B Y T TRNG I HC DC H NI V TH QUNH NH LNG NG THI CALCI ATORVASTATIN V SIMVASTATIN TRONG CH PHM BNG SC K LNG HIU NNG CAO KHA LUN TT NGHIP DC S H NI - 2013 B Y T TRNG I HC DC H NI V TH QUNH NH LNG NG THI CALCI ATORVASTATIN V SIMVASTATIN TRONG CH PHM BNG SC K LNG HIU NNG CAO KHA LUN TT NGHIP DC S Ngi hng dn: 1. ThS. ng Th Ngc Lan 2. DS. Phm L Minh Ni thc hin: B mn Ha phn tch c cht H NI - 2013 LI CM N Kha lun tt nghip ny c hon thnh ti b mn Ha phn tch c cht, trngihcDcHNivishngdnvchbovgip tn tnh ca ThS. ng Th Ngc Lan v DS. Phm L Minh cng cc thy c trong b mn Ha phn tch c cht, trng i hc Dc H Ni. Ti xin php c by t lng bit nsu sc nht ti hai thy c trc tip hng dn ti, dnh nhiu thi gian, cng sc hng dn, du dt ch botinhngkinqubutrongsutqutrnhthchinkhaluntt nghip. Tixingi licm nsuscti PGS.TS. Thi Nguyn Hng Thuv lungipvchotinhngkinnggpqu butrongsutthi gian ti thc hin ti. Ti xin chn thnh cm n s gip ca cc thy c, cc k thut vin b mn Ha phn tch cchttoiukinttnhttihonthnh kha lun ny. Ti xin chn thnh cm n s quan tm ca ban gim hiu, phng o to nh trng v cc thy c truyn t kin thc cho ti trong sut thi gian ngi trn gh nh trng. Cuicngtivcngcmngianh, bnbvnhngngithn ngvinv htlng gip titronghctp cngnhtrong thigianti thc hin ti ny. H Ni, thng 5 nm 2013 Sinh vin V Th Qunh MC LC DANH MC CC K HIU, CH VIT TT DANH MC CC BNG DANH MC CC HNH NH, TH T VN ....... 1 Chng 1. TNG QUAN . 2 1.1. TNG QUAN V CHT NGHIN CU. 2 1.1.1. Tng quan v calci atorvastatin ... 2 1.1.1.1. Tnh cht ... 2 1.1.1.2. Tc dng dc l .. 2 1.1.1.3. Mt s phng php nh lng calci atorvastatin trong ch phm 3 1.1.2. Tng quan v simvastatin 4 1.1.2.1. Tnh cht ... 4 1.1.2.2. Tc dng dc l .. 5 1.1.2.3. Mt s phng php nh lng simvastatin trong ch phm . 5 1.1.3. Mt s phng php nh lng ng thi calci atorvastatin v simvastatin..6 1.2. TNG QUAN V PHNG PHP SC K LNG HIU NNG CAO .... 7 1.2.1. Khi nim HPLC ..... 7 1.2.2. Nguyn tc ca qu trnh sc k ..... 7 1.2.3. Sc k phn b hiu nng cao . 8 1.2.4. Cc thng s c trng trong HPLC ... 9 1.2.4.1. H s phn b K ... 9 1.2.4.2. Thi gian lu tR .... 9 1.2.4.3. H s dung lng k ... 10 1.2.4.4. H s chn lc . 10 1.2.4.5. Cc h s lin quan ti i xng ca pic sc k .... 10 1.2.4.6. S a l thuyt N .. 11 1.2.4.7. phn gii RS .. 11 1.2.5. ng dng ca HPLC . 11 1.2.5.1. nh tnh 11 1.2.5.2. nh lng . 12 Chng 2. I TNG V PHNG PHP NGHIN CU .. 14 2.1. I TNG, NGUYN VT LIU V TRANG THIT B ...... 14 2.1.1. i tng nghin cu ... 14 2.1.2. Ha cht v thit b ....... 14 2.2. NI DUNG NGHIN CU .... 15 2.3. PHNG PHP NGHIN CU .... 16 2.3.1. X l v chun b mu th 16 2.3.2. Chun b dung dch i chiu gc . 16 2.3.3. Kho st v xc nh iu kin sc k .. 16 2.3.4. Thm nh phng php ... 17 2.3.4.1. Tnh tng thch h thng .. 17 2.3.4.2. Tnh c hiu / chn lc . 17 2.3.4.3. Khong tuyn tnh, ng chun ... 18 2.3.4.4. lp . 18 2.3.4.5. ng .. 19 2.3.4.6. Phng php x l kt qu . 19 Chng 3. THC NGHIM, KT QU V BN LUN ... 21 3.1. X L V CHUN B MU NGHIN CU .. 21 3.1.1. X l v chun b mu th 21 3.1.2. Chun b dung dch chun i chiu . 21 3.2. KHO ST V XC NH IU KIN SC K .. 22 3.2.1. La chn ct sc k ... 22 3.2.2. La chn bc sng pht hin ...... 22 3.2.3. La chn pha ng 23 3.2.4. T l pha ng v tc dng ... 25 3.3. THM NH PHNG PHP .. 26 3.3.1. Kho st tnh tng thch h thng ... 26 3.3.2. Kho st tnh chn lc ca phng php .. 28 3.3.3. Kho st khong nng tuyn tnh . 28 3.3.4. Kho st lp li ca phng php 30 3.3.5. Kho st ng ca phng php .. 31 3.4. NGDNGNHLNGCALCIATORVASTATINVSIMVASTATIN TRONG CH PHM ..... 33 3.5. BN LUN . 35 KT LUN V XUT 37 KT LUN . 37 XUT ... 38 TI LIU THAM KHO PH LC 1 PH LC 2 PH LC 3 PH LC 4 DANH MC CC K HIU, CC CH VIT TT STTCh vit ttCh y 1ASEANAssociation of Southeast Asian Nations 2HPLCHigh performance liquid chromatography 3DMSODimethyl sulfoxid 4FDAFood and Drug Administration 5DVNDc in Vit Nam 6VNVit Nam 7LSXL sn xut 8HSDHn s dng 9NSXNgy sn xut 10SKS ng k 11KNTTWKim nghim thuc trung ng 12SKSS kim sot 13CYP3A4Cytochrome P450, family 3, subfamily A, polypeptide 4 14HDLHigh-density lipoprotein 15LDLLow-density lipoprotein 16ddDung dch 17SIMSimvastatin 18CACalci atorvastatin 19NXBNh xut bn DANH MC CC BNG STTK hiuTn bngTrang 1Bng 3.1Kt qu kho st pH m24 2Bng 3.2Kt qu kho st t l pha ng v tc dng25 3Bng 3.3Tnh tng thch h thng27 4Bng 3.4 Kt qu kho st khong tuyn tnh ca simvastatin 29 5Bng 3.5 Kt qu kho st khong tuyn tnh ca calci atorvastin 30 6Bng 3.6 Kt qu kho st lp li ca phng php i vi calci atorvastatin v simvastatin 31 7Bng 3.7Kt qu kho st ng32 8Bng 3.8 Kt qu nh lng mt s ch phm c calci atorvastatin, simvastatin 34 DANH MC CC HNH NH, TH STTK hiuTn hnhTrang 1Hnh 1.1Cng thc cu to calci atorvastatin2 2Hnh 1.2Cng thc cu to simvastatin4 3Hnh 3.1 Hnh nh qut ph dung dch chun ca mu simvastatin (a) v ca mu calci atorvastatin (b) 22 4Hnh 3.2 Sc k mu chun kp ti hai bc sng 238 nm v 246 nm 23 5Hnh 3.3 Sc k mu kho st la chn pha ng vi m pH 4,5 ca mu simvastatin (a) v mu calci atorvastatin (b) 24 6Hnh 3.4Sc k ca mu chun kp pH 2,525 7Hnh 3.5 Sc k mu chun kp theo chng trnh chy xy dng 26 8Hnh 3.6 th biu din s ph thuc tuyn tnh gia nng v din tch pic ca simvastatin 29 9Hnh 3.7 th biu din s ph thuc tuyn tnh gia nng v din tch pic ca calci atorvastatin 30 10Hnh 3.8 Sc k nh lng mt s ch phm vi mu chun kp (a), mu th ca ch phm Simvastatin (b) v mu th ca ch phm Atostin (c) 35 - 1 - T VN Cngvisphttrincaxhi, mhnhbnhttccnctrnthgii ngycngadngvphctp,tlnhngngimcccbnhtimmch, tiu ng, ung th ngy cng cao dn thay th cho cc bnh nhim trng trc y. iu ny l do s thay i ca kh hu, thi quen sinh hot khi i sng ang ngy cng nng cao. VitNam cng khng nm ngoi quy lut ny. Trc y, m hnh bnh tt Vit Nam ch yu l cc bnh nhim trng th nay m hnh bnh tt hontonthayi: ch c 27% l cc bnhdovitrnggynn,cn 62% cc bnh khng phi do vi trng (cc bnh ly nhim do siu vi trng) nh:huyt p, tm thn, tim mch, suy dinh dng, tiu ng... cn li 11% loi bnh do tai nn thng tch [4].Theo s liu ca Cc Qun l Dc, nguyn nhn gy t vong chnh Vit Nam hin nay thuc v nhm bnh tim mch (13%) [4]. Trong phi ni n cc bnh x va ng mch, vi cc biu hin lm sng nh suy mch vnh, t t, nhi mu ctim,nhi mu no... Nguyn nhn ch yu l do cc ri lon lipid mu. Calci atorvastatin v simvastatin l mt trong nhng thuc h lipidmuc s dng nhiu hin nay. Tuy nhin, trong cc ti liutrong nc nhDVNIV v mt s tiliuncngoichngtithamkho[3,12,15,16,17,23],chacnhiu phngphpchun nhlng hai cht nycng nhccstatinkhchoc quy trnh cn phc tp. V vy, nghin cu xy dng mt quy trnh nh lng cc statin ny l ht sc cn thit. Tcccstrnchngtitinhnhti:nhlngngthicalci atorvastatinvsimvastatintrongchphmbngscklnghiunngcao vi hai mc tiu c th sau: Xydngcmtquytrnhnhlngngthicalciatorvastatinv simvastatin bng sc k lng hiu nng cao ng dng nh lng calci atorvastatin v simvastatin trong ch phm bng sc k lng hiu nng cao - 2 - Chng 1. TNG QUAN 1.1. TNG QUAN V CHT NGHIN CU 1.1.1. Tng quan v calci atorvastatin 1.1.1.1. Tnh cht a. Cng thc cu to [5, 23] Calci atorvastatin l mt thuc trong nhm statin, thuc dn cht acid heptanoic (khng ng vng -lacton). Thuc c cng thc cu to nh sau: Hnh 1.1. Cng thc cu to calci atorvastatin Cng thc phn t: C66H68CaF2N4O10 Phn t lng: 1155,34Tnkhoahc:Calcium( R, R)-2-(p-fluorophenyl)-,-dihydroxy-5-isopropyl-3-phenyl-4-(phenylcarbamoyl)pyrrole-1-heptanoate(1:2),trihydrate [344423-98-9]. Anhydrous [134523-03-8] b. Tnh cht vt l [16, 21, 23] Calciatorvastatincdng btkttinh trnghoctrngnht.Tannhiutrong methanol, hi tan trong ru, khng tan n t tan trong nc ct, m phosphat pH 7,4 v trong acetonitril Hp th UV vi cc cc i hp th 194, 224 v 246 nm Nhit nng chy: 176oC 178oC Gc quay cc: [o] - 7,4o (C =1 trong DMSO) 1.1.1.2. Tc dng dc l [10, 11] Calci atorvastatin l mt thuc thuc nhm c ch enzyme kh HMG CoA, hot ng bng cch c ch cnh tranh men kh HMG CoA reductase mt enzyme xc - 3 - tcchophnngbini 3-hydroxy-3-methylglutaryl-coenzyme A(HMGCoA) thnh mevalonate, y l bc trung giantrong qu trnh sinh tng hp cholesterol ti gan do lm gim lng cholesterol. c ch cnh tranh vi enzyme kh HMG CoA lm gim tng hp cholesterol, tng biu hin ca cc th th lipoprotein mt thp(ththLDL) trnt bogan, iunylmtnghpthuLDL bicct bo gan, lmgimlngLDL- cholesterol trong mu. Nhccstatinkhc, calci atorvastatincng lm gimnngtriglyceridetrongmuvhilmtnglng HDL - cholesterol. Dc ng hc: Calci atorvastatin hp thu nhanh sau khi ung, vi thi gian xp x nng tia(T max) 1 - 2 gi. Sinhkhdngtuyti ca thuc l khong 14%,tlgnviproteincao(98%).Cchchuynhachnhcacalci atorvastatin lthngquacytochromP4503A4hydroxyl tothnhorthov parahydroxylated hot ng. Calci atorvastatin ch yuothi qua gan mt, c t hn2%thuhitrongnctiu. Thigianbnthicathucl14 gi, hunh khng c chu k gan rut. 1.1.1.3. Mt s phng php nh lng calci atorvastatin trong ch phm Phng php 1 [16]:-Ct C18 (25 cm x 4,6 mm, 5 m) -Pha ng:+Dung dch m: ha tan 1,54 g ammonium dihydrogen orthophosphat vo 1000 ml nc, iu chnh pH v 4,0 bng acid acetic bng. +Dung mi A: hn hp ca acetonitril : tetrahydrofuran (92,5 : 7,5) +Pha ng l hn hp ca dung dch m pH 4,0 v dung mi A vi t l 50 : 50-Tc dng: 2 ml/pht, th tch tim mu: 20 l, bc sng ghi: 246 nm Phng php 2 [19]:-Ct: ct thp khng g Waters Symmetry C18 (250 mm x 4,6 mm, 5 m) iu nhit ct 40 oC - 4 - -Phang: Dungdchmkali dihydrogenphosphatpH3,0 : acetonitril (60 : 40) -Tc dng: 1 ml/pht, th tch tim mu: 20 l, bc sng ghi: 246 nmPhng php 3 [23] -Ct: Ct L7 ( 25 cm x 4,6 mm x 5m), iu nhit ct 35 oC Pha ng: chng trnh gradient dung mi nh sau: +Dung mi A: acetonitril : tetrahydrofuran : m ammonium acetat pH 5,0 0,1 (21 : 12 : 67) +Dung mi B: acetonitril : tetrahydrofuran : m ammonium acetat pH 5,0 0,1 (61 : 12 : 27) Thi gian (pht)Dung mi A (%)Dung mi B (%) 01000 401000 702080 850100 1000100 1051000 1151000 1.1.2. Tng quan v simvastatin 1.1.2.1. Tnh cht a. Cng thc cu to [6, 22, 23] Hnh 1.2. Cng thc cu to simvastatin - 5 - Cng thc phn t: C25H38O5 Phn t lng: 418,57Tnkhoahc:Butanoicacid,2,2-dimethyl-,1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl ester, [1S-[1,3,7,8 (2S*,4S*),8a]] b. Tnh cht vtl [13, 21, 22, 23] Simvastatincdngbtkttinhmutrng, khng mi, ttantrongnc, tan nhiu trong ethanol, methanol, DMSO, chloroform. Gc quay cc: [o]25 +292o (C =0,5% trong acetonitril) Nhit nng chy: 135oC 138oC Hp th UV: dung dch trong acetonitril c cc cc i 231, 238, 247 nm1.1.2.2. Tc dng dc l [10, 11] Lmttrong ccstatin nhcalciatorvastatin, simvastatin cng hot ngtheo c ch c ch enzyme kh HMG-CoA (3- Hydroxymethyl- 3- Glutaryl Coenzyme A reductase inhibitors) tronggan.y l mt enzyme kh cn thit cho vic sn sinh cholesterol giai on sm. Trong mu, simvastatin lm gim cholesterol ton phn v lipoprotein mt thp (LDL) hay cholesterol "xu" cng nh triglycerid. Simvastatincnglmtnglipoproteinmtcao(HDL)haycholesterol "tt". Tng mc cholesterol HDL, cng nh lm gim cholesterol LDL c th lm chm s pht trin ca bnh ng mch vnh. Dcnghc: Simvastatinhpthnhanhsaukhiung, quachuynhaln u quagan vcsinhkh dngtuytil 5%, nng nhtrong huyttng t c sau 4 gi. Phn b ch yu n gan, qua c hng ro mu no, 95% lin ktviproteinhuyttng. Qua gan, simvastatincchuynhabiCYP3A4 thnh dngc hot tnh. Thi gian bn thi ca simvastatin l 2 gi v 1,9 gi vi simvastatin dng acid. Thi tr ch yu qua phn (60%) v thn (13%). 1.1.2.3. Mt s phng php nh lng simvastatin trong ch phm Phng php 1 [12]:-Ct Hypersil ODS 25 cm x 4.,6 mm, 5 m, iu nhit ct 45oC - 6 - -Pha ng: m phosphat pH 4,5 : acetonitril (7 : 13) -Tc dng: 1,5 ml/pht, th tchtim mu: 20 l, bcsngghi: 238 nmPhng php 2 [14]:-Ct: C18-Hypersyl (250 mm x 4,6 mm, 5 m) -Pha ng: acetornitril : dung dch m phosphat pH 4,5 : methanol (5 : 3 :1) -Tc dng: 2,5 ml/pht, th tchtim mu: 20 l, bcsngghi: 230 nm Phng php 3 [22, 23]:-Ct L1 4,6 mm x 25 cm, iu nhit ct 45oC -Pha ng: acetonitril : dung dch m phosphat pH 4,5 (65 : 35) -Tc dng: 1,5 ml/pht, th tchtim mu: 10 l, bcsngghi: 238 nm1.1.3.Mtsphngphpnhlngngthicalciatorvastatinv simvastatinPhng php 1 [8]: Phng php: nh lng ng thi atorvastatin, lovastatin v simvastatin bng k thut sc k mixen in ng: - Dung dch in ly nn dinatri tetraborat 15 mM pH 8,0 cha 50 mM cht hot ng b mt sodium deoxycholat v 15% methanol - Ct mao qun silica nung chy, chiu di tng cng 57 cm, chiu di hiu qu 48,5 cm, ngknh trong 50 m; nhitct30oC;inth30 kV. Kiutim mu 50 mbar x 5 giy. Bc sng pht hin 237 nm -Calciatorvastatincdnglmchunnikhiphntchnhlngcho simvastatin v lovastatin. Lovastatin dnglmchun nikhi phntch nhlng calci atorvastatin - 7 - Phng php 2 [18]: - nh lng atorvastatin, simvastatin, lovastatin, pravastatin s dng chun ni l mevastatin: Mu chit pha rn trong nc Ct Genesis C18 (2,1 x 50 mm, 3m) Pha ng: gradient dung mi ca dung mi A (acetonitril +methyl acetat 2 mM +acid acetic 0,1%) v dung mi B (nc +methyl acetat 2 mM +acid acetic 0,1%) H thng sc k lng ghp u d khi ph ESI+MS-MS SRM -nhlngatorvastatin,simvastatin,lovastatin,pravastatin,rosuvastatins dng chun ni l theophyllin: Mu: chit t dch huyt tng Ct: Intersil ODS 3V (4,6 x 250 mm, 5m) Pha ng: ammonium acetat 0,01 M pH 5,0 +acetonitril +methanol Bc sng ghi: 238 nm 1.2. TNG QUAN V PHNG PHP SC K LNG HIU NNG CAO 1.2.1. Khi nim HPLC [2] HPLC l k thut phn tch da trn c s ca s phn tch cc cht trn mt pha tnhcha trong ct, nh dng di chuyn capha ng lngdi p sut cao. Sc k lng da trn c ch hp ph, phn b, trao i ion hay loi c ty vo loi pha tnh s dng.1.2.2. Nguyn tc ca qu trnh sc k [1, 7] Mu phn tch c ha tan trong mt pha ng. Pha ny c th l mt cht kh, cht lnghocchtlngsiutihnc cho qua pha tnhmt cch lin tc v khng ha ln vi n. Pha tnh c c nh trong ct hay trn b mt cht rn. Cc cht tan l thnh phn ca mu s di chuyn qua ct theo pha ng vi tc khc nhau ty thucvotngtcgia pha tnh,phang v cht tan. Nh tc di chuyn khc nhau, cc thnh phn ca mu s tch ring bit thnh di,lm cs cho phn tch nh tnh v nh lng. - 8 - Phatnh c nhivocttchtheo mtk thut nht nhvlyut quyt nh bnchtca qutrnhsck. C 4 k thut sckcn bn: Sc k phn b, hp ph, trao i ion v sc k ry phn t. Trong sc k phn b c s dng nhiu nht trong kim nghim thuc do ti xin trnh by k v sc k phn b. 1.2.3. Sc k phn b hiu nng cao [1, 2] Sc k phn b l phng php phn tch da trn khc bit v phn b ca cc cu t gia pha tnh v pha ng. Sc k phn b c chia lm 2 loi ty thuc vo pha tnh: sc k lng - lng v sc k pha lin kt. Pha tnh: Sc k lng - lng: pha tnh gm mt lp mng pha lng hu c bao trn b mt cc tiu phn cht mang silica hoc cc cht liu khc. Cc tiu phn ny thng c ng knh t 3 n 10m(kchthc ht c th n 50 m hocln hntrong sc k iu ch). Pha tnh kiu ny c nhc im l: b ra tri dn theo dng pha ng, hiu lc ct s gim dn trong qu trnh s dng. Sc k pha lin kt: pha tnh c lin kt ha hc vi cht mang nn khc phc cnhcimca scklng- lng. Trongphatnh loiny, cc nhm chc hu c lin kt vi b mt ca cc tiu phn silica qua nhm silanol. Tnh phn cc ca loi pha tnh ny ph thuc vo tnh phn cc ca cc nhm chc lin ktPha ng: Pha ng trong sc k phn b c th l dung mi n hay hn hp nhiu dung mi.Ngi ta c th thay i phn cc ca pha ng bngcchthayi t l cc thnh phn dung mi. Ty thuc vo vic s dng pha ng v pha tnh, ngi tachiasc k phn b thnh 2 loi: sc k pha thun v sc k pha o. -Sck phathun: H baogmphatnhphnccvpha ng khng phn cccgilsckphathun.Dungmiphangthnglcc hydrocacbonmchthngnh:pentan,hexan,heptanTrongsckpha thun cc cht khng phn cc s c ra gii sm, th t ra gii s chm dn theo chiu tng ca phn cc ca cc thnh phn trong mu th. - 9 - -Sc k pha o: H pha ng phn cc v pha tnh khng phn cc gi l sc kphao.ylphngphpcsdngnhiunhttrongkim nghim thuc. Cht cng tan tt trong dung mi phn cc th cng c ra gii sm.Dung mi pha ngthngl: nc, methanol, acetonitrilVic ui kh ha tan trong pha ng rt quan trng trong sc k pha o. 1.2.4. Cc thng s c trng trong HPLC [1, 2, 7] 1.2.4.1. H s phn b K Tc di chuyn ca cht tan qua pha tnh c xc nh bi h s phn b K: K =C5CM Trong : CS l nng mol ca cht tan trong pha tnh (mol/lit) CM l nng mol ca cht tan trong pha ng (mol/lit) HsKphthuc bnchtcaphang, phatnhvchthatan. TrsK cng ln, s di chuyn ca cht tan qua pha tnh cng chm. Nu cc cht trong hn hpchngsKkhcnhaucngnhiu, thkhnngtchdinracngddng hn. 1.2.4.2. Thi gian lu tR ThigianlutR lkhongthigiantlctimmuvoctnkhipicn detector. Trongcngmtiukinsckchn, thigianlucamichtl hng nh, iu ny lm c s cho php nh tnh. Thi gian lu ca mi cht ph thuc cc yu t: -Bn cht ca pha tnh -Bn cht, thnh phn, tc ca pha ng -Cu to v bn cht phn t ca cht tan -Mt s trng hp cn ph thuc pH ca pha ng Trong mt php phn tch nu tR qu nh th s tch km, nu tR qu ln th pic bdong v lplicapic rtkm, thigianphntchdingthikotheo nhiu vn khc nh hao tn dung mi, ho cht, chnh xc ca php phn tch km. thay i thi gian lu chng ta da vo cc yu t m tR ph thuc. - 10 - 1.2.4.3. H s dung lng k H s k l mt thng s quan trng m t tc di chuyn ca cht phn tch A qua ct. H s k cn c gi l h s phn b khi lng gia hai pha: kA =C5.V5CM.VM =KA vSvM Trong : VS: th tch pha tnh (lt) VM: th tch pha ng (lt) CS: nng mol cht tan trong pha tnh (mol/lt) CM: nng mol cht tan trong pha ng (mol/lt) KA: h s phn b 1.2.4.4. H s chn lc c trng cho tc di chuyn t i ca 2 cht A v B, ngi ta dng h s chn lc : = KEKA =kE|kA| =tR,E|tR,A| Vi quy c KB > KA nn lun ln hn 1 tch ring 2 cht thng chn dao ng trong khong 1,05 2. Nu qu ln thi gian phn tch s di. 1.2.4.5. Cc h s lin quan ti i xng ca pic sc k nh gi tnh i xng ca pic sc k ngi ta dng cc i lng: H s bt i AF: AF =ba Trong :a: Na chiu rng pha trc pic b: Na chiu rng pha sau pic (c a v b c o 1/10 chiu cao pic) Gi tr AF cng gn 1, pic cng i xng. H s ko ui AS: AS =a+b2a - 11 - Vi a v b o 1/20 chiu cao pic Nu AS cng ln hn 1, pic ko ui cng nhiu, pic cng mt cn i. 1.2.4.6. S a l thuyt N S a l thuyt l i lng c trng cho hiu lc ct sc k N =16 tR2W2 =5,54 tR2W1/ 22 Trong : W: chiu rng o y pic W1/2: chiu rng o na chiu cao pic 1.2.4.7. phn gii RS RS =2.(tR,E-tR,A)WE+WA =1,18.(tR,E-tR,A)W1/ 2,E-W1/ 2,A Vi: tR,B, tR,A: thi gian lu ca 2 pic lin k nhau (B v A) WB, WA: rng pic o cc y pic W1/2,B, W1/2,A: rng pic o na chiu cao pic Cc gi tr trn c tnh theo cng mt n v phn gii RS cn c tnh theo cng thc:RS =N4 u-1u kE|1+kE| Yu cu RS >1, gi tr ti u RS =1,5 1.2.5. ng dng ca HPLC Sc knichung, HPLC ni ringlcc kthut phntchrtc hiuqu tchvnhlngcchpchtccutrchahcgnnhnhautrongmthn hp. V vyn c dng ph bin khi mu phn tch c ngun gct nhin hoc sinh vt. HPLC c cc ng dng ph bin sau: 1.2.5.1. nh tnh Sc k cho ta thi gian lu (tR) ca cht phn tch trong cng iu kin sc k (pha ng, pha tnh, nhit ), nhng thng tin nh tnh gip ta khng nh s c mtcacht phntchtrong mu. Vi mu nhiu thnh phn, vic nhtnh bng - 12 - quangphthnggpnhiukhkhn, doscktrongcHPLCthng c dng tch cc thnh phn trc khi phn tch bng quang ph. 1.2.5.2. nh lng D liu thc nghim dng trong nh lng l chiu cao pic hoc din tch pic. Tt c cc phng php nh lng bng sc k u da trnnguyn tc: nng ca cht t l vi chiu cao hoc din tch pic. Cc phng php nh lng trong HPLC [2] Phng php chun ngoi Nguyn tc: mu chun v mu th u c tin hnh sc k trong cng iu kin, so snh din tch (hoc chiu cao) pic ca mu chun v mu th s tnh c nng cc cht trong mu th. C th s dng phng php chun ha mt im hoc nhiu im. -Chun ha mt im: Chn nng ca mu chun xp x vi mu th, tin hnh sc k c hai mu trong cng iu kin. Tnh nng mu th theo cng thc: CX =CS SXS5

Trong :CX: nng mu th CS: nng mu chun SX (HX): din tch (chiu cao) ca pic mu th SS (HS): din tch (chiu cao) ca pc mu chun -Chun ha nhiu im: Chun b mt dy chun vi cc nng tng dn ri tin hnh sc k. Cc p ng thu c l cc din tch hoc chiu cao pic mi im chun. V th biu din s tng quan gia din tch (S) hoc chiu cao (H) ca pic vi nng ca cht chun (C). S dng on tuyn tnh ca ng chun tnh ton nng ca cht cn xc nh. Phng php chun ni Nguyn tc: thm vo c mu chun v mu th nhnglng bng nhau ca mt cht tinh khit ri tin hnh sc k trong cng iu kin. Cht cthm ny - 13 - gi l chun ni. T d kin v din tch (hoc chiu cao) pic v lng (hoc nng ) ca chun, chun ni v mu th, c th xc nh c hm lng ca cht cn phn tch trong mu th.Yu cu ca cht chun ni l: - Tch hon ton v c thi gian lu gn vi thi gian lu ca cht phn tch - C cu trc ha hc tng t cht th - C nng xp x nng cht th - Khng phn ng vi cc thnh phn trong mu th - C tinh khit cao v d kim Phng php thm chun Nguyntc:thmvomuthnhnglngbitcaccchtchun tng ng vi cc thnh phn c trong mu th ri tin hnh sc k trong cng iu kin. Nng cha bit CX ca mu th c tnh theo cng thc: CX =SX CS Trong :SX: din tch (hoc chiu cao) ca pic mu th C: lng cht chun thm vo S: s chnh lch din tch pic (hoc chiu cao) K thut thm ng chun Nguyn tc: chun b mt dy hn hp gm cc lng mu th ging nhau v cc cht chun vi lng tng dn. X l mu ri tin hnh sc k trong cng iu kin. Dngng chun biuth mitngquangia din tch (S) hoc chiucao(H) ca pic tng (th +chun) vilnghocnngcht chun thm vo (C). Giao im ca ng chun ko di vi trc honh l nng ca cht cn xc nh. - 14 - Chng 2. I TNG V PHNG PHP NGHIN CU 2.1. I TNG, NGUYN VT LIU V TRANG THIT B 2.1.1. i tng nghin cu Cc ch phm c cha simvastatin 10mg: -Vin nn Simvastatin 10mg (STADA - VN),SK:VD-7764-09, NSX: 10-07-2012, HSD: 10-07-2014, LSX: 020712 -Vinnnbaofilm Winthrop (SanofiAventis),SK:VD-11615-10,NSX: 09-05-2011, HSD: 09-05-2013, LSX: 010511 Cc ch phm c cha calci atorvastatin 10mg: -Vin nn Lipitad (STADA -VN),SK:VD-10728-10, NSX: 27-05-2012, HSD: 27-05-2015, LSX: 030512 -Vin nn Atostin (CngtyDPTrngTh),SK:VD-9565-09, NSX: 17-02-2012, HSD: 17-02-2015, LSX: 121 -VinnnHacortin(COHAVINA-XNDP150),SK:VD-4592-09,NSX: 30-11-2012, HSD: 30-11-2014, LSX: 28211 2.1.2. Ha cht v thit b a. Ha cht -Acetonitril, methanol tinh khit loi dng cho HPLC (Merck, c) -Chtchun:simvastatinhmlng99,14%(SKS:0210182.02)vcalci atorvastatin khan hm lng 98,77%, m 4,98% (SKS: WS. 0208225) do Vin KNTTW cung cp -Natriumdihydrogenphosphat dihydrat (NaH2PO4.2H2O) (Merck, c) -Acid ortho - phosphoric 85% (Merck, c) -Nc ct 2 ln dng cho HPLC b. Trang thit b -Myscklnghiunngcao Agilent1100 G 1314Aktni vi detector UV-Vis (M) -Ct sc k Apollo C18 5u (4,6 x 250 mm, 5m) - My khuy t WiseStir MSH - 20A (Hn Quc) - 15 - - My o pH Eutech Instrument pH510 (Singapore) -Autopipet 100 - 1000 l, 1000 - 5000 l (Nht) -My siu m Ultrasonic LC 60 H (c) -My li tm Harmonic Series (i Loan) -My ht chn khng Vacuubrand Membran (c) -Mng lc Satorius cellulose acetat 0,4 m (c) -Cn phn tch Sartorius TE214S c chnh xc 0,1 mg (c) -B mng lc mu 0,20 m -Bnh nh mc dung tch 10 ml, 20 ml, 100 ml -Pipet thy tinh chnh xc 5 ml, 10 ml -ng ong dung tch 100 ml, 500 ml -Cc c m dung tch 50 ml, 100 ml, 1000 ml -Bm tim, vial 2.2. NI DUNG NGHIN CU Xydngphngphpnhlngngthisimvastatinvcalciatorvastatin bng sc k lng hiu nng cao -La chn iu kin sc k La chn ct sc k La chn pha ng v cc iu kin pha ng Tc dng, th tch tim La chn bc sng pht hin Quy trnh x l mu -nh gi chng trnh sc k xy dng thch hp ca h thng sc k Tnh chn lc Khong tuyn tnh gia nng v phn trm din tch pic lp li ng ng dng phng php xy dng nh lng cc ch phm chn - 16 - 2.3. PHNG PHP NGHIN CU 2.3.1. X l v chun b mu th Chun b dung mi pha mu Qua nghin cu tnh cht ha l ca cc cht nghin cu, kt hp vi cc ti liu tham kho, kho st la chn ra dung mi pha mu thch hp. Tin hnh pha dung mi pha mu phc v cho qu trnh phn tch. X l mu v chun b mu th Mun nh lng c dc cht trong ch phm vn u tin cn quan tm l c c quy trnh x l mu hp l, m bo dc cht c chit tch hon ton vomuth, khngbbinitnhchtvkhnglnqunhiutpgysailch cho kt qu phn tch. Tham khocctiliukt hp vithc nghimkhostlachnra quytrnh x l mu thch hp. Tin hnh x l mu v chun b mu th. 2.3.2. Chun b dung dch i chiu cthnhlngcdcchttrongccchphm, vicchun bdung dch gc i chiu v cc mu chun l rt cn thit, cn c tin hnh ng thi vi vic chun b mu th Qua nghincutnhchthalcaccchtnghincu, kthpcctiliu tham kho la chn ra dung mi pha v bo qun dung dch chun gc. 2.3.3. Kho st v xc nh iu kin sc k Ct sc k Tcctiliuthamkhoviukinhinc, lachnctsck, phng php sc k s dng trong qu trnh phn tch. Bc sng pht hinTinhnhqutphdungdchchuncalciatorvastatinvdungdchchun simvastatin mt nng thch hp. Cn c ph thu c, kt hp cc ti liu tham kho v tin hnh kho st la chn bc sng pht hin ph hp. Pha ng - 17 - L mt trong nhng yu t quyt nh hiu sut tch sc k, quyt nh thi gian lu gia cc cht phn tch v hiu qu ca s tch sc k. Qua nghin cu tnh cht ha l ca cc cht nghin cu v mt s phng php nhlngcc chtnycctiliuthamkho, phangchngtilachnl hn hp ca acetonitril v m phosphat Tin hnhkhost pha ng v pH m, tl pha ngvtc dng la chn c cc iu kin ph hp. 2.3.4. Thm nh phng php [9] 2.3.4.1. Tnh tng thch h thngKhosttnhtngthchhthnglphpth nhmnhginnhca ton h thng phn tch bao gm: my mc thit b, cch tin hnh phn tch, mu thVickho st tin hnh bngcchtimlp li 6lncng mt dung dch chun vo h thng scktheochngtrnhchn. thch hp ca h thng HPLC c biu th qua h sko ui, s althuyt, lch chuntng i (RSD) ca thi gian lu v din tch pic. Yu cu: RSD khng ln hn 2 (%) S a l thuyt: N 2000 H s bt i: 0,8 AF 2,0 [1, 6] 2.3.4.2. Tnh c hiu / chn lc 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 Cthtrongphpphntchnhtnh lphichngminhc ktqul dngtnhkhicmtcht phntch, mtnh khikhngcmt n, ngthikt qu phi l m tnh khi c mt cc cht khc c cu trc gn ging cht phn tch. Trongphpphntchnhlng, lkhnngxcnhchnhxcchtphntch trong mu khi b nh hng ca tt c cc yu t khc, nhm hng n n kt qu chnh xc. - 18 - Tnh chn lc: L khi nim rng hn tnh c hiu, lin quan ti 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 c tnh chn lc. 2.3.4.3. Khong tuyn tnh, ng chun Khongtuyntnhcamtphngphplkhongnngcsph thuctuyntnhgia ilng ocvnng chtphntch. Khong tuyn tnh l khong t nng thp nhtn cao nht trong mtng chuncp ng tuyn tnh. Vicxcnhkhongtuyntnhthngckhostbtut giihnnhlng(imthpnht) vktthclgiihntuyntnh(imcao nht). xc nh khong tuyn tnh cn ti thiu l 6 nng khc nhau. ng chun biu din mi quan h gia p ng o c vi nng bit ca cht cn phn tch trong dung dch. Yucu: hs tng quan hi quytuyntnhkhixydng ngchunphi tha mn yu cu sau: 0,995 R 1 Hay 0,99 R2 1 [9] 2.3.4.4. lp chm th hinmc dao ngca cc kt quth nghim c lp quanh gi tr trung bnh. chm ch ph thuc vo sai s ngu nhin v khng lin quan n gi tr thc. chm c th c phn ra lm ba trng hp sau: - lp li - chm trung gian - ti lp Trongcngiukinvthitb, ngiphntch, dngchacht, iukin mi trng, phng th nghim, ch khc nhau v nn mu, nng th chm th hin bng lp li. xcnh lpli, tin hnhth nghimlplit nht 6 lntrncng mt mu(milnbtutcnhayong mu). Muphntchcthlmuchun, hoc mu trng thm chun, tt nht l lm trn mu th hoc mu th thm chun.- 19 - lp li c biu th bng lch chun (SD) hoc lch chun tng i (RSD) Yu cu: lp li chp nhn ti cc nng khc nhau hm lng 100% l c lch chun tng i RSD 1,3 (%) [9] 2.3.4.5. ng ngca mtphngphplkhi nimchmc gn nhaugiagitr trungbnhcakt quphn tch vigi tr thchocgitrcchpnhnl 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). Mun xc nh ng cn phi tm c gi tr ng, c nhiu cch khc nhau xcnhng, baogmvicsosnhktquthcnghimviktquthc hin bi mt phng php i chiu hoc s dng mu bit nng vphng phpxcnhthuhi. ivicchxcnhngbngcchsosnhvi phng php chun/i chiu hoc cch s dng vt liu chun u gp nhng kh khnnhtnh.Trongnhiutrnghpkhngthtmchocpdngmt phng phptiu chun so snh kt qu, cng khng d dng c c cc 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. Xc nh thu hi bng cch 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: R% =Cm+c- CmCc x 100 Trong : R%: thu hi Cm+c: Nng cht phn tch trong mu thm chun Cm: Nng cht phn tch trong mu th Cc: Nng chun thm (l thuyt) - 20 - Sau tnh thu hi chung l trung bnh ca thu hi cc ln lm lp li 2.3.4.6. Phng php x l kt qu -Cc kt qu sc k c x l bng cc phn mm Chemstation ca thit b sc k lng hiu nng cao. -S dng cc phng php x l thng k trong phn tch vi cc i lng c trng kt hp vi s h tr tnh ton ca Microsofl Office Exel Mt s cng thc tnh ton s dng trong x l thng k kt qu - Gi tr trung bnh:nXXnii 1 - lch chun: SD =) 1 () (12nX Xnii - lch chun tng i: RSD =100 XSD - 21 - Chng 3. THC NGHIM, KT QU V BN LUN 3.1. X L V CHUN B MU NGHIN CU 3.1.1. X l v chun b mu th a. Chun b dung mi pha mu Qua nghin cu ti liu tham kho v tin hnh kho st, ti la chn dung mi pha mu l hn hp: acetonitril : m phosphat pH 2,5 (70 : 30), y cng chnh l pha ng. Cchphamphosphat pH2,5: cnkhong3,9 g natriumdihydrogenphosphat dihydrat (NaH2PO4.2H2O) v ha tan vo 900 ml nc. iu chnh pH v gi tr 2,5 bng dung dch acid phosphoric 85%, b sung nc ti 1000 ml. msaukhi pha c lcqua mnglccelluloseacetat 0,45 mtrn my lc ht chn khng sau trn vi acetonitril theo t l: acetonitril : m phosphat pH 2,5 (70 : 30), sausiumtihtbtkh, chsdnghnhpdungminy trong ngy. b. X l v chun b mu th Cn khilng 20 vin, tnh khilng trung bnh 1 vin. Nghin mn thuc thnh bt, cn lng bt tng ng vi 3 mg dc cht cho vo bnh nh mc 10 ml. Thm methanol ti 2/3 bnh, em siu m trong 10 pht, sau yn 10 pht ri thm methanol ti vch. em ly tm vi vn tc 3000 vng/pht, trong thi gian 10 pht. Ht chnh xc 5 ml dch ly tm cho vo bnh nh mc 10 ml, thm dung mi pha mu ti vch, lck. Dch phac emlc qua mng 0,2 mtrckhi chysck. Dung dch ny c nng cht phn tch khong 0,15 mg/ml. Lm ng thi vi cc ch phm ca simvastin v calci atorvastatin. 3.1.2. Chun b dung dch i chiu -Vccchtkhostutantttrongmethanolnntiquytnhlachn methanol lm dung mi pha v bo qun dung dch chun gc. -Dung dch chun gc: Cnchnhxclngchtchuntngngvi25 mg dcchtchunchovobnhnhmcdungtch25ml,thmkhong20ml methanol, lc siu m 5 pht, thm methanol va n vch, lck. Lmng - 22 - thi vi c 2 cht chun. Dung dch chun gc c nng 1 mg/ml c bo qun theo yu cu trn nhn. Dungdchnyc phalongtinngthchhp kho st v xy dng dy dung dch chun. -Muchunn: ht chnh xc 1,5 ml dung dch chun gc cho vo bnh nh mc dung tch 10 ml, thm pha ng ti vch v lc k. Dung dch thu c em lc qua mng 0,2 m trc khi em chy sc k. Dung dch chun n ny c nng cht chun khong 0,15 mg/ml. Lm ng thi vi simvastatin v calci atorvastatin. -Mu chun kp: ht chnh xc 1,5 ml dung dch chun gc ca mi cht cho vo bnhnhmcdungtch10 ml, thmphangtivchvlck. Dungdchthu cemlc qua mng 0,2 mtrckhi emchysck. Dung dchchunkp ny cha c hai cht chun nng 0,15 mg/ml. 3.2. KHO ST V XC NH IU KIN SC K 3.2.1. La chn ct sc k Quaccti liu nghincuv nh lngcalciatorvastatinvsimvastatin, ti thyrngccphng phpus dng sckphn b pha o. Hin naysck phn b pha o l phng php c s dng kh ph bin vi nhiu tnh u vit. Do , ti la chn sc k phn b pha o trong nghin cu ny. S dng ct hin c l ct sc k Apollo C18 5u (4,6 x 250 mm, 5m). 3.2.2. La chn bc sng pht hin xc nh bc sng thch hp pht hin simvastatin v calci atorvastatin, ti thc hin qut ph UV mu chun n ca c hai cht. Mu chun n c chun b theo quy trnh mc 3.1.2. Kt qu thu c nh sau:

(a)(b) Hnh 3.1. Hnh nh qut ph dung dch chun ca mu simvastatin (a) v ca mu calci atorvastatin (b) - 23 - Kt quchothysimvastatinc hp th ln nht ti bc sng 238nm. Do lachnbcsngnynhlngsimvastatin. Vicalciatorvastatin, cc hpthcci ti3bcsng194nm,224nm,246nm. gim sai s do dung mi,tilachnbcsngphntchl246nmlbcsngmcalci atorvastatin hp th ln th 2. Tin hnh chy sc k cc mu chun n kho st ca c 2 cht 2 bc sng 238 nm v 246 nm thu c kt qu v chiu cao ca pic nh sau: -Simvastatin: 278 mAU (ti 238 nm) v 205 mAU (ti 246 nm) -Calci atorvastatin: 469,85 mAU (ti 238 nm) v 488,3 mAU (ti 246 nm) Hnh 3.2. Sc k mu chun kp ti hai bc sng 238 nm v 246 nm Ta thy chiu cao pic ca simvastatin thp hn nhiu so vi calci atorvastatin v ti bcsng 238 nmchiucaopic cacalciatorvastatin thayikhng ngk. Do u tin v ci thin chiu cao pic ca simvastatin ti la chn bc sng 238 nm nh lng ng thi 2 statin ny. 3.2.3. La chn pha ng Tin hnh kho st vi chng trnh sc k nh ti liu tham kho [22, 23] pha ng l acetonitril : m phosphat pH 4,5 (70 : 30), tc dng 1,2 ml/pht , bc sng 238 nm. Kt qu thu c sc k nh sau: - 24 - (a) (b) Hnh 3.3. Sc k mu kho st la chn pha ng vi m pH 4,5 ca mu simvastatin (a) v mu calci atorvastatin (b) Kt qu thu c cho thy pic ca simvastatin kh cn i, h s i xng cao (0,923) trong khi pic ca calci atorvastatin c h s i xng rt thp (0,633), pic b ko ui.Do chngtitin hnh khost pha ng vi m c iu chnh pH vi cc gi tr pH ln lt l 5,0; 4,0; 3,0 v 2,5. Kt qu thu c nh bng 3.1 Bng 3.1. Kt qu kho st pH m pH m H s bt i xng SimvastatinCalci atorvastatin 5,00,9780,614 4,50,9230,633 4,00,9570,739 3,00,9890,789 2,50,9920,888 T kt qu cho thy ti cc pH kho st h s bt i xng ca simvastatin duy tr n nh mc cao (trn 0,9). Trong khi , vi calci atorvastatin ti pH 5,0; 4,0 v 3,0 h s bt i xng khng thay i nhiu v vn thp (