B Y T TRNG I HC DC H NI

NGUYN BNH NGUYN

NGHIN CU XY DNG PHNG PHP NH LNG CAPTOPRIL

TRONG NGUYN LIU 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

NGUYN BNH NGUYN

NGHIN CU XY DNG PHNG PHP NH LNG CAPTOPRIL

TRONG NGUYN LIU BNG SC K LNG HIU NNG CAO

KHA LUN TT NGHIP DC S

Ngi hng dn : ThS Trn Th Lan Hng

Ni thc hin: B mn Ha Dc - i hc Dc H Ni

H NI - 2013

LI CM N

Trc ht, ti xin by t lng bit n su sc nht ti Ths TRN TH LAN HNG ngi thy tn tnh ch bo, hng dn, gip ti hon thnh kha lun ny.

Tixin gi li cm n chn thnh n cc thy c gio, cc anh ch k thut vin b mn Ha Dc, Dc l nhit tnh gip v to mi iu kin thun li cho ti trong sut thi gian lm thc nghim vic ti b mn.

Ti xin gi ti cc thy c gio, cc cn b Trng i hc Dc H Ni li cm n chn thnh v s dy bo, du dt tn tnh trong sut nm nm ti hc tp ti y.

V cui cng, cho php ti c by t lng bit n su sc nht ti cha m knh yu v nhng ngi thn trong gia nh, bn b lun ginh cho ti tnh yu thng, s quan tm, ng vin trong sut qu trnh hc tp v lm kha lun tt nghip.

H ni, ngy 16 thng 05 nm 2013 Sinh Vin

Nguyn Bnh Nguyn

MC LC Trang T VN 1 Chng I : TNG QUAN 2 1.1 i cng v Captopril 2

1.1.1 Cng thc, tnh cht 2 1.1.2 Tc dng, ch nh, chng ch nh 2

1.2 Mt s phng php nh lng captopril 3 1.2.1 inh lng Captopril bng php o Iod 3 1.2.2 nh lng Captopril bng phng php o quang 4

1.3 Tng quan v phng php sc k lng hiu nng cao 5 1.3.1 nh ngha 5 1.3.2 Cc thng s c bn ca qu trnh sc k 5 1.3.3 Nguyn tc cu to ca h thng HPLC 7 1.3.4 C s l thuyt ca vic la chn sc k 7 1.3.5 Phng php nh lng bng HPLC 11

1.4 Thm nh phng php phn tch 12 1.4.1 Yu cu chung 12 1.4.2 Ni dung thm nh phng php 12

Chng II: I TNG, HA CHT, THIT B, NI DUNG NGHIN CU 2.1 i tng nghin cu 14 2.2 Dng c, ha cht 14

2.2.1 Dng c v thit b 14 2.2.2 Ha cht 14

2.3 Ni dung v phng php nghin cu 14 2.3.1 Phng php nghin cu 14 2.3.2 Ni dung nghin cu 15 2.3.3 Phng php x l v nh gi kt qu 16

Chng III: THC NGHIM, KT QU,BN LUN 17 3.1 Kho st iu kin sc k 17

3.1.1 Kho st chn bc sng thch hp 17 3.1.2 Chn pha tnh 18 3.1.3 Kho st chn thnh phn pha ng v tc dng 19 3.1.4 Kho st chn dung mi ha tan Captopril 24 3.1.5 iu kin sc k chn 25

3.2 Xy dng v nh gi phng php nh lng Captopril trong trong nguyn liu bng phng php HPLC 25

3.2.1 Kho st tnh thch hp ca h thng sc k 25 3.2.2 Xy dng phng php nh lng 27

3.3 nh gi phng php nh lng 28 3.3.1 Kho st khong tuyn tnh ca phng php nh lng 28 3.3.2 nh gi tnh chnh xc ca phng php 30 3.3.3 nh gi tnh c hiu ca phng php 33 3.4 Bn lun 35 3.5 p dng phng php xy dng 36 Chng IV: KT LUN, NH GI 37 4.1 Kt lun 37 4.2 xut 37 TI LIU THAM KHO

CH THCH CC CH VIT TT

AcCN : Acetonitril

AF : H s bt i xng(Asymmetric) HPLC : Sc k lng hiu nng cao (High Performance Liquid Chromatography). MeOH: Methanol

RSD : lch chun tng i(Relative Standard Deviation) Rs : H s phn gii (Resolution) TB : Trung bnh

DANH MC CC HNH

Hnh Ni dung Trang 1.1 Cu trc ct LC-DB 9 1.2 Cu trc ct c gc Isopropyl 9

3.1 Ph hp th UV ca Captopril trong MeOH:m phosphat 0,01M pH=2,40 (65:35)

17

3.2 Sc k ca Captopril khi chy cc h m khc nhau v cc pha ng khc nhau. 19

3.3 Thng s phn tch v sc k ca Captopril khi chy pH =2,50 20 3.4 Thng s phn tch v sc k ca Captopril khi chy pH =2,40 20 3.5 Thng s phn tch v sc k ca Captopril khi chy pH =2,30 21

3.6 Thng s phn tch v sc k ca Captopril khi chy h MeOH:m pH 2,4 (60/40) 22

3.7 Thng s phn tch v sc k ca Captopril khi chy h MeOH:m pH 2,4 (65/35) 22

3.8 Thng s phn tch v sc k ca Captopril khi chy h MeOH:m pH 2,4 (70/30) 23

3.9 th biu din s ph thuc tuyn tnh gia nng dung dch v din tch pic ca Captopil 29

3.10 S tng quan gia lng Captopril ban u v lng tm li 32 3.11 Sc k ca mu trng 33 3.12 Sc k ca Captpril chun i chiu (100g/ml) 34 3.13 Sc k ca Captopril th (100g/ml) 34

DANH MC CC BNG

Bng Ni dung Trang 3.1 Kt qu kho st tnh thch hp ca h thng HPLC 26 3.2 Cch pha dy dung dch kho st khong tuyn tnh 28 3.3 Kt qu kho st khong tuyn tnh ca Captopril 29 3.4 Kt qu nh gi lp li ca phng php 30 3.5 Kt qu nh gi tnh ng ca phng php 31

3.6 Kt qu nh lng Captopril trong Captopril nguyn liu ca cng ty dc phm Traphaco 36

1

T VN Hin nay, tng huyt p l mt bnh mang tnh cht ph bin. iu ng ngi

chnh l c ti 50% ngi b tng huyt p khng bit mnh b tng huyt p, khong 30% ca nhng ngi bit mnh tng huyt p nhng vn khng c mt bin php iu tr no.Tng huyt p nu khng c iu tr ng v y dn n nhiu bin chng nng n nh suy thn, suy tim, tai bin mch no thm ch c th gy t vong hoc li cc di chng nng n nh hng n sc khe, sc lao ng ca ngi bnh. Mt trong nhng thuc u tay bc s hay dng iu tr tng huyt p trn bnh nhn chnh l nhm c ch men chuyn trong Captopril l mt thuc kinh in.

Mun kim sottt cc ch phm cha Captopril th i t vic nh nht l nh gi c ngun nguyn liu sn xut thuc Captopril. Hin nay, Vit Nam, phng php chnh nh lng Captopril trong nguyn liu l phng php oxy ha kh bng php o Iod. Trn thc t nu nh trong nguyn liu c cc cht kh v cc cht oxy ha th khi p dng phng php ny d dn n cc sai s. HPLC l mt phng php phn tch c tnh n nh,c chnh xc v chn lc cao, c s dng rng ri trong phn tch v dc phm.

Xut pht t thc t trn, chng ti tin hnh nghin cu ti: Nghin cu xy dng phng php nh lng Captopril trong

nguyn liu bng sc k lng hiu nng cao. Vi 3 mc tiu sau:

Nghin cu xy dng quy trnh nh lng captopril trong nguyn liu bng phng php sc k lng hiu nng cao.

Thm nh phng php xy dng theo cc tiu ch ca mt phng php nh lng, t ra quy trnh nh lng thng quy nh lng Captopril trong nguyn liu

p dng phng php mi xy dng nh lng Captopril trong nguyn liu.

2

CHNG I : TNG QUAN 1.1. I CNG V CAPTOPRIL 1.1.1. Cng thc, tnh cht Cng thc ha hc :

Tn khoa hc : Acid 1-(3-mercapto-2-methylpropanoyl)pyrolidin-2-carboxylic

Cng thc phn t : C9H15NO3S ; M=217,28; Tnh cht:

Bt kt tinh mu trng ng, 25oC: tan tt trong nc (160 mg/ml), ethanol,methanol,methylclorid, tan trong glyceryltriacetat (20 mg/ml),trong dung

dch NaOH long, rt t tantrong du ng, ethyl acetat (< 1mg/ml).Nhit nng chy: 105oC 108oC.Gc quay cc ring trong ethanol tuyt i l D25 = - 127,8o,hai ng phn R,S c gc quay cc ring lch nhau +5o. Hng s phn ly trong nc pKa ln lt l 3,7 (-COOH) v 9,8 (-SH). H s phn b logP(octhanol/nc) =0,34, logP(CH2Cl2/nc(pH=2))=1,39, logP(octhanol/HCl 0,1M)=1,9.[12]

1.1.2. Tc dng, ch nh, chng ch nh: 1.1.2.1. Dc ng hc :

Captopril hp thu tt qua ng tiu ha, c Ftuyt i l 60% - 75%, Cmax trong huyt tng t c khong 1 gi sau khi ung. Captopril lin kt vi protein khong 30%. Ch yu c bi tit trong nc tiu, khong 40%-50% o thi qua thn dng cn nguyn hot tnh, cn li chuyn ha qua gan thnh dng captopril disulfide v captopril-cystein.Captopril c t1/2= 23h, Cls=13,33

ml/min/kg.[14]

3

1.1.2.2. Dc lc hc Captopril c tc dng c ch enzym chuyn Angiotensin IAngiotensin II,

gim ti hp thu mui nc ng thn nn h huyt p, gim tit cc yu t gy ph i c tim v ph i mch mu. Captopril c ch nh trong iu tr tng huyt p, iu tr suy tim, au tht ngc, bnh thn do tiu ng ph thuc Insulin c protein niu > 300mg/24h.[14] 1.1.2.3. Ch nh v tc dng khng mong mun

Liu dng:iu tr tng huyt p : liu u 12,5mg /ln x 2 ln /ngy, s tng gim phthuc vo huyt p ca bnh nhn, nu s dng thm thuc li tiu th liu u l 6,25 mg/ln x 2 ln /ngy.iu tr bnh thn do tiu ng : 75-100 mg/ngy,vi bnh nhn suy thn cn c hiu chnh liu cho ph hp vi tng i tng. Mt s ch phm Captopril ang c trn th trng c trnh by bng1.1

Bng 1.1 : Mt s ch phm c cha Captopril Bit dc Nng /Hm lng Dng thuc Hng sn xut

Lopril Captopril 25 mg Captopril 50 mg

Vin nn

Bristol-myers Ssquibb

Ecazide Captopril 50 mg + Hydroclothiazid 25mg

Bristrol-myers Ssquibb

Captopril STADA25

Captopril 25 mg Stadapharm GmnH

DH-CaptoHasan Captopril 25 mg Hasan

1.2. MT S PHNG PHP NH LNG CAPTOPRIL 1.2.1. nh lng Captopril bng php o Iod

Nguyn tc : Oxy ha Captopril bng dung dch I2. Ha tan 0,15g ch phm trong 30,0 ml nc ct. Chun bng dung dch Iod 0,05 M. Xc nh im kt thc bng phng php chun o in th (dng in cc Platin).[1]

4

Phng trnh phn ng :

R SH2R S

S R-2e + H+2

Nhn xt :

Phng php c trong dc in Vit Nam (IV), dc in anh (BP2009) v nhiu tiu chun c s khc .

Phng php nh lng thc hin n gin. Tuy vy, nutrong nguyn liu c cc tp kh hoc oxy ha khc c th dn n sai s.ng thi, Captopril c nhiu mcoxy ha nnbc nhy th c th din ra nhiu mc khc nhau, iu ny cng s nh hng ti kt qu nh lng. 1.2.2. nh lng Captopril bng phng php o quang

Nguyn tc:Bratton-Marshall a ra phng php nh lng Captopril bng phng php o quang sau khi to dn cht.Captopril tc dng vi mt lng d acid nitoro(HNO2) to sn phm S-nitroso tng i bn vng ph hy HNO2 d c ph hy bng acid sulfamic. Thy phn S-nitroso gii phng acid HNO2. To mui diazoni bng cch cho thm sulfanilamine. Sau thm N-(1-napthyl)ethylenediamine to phm mu azo c mu .Tin hnh o mt quang ca dung dch.[10]

Phng trnh phn ng: R SH

HNO2 R S

N OHNO2

HNO2

Hg++

H2O

+ SNH2

OO

NH2H2NSO3H S

NH2

OO

N+N

S

NH2

OO

N+N

+NH

NH2

NH NH2

SNH2

OO

NN

Nhn xt :

5

Cch tin hnh nh lng phc tp v nhiu giai oan kh thc hin, sn phm o quang l cht trung gian d b phn hy,lm gim s chnh xc ca php nh lng.

Phng php sc k lng hiu nng cao l mt phng php phn tch c tnh n nh, chnh xc v chn lc cao c s dng rng ri trong phn tch v dc phm.V vy trong ti ny, chng ti nghin cu xy dng phng php nh lng Captopril trong nguyn liu bng HPLC. 1.3. TNG QUAN V PHNG PHP SC K LNG HIU NNG CAO 1.3.1. nh ngha Sc k lng hiu nng cao (HPLC) l k thut phn tch da trn c s ca s phn tch cc cht trn mt pha tnh cha trong ct nh dng di chuyn ca pha ng lng p sut cao. Pha tnh c th l cht rn c bao trn b mt mt cht mang rn, hoc l cht mang rn c bin i bng lin kt ha hc vi cc nhm hu c. Qu trnh sc k xy ra theo c ch : hp ph, phn tn, trao i ion, loi c hay tng tc ha hc trn b mt.[5] 1.3.2. Cc thng s c bn ca qu trnh sc k Thi gian lu tR : Thi gian lu ca mt cht l thi gian cn mt cht di chuyn t ni tim mu qua ct sc k, ti detertor v cho pic trn sc k tnh t lc tim n lc xut hin nh ca pic. Thi gian lu l thng tin v mt nh tnh ca sc k , n l hng s i vi mt cht nht nh khi tin hnh sc k trong iu kin khng i. Ngoi ra trong mt php phn tch cn thi gian lu hiu chnh, c tnh bng cng thc sau: tR = tR tM.

Vi: tM l thi gian lu ca 1 cht khng b lu gi bi pha tnh. Nu tR qu nh th s tch km, cn nu tR qu ln th pic b long, thi gian

phn tch ko di.[2]

H s phn b k : l h s phn b trng thi cn bng xc nh tc trung bnh ca mi vng cht tan do pha ng vn chuyn khi n qua ct

6

k = Trong : CS l nng mol ca cht tan trong pha tnh

CM l nng mol ca cht tan trong pha ng H s dung lng k : cho bit kh nng phn b ca cht phn tch trong hai

pha v c tnh theo sc cha ca ct:

k = k x = =

=

k : ti u trong khong : 1 k 8 [5] 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 :

= = =

Qui c : Cht B l cht b lu gi mnh hn cht A nn >1 tch ring 2 cht thng chn 1,05 2,0 [5]

H s i xng ca pic F : F =

Trong :W : chiu rng ca pic o 1/20 chiu cao ca pic

a : l khong cch t chn ng vung gc h t nh pic nmp ng cong pha trc ti v tr 1/20 chiu cao pic.

S a l thuyt v hiu lc ct N : Hiu lc ct c o bng thng s : S a l thuyt N ca ct N = 5,54 x

/[5]hoc16 x

[5]

Trong : W1/2h l chiu rng pic o na chiu cao ca nh pic. W l chiu rng ca pic o y pic.

phn gii Rsca ct : Rs =

. [5]hoc

,.// [5]

Trong : t(R)A ; t(R)B : thi gian lu ca 2 pic lin k nhau (B v A).

7

WB ; WA : rng pic o cc y pic. W1/2A ; W1/2B : rng pic o na chiu cao pic

Yu cu : RS> 1 ; gi tr ti u Rs = 1,5.[5]

1.3.3. Nguyn tc cu to ca h thng my HPLC H thng HPLC n gin v lm vic theo k thut HPLC bao gm

cc b phn chnh sau : Bm cao p :

bm pha ng vo ct sc k, bm ny phi iu chnh c p sut (thng 0 400 bar) y pha ng qua h thng vi mt tc dng n nh, ph hp vi qu trnh sc k. Bnh cha dung mi v h thng x l dung mi :

Bnh cha dung mi thng bng thy tinh i khi bng thp khng g. Dung mi chy sc k c lc qua mng lc (thng dng mng lc c 0,45 m) v ui kh ha tan. H tim mu :

a mu phn tch vo ct c th tim mu bng tay hay tim mu bng h thng tim t ng . Ct sc k:

Qu trnh tch cc cht din ra ct. Ct c ch to bng thp c bit tr vi ha cht, chu c p sut cao n vi trm bar. Trong ct c nhi pha tnh. Ct sc k c nhiu c khc nhau ty thuc vo mc v mc ch ca qu trnh sc k. Ni chung, cc ct phn tch thng c chiu di t 10 25 cm, ng knh 2 5 mm .

Detector :

L b phn pht hin cht phn tch. Ty theo bn cht ca cc cht cn phn tch m s dng detector thch hp bao gm cc loi sau: Detector hp th UV VIS, Detector ph pht x nguyn t, Detector hunh quang.[5] 1.3.4. C s l thuyt ca vic la chn iu kin sc k

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1.3.4.1. La chn pha tnh cho HPLC Pha tnh trong HPLC l cht nhi ct, l yu t quan trng quyt nh s tch

ca mt hn hp nhiu cht. Ty vo phn cc ca pha tnh v pha ng sc k c chia thnh 2 loi :

Sc k pha thun: pha tnh phn cc, pha ng khng phn cc, cht phn tch thng l cc cht phn cc hoc l cc cht c th phn cc.

Sc k pha o: pha tnh t phn cc, pha ng phn cc, cht phn tch thng l cc cht c th phn cc hoc t phn cc .

Pha tnh thng c ch to trn nn Silica (SiO2), nn oxyd nhm (Al2O3), nn hp cht cao phn t (cellulose) hay trn mch carbon.Trong sc k hp ph pha o: pha tnh trn nn silica c nhiu u vit c s dng nhiu nht.

C th phn loi cht nhi ct theo gc Siloxan:

R l cc nhm phn cc (a nc): nhm OH hoc cc alkylamin CH2-NH2, alkyl nitril CH2 CN, s dng lm pha tnh trong sc k hp ph pha thun phn tch cc cht t hoc khng phn cc.[15]

Vi R l cc nhm t phn cc nh octyl, octadecyl, phenyl, c iu ch bng cch alkyl ha cc nhm OH b mt silica trung tnh bng cc gc alkyl-R ca mch carbon (C2;C8;C18) hay cc gc carbon vng phenyl. Do cc nhm OH thn nc c thay th bng cc gc R k nc nn b mt tr nn t phn cc. Silica alkyl ha c s dng trong sc k hp ph pha o phn tch cc cht phn cc, t phn cc hay sc k to cp ion.[5]

9

Tuy nhin do hiu ng lp th nn trong cu trc ca pha tnh cn nhng nhm OH gy nh hng xu n qu trnh tch sc k ty theo mi trng pH phn tch .

Trong mi trng qu acid pH< 2 th c s phn ly cc nhm ether ra khi nn. Lc ny ct s mt hot tnh dn n cht cn phn tch khng th tng tc vi ct .

Trong mi trng Base (pH>7), cc nhm OH trong ct s tng tc vi cht tan c tnh base. Tng tc ny khc vi kiu tng tc ca cht tan vi nhm Si-C18, dn n hin tng cng mt cht nhng s c nhng nh ra khc nhau hay l hin tng ko ui. Kt qu gimi chnh xc.

Mt trong nhng cch khc phc hin tng ny l dng cc hp cht nh trimethylclorosilan ClSi(CH3)3 hoc hexametyldisizan (t s dng hn) tng tc vi nhm OH ny. Cu trc pha lin kt c trnh by hnh 1.1

Hnh 1.1: Cu trc ct LC-DB

C mt cch khc loi tr bt s nh hng ca nhm OH m khng cn tng tc vi n l thay nhng nhm methyl ca Si(CH3)2-C18 bng nhng nhm th ln hn nh isopropyl nhng nhm ny sche chn i nhng nhm OH, c trnh by hnh 1.2.

10

Hnh 1.2 : Cu trc ct c gc Isopropyl

Ngoi ra, trong mt s trng hp cn ghp ln dy C18mt s nhm phn cc tng thm phn cc ca dy C18 lm ct c kh nng tch chn lc hn i vi nhng hp cht phn cc mnh (Ct EPS Expended Polar Selectivity)[15] 1.3.4.2. La chn pha ng cho HPLC

Pha ng l dung mi ra gii cht phn tch ra khi ct sc k, l yu t th hai quyt nh hiu sut phn tch ca mt hn hp. Trong sc k pha o, pha ng l dung mi phn cc nh : nc, methanol, acetonitril hoc hn hp ca chng.Cc dung mi ny c th ha tan thm mt lng nh acid hay base hu c.

Pha ng trong HPLC nh hng ti rt nhiu thng s ca qu trnh sc k nh : chn lc , thi gian lu tR, phn gii Rs, rng ca y pic.Nn vic phn tch, la chn pha ng thch hp l rt quan trng . Yu cu ca 1 pha ng :

Tr vi pha tnh v bn vng theo thi gian. Ha tan cht cn phn tch . C tinh khit cao. Nhanh t cn bng trong qu trnh sc k . C tnh kinh t v m bo mi trng.

C cc yu t quan trng ca pha ng nh hng n kt qu : Bn cht ca dung mi pha pha ng . Thnh phn cc cht trong pha ng . pH ca pha ng . Tc dng ca pha ng. [7]

11

Chn m pH : Trong sc k hp ph m cht tan c tnh cht acid hoc base thng phi s

dng m pha ng n nh pH cho qu trnh sc k. Gi tr pH thch hp s lm tng hiu lc tch ca sc k.[7] Tc dng ca pha ng :

Sau khi c pha tnh, pha ng,pH thch hp, tng hiu lc tch ca qu trnh phi chn tc pha ng ph hp, m bo vic tch cc cht phn tch c tt nht m t hiu qu cao v thi gian v kinh t.[7] 1.3.4.3. La chn Detector

Khi pha ng ra gii cc cht ra khi ct s ghi nhn bi Detector chuyn thnh tn hiu v c ghi trn sc k . Detector ph bin nht l Detector UV-VIS. Ty theo cht cn phn tch m ngi ta t cc bc sng pht hin khc nhau.[15]

1.3.5. Phng php nh lng bng HPLC 1.3.5.1. Cch nh gi pic

nh gi din tch pic : tnh din tch pic c th dng tch phn k hoc dng my tnh ci t sn chng trnh.

nh gi chiu cao ca pic vi iu kin cc ch s k l hng nh. o chiu cao ca pic ch thch hp khi nng mu th t thp n trung bnh . 1.3.5.2. Phng php nh lng v cch tnh kt qu trong HPLC

Phng php nh lng cht phn tch trong HPLC da trn nguyn tc nng ca cht phn tch t l thun vi chiu cao hoc din tch pic ca cht phn tch. Ccphng php tnh kt qu thng dng

Phng php chun ni. Phng php chun ha din tch. Phng php chun ngoi.

12

Da trn vic so snh p ng pic ca mu th vi mu chun c phn tch trong cng mt iu kin. Nng (hm lng)ca cht cha bit c tnh ton da trn nng (hm lng) cht chun bit hoc suy ra t ng chun.

Trong kha lun ny, chng ti s dng phng php chun ngoi, chun ha mt im:chun b mt mu cht chun v mt mu cht th. Tin hnh song song vi nhau.Cn c vo din tch pic ca cht chun, cht th v nng cht chun, tm c nng ca cht th.

iu kin p dng phng php chun ha mt im: trong khong nng kho st, chng minh lun c tng quan hi quy tuyn tnh gia nng cht phn tch vi din tch pic ca cht phn tch.[7] 1.4. THM NH PHNG PHP PHN TCH 1.4.1. Yu cu chung

Thm nh mt phng php phn tch trong nguyn liu l mt qu trnh xc nh bng nghin cu trong phng th nghim nhng c trng c bn ca phng php m bo phng php t yu cu vi cc ng dng phn tch thc t, nhm chng minh rng phng php l ng tin cy v c kh nng thc hin phn tch nhng mu c kho st . 1.4.2. Cc tiu ch trong thm nh phng php 1.4.2.1. Khong tuyn tnh, ng chun

ng chun biu din mi quan h p ng ca pic (din tch pic) vi nng cht phn tch trong dung dch.

Tnh tuyn tnh ca phng php phn tch l kh nng suy ra cc kt qu th mt cch trc tip hoc thng qua php bin i ton hc, n t l thun vi nng ca cht cn phn tch c trong mu th trong mt gii hn nng cho.

Khong tuyn tnh: l khong nng t nng thp nht n nng cao nht trong mt ng chun c p ng tuyn tnh. Thng thng i vi nguyn liu th kho st tnhtuyn tnh trong khong t 80% n 120% nng so vi nng

13

nh lng. ng chun phi c t nht l 5 nng ca cht chun pha trong dung dch v t nht 4/5 im trong dy chun c ng v c chnh xc t yu cu . 1.4.2.2. Tnh chnh xc : bao gm cc tiu ch v lp li v ng lp li

lp li ca phng php phn tch l mc ph hp vi nhau gia cc kt qu ring r khi p dng phng php nh lng lp i lp li nhiu ln ly mu t mt mu trn ng nht.

Phng php l lp li nu lch chun tng i RSD 2,0%. Tnh ng

Tnh ng ca phng php l s gn st ca kt qu nh lng so vi gi tr thc. Do hm lng mu th cha c xc nh chc chn thi im no, v vy tnh ng phi c xc nh trong ton b vng cn xc nh. i vi nguyn liu, tnh ng c xc nh trong khong 80% n 120% nng phn tch.

Phng php l ng nu : Trung bnh tm li c t 98% n 102%. Khong tin cy ca dc cha 1 (khng c sai s h thng t l).

Khong tin cy ca b (y=ax + b) cha gc ta (khng c sai s hng nh).

1.4.2.3. Tnh c hiu Tnh c hiu ca phng php l kh nng xc nh cht cn phn tchm

khng b nh hng bi s c mt ca cc cht khc nh tp cht, sn phm phn hy v cc thnh phn khc.

14

CHNG 2 : I TNG HA CHT THIT B NI DUNG V PHNG PHP NGHIN CU

2.1. I TNG NGHIN CU Captopril nguyn liu ca cng ty dc phm Traphaco

2.2. THIT B, HA CHT 2.2.1. Dng c, thit b - My sc k lng hiu nng cao : DIONEX detector UV diode aray - My lc siu m SONOREX - My o pH : METTLER TOLEDO MP 220 - My lc chn khng : SATORIOUS - Cn phn tch :SATORIOUS

- My khuy t: HEIDOLPH MR 1000 - Bnh nh mc 10,25,50,100 ml - ng ong 100ml, cc c m 100, 500, 1000 ml - Pipet chnh xc cc loi, a thy tinh, mng lc 0,45m 2.2.2. Ha cht - Captopril nguyn liu ca cng ty Traphaco (ngy ht hn 11/2015) - Captopril chun i chiu ca Vin kim nghim B Y T (hm m 3%, hm lng 95,3%, ngy ht hn 11/2015). - Mui KH2PO4, H3PO4 (85%) loi tinh khit dng phn tch - MeOH dng cho HPLC (hng Honeywell)

15

- Nc ct 2 ln dng cho HPLC 2.3. NI DUNG V PHNG PHP NGHIN CU 2.3.1. Phng php nghin cu - Tham kho cc phng php nh lng Captopril cng b - Da vo cu to phn t, tnh cht l ha hc ca Captopril cng nh iu kin thc nghim nc ta hin nay nghin cu, xy dng quy trnh nh lngCaptopril trong nguyn liu bng phng php HPLC c tnh chnh xc, c hiu cao. 2.3.2. Ni dung nghin cu 2.3.2.1.Nghin cu cc iu kin tin hnh xy dng phng php

Kho st iu kin sc k Chn bc sng pht hin, chn ct, chn pha ng, tc dng thch hp c pic captopril cn i v thi gian lu hp l.

Chng ti tin hnh kho st trn ct Gemini C18 (250mm x 4,6mm, 5m) v ct Luna C8 ( 250 mm x 4,6 mm, 5m ) l nhng ct thng dng trong cc c s kim nghim hin nay. Tin hnh kho st cc iu kin khc chn chng trnh sc k ph hp.

La chn dung mi ha tan Captopril hon ton t nguyn liu Da vo tnh cht ca Captopril v qua tham kho ti liu, bng thc nghim chn ra dung mi thch hp ha tan captopril v m bo Captopril khng b thy phn. 2.3.2.2. Xy dng, nh gi phng php nh lng Captopril trong Captopril nguyn liu .

Kho st tnh thch hp ca h thng sc k Xy dng phng php nh lng nh gi phng php xy dng bng cc tiu ch

S ph thuc tuyn tnh gia nng Captopril v din tch pic trn sc k

Tnh chnh xc ca phng php:

16

- lp li ca phng php - Tnh ng ca phng php

Tnh c hiu ca phng php 2.3.2.3. p dng phng php xy dng nh lng Captopril trongnguyn liu. p dng phng php xy dng tin hnh nh lng Captopril trong nguyn liu ca Cng ty dc phm Traphaco Tin hnh sc k theo chng trnh chn trn cc mu th, song song vi mu chun. Trong khong tuyn tnh, nng cht phn tch t l thun vi din tch pic trn sc k . So snh din tch mu chun v mu th nh lng cht th trong dung dch phn tch, t tnh ra hm lng cht th trong mu cn nh lng. 2.3.3. Phng php x l v nh gi kt qu 2.3.3.1. nh gi dung mi ha tan Dng phng php HPLC xy dng nh gi s ph hp, tnh dc hiu ca dung mi s dng ha tan hon ton Captopril trong nguyn liu da trn kt qu nh gia tnh chnh xc, tnh ng ca phng php. 2.3.3.2.nh tnh Thi gian lu v hnh dng pic ca captopril trn sc k ca mu th v mu chun (i chiu) phi nh nhau trong cng iu kin sc k chn. 2.3.3.3 nh lng Cc s liu thc nghim c x l bng phng php ton thng k, s dng mt s cng thc sau :

Gi tr trung bnh: xtb = x (2.1)

lch chun : S = (2.2)

lch chun tng i: RSD(%)= (2.3)

3.1

hn

ph

Cap

3.1

c

thbb

kho

Sa

Sa

K

CHN

1. KHOQua th

nh kho stt hin Capptopril chu

1.1 Kho sTrong c

nhm CO

i c nhm c sng() c sng ()

Tin hong 4 g/m

ai s chun

ai s tng

Khong tin c

NG 3. TH

O ST Iham kho ct la chn ptopril, pha

n. st chn bcng thc c

OOH, nit carbonyl

di, Captop

ngn. nh qut ph

ml. Kt qu

:

i :

cy :

C NGHILUN

U KINc ti liudung mi h

a tnh, pha

c sng thcu to ca v tr s 1 v tr thpril khng c

h i vi ph hp th

17

M, KT N

N SC Kv phn tc

ha tan v

ng, tc

hch hp a captopril t

1 ca vng nht ca mcho ph

Captopril

h ca Capt

=

(%) =

QU, B

ch cu trccc iu ki dng tro

th Carbopyrolidin c

mch nhnhc trng, tuy

trong dung

opril c t

x 100%

x

N

c Captopril,

in sc k ong dung d

on s 2 ca c cp inh, nn v my nhin vn

g mi ha t

trnh by h

(2.4

% (2.5

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, chng ti

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vng pyro

n t t do, mt l thuyn cho p

tan c nngnh 3.1.

4)

5)

6)

tin sng

cha

lidin

ng yt ng

g

18

Hnh 3.1: Ph hp th UV ca Captopril trong MeOH:m phosphat 0,01M

pH=2,4 (65:35)

Nh vy, vi dung dch Captopril pha trong pha ng vi nng khong 4 g/ml th bc sng = 220 nm,Captopril c hp th l khong 0,4 v = 210 nm c hp th khong 1,2

Tuy nhin, cc dung mi hu c nh MeOH c nh hp th 205 nm, cn AcCN c nh hp th 195 nm. Nu chn bc sng gn 205nm hoc 195 nm th hp th dung mi hu c cao, th cc cm bin pht hin cht phn tch b qu ti, dn n sai s trong nh lng. V cc l do trn chng ti d kin bc sng pht hin Captopril l 220nm . Qua kho st, chng ti thy bc sng 220nm, pic ca Captopril cho p ng cao, nhn v i xng. V vy bc sng pht hin Captopril trong quy trnh nh lng chng ti xc nh l 220 nm . 3.1.2 Chn pha tnh

Do Captopril c cha nhm carboxylic (-COOH) hot ng mnh c pKa = 3,7 nn Captopril rt tan trong nc. tan trong nc 25oC l 160mg/ml. Mt khc c logP (octhanol/nc) = 0,34; logP (CH2Cl2/nc(pH=2)) =1,39; logP (octhanol/HCl 0,1M) =1,9. Nh vy, nu chn 1 pha tnh phn cc th c th m bo c s lu gi captopril tch cc tp khc c lin quan trong nguyn liu,

19

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 15.7-5.0

10.0

20.0

30.0

40.0

50.0 KS C18 #10 [modified by Nam Hung] UV_VIS_1mAU

min

1 - 6.073

2 - 6.780

3 - 9.033

WVL:220 nm

tuy nhin s dng pha tnh phn cc tc l dng sc k pha thun, trong khi dung mi dng cho sc k pha thun l dung mi khng phn cc nh : benzen, toluen. l rt c. Chnh v vy, chng ti vn nh hng nghin cu nh lng Captopril bng phng php sc k lng pha o.

Tin hnh kho st chn ct,trc ht vi ct Gemini C18.S dng cc h m khc nhau v thay i thnh phn pha ng khc nhau trn ct C18 (250mm x 4,6 mm, 5m).Kt qu kho st trn ct C18 c minh ha hnh 3.2.

Hnh 3.2: Sc k ca Captopril khi chy cc h m khc nhau v cc pha ng khc nhau.

Trn cc sc k thu c, pic ca Captopril khng tch ra khi pic tp,pic tp lun ra sau pic Captopril, chng t Captopril v tp cht c i lc vi pha tnh C18 kh ging nhauv mc thn ncca tp km hn Captopril. Chnh v vy,nu gim mc khng phn cc ca pha tnh th Captopril s b lu gi hn, v s lu gi vi cc tp cht lin quan cng tngv tng nhiu hn so vi Captopril, to iu kin c th tch c Captopril ring r .

V nhng l do trn nn chng ti quyt nh thay i pha tnh hay chnh l thay ct Gemini bng ct Luna C8c mc phn cc cao hn ct C18. 3.1.3 Kho st chn thnh phn pha ng v tc dng i vi ct LunaC8 (250 mm x 4,6 mm, 5m)

Trong HPLC pha o th pha ng thng dng nht l acetonitril, methanol, nc v hn hp ca cc thnh phn trn. Do Captopril d tan trong MeOH mt khc dung mi AcCN c nht thp mun t p sut u ct trong khong thch hp t 80-150 bar th tc dng tng cao, khi tc dng tng cao th thi gian lu b rt li rt ngn khng tch c cc tp khc ra. ng thi: mc nh

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.5-10.0

0.0

12.5

25.0

37.5

50.0

70.0 KS C18 #11 [modified by Nam Hung] UV_VIS_1mAU

min

1 - 4.193

2 - 5.207

WVL:220 nm

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.9-2.0

2.5

5.0

7.5

10.0

12.5

16.0 KS C18 #7 [modified by Nam Hung] capto lan 2 UV_VIS_1mAU

min

1 - 9.213

2 - 10.553

WVL:220 nm

20

hng ti s thay i pH ca MeOH t hn l khi dng AcCN, hp th 220 nm ca MeOH thp hn AcCN,v gi thnh ca MeOH ch bng 1/3 so vi gi thnh ca AcCN nu cng th tch nn chng ti s dng MeOH l mt thnh phn trong pha ng. 3.1.4.1 Chn h m pH

Trn c s l thuyt, Captopril c pKa =3,7, nn khong pH Captopril dng phn t gn nh hon ton l 1,7 2,7. Trong khong pH ny, m phosphat c pKa1 = 2,15. V th, chng ti s dng m phosphat phn tch Captopril

Do gii hn chu ng ca ct vi pH l t 1,5 12,5,ng thi pH< 2 th captopril tng tc vi nhau to ra cc thiocetal dn n nh lng khng chnh

xc nn chng ti khng s dng pH thp hn pH = 2

Chng ti chn pH ca m l pH = 2,5 khi u.Kt qu phn tch c trnh by trong hnh 3.3.

Hnh 3.3: Thng s phn tch v sc k ca Captopril khi chy vi pH = 2,5 Qua sc k v cc thng s phn tch pic hnh 3.3, ta thy vi m pH

2,5: pic ca Captopril b ko ui v khng cn xng (AF = 2,17), chng t t l Captopril dng phn ly/dng phn t cn ln nn t l ny gim th cn gim pH ca pha ng.Chng ti gim pH ca h m xung pH 2,4 tip tc nghin

No. Ret.Time Width Resol. Asym.min Min (EP) (EP)

1 6,300 0,922 2,48 2,17 2 9,007 1,239 n.a. 1,65

0.0 2.0 4.0 6.0 8.0 10.0 13.3-10.0

0.0

12.5

25.0

37.5

50.0

70.0 KS C8 CP #9 [modified by Nam Hung] UV_VIS_1mAU

min

1 - 6.300

2 - 9.007

WVL:220 nm

21

cu. Thng s phn tch v sc k ca Captopril khi s dng h m pH 2,4 c ghi hnh 3.4

Hnh 3.4: Thng s phn tch v sc k ca Captopril khi chy vi pH = 2,4 Nh vy pH = 2,4 th nh pic Captopril nhn, h s bt i lc ny l 1,62

chng t pic Captopril cn i hn so vi pH = 2,5, ng thi rng chn pic lc ny cng gim ng k.

Tuy nhin, h s bt i ca pic Captopril pH = 2,4 l AF = 1,62, trong khi gi tr bt i tt nht l xp s 1, nn chng ti gim pH ca h m xung pH = 2,3 chy nhm mc ch tng tnh i xng ca pic captopril. Kt qu chy Captopril m pH = 2,3 cho kt qu hnh 3.5

Hnh 3.5:Thng s phn tch v sc k ca Captopril khi chy pH = 2,3 Nh vy, vic gim pH ca chng ti t c mc ch : tng tnh i

xng ca pic Captopril th hin qua cc thng s : h s bt i l AF = 1,59, rng y pic l 0,683.

No. Ret.Time Width Resol. Asym.Min Min (EP) (EP)

1 6,360 0,698 2,76 1,62 2 8,133 0,597 n.a. 1,24

No. Ret.Time Width Asym.min min (EP)

1 6,367 0,683 1,59

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.6-20

0

25

50

75

100

140 KS 08112012 #51 [modified by Nam Hung] UV_VIS_1mAU

min

1 - 6.367WVL:220 nm

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.1-20

0

25

50

75

100

140 KS 08112012 #46 [modified by Nam Hung] UV_VIS_1mAU

min

1 - 6.360

2 - 8.133

WVL:220 nm

22

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 15.6-50

0

50

100

150

200

250

300 KS 08112012 #35 [modified by Nam Hung] UV_VIS_1mAU

min

1 - 8.467

2 - 9.600

WVL:220 nm

Tuy nhin pH gim t 2,4 xung 2,3 th thm 1 lng acid ln, trong khi h s bt i AF gim 1 lng khng ng k t 1,62 xung 1,59.Mt khc: cng gim pH th tui th ca ct cng gim theo v lm tng tc thy phn pha tnh, v ct Luna C8 chng ti ang s dng qua s dng t lu, nn chng ti chn h pha ng l hn hp MeOH: m phosphat 0,01M pH = 2,4 tip tc kho st. 3.1.4.2 Chn t l pha ng

Thay i thnh phn pha ng vi mc ch tch pic Captopril ra khi pic tp sn c v chn pha ng c kh nng tch tt nht, chng ti tin hnh kho st h pha ng l hn hp MeOH : m phosphat 0,01 M pH = 2,4. Theo t l MeOH tng dn nh sau:

H I : MeOH : m phosphat 0,01M pH 2,4 (60:40) H II : MeOH : m phosphat 0,01M pH 2,4 (65:35) H III : MeOH : m phosphat 0,01M pH 2,4 (70:30)

Sc k v thng s phn tch Captopril khi chy h I c ghi hnh 3.6

Hnh 3.6 : Sc k v thng s phn tch Captopril chy h MeOH : m pH 2,460:40

Sc k v thng s phn tch thu c cho ta thy pic ca Captopril v pic ca tp cht cha tch ra khi c nhau (tCaptopril = 8,467 pht, ttp cht = 9,600 pht ) v thi gian lu ca Captopril di. Trong trng hp ny, nu gim t l MeOH trong pha ng th thi gian lu cng tng, trong khi thi gian lu ca pic tp l

No. Ret.Time Width Resol. Asym.Min Min (EP) (EP)

1 8,467 0,659 n.a. n.a. 2 9,600 1,285 n.a. n.a.

23

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 12.2-10

20

40

60

80

100 KS 08112012 #79 [modified by Nam Hung] UV_VIS_1mAU

min

1 - 5.280

2 - 7.540

WVL:220 nm

9,600 pht nn nu gim t l MeOH trong pha ng th thi gian phn tch s ko di. Khi tng t l dung mi hu c trong pha ng th thi gian lu ca Captopirl v tp cht s rt ngn li. V th chng ti tip tc kho st vi h MeOH : m phosphat 0,01M pH 2,4 (65:35).Kt quphn tch th hin hnh 3.7

Hnh 3.7 :Sc k v thng s phn tch ca chy hMeOH : m 0,01M pH 2,4 (65:35)

Kt qu thu c cho ta thy vi t l 65:35 thi gian lu ca Captopril v tp cht gim xung ng k ( h MeOH/(m phosphat 0,01M pH = 2,4) = 60/40 th tCaptopril = 8,467 pht, cn h MeOH/(m phosphat 0,01M pH = 2,4) = 65/35 th tCaptopril = 6,360 pht), ng thi pic ca Captopril v pic ca tp cht tch hon ton ra khi nhau ( phn gii Rs = 2,76), t c mc ch tch Captopril v tp cht ra khi nhau. Tin hnh kho st vi h 70:30 chn pha ng thch hp nht trong phn tch Captopril. Kt qu c trnh by hnh 3.8

No. Ret.Time Width Resol. Asym.Min Min (EP) (EP)

1 6,360 0,698 2,76 1,62 2 8,133 0,597 n.a. 1,24

No. Ret.Time Width Resol. Asym.Min Min (EP) (EP)

1 5,280 0,744 2,63 1,89 2 7,540 0,988 n.a. n.a.

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.1-20

0

25

50

75

100

140 KS 08112012 #46 [modified by Nam Hung] UV_VIS_1mAU

min

1 - 6.360

2 - 8.133

WVL:220 nm

24

Hnh 3.8:Sc k v thng s phn tchCaptopril trong hMeOH : m pH 2,470:30

Nh vy vi h 70:30 ta thy rng thi gian lu ca Captopril v tp cht u gim ( h MeOH/(m phosphat 0,01M pH = 2,4) = 65/35 th tCaptopril = 6,360 pht, cn h MeOH/(m phosphat 0,01M pH = 2,40) =70/30 th tCaptopril = 5,280 pht), ng thi phn gii RS ca Captopril v tp cht l Rs = 2,63, cng t c, nhng so vi h 65/35 chy th pic Captopril chy h 70/30 c tnh i xng km hn (AF h 65/35 ca Captopril l AF = 1,62, cn h 70/30 th c AF =1,89).

Do vy, chng ti thy h dung mi MeOH : m phosphat 0,01 M pH2,40 t l 65:35 l h ti u nht phn tch Captopril, cho pic Captopril tch r rng,cn xng, ng thi thi gian chy ph hp. 3.1.4.3 Kho st tc dng

Kho st kh nng tch Captopril trong mu chun vi pha ng chn : MeOH/m phosphat 0,01 M pH=2,40 (65:35) vi cc tc dng kho st l0,6 ml/min v 0,7 ml/min nhm chn c tc dng thch hp, m bo vic tch pic Captopril ra khi pic tp, ng thi rt ngn ti a thi gian phn tch .

Qua kt qu thc nghim, chng ti thy vi tc dng 0,6 ml/min th tCaptopril = 6,360 pht v ttp cht = 8,133 pht, phn gii gia 2 pic ny l Rs = 2,76. Vi tc dng 0,7 ml/min th pic Captopirl v pic tp u ra sm, tch ra c khi nhau, nhng h s phn gii gia hai pic gim, h s bt i ca Captopril tng. Chnh v vy chng ti chn tc dng l 0,6 ml/min phn tch Captopril. 3.1.4 Kho st chn dung mi ha tan Captopril

Captopril l acid-2-pyrolidincarboxylic pKa = 3,7,nnCaptopril l cht phn cc d tan trong nc, ethanol, MeOH . Trong ti ny, vi mc ch tm c dung mi ha tan hon ton captopril c trong nguyn liu m khng b thy phn v oxy ha, chng ti tin hnh kho st mt s dung mi nh sau :

S dng MeOH 100%.

25

S dng pha ng : MeOH v m phosphat 0,01M pH=2,40 (65 :35). S dng hn hp MeOH : H2O theo t l 65 :35. S dng H2O 100%.

Bng kt qu thc nghim, chng ti nhn thy : Khi s dng MeOH 100% lm dung mi ha tan Captopril: c s thay i

thnh phn pha ng lc tim mu nn trn sc k ng nn khng n nh. Khi s dng H2O 100% lm dung mi ha tan Captopril: mu pha khng n

nh, Captopril d b thy phn, ng thi khi chy sc k th c s khc bit gi thnh phn pha ng v h pha mu nn ng nn khng n nh.

Khi s dng hn hp MeOH : H2O theo t l ging pha ng (65:35), khi chy cho ng nn n nh, pic ca Captopril cn i, sc nt , tch c pic tp, nhng khi mt thi gian lu d b thy phn to ra cc tp cht khc.

Khi s dng hn hp MeOH : m phosphat 0,01M pH=2,40 (65 :35) ng nn n nh, pic ca Captopril v pic tp tch ra khi nhau, cn i, kt qu c ng v chnh xc cao. V vy, chng ti la chn dung mi ha tan Captopril c trong nguyn liu l hn hp MeOH : m phosphat 0,01M pH =2,4 (65 :35).

3.1.5 iu kin sc k chn Nh vy, qua qu trnh kho st chng ti a ra chng trnh sc k nh

lng Captopril trong Captopril nguyn liu nh sau: Ct LunaC8 (250mm x 4,6mm, 5m) Pha ng l MeOH/m phosphat 0,01M pH=2,4 (t l 65 :35) Tc dng 0,6 ml/min Th tch tim 20l Detetor UV 220nm

Nhit phn tch : nhit phng. 3.2. XYDNG PHNG PHP NH LNG CAPTOPRIL TRONG NGUYN LIU BNG PHNG PHP HPLC 3.2.1 Kho sttnh thch hp ca h thng sc k

26

Cn chnh xc khong 50,0mg Captopril chun i chiu (nng 95,3%, hm m 3%) do Vin Kim nghim sn xut vo bnh nh mc 50ml, thm hn hp MeOH : m phosphat 0,01M pH = 2,4 (t l 65 :35) n vch v siu m 2 pht. Ly chnh xc 1,00ml dung dch trn, cho vo bnh nh mc 10ml, thm hn hp dung mi n. Siu m trong 3 pht. Lc qua mng lc 0,45m, dung dch lc tim sc k(nng captopril trong dung dch khong 100g/ml).

Tim 6 ln mu chun vo h thng HPLC. Tin hnh sc k vi iu kin chn. Ghi thi gian lu, din tch pic v h s bt i xng ca pic captopril thu c trn sc k .

Tnh thch hp ca h thng HPLC c biu th thng qua h s bt i xng, lch chun tng i (RSD%) ca 6 php th song song i vi thi gian lu v din tch pic. Kt qu kho st tnh thch hp ca h thng sc k c trnh by bng 3.1

Bng 3.1. Kt qu kho st tnh thch hp ca h thng HPLC

STT Thi gian lu(pht) Din tch

pic(mAU*min) H s bt i xng

1 6,360 35,3301 1,62 2 6,356 35,3292 1,62 3 6,392 35,3368 1,63 4 6,378 35,3347 1,62 5 6,362 35,3303 1,62 6 6,365 35,3307 1,62

RSD(%) 0,21 0,01 0,25

Kt qu kho st tnh thch hp ca h thng HPLC cho thy lch chun tng i (RSD%) ca thi gian lu v din tch pic, h s bt i ca Captopril trong 6 php th song song u < 2%. iu ny chng t h thng HPLC m chng

27

ti s dng l ph hp v m bo n nh cho php phn tch nh lng Captopril.

3.2.2 Xy dng phng php nh lng 3.2.2.1 Chun b mu th, mu i chiu v pha ng

Dung dch th Cn chnh xc mt lng bt Captopril nguyn liu tng ng vi 50,0mg

captopril cho vo bnh nh mc 50ml, thm 40ml hn hp MeOH:m phosphat 0,01M pH = 2,40 (t l 65:35). Lc k ri siu m 2 pht. Thm hn hp dung mi n vch, lc u. Ht chnh xc 1,00 ml dung dch va pha cho vo bnh nh mc 10ml, thm hn hp dung mi n vch, lc u siu m trong 3 pht. Lc qua mng lc 0,45m, b khong 2ml dch lc u, sau dch lc c em tim sc k (nng Captopril trong dung dch khong 100g/ml).

Dung dch chun Cn chnh xc khong 50,0mg Captopril chun i chiu (nng 95,3%)

cho vo bnh nh mc 50ml, thm 40ml hn hp MeOH:m phosphat 0,01M pH = 2,40 (65:35). Lc k ri siu m 2 pht. Thm hn hp dung mi n vch, lc u. Ht chnh xc 1,00 ml dung dch va pha cho vo bnh nh mc 10ml, thm hn hp dung mi n vch, lc u siu m trong 3 pht. Lc qua mng lc 0,45m, sau dch lc c em tim sc k (nng Captopril trong dung dch khong 100g/ml).

Pha ng + Dung dch m phosphat 0,01M pH = 2,40

Ha tan 1,36g KH2PO4 trong 1 lt nc ct 2 ln, iu chnh v pH 2,40 bng dung dch H3PO4 (8,5%). Lc qua mng lc 0,45m, sau siu m ui kh 30 pht.

+Methanol : siu m ui kh 20 pht 3.2.2.2 Chng trnh sc k

Ct Luna C8 (250 mm x 4,6 m, 5m) Pha ng l MEOH/m phosphat 0,01M pH = 2,40(t l 65/35) Th tch tim 20l Tc dng 0,6 ml/min

28

Detertor UV 220 nm Nhit phn tch l nhit phng

3.2.2.3 Tin hnh sc k nh lng. Chun b v cho h thng HPLC chy n nh vi pha ng trong 30 pht.

Tim ln lt 20l mi dung dch chun b vo h thng sc k. Tin hnh sc k trong iu kin chn. Ghi cc gi tr din tch pic thu c trn sc k ca mu th v mu chun tng ng. 3.2.2.4 Tnh kt qu

Hm lng phn trm so vi nhn ca captopril trong nguyn liu c tnh theo cng thc:

C(%) = x Cch x (100 - a) (3.1)

Trong : a(%) : Hm m ca Captopril trong mu chun. C(%) : Hm lng phn trm ca captopril trong mu th. Cch(%) : Hm lng phn trm ca captopril trong mu chun. mth(gam) :Khi lng ca bt nguyn liu cn pha dung dch th. mch(gam) : Khi lng ca captopril chun cn pha dung dch chun. Sth v Sch : Din tch pic ca dung dch th v dung dch chun.

3.3. NH GI PHNG PHP NH LNG CAPTOPRIL TRONG NGUYN LIU BNG PHNG PHP HPLC 3.3.1Kho st khong tuyn tnh ca phng php nh lng

Pha mt dy nng dung dch ca captopril c nng bin thin t 80g/ml n 120g/ml.

Trong bnh nh mc 50 ml, cn chnh xc mt lng bt Captopril chun i chiu (nng 95,3%) cha khong 60,0mg Captopril. Thm 40ml hn hp MeOH:m phosphat 0,01M pH 2,40 (65:35), lc k siu m trong 2 pht. Thm hn hp dung mi n vch, lc u. Ht chnh xc 5,00 ml dung dch va pha, cho

29

vo bnh nh mc 50ml, thm 40 ml hn hp pha ng, lc u, thm tip hn hp pha ng cho ti vch lc v lc u. Dung dch thu c c nng Captopril khong 120g/ml. Tin hnh pha long theo bng 3.2 thu c cc dung dch c nng ln lt l 80g/ml, 90g/ml, 100g/ml, 110g/ml. Lc qua mng lc 0,45m. Cc dch lc thu c em tim sc k.

Bng 3.2. Cch pha dy dung dch kho st khong tuyn tnh. Dung dch s 1 2 3 4 5 s ml dung dch gc (120g/ml) 2 3 5 11 1 S ml hn hp pha ng 1 1 1 1 0 Nng dung dch (g/ml) 80 90 100 110 120

Tim ln lt 20l mi dung dch trn vo h thng HPLC chy n nh vi pha ng 30 pht. Tin hnh sc k. Ghi cc gi tr din tch pic o c ng vi cc nng ca Captopril. Kt qu kho st tuyn tnh c trnh by bng 3.3.

Bng 3.3. Kt qu kho st khong tuyn tnh ca Captopril Dung dch 1 2 3 4 5 Nng (g/ml) 80 90 100 110 120 Din tch pic (mAU*min) 28,304 31,752 35,330 38,978 42,438

T kt qu thu c bng 3.3, chng ti thit lp mi tng quan gia din tch pic thu c trn sc k v nng ca dung dch. Phng trnh hi quy tuyn tnh biu th mi tng quan ny l: y = 0,3549 x 0,1336 vi h s tng quan r=0,9999, R2= 0,9999

lch chun ca y-intercept : Sb = 0,1960 Khong tin cy ca y-intercept : b = 0,6238 ;-0,7575 b 0,4902 cha

gc ta .

30

th biu din s ph thuc tuyn tnh gia nng dung dch v din tch pic ca Captopril c ghi hnh 3.9.

Hnh 3.9. th biu din s ph thuc tuyn tnh gia nng dung dch v din

tch pic ca Captopril T kt qu thu c hnh 3.9 cho ta thy: tt c cc gi tr o c nm

trn ng hi quy 80% n 120% nng nh lng tng ng vi 80g/ml n 120 g/ml.

Nh vy, trong khong nng t 80g/ml n 120g/ml c s ph thuc tuyn tnh gi nng dung dch v din tch pic ca Captopril. S ph thuc ny l cht ch. Khong nng kho st l rng nn thun tin cho vic phn tch nh lng trong nguyn liu. 3.3.2 nh gi tnh chnh xc ca phng php 3.3.2.1nh gi lp li ca phng php

Chun b 6 mu th nguyn liu Captopril m cng ty dc Traphaco dng cho sn xut v 6 mu chun dng cht chun i chiu Captopril hm lng 95,3% ca Vin kim nghim.

Tim ln lt tng mu th v mu chun vo h thng HPLC. Tin hnh sc k theo iu kin chn, tnh ton hm lng cc hot cht trong mu th theo cng thc 3.1. lp li ca phng php c nh gi bng lch chun tng

y = 0.354x - 0.133R = 0.999

25

30

35

40

45

70 80 90 100 110 120 130

Din

tch

pic

(m

AU

*min

)

Nng (g/ml)

31

i (RSD%) ca 6 php th c lp. Phng php llp li khi RSD 2%. lp li ca phng php c nh gi bng sai s tng i ( %) ca 6 php th song song. Khi cng nh, th lp li ca phng php cng cao. Kt qu nh gi lp li ca phng php c ghi li bng 3.4

Bng 3.4 : Kt qu nh gi lp li ca phng php

STT

Mu th Mu chun Hm lng

Captopril chun i chiu (%)

Hm lng

Captopril th (%)

Khi lng cn (gam)

Din tch pic

(mAU*min)

K/l Captopril chun i

chiu (gam) Din tch

pic (mAU*min)

1 0,0502 34,980 0,0545 35,330 95,30 99,622 0,0506 35,102 0,0548 35,346 95,30 99,513 0,0508 35,425 0,0540 35,285 95,30 98,504 0,0504 34,989 0,0548 35,342 95,30 99,59 5 0,0503 35,209 0,0544 35,312 95,30 99,746 0,0507 35,248 0,0546 35,324 95,30 99,41

S liu thng k

xtb = 99,40(%)S = 0,4512 Sx = 0,1842RSD = 0,4539(%)x = 0,4735 = 0,4764(%)

Kt qu nh gi tnh chnh xc ca phng php cho thy, trong cc iu kin sc k chn, lch chun tng i ca cc kt qu rt nh (RSD =0,4539); vi tin cy p = 0,95, sai s tng i ca 6 php th song song ( = 0,4764%) cho thy phng php nh lng nguyn liu Captopril xy dng l chnh xc v c chnh xc cao. 3.3.2.2 nh gi tnh ng ca phng php

nh gi tnh ng ca phng php c tin hnh da trn vic kho st kh nng tm li hot cht trong cc mu chun nng 80% n 120% nng nh lng.

Cn 9 mu captopril chun, chia thnh 3 nhm, mi nhm 3 mu nh sau : Nhm I : cn chnh xc mi mu khong 40,0mg Captopril chun i chiu

(hm lng 95,3%) do Vin kim nghim sn xut. Nhm II :cn chnh xc mi mu khong 50,0mg Captopril chun i chiu.

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Nhm III :cn chnh xc mi mu khong 60,0mg Captopril chuni chiu. Tin hnh nh lng cc mu ny (theo cc bc mc 3.2.2). Kt qu c

trnh by bng 3.5. Bng 3.5: Kt qu nh gi tnh ng ca phng php

Nhm Mu K/l Captopril

chun i chiu cho vo

(gam)

K/l captopril c trong chun

i chiu (gam)

Din tch pic

(mAU*min)

Khi lng Captopril

tm li(gam)

Phn trm Captopril

tm li (%)

I 1 0,0440 0,0407 28,083 0,0400 98,28 2 0,0434 0,0401 27,625 0,0393 98,003 0,0427 0,0395 27,328 0,0389 98,48

II 4 0,0552 0,0510 35,459 0,0505 99,025 0,0539 0,0499 34,546 0,0491 98,406 0,0547 0,0506 35,285 0,0502 99,21

III 7 0,0657 0,0608 42,396 0,0603 99,18 8 0,0654 0,0605 41,718 0,0593 98,029 0,0658 0,0609 41,959 0,0597 98,03

Chun 0,0544 0,0503 35,342 0,0503 100,00

S liu thng k xtb =98,51(%)S = 0,4988 RSD = 0,5063(%) x = 0,3834 = 0,3892(%)

T kt qu bng 3.5, ta c : Gi tr trung bnh ca t l thu hi l 98,51% ; lch chun tng i l

RSD = 0,5063% < 2% ; vi tin cy p = 0,95 th sai s tng i ca 9 php th nh ( = 0,3892%). Khong tin cy ca t l thu hi l : 98,31 x 98,89. Kt qu ny cho thy phng php c ng cao.

nh gi sai s t l v sai s h thng trong phng php, chng ti thit lp mi tng quan gia lng hot cht ban u v lng hot cht ban u v lng hot cht tm li. th biu din s tng quan gia lng hot cht v lng hot cht tm li hnh 3.10

Phng trnh hi quy v mi tng quan gia lng hot cht ban u v lng hot cht tm li l :

33

y = 0,9887x 0,0002 vi h s tng quan r=0,9999, R2 = 0,9998 lch chun ca dc l : Sa = 0,0121 lch chun ca y-intercept l : Sb = 0,0006 Khong tin cy ca dc l : 0,8340 a 1,01434 cha 1 vi tin

cy l p = 0,95. Khong tin cy ca y-intercept : -0,0080 b 0,0077 cha gc ta vi

tin cy l p = 0,95.

Hnh 3.10 : S tng quan gia lng Captopril ban u v lng tm li

Nh vy ta thy, trong khong nng kho st, phng php xy dng c ng cao, khng mc sai s t l (khong tin cy ca dc cha 1) v khng c sai s h thng (khong tin cy ca y-intercept cha 0) 3.3.3nh gi tnh c hiu

Trong Captopril nguyn liu, ngoi hot cht chnh l Captopril cn c nhiu cht khc. Trc tin phi k n cc loi tp cht to ra do Captopril b phn hy trong qu trnh sn xut v bo qun nguyn liu ban u dng tng hp Captopril nh Captopril disulfide Tp cht ny nh hng n kt qu nh lng Captopril bng phng php hin ti ang p dng. Do vy trong ti ny, chng ti tin hnh nghin cu xy dng mt phng php nh lng Captopril nguyn liu m s c mt cc tp cht khng nh hng ti kt qu nh lng.

y = 0.988x - 0.000R = 0.999

0.000.010.020.030.040.050.060.070.080.090.10

0.0000 0.0200 0.0400 0.0600 0.0800 0.1000Kh

i l

ng c

apto

pril

tm l

i (g

am)

Khi lng captopril c trong chun i chiu (gam)

34

kho st tnh c hiu ca phng php, chng ti tin hnh nh sau: - Trong bnh nh mc 50 ml, cn chnh xc khong 50,0mg Captopril chun

i chiu ca Vin kim nghim (hm lng 95,3%), ha tan v thm dung mi (h MeOH/m phosphat 0,01M pH=2,40 65/35) va ti vch v lc u. Lc qua mng 0,45 m.

- Trong bnh nh mc 50 ml, cn chnh xc khong 50,0 mg Captopril th, ha tan v thm dung mi (h MeOH/m phosphat 0,01M pH=2,40 65/35) va ti vch v lc u. Lc qua mng 0,45 m.

Tim ln lt 20l mi dung dch chun b vo h thng HPLC chy n nh vi pha ng 30 pht. Tin hnh sc k theo chng trnh chn. Song song tin hnh ghi sc k ca mu trng. Sc k ca mu trng, dung dch chun i chiu, thc ghi li cc hnh 3.11, 3.12 v 3.13.

Hnh 3.11: Sc k ca mu trng

35

Hnh 3.12: Sc k ca Captpril chun i chiu (100g/ml) Sc k ca Captopril chun i chiu (100 g/ml) cho mt pic chnh ti

thi gian lu t = 6,360 pht.

Hnh 3.13: Sc k ca Captopril th (100g/ml)

Sc k ca Captopril th (100 g/ml) cho 2 pic, pic chnh ti thi gian lu t = 6,362 pht, pic tp ti thi gian lu t = 8,131 pht, Rs = 2,63.

Sc k ca mu trng ko cho pic l no ti cc khong thi gian lu ca Captopril, iu cho thy tp cht khng nh hng n kt qu nh lng Captopril.

3.4. BN LUN Phng php sc k lng hiu nng cao (HPLC) l mt phng php phn

tch hin i, ang c s dng ti cc phng v trung tm kim nghim ti Vit Nam. u im chung ca phng php HPLC l c s n nh v chn lc cao, tin hnh nhanh chng, c th p dng nh tnh v nh lng ng thi nhiu hot cht m khng cn phi tch ring bit.

36

Kt qu thc nghim v x l thng k cho thy phng php xy dng p ng y cc ch tiu ca mt phng php nh lng. Trn sc k , pic Captopril u chn lc, cn xng, c phn gii r rng vi cc pic khc.

Kt qu kho st tnh thch hp ca h thng HPLC cho thy lch chun tng i RSD ca thi gian lu, din tch pic ca Captopril trong 6 php th song song u < 2%v h s bt i ca Captopril trong 6 php th song song dao ng t 1,62 n 1,63. iu ny chng t h thng HPLC m chng ti s dng l ph hp v m bo s n nhcho php phn tch nh lng Captopril.

Kt qu nh gi tnh chnh xc ca phng php cho thy, trong cc iu kin sc k chn, lch chun tng i ca cc kt qu rt nh (RSD = 0,4539%) ; vi tin cy p = 0,95, sai s tng i ca 6 php th song song = 0,4764% cho thy phng php nh lng Captopril nguyn liu xy dng l chnh xc v c chnh xc cao.

Trong khong nng kho st, phng php xy dng m bo tnh ng v c ng rt cao. Gi tr trung bnh ca t l thu hi l 98,51% ; lch chun tng i l RSD = 0,5063% < 2% ; vi tin cy p = 0,95 sai s tng i ca 9 php th l nh ( = 0,3892%), khong tin cy ca t l thu hi l 98,31 x 98,89; khng mc sai s t l v khng mc sai s h thng.

Tnh tuyn tnh c kho st trong khong nng (t 80% n 120% nng nh lng) cho thy c s ph thuc tuyn tnh cht ch gia nng dung dch v din tch pic ca Captopril (h s tng quan r = 0,9999, khong tin cy ca y-intercept cha gc ta ). Phng php x l mu n gin d thc hin trong thi gian ngn, tit kim thi gian v dung mi, thun tin cho cc c s c trong b my HPLC 3.5 P DNG PHNG PHP XY DNG TIN HNH NH LNG CAPTOPRIL NGUYN LIU CA CNG TY DC PHM TRAPHACO

p dng phng php va xy dng, chng ti tin hnh nh lng Captopril trong Captopril nguyn liu ca cng ty dc phm Traphaco( ngy ht hn 11/2015)

37

S dng phng php xy dng nh lng Captopril trong Captopril nguyn liu ca Cng ty dc phm TRAPHACO, dng cht chn i chiu l Captopril hm lng 95,3% do vin kim nghim sn xut pha dung dch chun i chiu. Tin hnh nh lng 3 ln. Kt qu nh lng Captopril trong nguyn liu c trnh by bng 3.6.

Bng 3.6. Kt qu nh lng Captopril trong Captopril nguyn liu ca cng ty dc phm Traphaco

STT Khi lng cn (gam) S ln tim

Din tch pic (mAU*min)

Din tch pic trung bnh

Hm lng Captopril trong nguyn liu (%)

1 0,0506

1 35,310 35,321 99,59 2 2 35,334

3 3 35,319

Chun 0,0548 1 35,452

35,536 95,3 2 35,483 3 35,674

T cc kt qu kho st chng ti thy : cc mu nguyn liu t tiu chun v hm lng qui nh (nm trong khong 98,0% n 101,5%)

Pic captopril trn sc k cao, cn xng (h s bt i dao ng t 1,61 n 1,63), tch hon ton vi cc pic tp ( phn gii RS ca Captopril vi cc pic tp l 2,76).

CHNG 4. KT LUN V XUT 4.1 KT LUN

T cc kt qu thc nghim thu c, chng ti t c mc tiu ra l xy dng phng php nh lng Captopril trong nguyn liu Captopril m khng b nh hng bi s c mt ca cc tp cht hay gp. Cc kt qu thu c c th nh sau:

38

Cch chun b mu th v mu chun n gin, nhanh chng : dung mi s dng c thnh phn n gin, khng phc tp, khng gy tn km, chun b mu bng phng php ha tan trc tip nn tin hnh n gin, nhanh chng.

Chng trnh sc k lng hiu nng cao xy dng ph hp v n nh nh lng Captopril trong nguyn liu Captopril. Chng trnh l:

Ct LunaC8 (250mm x 4,6mm, 5m) Pha ng l MeOH/m phosphat 0,01M pH=2,4 (t l 65 :35) Tc dng 0,6 ml/min Th tch tim 20l Detetor UV 220nm

Nhit phn tch : nhit phng nh gi phng php HPLC xy dng trn 4 ch tiu: kho st tnh tuyn

tnh, nh gi tnh chnh xc, tnh ng. Kt qu cho thy trong khong nng kho st, phng php xy dng c tnh tuyn tnh, tnh chnh xc, tnh ng cao, m bo yu cu nh lng Captopril trong nguyn liu Captopril.

p dng phng php HPLC xy dng, nh lng c hm lng Captopril trong nguyn liu Captopril ca cng ty dc phm Traphaco. Cc kt qu cho thy cc mu nguyn liu nh lng u t tiu chun v hm lng 4.2 XUT - Nghin cu k hn v n nh ca dung dch nh lng - Tip tc nghin cu c th nh lng ng thi cc tp cht lin quan nh : captopril disulfid.

Ti liu Tham Kho Ting Vit 1. B Y T, Dc in Vit Nam IV, Nh xut bn Y hc, trang 124-126 2. B Y T, Dc th Vit Nam, 2006, trang 142 - 144 3. PGS.TS Mai Tt T, TS V Th Trm (2007), Dc l hc tp II, Nh xut bn

Y hc, trang 26-28 4. B Y T(2007), Kim nghim dc phm, Nh xut bn Y hc, trang 84 110. 5. PGS.TS Trn T An, PGS.TS Thi Nguyn Hng Thu, Ha Phn Tch 2, Nh

xut bn Y hc, trang 168 200. 6. PGS.TS Trn c Hu (2006), Ha dc tp 1, Trng i hc Dc H Ni,

trang 68 - 69

7. Trn Th Lan Hng (2005), Nghin cu nh lng Artesunat khi c mt Dihydro Artemisinin v/ hoc Acid Succinic trong thnh phm vin nn Artesunat 50mg bng phng php sc k lng hiu nng cao (HPLC), Lun vn thc s dc hc, Trng i hc Dc H Ni, trang 10 17, 32 37 .

Ting Anh 8. Australian Pharmaceutical Formulary and Handbook 21, p.11, p.91, p.324,

p.473

9. Bristish pharmacopeia(2009) volume I&II, p.1020 p.1022.

10. Chromatographia, volume 55, issue 9, p.565 p.571.

11. Clarke's Analysis of Drugs and Poisons 4th ed. - A. Moffat, et al., (Pharma.

Press, 2011) BBS, p.1038 p.1039

12. Profiles of Drug Substances Vol 11, p.80 p.130

13. The United States Pharmacopeia thirty edition, USP Monographs, C, Captopril.

14. Martindale Thirty-six edited by Sean C Sweetman, p1239 p.1240.

15. HPLC for Pharmaceutical Scientists(2007), Yuri Karakevich & Rosario

Lobrutto, p.139 p.188 & p.75 p.132.