TAP CHi y - Diroc HOC OUflN SV s o 8-2014
DjNH LUONG METFORMIN TRONG HUYET TUONG CHO BANG SAC KY LONG HIEU NANG CAO
Trin Thj VSn Anh'; Nguyin Van Thinh": VO Tuan Anh"
Dao Wit Hung"; Pham Thj Minh Hue"'; Bill Thj Thu Hi"
TOM T A T
Metformin la dupe chit thuOc nhdm biguanid dung theo dudng u6ng cd tac dyng iam giSm nlng dd glucose trong mau dli vdi benh nhSn (BN) dai thao du'dng (BTB) typ 2. Mgc dich ciJa nghien cO'u nhlm thlm dinh phu-ong phap djnh lu'png metformin trong huylt tuong chO blng sic ky long hi^u nSng cao (HPLC) vdi nOi chuan la ranitldin Sau khi kit tua protein trong huyet tuong blng acetonitril, metformin va nOi chuan dupe phan tach vdi dieu kiSn sic ky sau. cOt fortis phenyl (5 pm, 150 x 4,6 mm) d 25''C vdi pha ddng glm acetonitnl - d6m Na2HP04 0,02 M, pH 7,0 t} l# 60:40 (v/v), toe dO ddng 1,0 ml/phiit Phu'ang phap phSn tich c6 gidi han djnh luong dudi la 0,05 pg/ml, vdi RSD < 20%; khoang tuyen tinh 0,05 - 2 pg/ml, dS chinh xSo trong ngSy va khac ngay lln Iupt 2,70 - 6,35% vS 3,59 - 6,78%; dd diing trong ngay va khac ngSy lln Iupt 97,43 -100,96% va 94,62 -103,29%. Phuang phap niy cd the sil dung trong danh gia sinh khS dgng metformin trdn chO thuc nghiem.
* Til khda: IVIetformin; Huylt tuong; Sic ky long hi§u nang cao.
DETERMINATION OF METFORMIN IN DOG PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
SUMMARYMetformin is an oral biguanide medication, it has effective in reducing blood glucose levels in patients with type 2 diabetes. The aim of this study was to validate the analytical method of metformin in dog plasma by high pressure liquid chromatography (HPLC) with detector UV (at 232 nm) using ranitidin as an intenal standard. After precipating protein in plasma by acetonitrile, metformin and internal standard were eluted from fortis phenyl column (5 /jm, 750 x 4 6 mm) at 25 'C with a mobile phase consisting of acetonitrile and 20 mM Na2HP04 (60:40, v/v), at a flow rate of 1.0 ml/min. The tower limit of quantitation was 0.05 pg/ml, with a relative standard deviation of less than 20%. A linear range of 0.05-2 pg/ml was established. This HPLC method was validated with between - batch and within - batch precision of 2.70 - 6.35% and 3.59 - 6.76%, respectively. The between - batch and within - batch accuracy was 97.43 - 100.96% and 94.62 - 103.29%, respectively. This validated method is sensitive and repeatable enough to be used in bioavailability studies in dogs.
* Key words: Metformin; Plasma: HPLC.
* TnJdng Cao ding Duac PhO Tho
*' Hoc vi§n QuSn y
**' Trudng Dai hoc Duac Ha N6i
Ngumphan h6f (Corresponding): Nguyin Van Thfnh ([email protected]) NgAy nhSn bdi: 12/08/2014; Ngiy phan bien danh gia bai bio: 16/09/2014
Ngiy bai bao dwpc dang: 25/09/2014
TAP CHl V . Diroc HOC QURN SIT sS 8-2014 D A T VAN O i
Metformin la duae chit ed tdc dgng chong DTD nhdm biguanid, cd cp e h l tdc dung khae vdi cdc thuoc chlng DTD nhdm sulfpnylure. Khdng g i l n g sulfonylure, metformin khdng kich thich giai phdng insulin tu' eac t l bao beta tuyln tyy.
Thulc khlng ed tdc dung ha dudng huylt d ngu'di khlng bj DTD. Q ngudi DTD, metformin Idm giam tang du'dng huyet, nhung khdng gay tai biln ha dudng huylt (trLP tru'dng hap nhjn ddi hoac phli hap thulc hipp dong tdc dgng) [1].
Metformin la dupe ehlt n l m trpng danh muc dupe chat yeu c l u bao cao so li#u nghien oi>u tu'ang du'ang sinh hoc khi dang ky thulc {2]. Do dd, c i n phai t h l m djnh quy trinh phdn tich metformin trong djch sinh hpc theo quy djnh cua Cyc QuSn ly Thyc phlm va Du'pc phlm Hoa Ky (FDA). Vi vay, chung tdi t i l n hdnh nghidn cuu nay nham iam ca sd d l danh gia sinh kha dung cua nhu'ng e h l phlm chira metfomiin bao g l m ca dgng bao e h l thdng thudng va dgng giai phdng keo dai.
VAT LIEU VA PHUONG PHAP NGHIEN Ciru 1. Nguyen vgt U#u va thiet bj, Huylt luang ehd: do Ban Cung d p Dpng vgt thi nghiem, Hpc vi?n QuSn y cung d p .
Hda c h i t : metfermin c h u l n , ranitidin chuln do Vidn Kilm nghi#m Thulc TW eung d p , duae giu' d -25°C tru'dc khi su- dung; acetonitril, methanol vd nu'dc c i t (HPLC-Merek), eac dung moi hoa chit khae dat tieu chuln PA.
Thilt bj: hp thing sac ky Idng hidu nang cao HPLC Waters, autosampler (MJ), detector UV, mdy ly tam Ignh UNIVERSAL 320 (Hettich, DLPC), tu Ignh am sau DF8514 (llshinlab, Han Quoc).
2. Phu'ang phap nghien cu>u.
Xay dung phuang phap ilnh luvng glm:
khao sat d i l u kipn sac ky, quy trinh xu ly m l u va ehlt npi chuln.
Thlm djnh quy trinh djnh lupng: theo hydng d i n cua FDA (2001) (dp dac hieu, gidi han djnh lygng dudi, dp tuyln tinh, ty I? thu h l i , dp I n djnh) [3].
KS'T Q U A N G H I E N CLTU VA BAN LU/SkN
1. K i t qua xay dvmg phu'O'ng phap phan tich.
Phuang phdp djnh lygng metfarmm trong huyet t y g n g ehd dygc xay dyng nhy sau:
Lly 0,2 ml huylt tuang (mlu trang/chuin/
thy) eho vao Ing ly tam. Them 50 pi dung djch ranitidin 7,5 pg/ml (nil chuan) va 50 jjl nude elt; 0,4 ml dung djch acetonitril, l i e xoay 30 giSy. Sau dd, ly tam 10.000 vong/phut trong 10 phOt. Lay djch n i l , Ipc qua mdng Ipc 0,45 ^m. Djch Ipe thu dypc diing de tiem s i c ky.
K i t qua lya chpn dieu kien s i c ky:
cpt fprtis phenyl (5 nm, 150 x 4,6 mm).
Nhi?t dd cpt: 25°C. Pha dpng: aeetpnitril - dpm NazNPOa 0,02 M, pH 7 ty Ip 60:40 (v/v). Toe d l ddng 1,0 ml/phut. Deteetpr UV do d budc sdng 232 nm. T h i tich tiem m l u : 50 nl. N|i c h u l n ranitidin ed nlng dp 7,5 ng/ml.
TdP CHi V - Dir^c Hpc Q U S N sir sd 8-2014
2. Kit qua thlm dinh quy trinh djnh luv'ng metfonmin trong huylt tuwng ehd.
* Dp (S§c hi§u - chpn IQC:
Phan tich cae m l u huylt tuang trang vd huylt tuang cd metformin chuln (0,05 pg/ml) vd npi chuln ranitidin (7,5 pg/ml).
l-iinh 1: Sac ky d l m l u huyet tuang trang (a) va sac ky dc m l u huylt tuang ehd chira metformin vd ranitidin (b).
Tren s i c d l m l u chuln, xult hi#n 2 pic dygc xac dinh la metfomiin va ranitidin; tai thdi gian lyu cua hai ehlt trin m l u tring khdng cd pie xult hien. Dya vdo kit qua do pho hap thu ciia 2 pic blng detector UV, ed t h i khang dnh: 2 pic nay Ian lygt la ranitidin va metfomiin.
Hlnh 2: P h i hip thu cua oac pic cd tR= 3,33 (ranitidin-a) va IR = 4,26 (metformin-b).
Bang 1: K i t qua k i l m tra dp dac hipu - chpn Ipc cua phuang phap.
STT 1 2 3 4 5 6
DieN TlCH PIC MAU TRANG (mAU's) Rt tLF 4,0 - 4,4 phot
328
R, tip 3,1 - 3,7 phot
276 652
Ty" L$ DAP ONG PIC Tr^ng/metformin
< 20%
< 20%
< 20%
< 20%
< 20%
< 20%
Trang/ranltldin
<5%
<5%
<5%
<5%
<6%
<5%
(•"-"• khong co tin hi$u tap irng)
TAP CHl V - Diroc HOC OUAN SV S6 8-2014 Tai thdi diem trung vdi thdi gian luu ciia metformin (4,26 phtit), dap yng pic eua mau tring s 20% didn tich pie cua mlu chuln (dipn tieh pic trung binh cua mlu chuln metformin cd nong d l g i n gidi han djnh lygng dydi Id 3.028 mAu.s).
Trong khoang thdi gian lyu tir 3,1 - 3,7 phiit, dien tich pic m l u trang £ 5% dien tich pic npi chuln (dien tich pic ranitidin trung binh 68.279 mAu.s).
Nhu vay, phupng phap phan tieh da dam bap nhan dien, phan biet dygc metformin
va ndi chuln ranitidin va khlng bi anh hydng bdi ode tap ehlt cd trong huylt tyang, e l dp chpn Ipc dat yeu e l u .
* Bo tuyen tinh:
Phan tich cae m i u chuln metfomiin trong huylt tyang ed nong dp x I p xi 2,0;
1,0; 0,5; 0,2; 0,1; 0,05 pg/ml thep quy trinh da d y g c xay dyng. Xdc dinh tyang quan giya nong dp metformin trpng huylt tugng va ty le dien tieh pie metfermin/
ranitidin.
B^ng 2: Sy phu thupc giu'a ty le dien tich pie ehuln/npi chuan vd nlng dp metfermin chuln trong huylt tygng cho.
TEN MAU N6ng do thi,FC (pg/ml) Di?n tich pic metformin (mAU.s) Dien tich pic ranitidin (mAU.s) Ty le di$n tich chuln/noi chuan Phuang trinh hoi quy (Y = aX + b)*
Nong dp xac dmh t u duong chuan so v6i gia tri thuc (%)
SI 1,95 154.984 70.580
2,196 S2 0,98 80.963 74.280 1,090
S3 0,44 33.419 70.937 0.471
S4 0,22 16 853 74.727 0,226
S5 0,11 7.086 71 096
0,100 S6 0,05 1 549 69.951 0,022 Y = 1,1438X - 0,0303; R^ = 0,998 97,32 97,94 87,67 111,82 113,60 91.65
('Y: ty le di$n tich pic metformin/ranitidin; X: nong <30 metformin trong mau)
Trong khoang nlng dp tir 0,05 - 2 pg/ml, ed m l i tyang quan tuyln tinh giu'a nlng dp metformin vd ty le didn tieh pie ehuln/npi chuln vdi he so tyang quan = 1. Nong dO metformin xdo djnh tir dydng chuan so vdi gid tri thyc deu dgt 87,67 -113,6% va nam trong gidi han cho phep (80 - 120% doi vgi nlng dp t h l p nhlt va 85 - 115% cho cac nlng dp edn lai).
* Giui hsn anh lirgng dmi (LLOQ):
Xac fflnh dap ling pic cua m l u chuln, mlu trang. Tinh nong dp metfprmin trong mlu ehuan ttr ty l# dien tich pic cua metformin/ranitidin vd dydng chuln; tir dd xac djnh dO dung blng each so sdnh vdi gid tri thyc cd trong mlu.
T(IP CHl V - Diroc HOC OUAN Sir s S 8-2014 Bing 3: K i t qua xdc fflnh gidi han fflnh lygng dydi.
STT
1 2 3 4 5 X SD RSD (%)
DieNTiCHPie(mAUs) Mau chuln (metformin 0,05 pg/ml) Metformin
(Rt = 4,3 phot) 2.483 3.764 3.061 3.742 3.471 3 302 541 16,37
Ranitidin (Rt = 3,3 phut)
68944 64835 68432 68211 68552
M§u trang Rt tip 4,0-4,4 phut
328
RttO" 3,1-3,7 phut
276
652
Tai vj tri ed thdi gian lyu khoang 4,3 phiit, ddp irng mau chuan ed nong dd 0,05 pg/ml ldn han 5 lln ddp U'ng cua m l u trang va dp lap Igi sau 5 Ian phan tich vdi RSD = 16,37%
(< 20%). Nhy vgy, mau chuln chira metformin cd nlng dp 0,05 pg/ml dap irng yeu elu la gidi hgn djnh lygng dydi eua phygng phdp phan tich dich sinh hpc.
* 00 dOng, dQ chinh xac trong ngay va l<hiic ngiy:
Tiln hanh phan tich eac m l u kilm chirng g 3 mu'c nlng dp LQC (0,15 pg/ml), MQC (0,4 pg/ml) vd HQC (1,5 pg/ml). M i l Ipgi mlu QC g l m it nhat 5 m l u ddc Igp cc cung nlng dp va l|p Igi trpng 4 ngay khac nhau. Tiln hanh xCr ly m l u va ehgy sac ky theo nhO'ng dilu ki#n da lya chpn. K i t qua: phyang phdp djnh lygng ed dp diing va dp chinh xdc trpng ngdy la 97,43 - 100,96% (nam trpng gidi hgn chp phep 85 - 115%) va 2,70 - 6,35% (nlm trpng gidi hgn cho phdp < 15%), dp dung khde ngay la 94,62 - 103,29%, dp chinh xac 3,59 - 6,76%. Phygng phap dap u'ng yeu c l u v l dd dung vd dp chinh xdc khi phan tich trong djch sinh hpe.
* Tf 1$ thu hoi:
Xic dinh ty Ip thu hei ciia ehlt chuan vd npi ehuan bang each so sanh dien tich pic cua metformin va ranitidin tir mlu huylt tygng vdi dien tich pic cua chung trong mau pha dong cd chira ciing nlng d l . Kit qua: ty Id thu h l i cua npi chuln ranitidin la 90,23% (n = 4), ciia metfprmin g cac nlng d l LQC, MQC va HQC lln lygt Id 70,94%, 75,42% va 90,84%. Ty le thu hli cua chuln vd n|i chuln ed dp Igp Igi tot (RSD < 5%i vdi n i l chuln, RSD < 10% vdi chuln).
* Dp on dinh:
- Dp on dinh ciJa dung djch chuln glo, n i l chuln g l c treng thdi gian ngin: tiln hanh phan tich xac dinh nong do metfomiin va ranitidin cd trong mlu de g nhiet dp phong va mlu bao quan lanh g nhidt dp -80°C doi vgi metformin va d -20°C d l i vdi ranitidin.
Ti)p CHl V - Dirge HOC OUfiN sir s6 8-2014
Bang 4: O l on fflnh thdi gian ngan oiia dung dich chuan va n l i chuln gee.
GIAI DOAN
Bao quan lanh
Sau 4 gi6'/26°C STT
1 2 3 4 1 2 3 4
K^T QUA DINH LUONG Chulin goc (mAU.s)
Di$n tich pic 12.896 13.525 11.591 13.495 12.759 12.784 13.181 11.814
Th6ng ke X = 12877 SD = 904,8
X = 12635 SD = 680,2 p = 0,668
NOi chuln goc (mAU.s) Di$n tich pic
83.551 87.965 87.526 88.530 85.609 83.628 86.606 79.966
Th6ng k i X = 86.893 SD = 2.265,6
X = 83 952 SD = 2.931,6 p = 0,164
Nlng dp tmng binh ciia dung djch chuln g l e vd npi chuln g l c d l a nhidt dp phdng va bao quan lanh khac nhau khdng cd y nghTa thong ke (p > 0,05). Nhy vgy, dung dich chuln g l c vd dung djch npi chuln gee b i n vQ'ng d dilu kipn nhipt d l phdng trong vdng 4 gid.
- Dp I n djnh oiig m l u trong thdi gian dai: phan tich xdc djnh nlng d | dugc ehlt c6 trong cae mau, theo ffleu kien da chpn tai thgi dilm ngay sau khi pha va sau khi bao quan Ignh tir 1 - 20 ngay d -80°C d l i vdi metfomiin va d -20°C doi vdi ranitidin.
Bing 5: Dp I n djnh ddi ngay eiia dung dich chuln va npi chuln gle.
GIAI BOAN Ban dau Sau 3 ng^y
Sau 5 ngSy
Sau 7 ng^y
Sau 9 ngdy
Sau 20 ngSy
KET QUA BINH LUONG TRUNG BlNH (n = 4) Chuln g6c (1.000 pg/ml)
X ± S D X + SD Po-3 X ± S D
PO-5 X ± S D
PO-T X ± S D
PO-9 X ± S D
P Q - 1 5
1.000 + 32,06 973,80 + 15.23
0,190 974,29 ±4,16
0,163 952,66 ±29,18
0,072 1 033,91 ±21,02
0,127 970,19 ±38,33
0,278
N5i chuln goc (1.500 pg/ml) X ± S D
X ± S D Po-3 X ± S D
PO-5 X ± S D
Po-7 X ± S D
PO-9 X ± S D
Po-is
1.500 ± 59,76 1.575,76 ± 58,01
0,119 1.491,99 ±11,85'
0,801 1.465,02111,37
0,294 1.449,47 ±32,80
0,189 1.479.82 ±20,43
0,546 - 0 | I n djnh cua m l u huylt tygng sau 3 chu ky ding - ra ddng
THP CHi y - Dirge HOC OUAN sir so 8-2014 Bing 6: K i t qua nghidn ciru d | I n djnh cua m l u sau 3 chu k^ ding - ra ding.
GIAI BOAN
Ph§n tich ngay (ban dlu)
Sau 3 chu ky dong - ra dong
STT
1 2 3 4 1 2 3 4
N 6 N G B O METFORMIN (pg/ml) Miu LQC
Ndng dp 0,171 0,170 0,149 0,169 1,292 1,297 1,312 1,310
Th6ng ke X = 0,165 SD = 0,010
X = 0,166 SD = 0,006 p = 0,831
M l Nong do
0,170 0,165 0,172 0,157 1.285 1.349 1,299 1,296
J HQC Thing kO X = 1,302 SD = 0,010
X = 1,308 SD = 0,028
p = 0,746
Mlu I n djnh sau 3 ehu kji ding - ra ddng (p > 0,05).
- Dp I n dinh trpng qud trinh xir ly m l u : so sanh nlng d | metformin trong cdc mau LQC, HQC xii ly m l u theo quy trinh da dugc lya chpn ngay sau khi d l tan chay hoan toan g nhipt d | phdng, ede m l u LQC, HQC ed cung nlng d | , nhung cht tiln hdnh xir ly m l u sau khi d l tan chay g nhiet dp phdng 4 gig.
Bing 7: D l on djnh trong qua trinh xia ly mau
GIAI DOAN
Mlu dup'C xu* ly ngay
Mlu du'O'c xir ly sau 4 gifr d l tan chay 6 nhi$t do phOng
STT
1 2 3 4 1 2 3 4
N 6 N G o d METFORMIN (pg/ml) M l
N6ng do 0,171 0,170 0,149 0,169 0,165 0,169 0,169 0,164
J LQC Th6ng k§
X = 0,165 SD = 0,01&
X = 0,167 SD = 0,002
p = 0,697
Miu Nong do
1,292 1,297 1,312 1,310 1,303 1,310 1,331 1,335
HQC Thong ke X = 1,302 SD = 0,010
X = 1,320 SD = 0,016 p = 0,111
Nlng dp metfprmin trong m l u LQC vd HQC dygc xir ly ngay so vdi m l u dugc xir ly sau 4 gid de tan chay d nhipt d | phdng khde nhau khdng od y nghla thing ke (p > 0,05).
- Dp on djnh ddi ngdy: ddnh gia d | I n djnh eua m l u huylt tygng trdn cae m l u LQC va HQC. Xdc dinh nlng dp metformin od trong m l u tgl eac thdi diem nggy sau khi pha va sau 7, 17 va 27 ngay bao quan m l u d -20°C.
TAP CHf V - DUrOC HOC QUHN Sy SO 8-2014
Bing 8: K^t qua 36 6n dmh dai ngay cua m i u huy4t ticcyng.
GIAI DOAN
M l u ban dau
Sau 7 ngay
Sau 17 ngay
Sau 27 ngay STT
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
N I N G Dd METFORMIN (pg/ml) M i
0,157 0,149 0,153 0,146 0,156 0,148 0,168 0,172 0,150 0,152 0,156 0,158 0,155 0,164 0,171 0,156
uLQC X = 0,151 SD = 0,005
X = 0,159 SD = 0,010
p = 0,221
X = 0,154 SD = 0,004 p = 0,377
X = 0,161 SD = 0,007 p = 0,060
Mau HQC 1,373 1,553 1,480 1,332 1,366 1,349 1,371 1,435 1,510 1,421 1,571 1,540 1,333 1,289 1,343 1,304
X = 1,434 SD = 0,101
X = 1,380 SD = 0,038 p = 0,353
X=1,511 SD = 0,065
p = 0,251
X = 1,317 SD = 0,025 p = 0,064
Nong dp metformin trong cdc m l u bao quan g dilu kipn -20°C trong vdng 7, 17 va 27 ngdy so vgi nlng dp metfprmin trong m l u mdi pha khae biet khlng cd y nghla thing kd (p > 0,05). Nhy v|y, metformin I n djnh trpng huylt tygng trong vdng 27 ngSy g dieu kidn bap quan -20°C.
KfiT LUAN
Qua khap sat va thlm djnh phygng phap djnh lygng metfprmin trpng huylt tygng chl, da iya chpn dygc quy trinh xir ly m l u va dieu kien s i c ky cu the nhy sau: l l y 0,2 mi huylt tugng (mlu trang/ehuln/thO') cho vao Ing ly tam. Them 50 pi dung djch ranitidin 7,5 pg/ml (n|i chuln) va 50 pi nydc elt, 0,4 ml acetonitril, lie xody 30 giiy.
Sau dd, iy tam 10.000 vdng/phut trong 10 phut va lay dich noi Ipc qua mang Ipc 20
0,45 nm. Djch Ipc thu dygc diing d l tiem sac ky. Dieu kidn sac ky: cpt fortis phenyl (5 pm, 150 X 4,6 mm). Nhipt dp cdt: 25°C, Pha ding: aeetpnitril - ddm Na2HP04 0,02 M, pH 7 ty Ip 60:40 (v/v). Too d | d i n g 1,0 ml) phut. Detector UV g 232 nm. T h i tich tidm mlu: 50 nl. N|i chuln lam viee {ranitidin) nlng dp 7,5 ng/ml.
Quy trinh dinh lygng metfprmin tronj djch sinh hoe dS xdy dyng dap irng t i t ci oac ySu c l u theo quy djnh cua FDA (My
T9P CHI V - Diroc HOC QURN SV SO 8-2014 ve t h l m dinh phu-cng phap phan tich
trong djch sinh hpc. Nhu- vay, c6 the ap dung quy trinh djnh lu'p'ng nay de danh gia sinh l^ha dgng cua vien metfomnin giai phong k6p ddi trong huyet tu'ang cho.
TAI L l | U THAM K H A O 1. Duqrc thw qu6c gia Viet Nam. B6 Y t4.
2002
2. Thdng tw huCfng d§n bao c^o s6 li$u nghi&n cCeu sinh i<hS dungAuung duang sinh hoc trong dSng /cy thuoc. Bo Y te. 2010.
3. Center for drug evaluation and research.
Guidance for industry, bioanalytical method validation FDA, US, Department of Health and Human Service. 2001.
4 Himal Paudel Chhetn. Simple HPLC-UV method for the quantification of metformin in human plasma with one step protein precipitation. Saudi Pharmaceutical Journal.
2014, pp.1-5-
5. Valentine Porta. HPLC-UV determintaion of metfomiin in human plasma for application in phanrtacokinitics and bioequivalence studies 2007, pp.143-147.
