch) c cc dung dch T1+C T6+C. Tin hnh sc k cc dung dch trn trong cng iu kin, tnh thu hi theo cng thc:19 H% = Trong :H%: thu hi C t + c : Nng cht phn tch trong mu thm chun C t : Nng cht phn tch trong mu th C c : Nng chun thm (theo l thuyt)- Theo AOAC, gii hn h s thu hi i vi 1 phng php nh lng l t 92 105% nng cht phn tch 1%; t 90 108% nng cht phn tch 0,1% [9]. 2.2.3. ng dng phng php trn mt s ch phm thuc lu hnh trn th trng -Ccchphmcpdngphngphp:Nebamin,TervitH, Neurobion. - Vi cc ch phm l vin nn: x l theo quy trnh xy dng; vi ch phm thuc tim: pha long ch phm vi EtOH t nng cc vitamin trong khong kho st v tin hnh sc k.2.3. Phng php nghin cu. - Phng php sc k lp mng hiu nng cao (HPTLC). -Sau khi trin khai sck,hinvt sc k vchpnh bcsng 254nmquanstc3vt:B1,B6,B12;bcsng366nmbnnh lng vitamin B1, B6 v nh sng kh kin bn nh lng vitamin B12. - nh chp sc k c chuyn sang x l bng phn mm VideoScan. Phn mm ny s da trn hnh nh sc k va c chp ghi li thnh cc pic v tnh ton din tch pic (hoc chiu cao) tng ng. C t + c C t 100% C c 20 - Phng php x l kt qu: gi tr trung bnh, lch chun tng i, phng trnh hi quy, h s tng quan xc nh bng phn mm Microsoft Office Excel. - Nng B1, B6, B12 trong mu th c xc nh da vo phng trnh hiquybiudinmi tngquangiadin tch picv nngB1,B6,B12 trong mu chun c xy dng trong mi ln phn tch. 21 Chng 3. THC NGHIM, KT QU V BN LUN. 3.1. Chun b mu trng, dung dch chun v th. 3.1.1. Pha mu trng v dung dch chun gc - Mu trng: ly 1 nang mm KO-GINSZHI, b v nang, ha tan trong 2 ml chloroform ri thm EtOH thnh khong 10 ml, em siu m trong 5 pht. yn 20 pht, sau ly tm ly dch lm mu trng. -ChungcB1:Cnchnhxckhong125mgthiaminhydrocloridvo bnh nh mc dung tch 50 ml, thm 10,0 ml nc ct, lc u ri em siu m trong 5 pht cho tan hon ton. Sau pha long bng EtOH ti vch nh mc, lc u ta c dung dch chun gc B1 c nng khong 2,5 mg/ml.- Chun gc B6: Cn chnh xc khong 100 mg pyridoxin hydroclorid vo bnh nh mc dung tch 50 ml, thm 10,0 ml nc ct, lc u ri em siu m trong 5 pht cho tan hon ton. Sau thm EtOH ti vch nh mc, lc u ta c dung dch chun gc B6 c nng khong 2 mg/ml. - Chun gc B12: Cn khong 25 mg cyanocobalamin vo bnh nh mc dung tch 50 ml, thm 10,0 ml nc ct, lc u ri em siu m trong 5 pht chotanhonton.ThmEtOHtivch,lcucdungdchchungc B12 c nng khong 0,5 mg/ml. DoB12 rt d htm v d phn hy, li c cn vi lng nh, nn gim sai s trong qu trnh cn, nng B12 c xc nh li bng phng php o quang bc sng 361 nm, E (1%, 1cm) bc sng ny l 207 [4]. 3.1.2. Pha dung dch chun- Chun hn hp B1, B6, B12: Ly 2,0 ml dung dch chun gc B1, 5,0 ml dung dch chun gc B6, 1,0 ml dung dch chun gc B12, 0,2 ml nc ct cho vobnhnhmcdungtch10ml,thmEtOHtivch,lcuthuc 22 dungdchchunhnhpcnngB1khong0,5mg/ml,B6 khong1 mg/ml, B12 khong 0,05 mg/ml. -MutrngcthmvitaminB1:dngpipetchiavchly0,2mldung dch chun gc B1 v 0,8 ml mu trng cho vo ng nghim nh, lc u. - Mu trng c thm vitamin B6: ly 0,5 ml dung dch chun gc B6 v 0,5 ml mu trng cho vo ng nghim nh, lc u. - Mu trng c thm vitamin B12: ly 0,1 ml dung dch chun gc B12 v 0,9 ml mu trng cho vo ng nghim nh, lc u. - Mu trng c thm B1, B6 v B12: ly 0,5 ml dung dch chun hn hp v 0,5 ml mu trng cho vo ng nghim nh, lc u. 3.1.3. Pha dung dch th - Cn chnh xc 20 vin ch phm, xc nh khi lng trung bnh ca 1 vin, nghin mn.- Dung dch th 1 (bn nh lng B1 v B6): cn chnh xc mt lng bt vin tng ng vi 50 mg B1 vo bnh nh mc dung tch 100 ml, thm 20,0 ml nc ct, lc u, em siu m trong 5 pht, sau thm EtOH ti vch, lc u. Lc b 20 ml dch lc u, ly dch lc sau. - Dung dch th2 (bn nh lng B12): cn chnh xc mt lng bt vin tng ng vi 200 g B12 vo ng nghim, thm 5,0 ml hn hp gm EtOH H2O (4:1), lc u, em siu m trong 5 pht, sau em ly tm ly dch. 3.2. Kho st iu kin sc k Bn mng s dng l HPTLC silica gel 60 F254 (MERCK). 3.2.1. Kho st thnh phn pha ng - Tin hnh kho st 2 h pha ng:23 1. EtOH chloroform aceton amoniac, 2:2:2:1. 2. EtOH chloroform acid acetic amoniac nc, 6:5:5:1:1. -Mualn bnmng l dung dchchun hn hpc cha 3 vitamin B1, B6, B12. Hin vt sc k di n UV VIS bc sng 254 nm, 366 nm v nh sng kh kin, kt qu thu c hnh 3.1 v 3.2: Hnh 3.2: Sc k khi trin khai bng pha ng 1 abc a b c a:Quanstdi nh sng kh kin. b:Quanstdi bc sng 254 nm. c:Quanstdi bc sng 366 nm a:Quanstdi nh sng kh kin. b:Quanstdi bc sng 254 nm. c:Quanstdi bc sng 366 nm Hnh 3.1: Sc k khi trin khai bng pha ng 2 24 -Nhnxt:Sckthuckhitrinkhaibngphang1cB1 b ko vt, vt B12 c Rf qu thp. Pha ng 2 cho sc k gn, tch tt, h s lu gi Rf ca cc vt thch hp. Do , chng ti chn h dung mi ny cho cc nghin cu tip theo. 3.2.2. La chn bc sng pht hin - Khi quan st sc k bc sng 254 nm c th pht hin c c 3 thnhphnB1,B6,B12,tuynhinkhalunchnnhsngkhkinbn nh lng B12 v chn bc sng 366 nm bn nh lng B1, B6 v cc l do sau: Do hm lng B12 qu chnh lch vi B1, B6 nn vic nh lng ng thi c 3 thnh phn bc sng 254 nm l khng kh thi. Di nh sng kh kin ch quan st thy vt B12, bn nh lng B12 nh sng ny s loi c nh hng bi cc vt khc (vt B1, B6 nng m c trong dung dch th, vt t dc) thy c khi soi di n UV VIS bc sng 254 hoc 366 nm.Khi bn nh lng ring B12 nh sng kh kin th B1, B6 c th bn nh lng bc sng 366 d quan st v khng lmrisc k bi vt B12. 3.2.3. Kho st th tch dung dch mu a ln bn mng v cc iu kin sc k khc. Kho st qua nhiu ln thc nghim, chng ti nhn thy cc thng s sau l thch hp sc k tch vt hon ton, r nt, pic thu c p, cn i, h s Rf nm trong khong tin cy: -Hot ha bn mng: 100oC trong 30 pht. -Th tch mu a ln bn mng: 5l. 25 - rng vt mu khi chm: 4 5 mm. -Khong cch t vt mu u tin n mp bn ca bn mng ti thiu 1 cm trnhhiung b,khongcch t ngxut pht ncnh di ca bn mng l 1cm, qung ng pha ng chy tnh t im xut pht l 7 cm. -Tc phun mu t ng l 30 nl/s. -Nhit phn tch: 20 25oC. Nh vy, iu kin ti u bn nh lng 3 thnh phn B1, B6, B12 l: -BnmngHPTLCsilicagel60F254(MERCK),hotha100oCtrong 30 pht. -Pha ng: EtOH chloroform acid acetic amoniac nc, 6:5:5:1:1. -Th tch mu a ln bn mng: 4 5l. - rng vt chm, v tr vt chm: nh hnh 3.3. -Tc phun mu t ng l 30 nl/s. -Nhit phn tch: 20 25oC. -Pht hin vt: vt B1, B6 pht hin bc sng 366 nm, vt B12 pht hin nh sng kh kin. -X l kt qu: bng phn mm VideoScan. 7 cm 1 cm 1cm 0,5 cm Tuyn dung mi Vt mu chm Hnh 3.3: V tr chm mu trn bn mng HPTLC silica gel 60 F254 kchthc 20 x 10 cm 26 3.3. nh gi phng php phn tch 3.3.1. Bn nh lng B1 v B6 3.3.1.1. c hiu chn lc - Trn mt bn mng chm 5 vt: mu trng, mu trng c thm B1, mu trng c thm B6, mu trng c thm B12 v mu trng c thm c B1, B6, B12. - Khai trin sc k vi cc iu kin ti u kho st trn. - Sau khi khai trin, quan st sc k bc sng 366 nm, kt qu thu c hnh 3.4: Nhn xt: Kt qu thc nghim cho thy: - Cc vt trn sc k tch ri nhau. 1. Mu trng 2. Mu trng c thm vitamin B1 3. Mu trng c thm vitamin B6 4. Mu trng c thm vitamin B12 5. Mu trng c thm B1, B6, B12 Hnh 3.4: Sc k th c hiu B1, B6 khi soi di n UV bc sng 366 nm. B 6 B 1 27 - Mu trng c thm B1 hoc B6 xut hin ln lt 2 vt tng ng vi cc vitamin ny, c Rf = 0,38 v 0,65. Mu trng cha B1, B6 v B12 xut hin c 2 vt tngng vi 2 vitaminB1, B6, ring B12 khng quan st thy bc sng ny. Ccvt B1, B6 u tch nhau v khng trng vi vt no ca mu trng. Kt lun: phng php c tnh c hiu chn lc i vi B1 v B6 . 3.3.1.2. ng chun khong tuyn tnh. -Thitlpngchun:Chm5vtdungdchchunhnhpvith tch mu a ln bn mng ln lt t 3 7 l. Sy cho dung mi pha mu bay hi ht, sau tin hnh sc k iu kin chn. -HinvtvitaminB1 vB6 bcx366nm,xlnhchpbngphn mm VideoScan. Kt qu c trnh by hnh 3.5 v bng 3.1. B1 B6 Hnh 3.5: Sc k 1 vt ca ng chun B1v B6 thu c vi phn mm VideoScan 28 Bng 3.1: Kt qu phn tch vitamin B1 v B6 trn dy dung dch chun Th tch dung dch chun (l) Vitamin B1Vitamin B6 Nng tng ng (mg/ml) Din tch pic (mAU) Nng tng ng (mg/ml) Din tch pic (mAU) 30,304689350,634258708 40,4061157340,845677815 50,5076219511,057092653 60,6091271741,2684103643 70,7106322731,4798113425 - Thit lp ng chun vitamin B1 v B6 da trn kt qu thu c (hnh 3.6 v 3.7): y = 57257x - 7850r = 0.995050001000015000200002500030000350000 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8Din tch pc (mAU)Nng (mg/ml)ng chun vitamin B1Hnh 3.6: ng chun vitamin B1 29 -KtquchothyngchundungdchvitaminB1vB6tuyntnh trong khong nng kho st vi h s tng quan (r) ln lt l 0,995 v 0,980. Nh vy, khong nng kho st l ph hp bn nh lng cc vitamin B1, B6 trong hn hp vitamin. 3.3.1.3. lp li v ng. - Thit k th nghim trn mt bn mng (hnh 3.7): Xy dng ng chun: 5 vt dung dch chun hn hp (vt s 1 5). nh gi lp li: 6 vt dung dch th: T1 T6 (vt s 6 11). nhging:6vtdungdchthcthmchunhnhptheot l 1:1 v th tch: T1+C T6+C (vt s 12 17). - Kt qu c th hin trong hnh 3.8 v bng 3.2: y = 63984x + 21618r = 0.9800200004000060000800001000001200001400000 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6Din tch pc (mAU)Nng (mg/ml)ng chun vitamin B6Hnh 3.6: ng chun vitamin B6 30 Xy dng ng chunnh gi lp linh gi ng Hnh 3.8: Sc k nh gi lp li v ng B1, B6 bc sng 366 nm Bng 3.2: Kt qu kho st lp li B1, B6 Mu Vitamin B1Vitamin B6 Din tch pic (mAU) Nng (*) (mg/ml) Din tch pic (mAU) Nng (*) (mg/ml) T1190750,4801855161,0198 T2211110,5109877991,0446 T3209430,5027873331,0267 T4202240,4805871271,0034 T5192310,4709859221,0007 T6193420,4986870321,0734 TB0,49061,0281 RSD (%)3,192,43 (*): tnh t phng trnh hi qui v hiu chnh cho lng cn 31 Nhnxt:lchchuntngicaphngphpbnnhlng vitaminB1 =3,19 3;ringpdngphntch vitaminB6 c lp licao vi RSD = 2,43 < 3. Nh vy phng php bn nh lng cc vitamin B1, B6 trong hn hp bng HPTLC c lp li kh cao. -Ktqukhostngcaphngphpctrnhbytrongbng 3.3: Bng 3.3: Kt qu kho st ng B1, B6 Mu Vitamin B1Vitamin B6 Din tch pic (mAU) Nng chun (C) tm li (mg/ml) ng(**) (%) Din tch pic (mAU) Nng chun (C) tm li (mg/ml) ng(**) (%) T1 + C196550,490596,6878071,0703101,3 T2+ C202230,474893,5897061,0939103,5 T3+ C204600,486095,8898301,1051104,6 T4+ C200430,484095,4878261,045798,9 T5+ C206310,5219102,8872481,046499,0 T6+ C202890,5080100,1895581,1013104,2 TB97,4101,9 (**): So vi nng thc bit ca chun (h s thu hi). Kt qu bng cho thy: 32 -ngcaphngphpkhibnnhlngvitaminB1nmtrong khong 93,5% n 102,8%. -ngcaphngphpkhibnnhlngvitaminB6nmtrong khong 98,9% n 104,6%. Nhvyngcaphngphpbnnhlngkhiphntchcc vitamin B1, B6 trong hn hp u nm trong khong gii hn 92 105%, l ngchpnhncaAOACivimtphngphpnhlngkhong nng cht phn tch 1% (tng ng nng 0,1 mg/ml) [9].3.3.2. Bn nh lng B12 3.3.2.1. c hiu chn lc Quan st sc k sau khi trin khai mt bn mng c 5 vt: mu trng, mu trng c thm B1, mu trng c thm B6, mu trng c thm B12 v mu trng c thm c B1, B6, B12 (l sc k dng xc nh c hiu ca phng php khi bn nh lng B1, B6) nh sng kh kin (hnh 3.9): Hnh 3.9: Sc k th c hiu - B12 khi quan st di nh sng kh kin. 1. Mu trng 2. Mu trng c thm vitamin B1 3. Mu trng c thm vitamin B6 4. Mu trng c thm vitamin B12 5. Mu trng c thm B1, B6, B12 B 12 33 Nhn xt: Trn sc k xut hin 2 vt mu hng ca B12 c Rf = 0,56 mu trng thm B12 v mu trng thm c 3 vitamin, cc mu cn li khng xuthinvtny.Vyphngphpbnnhlngnhsngkhkinl chn lc vi vitamin B12. 3.3.2.2. ng chun khong tuyn tnh -Thitlpngchun:Chm5vtdungdchchunhnhpvith tch mu a ln bn mng ln lt t 3 7 l. Sy cho dung mi pha mu bay hi ht, sau tin hnh sc k iu kin chn. -HinvtvitaminB12 nhsngkhkin,xlnhchpbngphn mm VideoScan. Kt qu c trnh by hnh 3.10 v bng 3.4. Hnh 3.10: Sc k 1 vt ca ng chun B12 thu c vi phn mm VideoScan B12 34 Bng 3.4: Kt qu phn tch vitamin B12 trn dy dung dch chun Th tch dd chun (l) Nng tng ng (mg/ml) Din tch pc (mAU) 30,028312440 40,037714561 50,041716570 60,050018850 70,058420610 -ThitlpngchunvitaminB12datrnktquthuc(hnh 3.11): Hnh 3.11: ng chun vitamin B12 -Nhnxt:KtquchothyngchundungdchvitaminB12tuyn tnh trong khong nng kho st vi h s tng quan r = 0,986. Nh vy, y = 281117x + 4456r = 0.98605000100001500020000250000 0.01 0.02 0.03 0.04 0.05 0.06 0.07Din tch pc (mAU)Nng (mg/ml)ng chun vitamin B1235 khong nng kho st l ph hp bn nh lng vitamin B12 trong hn hp vitamin. 3.3.2.3. lp li v ng -Thitkthnghimtrnmtbnmngtngtnhthnghimxc nh lp li v ng ca phng php i vi B1 v B6: Xy dng ng chun: 5 vt dung dch chun hn hp (vt s 1 5). nh gi lp li: 6 vt dung dch th: T1 T6 (vt s 6 11). nh gi ng: 6 vt: T1+C T6+C (vt s 12 17).- Hin vt sc k nh sng kh kin v x l kt qu (hnh 3.11 v bng 3.5, bng 3.6): Xy dng ng chunnh gi lp li nh gi ng Hnh 3.12: Sc k nh gi lp li v ng ca B12 nh sng kh kin 36 Bng 3.5: Kt qu kho st lp li B12 MuDin tch pic (mAU)Nng (*) (mg/ml) T1141270,0321 T2146400,0336 T3136760,0318 T4142140,0324 T5140000,0295 T6122750,0328 TB0,0320 RSD (%)4,34 (*): tnh t phng trnh hi qui v hiu chnh cho lng cnNhn xt: - Phng php bn nh lng p dng cho vitamin B12 c lp li trung bnh vi RSD = 4,34 < 10%. 37 Bng 3.6: Kt qu kho st ng B12 MuDin tch pic (mAU) Nng chun (C) tm li (mg/ml) ng(**) (%) T1 + C152800,0426102,2 T2 + C153430,041298,9 T3 + C148460,041198,6 T4 + C152680,0422101,2 T5 + C144750,037389,5 T6 + C144320,0431103,5 TB99,0 (**): So vi nng thc bit ca chun (h s thu hi). -ngcaphngphpkhibnnhlngvitaminB12nmtrong khong89,5%n103,5%(sovingchpnhncaAOACchomt phngphp nh lngl t90108%nng cht phntch0,1%, tng ng nng 0,01 mg/ml [9]). Nhvy,vicB1, B6v B12,ccphngphpxutmbo chnlcvchnhxccnthitbnnhlngccthnhphntrn trongchphm.Trncsny,phngphp cp dng kho st thm mt vi ch phm mua c trn th trng. 3.4. ng dng bn nh lng vitamin trong mt s ch phm thuc 38 - Kt qu bn nh lng cc vitamin trong cc ch phm c kho st th hin trong bng 3.7: Bng 3.7: Phn trm hm lng cc vitamin trong cc ch phm so vi nhn Ch phm Thnh phn Hm lng trn nhn Hm lng pht hin % so vi hm lng trn nhn Nebamin (vin nn bao ng) B150 mg55,8336 mg111,7 B6250 mg280,1075 mg112,0 B12250 g208 g83,2 Tervit H (vin nn bao phim) B1250 mg258,8702 mg103,6 B6250 mg269,5438 mg107,8 B12250 g223 g89,2 Neurobion (thuc tim) B1100 mg/3ml98,7014 mg/3ml98,7 B6100 mg/3ml102,3583 mg/3ml102,4 B125000 g/3ml4586 g/3ml91,7 - Qua kho st thy rng phng php bn nh lng cc vitamin B1, B6 vB12tronghnhppdngcchoccchphmthuc3Btrnth trng c cha 3 loi vitamin ny (cc ch phm kho st cho cc vt B1, B6 vB12 trn sc ktchri nhau v khngln viccvttp khcc trong ch phm), c th bn nh lng c ng thi nhiu ch phm trn mt bn mng, cho php tit kim thi gian v kinh ph thc hin.39 -HmlngcaB12 tmlictrongccchphmthpdoB12db phnhybinhiutcnhn:nhsng,nhit,m,cctcnhnha hc... trong khi iu kin kh hu nc ta li c nhit , m rt cao. Mt khc hm lng ca cht ny trong thuc thng rt nh, ch bng khong vi phntrmsovi2vitaminB1vB6.Vvy,dcccbinphpngn nga cc tc nhn c hi nh dng bao b kn trnh m v nh sng, bao vin, la chn t dc t gy tng tc... th cc vitamin (nht l B12) vn b phn hy lm gim hm lng. 40 KT LUN V XUT Kt lun: Trongkhalunny,quaqutrnhnghincuthcnghim,chngti hon thnh c cc mc tiu: 1.XydngphngphpbnnhlngccvitaminB1,B6,B12trong hn hp bng HPTLC. 2. nh gi c phng php va xy dng:-Phng php c chn lc cao vi cc vitamin B1, B6, B12. -ng chun tuyn tnh trong khong nng kho st. -Phng php c chnh xc tt vi ng: 93,5% - 102,8% khi bn nh lng vitamin B1; 98,9% - 104,6% khi bn nh lng vitamin B6; 89,5%- 103,5%khi bnnhlngvitaminB12 v lpli:RSD= 3,63%khibnnhlngvitaminB1,RSD=2,50%khibnnh lng vitamin B6, RSD = 4,34% khi bn nh lng vitamin B12. xut: Dothigianviukinchnnnkhalunmikhostccmu thuc ch cha hn hp 3 vitamin B1, B6, B12, v vy chng ti xin xut:- S dng phng php xy dng tip tc kho st cc ch phm thuc multivitamin cha 3 loi vitamin trn cc dng bo ch khc. -Tiptcnghincu,phttrinphngphpxydngbnnh lng ng thi c cc vitamin khc (B2, B3, B9, C, A, D, E,...) trong hn hp. 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