• Nghien CIFU - Ky thuat
Dinh liroTig rhein trong huyet tirong ngiroi
•^ r
bang phtfong phap sac ky long hieu nang cao
Nguyen Thj HiroTig Giang', Le Thi Anh Nguy§t, Huynh Ha Ngoc Dung Nguyen Huynh Lien Khinmg, Tran Thf Thu Ha, Nguyin Ngoc Vinh
Vien Kiem nghiem thuoc TP Ho Chi Minh'E-mail: nthgiangl23@gmail com
Summary
An HPLC method was developed for quantitative determination of Rhein in human plasma with sodium diclofenac as the internal standard. The injected sample was prepared by protein precipitation with acetontrii. The chromatography was performed on a C18 column (250 x 4.6 mm; 5 pm) with a mobile phase consisting of pH 2.7 acetic acid buffer and acetonitrile (45:55). The linearity covered the concentration range of 0.1 - 9.5 pg/mL (r'= 0.9978). The LLOQ was 0.1 pg/mL. The intra-day and inter-day accuracy was within ± 15% of the theoretical value (102.05 % - 107.43 %). The intra- and inter-day precision was less than 15% of the CV (101.98 % -106.72 %). The method proved applicable to pharmacokinetic studies and estimation of in vivo bioavailability of diacerhein preparations in healthy volunteers after oral administration, and Rhein itself as its active metabolite in human plasma.
Keywords: Rhein, Diacerhein, HPLC.
Dat van de
Diacerhein thu6c nhom thuoc trj lipu mdi dc c h l interleukin 1a (IL-lbeta) co tac dung chong viem d i l u tri viem XLrang, thodi hoa khdp.
Dgc diem quan trpng nhat v l du'yc dpng cua diacerhein Id sau khi u i n g du'yc c h u y i n hda hoan toan thdnh rhein tru'dc khi vao mau bang phan ling deacetyl h6a i^"*'.
Hi#n nay, trcing nirdc co r i t n h i l u c h l pham chda diacerhein vdi gia thdnh re han so vdi cac thulc nhgp t d nu'dc ngoai. Tuy nhiSn, c h l pham ndy chLca du'yc ddnh gia tu'ang dipang sinh hgc so vdi thuoc g l c . Cho d i n nay, d Viet Nam van chua CO c6ng trinh nghien cdu v l phLpang phap djnh lu'yng rhein ( c h i t c h u y i n hoa cua diacerhein) trong h u y l t tu'ang ngu'di d l danh gid c h i t lu'yng cdc c h l p h i m chda diacerhein. Nghidn cdu nay n h i m mye dich dng dyng trong nghien cdu sinh kha dyng vd ddnh gid tu'ang du'ang sinh hpc cho cac c h l p h i m chda diacerhein d Vipt Nam.
Nguyen lieu, doi tu'O'ng va phuang phap nghien CIFU
Ooi t u ^ n g nghien ciiu
Rhein (hinh 1) la sdn pham deacetyl hoa cua diacerhein.
Hinh 1 ; Ciu true ciJa rhein Dung moi, hoa chat
Chuan Idm viec (AS): Rhein, so 16 110M1180V, ham lu'yng 99,64% cua Sigma-Aldrich. NOi chuan (IS): diclofenac natri, QT005 080111, ham l u y n g 99,50 % cua Vien Kiem nghipm thuoc TP. Ho Chi Minh, Viet Nam.
Dung m6i, hda c h i t : Dgt tieu chuan tinh k h i l t dung cho HPLC (J. T. Baker).
Huylt tuang trang do Benh vien T r u y i n mdu va Huylt hpc TP. Ho Chf Minh cung cap.
Trang thiet bj va dieu kipn phan tich May ly tam Ignh Ivlikro 220R Hettich (Ddc), can phdn tich GH 202 (Nhgt), tu d6ng -20''C Sanyo (Nhgt).
He t h i n g sac ky I6ng hipu nang cao LC-20A Shimadzu (Nhat) d u a c hieu chuan theo quy djnh cua ISO/IEC va GLP vdi cac d i l u kien nhir sau:
Pha dgng: dung djch acid acetic pH 2,7 - acetonitril (45:55); Cgt: C18 ( 2 5 0 x 4 , 6 mm; 5 pm) T^P CHI DUOC HQC - 9/2015 (SO 473 NAM 55)
Nghien CIPU - Ky thuat
hay tufong du'ang; Toe dp dong: 1 m U phut; T h i tfch tiem: 50 yL; D i u dd UV: bLrdc song 256 nm.
Chuan bj va x i i ly mau h u y l t t u ' o n g Chuin bj mau
Dung dich gdc rhein co nong dp 100 yg/mL vd diclofenac natri c h u i n IS cd nong dp 200 pg/mL trong nu'dc.
Mau huyit tuong c/iua rhein: Cho dung dich chuin chda rhein vdo m i u huylt tLFang trdng de cd cac mau huylt tLPang chiia Rhein co n i n g dp khdc nhau t d 0,1 d i n 9,5 pg/mL. Bdo qudn d -20 "C cho den khi phan tich.
Xu-1^ miu huyit tircmg
Dung djch mdu t h d : Lay chinh xac 500 yL h u y l t tLrang cd rhein cho vao I n g eppendorf, them 50 pL dung dich npi chuan diclofenac natri nong dp 200 pg/mL trong nu'dc, lac d i u , k i l m hda bang 50 pL dung djch kali hydroxid 0,1 N, them 750 pL methanol, l i e xoay trong 60 giay, ly tam
Hinh 2: Sdc ky do cua miu huyit tucmg tring Tinh tuyen tJnh
K i t qud t6m t i t d bang 1 cho t h i y co s y tu'ang quan t u y l n tinh gida n i n g dp rtiein va n6i chuan diclofenac natri trong huyet tu'ang vd ty lp dipn tich dinh rhein va diclofenac Natri ^ong khoang n i n g dp khdo sat khodng t d 0,1 d i n 9,5 pg/mL.
Bang 1 ; Tuang quan giOa nong do vi ti le dign tich dinh cua AS/IS Nong do (pg/mL) Vie dien b'ch dinh
0,100 0.294 0.478 0,923 2,512 4,536 7,872 9,514
0,0183 0,0539 0,0877 0,1693 0,4607 0,8319 1,4437 1,7450 Phuong tiinh hoi quy: y = 0,1834x
W$ s6 tuang quan ff = 0,9978
17000 vdng/7 phut, loc qua g i l y lpc millipore 0,45 pm va tiem sac ky.
T h a m d j n h p h u v ' n g phdp
T i l n hdnh t h i m djnh d6 dac hieu - chpn lpc, gidi hgn djnh lu'yng dLPdi, dp dung, dp chinh xac, ty lp thu hoi hogt chat, dp on djnh... cua phLrang phdp theo hLPdng ddn tham djnh phu-ang phdp djnh lu'yng thuoc trong djch sinh hpc cua US- FDA vd E M E A ' ^ i .
Ket qua va ban luan
Khao sat t i n h dgc hipu cua phuxyng phap s i c ky do mau huyet tirang chda chuan rhein vd nOi c h u i n diclofenac natri co 2 dinh tdch rdi nhau vdi thdi gian ILPU tirang dng 7,1 phiit (rhein) vd 10,2 phut (diclofenac natri). Trong khi m i u h u y l t t u a n g t r i n g kh6ng cd dinh trung vdi thdi gian ILPU cua hai c h i t c i n khao sat (hinh 2,3).
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i 1
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Hinh 3: Sic ky di cua miu huyit tuvmg ndng dd 4,5 pg/mL rhein vi 200 pg/mL diclofenac natri
D p d u n g va d o c h i n h xdc
Xdc djnh cdc m i u huylt tLPang co rhein d 4 nong dp khoang 0,1; 0,3; 4,5; 7,9 pg/mL Chuan bj 6 m i u d moi nong dp. K i t qud tdm t i t d bang 2 cho t h i y phuwng phap cd dp dung vd dp chinh xac dgt theo yeu cau trong phan tich m i u sinh hpc.
Bang 2 ; Kit qui khao sat do dung vi dg chinh xic
Trong ngay Tnjng binh D$ diinh xac % Dp dung % Ktiacngiy Taing binh B^ diinh xac % OQQung%
0,10 0,10 1,71 7,43 0,10 3,52 6,72
Nongd^pgtmL 0,28 0,30 1,12 4,35 0.30 1.34 4.99
4,54 4,91 1,87 2,05 4,90 2.54 1.98
7.S7 8.26 2.56 7.39 7.96 2.28 3.44
T^P CHI D i r p c HQC - 9/2015 (SO 473 NAM 55)
• Nghien ciru - Ky thuat
Gidi han djnh lu-yng cua phirang phap 6 nong dp 0,1 yg/mL du'yc chap nhdn id gidi hgn djnh iLrang du'di ciJa phirang phap vi sau khi phan tich cho t h i y ti so S/N > 10 va dgt do dung, dp chinh xdc theo quy dinh.
Ty le phuc hoi
Ojnh iLFpng cac m i u rhein d cac n i n g dg khodng 0,3; 4.5; 7,9 yg/mL trong huyet tu-ang.
K i t qua tdm t i t d bang 3 cho t h i y dp phuc h i i CLia phu-ang phdp dat tren 90%.
Bdng 3; Kit qua khao sat ty lg phuc hii (n = 6) Ning do (pg/mL) Ti /# phuc h6i (%) RSD (%}
0,29 91,96 2,95 4,54 94,96 1,99 7,87 99^20 2.12 Dp on djnh cua mau
Khao sat dp on djnh cua rhein trong h u y l t tuang d n i n g dp khoang 0,3 va 7,9 pg/mL trong
^ l u kipn: bao quan d nhiet do phong trong 6 gid;
trong 24 gid trong bam mSu t y dpng d 15°C, sau 3 chu ky ddng - ra dong; bdo qudn d nhi$t do -20 °C trong 30, 60, 90 ngdy, Mdu d u y c xem Id I n djnh khi k i t qua djnh luyng cdc m i u sau thdi gtan bao qudn dat t d 85 -115 % so vdi gid tri. ban d i u .
K i t qud tdm t i t trinh bdy trong bdng 4 cho thay phuang phdp phCi h y p vdi yeu c l u do I n djnh cCia nghi6n cdu.
Bdng 4: Kit qua khio sat dg on djnh cua ilieln trong huyit twang
Dieu kien dg on djnh Og on d|nh ngSn hgn Sau 6 gi6' a nhi§t i30 pli6ng Sau24gicfcrnhi0t<3O15=C trong bom miu ti,f dpng DO 6n dinh dong-ra dflng
£)9 fin djnh dSi hgn d-20»Csau15ngdy d-20°Csau35ngay d-20°Csau60ng^y
Do l^ch C/o) 0,29pg/mL
5.99
8.67 11,18 0,87 0,08
'0 v6i ban dau 7,87pg/mL
6,81
5,92 8.62 8.72 4,21
Ban luan
PhLPang phdp HPLC dung djnh lu'yng rhein trong h u y l t t u a n g ngudi du'yc xay dyng vdi dp nhgy vd dO chlnh xac cao. Theo Wei Zhu va Xue- Mei Wang i^' da s d dung phu-ang phap c h i l t long-
long bdng hon hyp diethylether-cyclohexan ty le 4 : 1 , trong nghien c d u chung tdi s d dung phu'ang phdp chiet tua protein vdi methanol v i n cho hieu s u i t c h i l t t i t (91,96 - 99,20 %) va dd lap lai cao (1,99 - 2,95 % ) . Phuang phdp dat do nhgy cao (0,1 pg/ml) so vdi quy trinh du'yc Wei Zhu va Xue-I^ei Wang ™ xdy d y n g (0,4 M9/ml). S d dyng pha dpng Id hdn hap dung djch acid acetic pH 2,7 va acetonitnl vdi ty le 45:55, khong dung m u l i phosphat '^' nen it dnh hu'dng d i n c h i t lu'yng cua cpt phdn tich khi dp dung tryc t i l p trong nghien cdu du'yc dpng hpc, t h d tu'ang du'ang sinh hpc.
D6 I n djnh cua rhein trong huyet tu'ang d u a c duy tri trong 60 ngay du thdi gian d l t h y c hien cdc nghien c d u d u a c dpng hpc, thd tu'ang dLPang sinh hgc tren cac che pham chda diacerhein.
Ket luan
Nhdng d i l u ki^n cua phipang phap HPLC dng dung de danh gia rhein trong h u y l t tu'ang da du'yc xdy dyng. S d dung phu-ang phdp c h i l t tda protein vdi ngi chuan Id diclofenac natri.
Dieu kien sac k^ tdch tren cgt C18 (250 x 4,6 mm, 5 pm) vdi pha dpng Id hdn hyp acid acetic pH 2,7 vd acetonitril vdi ty lp 45:55. Phu'ang phap djnh lu'yng da du-yc t h i m dinh mpt cdch d i y dii. Tinh t u y l n tinh du'yc xdc djnh trong khoang nong dp 0.1 - 9,5 pg/ml (r^ = 0,9978) vdi LLOQ la 0,1 pg/ml. K i t qua dp chinh xac, dp dung trong ngay va gida cac ngdy dat yeu cau cho cho phep phdn tich rhein trong h u y l t tu'ang. Phuang phap CO the ap dung trong nghidn c d u du'yc dpng hpc, t h d tuang difang smh hpc tren cdc c h l pham chda diacerhein.
Tai lieu tham khao
1 G6raldine Falgarone, Maxime Dougados (2001), "Diacerein as a disease-modulating agent in osteoarthritis", Current Rheumatology Reports, 3(6), 479-483.
2. Nicolas P., Tod M., Padoin C , Petltjean O.
(1998), 'Clinical pharmacokinetic of diacerein", Clinical Pharmacokinetics, 35(5), 547-359.
3. Uday S. Chakrabarty, Uttam Mandal, Uttam Bhaumik, Bappaditya Chatterjee, Animesh Ghosh, Anirban Bose, Japan K. Pal (2008), "Bioequivalence study of two capsule formulations containing diacerein 50 mg in healthy human subjects", Drug Research, 58(8), 405-409.
4 Wei Zhu, Xue-Mei Wang. Li Zhang, Xiao-Ye Li, Bao-Xiu Wang (2005), "Pharmacokinetic of rtiein
Ti^P CHI DUgfC HQC - 9/2015 (SO 473 NAM 55)
