DjNH Ll/aNG GLIBENCLAMID TRONG HUVeiTU'aNG B A N G PHiraNG PHAP
HPLC GHEP N 6 | DETECTOR HUYNH QUANG
Nguyen Thi ^?t Trinh', Nguyin Hiht Tiit^
(1) LdpD5. khoa 2011-2016, Trudng Dgi hgc YDugcHui (2) Khoa Dugc, Trudng Dgi hgc YDugc Hui Tomtit
Dat vin de: GUbenclamid li mdt sulfonylure the hd 2 dugc diing trong dieu trj dli thio dudng ^rpe II vi II mgt trong 12 dugc chat co trong danh myc dugc chat cin bao cao so lieu nghien cihi tucmg duang sinh bgc khi dang ky thudc. Do do viec xiy dyng mgt phuang phip dinh lugng Glibenclamid trong huyet tuang ll rat cin ibiet. Nghien cuu dugc thyc hien vdi muc tieu: Xiy dyng vl tham djnh phuang phip dinh lugng Glibenclamid trong huydt tuang bang HPLC ghep ndi detector huynh quang.
Doi tugng v i phinmg phap nghien ciru: ddi tucmg nghten ciiu la huyet tuang tring sau khi them chuan Glibenclamid. Sau khi lya chgn dugc quy trinh, phuang phip dugc thim dinh theo huong dan cua US-FDA. Kdt qui: Dilu kidn sic ky nhu sau: cgt Agilent Eclipse XDB-C18 (150 x 4,6mm, 5fmi);
pha dgng gom Acetonitril vi Dgm diamoni hydrophosphat 0,05 M (pH 5,5 dugrc didu chinh bang Acid phosphoric) t>' Id 40:60 (v: v), tdc dd dong 1 ml/ph6t, thd tich tiem 50 |il, detector huynh quang cd buoc song kich thich v i buoc song phit xa lin lugt la 235 nm vi 354 nm. Phuang phip dugc tham djnh theo cic tidu chi: dd chgn lgc, khoing ndng dd tuydn tinh, dd dung, dd chinh xac, hieu suat chiet va dg dn djnh. Ket lu^n: phuong phap djnh lugng Glibenclamid trong huyet tuong bing HPLC ghep ndi detector hujnh quang c6 the umg dyng de djnh lugng Glibenclamid trong huyet tuomg.
Tukhod: Glibenclamid, HPLC, huyit luang.
Abstract
DETERMINATION OF GLIBENCLAMIDE IN HUMAN PLASMA BY HPLC WITH FLUORESCENCE DETECTOR
Nguyen Thi Viet Trinh'. Nguyen Huu Tiet^
(I) Class D5, Faculty of Pharmacy, Hue Universily of Medicine and Pharmacy (2) Faculty of Pharmacy, Hue Universily of Medicine and Pharmacy Background: GHbenclamide is a second generation oral sulfonylurea that is widely used to treat type II diabetes mellitus and one of 12 drugs in the list of drugs required to report data bioequivalence studies as drug registration. The development and validation of liquid chromatography method for the determination of glibenclamide in human plasma is essential. Materials and methods: Sample is the blank human plasma after adding glibenclamide standards. After selecting process, the method was validated as guidance of US-FDA. Results: The HPLC condition was optimized as follow: Agilent Eclipse XDB-C18 column (150 x 4.6mm, 5\im); mobile phase: acetonitrile and 0.05 M ammonium phosphate (pH 5.5) (40:60, v : v); flow rate 1.0 ml/min, injection volume: 50 pi, excitation and emission wavelengths were set at 235 nm and 354 nm, respectively. Method was validated with parameters: system suitability, specificity, linearity, exfraction efficiency, precision, accuracy and stability. Conclusion: The HPLC method can determine Glibenclamide in human plasma.
Afy words: Glibenclamide, HPLC, plasma.
-Dfa chi liin hi: Ngi^in Hihi Tiin, email: [email protected] •'^^,\. ,^. ' • ^ -Ng^ nhgn bdi: 25/3/2016 * Ngdy dingy ddng: 15/4/2016* Ng^xudtbdn:_29/4/2016 , ' - J
120 T9P chi Y Dugc hgc - Truimg D?i hgc Y Dugc Hui - So Die bi^t
l.B^T VkNI>t
Glibenclamid (5-chlon>^ - P - [ 4 - (cycIohe:Q'l carbamoyl sulfamoyl) phenyl] ethyl] -2- methojo-benzamide) 11 thudc dieu tri dii thio dudng khdng phy thudc insulin nhdm sulfonylure [2] va 11 mgt trong 12 dugc chit co trong danh myc dugc chdt can bao cio sd Hgu nghidn cuu tuang ducmg sinh hgc khi dang k^ thudc [1]. Do do vide xiy dyng mdt phuong phip co the djnh lugng duge Glibenclamid trong huydt tuong 11 rat cin thiet, vira phyc vy cdng tic dinh gii tuang duang sinh hgc, viia giiip theo doi kiem soit ndng dd thuoc trong mau khi dieu trj. Mgt sd nghien cuu tren the gidi da ap dyng phuong phap sic ky long higu ning cao de djnh lugng Glibenclamid trong huyet tuong vdi cic lo^ii detector UV [3], [6], [7], huynh quang [4], khdi phd [5]... trong dd detector huynh quang kha dugc quan tam do co dg nhay cao, don giin va kinh te. Hign nay tai Vigt Nam chua co cdng trinh dinh lugng Glibenclamid trong huyet tuang bang detector huynh quang nio.
Bii bio nay gidi thidu ket qui nghidn cthi xiy dyng vl thim djnh phuang phip dinh lugng Glibenclamid trong huyet tuang bang detector huynh quang, nham de xuat mgt quy trinh phan tich thudng quy 6e djnh lugng Glibenclamid trong huydt tuong.
2. NGUYEN V^T LIEU, THIET BI VA PHU'ONG PHAP NGHEEN CiTU
2.1. Hoa ch^t - dung m&i
Cic chat chudn qudc gia Glibenclamid (GLI) (ham lugmg 100,10%, dg am 0,09%), Ketoconazol (KET) (him lugng 98,99%, dp dm 0,06%) ciia Vidn Kiem nghigm thudc Trung ucmg. Huyet tuong ti-Ing (SKS : 457041249. HD : 27 - 05 - 2016) ciia Vign huyet hgc truydn mlu Trung uang.
Methanol (MeOH), Acetonitril (ACN) tinh khi^t HPLC; Diamonihydrophosphat, Acid phosphoric deu cua Merck, Dure.
2.2. Thiet bi
Hd thdng HPLC Shimadzu 20AD kdt ndi detector huynh quang RF 20A, miy li tam Labnet Spectrafuge 24D, may lie xoiy Vortex mixer 250VM Hsi, tu l^nh am slu Sanyo MDF
293AT, can phan tich Mettler Toledo, may do pH sensIONTM PH3 HACH.
2.3. Phuorng phap ngbien ciru Chudn bf cdc dung dich
Dung djch chuan gdc GLI vl KET c6 ndng dg I mg/ml dugc pha trong MeOH. Tir do pha ra cic dung djch chu4n thur cap GLI 10 ^g/ml vi KET 1 ng/ml frong MeOH.
Chuan bi mdu dexdy dumg phucmg phdp CIc mau xay dyng duong chuan dugc chuan bj bing cich cho vao huydt tuang tring nhihig the tich dung djch GLI thii cap khic nhau dd dugc cic ndng dd GLI trong huydt tuong ll 10; 25; 50; 100; 200;
400 ng/ml. Cic mau kiem soat (QC) dugc chuan bj tuang ty mau xiy dyng dudng chuan nhung d cic ndng dg GLI trong huydt tuang 1110 ng/ml (LQC);
100 ng/ml (MQC); 400 ng/ml (HQC).
X&lymdu
100 nl dung djch KET 1 jig/ml dugc cho vao eppendorf (1,5 ml) chiia 0,5 ml huyet tuang cd GLI, lie vortex 30 giiy. Tiep tyc cho vio mdi mau 500 jil TCA 1,25% ti-ong MeOH, lie vortex 30 giay, li tim 7400 vdng/phiit x 10 phiit. Hiit 900 pi phin djch tren, lgc qua mang lgc nylon 0,45 nm, dung djch lgc dugc tiem vio h? thdng sic ky.
Diiu kifn sdc ky
Phucmg phip phin tich dugc tien hinh d nhigt dg phdng tren cgt Agilent Eclipse XDB-CI8 (150 X 4,6mm, 5nm); pha dgng gdm ACN vl ddm diamoni hydrophosphat 0,05 M (pH 5,5 dugc didu chinh bing Acid phosphoric) t^ Id 40 : 60 (v : v), tdc dd dong 1 ml/phiit, the tich tiem mlu 50 (il, detector huynh quang co budc song kich thich va budc sdng phit xg lan lugt li 235 nm vl 354 nm.
Cic sd ligu thdng kd dui?c tinh toln dya vio phin mem Microsoft Excel.
Phuang phip dugc thim djnh theo hudng din cua FDA ve thim djnh quy trinh phan tich djch sinh hgc [8].
Nghien ciiru dugc tien hinh tai khoa Dugc, trudng Dai hgc Y Dugc Hud frong thdi gian tii thing 09/2015 den thing 03/2016.
3. KET QUA VA BAN L U ^ 3.1. Khio sat didu ki|n sic ky
Qua tham khio tii lidu [3], [7] vl vdi dieu kign T^ip chl Y Dugrc hgc - Truimg D?i hgc Y Dugc Hue - Sd Die bi^t 121
hidn cd cia phdng thi nghigm, chung tdi siir dyng c0t Agilent Eclipse XDB-C18 (150 x 4,6mm, 5nm) trong nghien cim nay. Nghidn ciiu khio sit tren nhieu dung mdi pha dgng khic nhau: MeOH;
ACN; Ddm natri dihydrophosphat 0,01 M pH 3,5;
Ddm diamoni hyphosphat 0,05 M pH 5,5. Kit qui cho thiy vdi hg dung mdi Acetonitril - Dgm diamoni hyphosphat 0,05 M pH 5,5; pic hai ho^t chat sic dgp, cin ddi. De lya chgn tf Id pha ddng cho thdi gian phan tich hgp 1^, nghien cfru da khio
sit cic tJ Ig khic nhau ciia 2 dung mdi tren vi lya chgn dugc hg pha ddng: Acetonitril - Ddm diamoni hyphosphat 0,05 M pH 5,5 (40; 60 v/v).
Sau khi lya chgn dieu kign sic kf, chung tdi tidn hinh tham dinh phuang phip.
3.2. D$ phu hgrp ciia h$ thdng sic k^
Chuan bj miu GLI trong huyet tuong cd ndng dg 100 ng/ml, tidn hinh xii H miu, tiem l|p Igi 6 lin vio hd thdng sic kJ. Kdt qui dugc frmh bay tgi bing 1.
Bing 1. Khio sit tinh thich hgp hd thdng sic k^ (n = 6)
TB SD RSD%
t , K E T (phut) 11,87 0,05 0,45
t , G L I (phit) 15,37 0,07 0,43
(mAu.s) 281475,3 5059,9
1,80
(mAu.s) 288857,2 5502,1
1,90
^ K E T
1,03 0,02 1,62
N K I T
6912,7 41,98 0,61
N.„
6701,9 37,87
0,56 Rs
5,30 0,01 0,14 Nhgn xit: CIc so lidu thu dugc cho thay hg thong vl cic dieu kign sic k^ chgn lya li tuong thich cho vigc phan tich djnh lugng GLI frong huydt tucmg.
33. Tinb chgn Igc
Tidn hinh sic kf ciic mlu dung djch chuin trong MeOH, huydt tuang tring, huydt tuang frlng thdm chuin. Sic ky do dugc trinh biy d hinh la, lb, Ic.
»
s-
bua a.
iltansi
„ / J Glibcoclinid
1, ( / L
1 hUaiSSn-Xtn
vju.
Hinh 1. sic ky dd cac mlu: a. dung djch chuin MeOH; b. huyet tuomg tring;
c. huyet tuang tring them chuan.
Nhgn xit: Cac pic sic ggn, can ddi, tich nhau, tren mau tring tgi didi gian luu ciia 2 chat khong cd pic Ig.
Tpp chf Y Dugc hgc - 'mrtTig D»i hgc Y Dirge Hui - sd D$c bift
3.4. Khoang nong d$ tuyen tinh ng/ml. Xft li mau v4 tien hanh sic k^ theo dieu Chu^bjcScmSn GLI trong huyit tuong tiing ki$n ti xic djnh. Ket qua duoc trinh bay 4 CO nSng dO lan luot la 10; 25; 50; 100; 200; 400 Bing 2 vi Hinh 2.
Bang 2. Ket qui Idlio sit khoing nong dp tuyin tinh C (ng/ml)
^ K E T
10 0,45
25 0,60
50 0,77
100 1,07
200 1,67
400 2,99 Phuong trtah h6i quy: y = 0,0064x + 0,416
Hp so tuong quan: r^= 0,9993
Ndng d9 CLI (ng/ml)
Hinh 2. Dd thj bidu diln mdi tuong quan giu'a ndng dg vl t^ sd didn tich pic Ket qua cho thay, trong khoing nong dg khio
sat CO sy tuong quan tuydn tinh chit che giita ndng dg vdi tf Ig dign tich pic chuin vi chuin ndi (vdi i^> 0,995).
3.5. Gidi hf^n djuh lugrng dudi (LLOQ) Chuan bj cic miu huydt tuong chiia GLI co nong dg giim dan, tidn hanh xii ly v l phin tich miu. XIc djnh dugc tgi nong dd 5 ng/ml, dap iing
RSD dgt 6,17%. Nhu vgy, 5 ng/ml l i gii hi LLOQ cua phuang phip.
3.6. D$ chinh xic v i d$ diing
Tidn hinh sic ky cic mau QC gdm LQC (10ng/ml),MQC(lOOng/ml)vlHQC(400ng/ml), mdi 16 mlu gdm 5 miu ddc Igp co ciing ndng dd.
XIc djnh ket qua djnh lugng ciia cic mlu QC tir dudng chuin pha trong huyet tuang tring, tidn hinh ciia pic GLI cao gap 10 lan so vdi nhidu, ddng xic trong ciing dieu kign, trong 2 ngiy khic nhau. Ket djnh dugc dd diing dgt 89,72% va dd chinh xic vdi qua dugc frinh bay d Bing 3a vl Bing 3b.
Bing 3a. Ket qua danh gii dd diing vi dd chinh xac trong ngiy Man
LQC (n=5) MQC (n=5) HQC (n=S)
Nong <l9 GLI 1^
thuyet (ng/ml) 10 100 400
Nong d$ GLI (ng/ml) (TBiSD) 9,22*0,69 91,32 ±3,23 392,93 ±11,67
R S D % 7,52 3,54 2,97
D$ thu hoi (%) 91,24 90,32 97,16 Mlu
LQC (n=5) MQC (n=5) HQC (n-5)
Bang 3b. Ket qua danh gii dp dung va dp chinh xic khic ngiy Ning d$ GLI 1^
thuyet (ng/ml) 10 100 400
N6ng dp GLI (ng/ml) (TB±SD) 9,08 ± 0,77 93,73 ± 7,96 369,05 ±18,98
RSD % 8,47 8,49 5,14
D9 thu bbi (%) 93,14 94,67 93,19 Nhgn xel: Phuotig phip c6 dd dijng tdt vdi ty 1? thu hdi td 90,32% ddn 97,36% vi dd chinh xic vdi gii tri RSD ddu dudi 15% (2,97% den 8,49%), dip irng yeu ciu vd dd dung vi dd chinh xac ciia phucmg phap phin tich frong djch sinh hgc.
Tgp chf Y Dugc hgc - Tftrfrng Dgi hgc Y Dugc Hud - Sd D|c bift 123
3.7. Hi|u suit chidt
Chuan bj cic mlu GLI trong huydt tuang vl frong pha ddng d 2 mdc ndng dd LQC vi HQC.
Xur ly cic mlu huydt tuang vi tien hanh xic djnh ndng dd GLI trong cic mlu. XIc dinh dugc higu suit chiet tgi LQC li 55,13 ± 0,47% (n= 3) vi tgi HQC 1156,43 ± 0,31 % (n=3).
3.8. Dg dn djnh ciia mSu trong qua trinh xir ly vi bao quan
Dg dn djnh sau 3 chu ky ddng - rd dong
Chuin hi 6 miu LQC vi 6 miu HQC, 3 mau mdi nong dd dugc diing de dinh lugng ngay, cac mau con Igi dugc bio quin d nhidt dg -70°C frong 24 gid, liy ra vl dd rii ddng d nhigt dg phdng. Khi dl ra dong holn toin, miu dugc de ddng Igi frong 24 gid d dieu kidn tuomg ty. Thyc hign thdm 2 chu kf niia vl tidn hinh phan tich d lan thur 3. So sanh kdt qui djnh lugng GLI c6 frong miu sau 3 chu kjr ddng - ni vdi lugng GLI co trong miu ban diu, kdt qui frinh bay d bang 4a.
LQC (10 ng/ml) HQC (400 ng/ml)
Bang 4a. Ket qui dO 6n djnh sau 3 chu ky dong - Ning dp GLI (ng/ml) (TB ± SD) (n=3) Ban dau
8,66 ± 0,06 395,96 ±6,10
Sau 3 chu ky dong - ra 8,69 ±0,25 387,97± 11,02
ra dong Ty If so voi ban diu (%)
100,40 97,98
P 0,826 0,333 Nhdn. xel: Q cl 2 khoing ndng dd LQC va HQC, ndng dg GLI d miu sau 3 chu ky ddng - ra Idch 97,98% - 100,40% so vdi mlu ban dau, vdi p>0,05 cha thiy miu huydt tuang dn djnh sau 3 chu ky ddng ri.
Dg on dinh ngdn hgn
Chuin hi 6 mlu LQC vl 6 mlu HQC, 3 mlu mdi ndng dg dugc diing dd djnh lugng ngay, cac miu con Igi (sau khi xii If) dugc gii^ d dieu kign phdng trong 6 gid vi tidn hanh phan tich. So sinh ket qui djnh lugng GLI co frong mau ban dau va sau 6 gid, ket qui frinh bay d bing 4b.
Bing 4b. Kdt qui dg dn djnh ngin hgn
LQC (10 ng/ml) HQC (400 ng/ml)
Ning d$ GLI (ng/jnl) (TB ± SD) (n=3) Ban diu
8,72 ±0,24 391,86 ±8,69
Sau 6 gitf 8,25 ±0,13 373,55 ±13,42
Ty If so voi ban diu (%)
94,57 95,32
P 0,594 0,278 Nhgn xet: (5 c i 2 khoing ndng 6^ LQC vi HQC, ndng dg GLI d mau (sau khi xii ly) sau 6 gid d nhigt dd phong Igch 94,57% - 95,32% so vdi mau ban diu, vdi p>0,05 cho thiy mlu dn djnh sau 6 gid d nhidt dd phong.
Dg on dfnh ddi ngay
Chuin bj 6 mlu LQC vl 6 mau HQC, 3 mlu mdi ndng dg dugc diing de djnh lugng ngay, cic mlu con Igi dugc giu* d dieu kign -70 °C. So sinh ket qui djnh lugng GLI cd frong man ban dau vi sau 14 ngay, ket qua trinh biy d bing 4c.
Bang 4c. Ket qui dg dn djnh dii ngiy
LQC (10 ng/ml) HQC (400 ng/ml)
Ning dp GLI (ng/ml) (TB ± SD) (n=3) Ban dau
8,82 ±0,19 392,21 ±4,26
Sau 14 ngiy 8,38 ±0,28 377,44 ±10,62
Ty 1^ so voi ban diu (%)
95,00 96,23
P 0,092 0,089 Nhan xel: 0 ci 2 khoang nong dp LQC va HQC, nong dO GLI i> miu sau 14 ngay * dieu ki?n -20 "C, l§ch 95,00% - 96,23% so vin miu ban dau, v6i p>0,05 cho thay miu huyet tuong in djnh sau 14 ngiy odieuki?n-70"C.
124 T9P chf Y Dupe hpc - tyirfrng B?i hpc Y Dupe Hu^ • Si D$c bj^t
Dg on dfnh dung dich gdc.
Td dung djch chuan gdc GLI 1 mg/ml, pha 3 mlu GLI 100 ng/ml dd djnh lugng ngay. Phin dung djch chuan gdc cdn Igi dem bio quin d nhigt dd Ignh. Sau 48 gid bao quan, tien hinh sic kf 3 mlu chuan frong methanol d ndng dg 100 ng/ml.
Kdt qui dugc frinh biy d bing 4d.
Bang 4d. Kdt qua dd dn djnh dung djch gdc Ndng dg GLI (ng/ml)
(TB±SD)(n=3) Ban dau Mau sau
48 gid 99,29 ± 0,87
Tf I§ so vdi ban dau (%)
98,72 0,404 Nhgn xet: Ndng dd GLI d mlu giii d nhigt dg Ignh Igch 98,72% so vdi mlu ban diu, vdi p>0,OS cho thay dung djch chuan gdc dn djnh sau 48 gid d nhigt dd Ignh.
4. BAN L U ^
Phucmg phip phan tich bing HPLC de djnh lugng GLI frong huyet tuang phit hidn bing detector huynh quang cd tinh chgn lgc cao, khoing TAI H$U 1. Bg Y tl (2010), "Thdng fti 08/20 lO/TT-BYT Hudng dan bio d o sd li$u nghiSn cdu sinh khi dgng/ tuong duong sinh hgc frong ding k^ thudc", HI Ngi, tr. 8.
2. Dio van Phan (2007). Dugc ly hgc. Nhi xuit bin Y hgc, HI Ngi, tr. 378-379.
3. Ayesha Rashid, Mahmood Ahmad, Muhammad Usman Minhas, Ijaz Javed Hassan and Muhammad Zubair Malik. (2014), "Pharmacokinetic studies of metformin and glibenclamide in normal human volunteers"./"ai. /. Pharm. Sci.. Vol.27, No.!, pp.153-159.
4. Ahmed Yusuf, Muhammad M Hammami. (2009),
"Validation of a new high performace liquid chromatography assay for glibenclamide in human plasma". Fabad J.Pharm. Sci., 34, pp.
119-125.
5 Albu F, Georgita, David V, Medvedovici
ndng dg tuyen tinh khi rdng, dd diing vi dd chinh xic cao, thdi gian phin tich khdng qua dai. Dgc bidt phuang phip c6 gidi hgn djnh lugng dudi (LLOQ) khi thap 5 ng/ml, phii hgp cho vide djnh lugng nhiing mlu huyet tuang co ndng dd GLI nhd khi xay dyng dudng cong ndng dO thudc frong miu (gii trj C^^^^khi udng vien Smg li 131,856 ng/
ml) [3]
Cich xur ly miu dan giln, nhanh, tign Igi, tidt kigm dung mdi vl huyet tuang, thich hgp cho nghidn cdu tuang duang sinh hgc ciia thudc.
5. K E T L U ^
Dl xiy dyng dugc phuang phip djnh lugng Glibenclamid trong huyet tuang bing HPLC ghep ndi detector hu^^nh quang co dg chfnh xic cao, dg diing tdt, khoing tuydn tinh phii hgp, tinh chgn lgc cao vl quy frinh xii ly mau khi don giin.
Phuang phip nly cd thd img dyng de djnh lugng Glibenclamid frong huyet tuong nhim dinh gia ndng dg thudc trdn bdnh nhin ciing nhu phyc vy dinh gii tuomg duang sinh hgc ciia thudc.
THAM KHAO
A.(2007),"Determination of glibenclamide in human plasma by liquid chromatography and atmospheric pressure chemical ionization/MS-MS detection". Journal of Chromatography B, 846, pp.222-229.
S.D.Rajendran, B.K.Philip, R. Gopuiath, B. Suresh.
(2007), "RPHPLC Method for the esrimation of glibenclamide in human serum". Indian Journal of Pharmaceutical Siences, pp. 796-799.
S. W. Coppack, A. F. Lant, C. S. Mcintosh &
A. V. Rodgers. (1990), "Pharmacokinetic and pharmacodynamic studies of glibenclamide in non- insulin dependent diabetes mellitus". Br. J. dm.
Pharmac. 29, pp. 673-684.
U.S. Department of Health and Human Services, Food and Dmg Administration, CDER, CVM.
(2013),"Guidance for industry: Bioanalytical Method Validation".
Tgp chl Y Dirge hgc - Trtsimg Dgi hgc Y Dug-c Hue - Sd D$c bi$t 125
