NGHIEN cvfu OINH Ll/GfNG GINScivosiu - n» u
(VJ-KD•;
TRONG HUYET fl/ONG
C H 6 B A N G P H I / O N GPHAP HPLC
TRINH VAN LAU Vien Kiem nghiem thuoc TW
NGUYEN VAN MINH Hpc Vipn Qudny
PHAM THANH SUOL, PHAM HUNG LlTC- Trifomg Dai hoc Ydwac Cdn Thar
1. Dat van de
Nhdn sdm la loai thude quy, dupe nhdn dan nhifu nude trfn thf gidi su dung [1]. Nhieu nha khoa hpe trong va ngodi nudc da nghifn ciiu va khang dinh:
Nhdn sam cd tdc dung chdng mft mdi, tang cudng sinh luc, tdng sue bfn, tang sinh thich nghi cila ca thf, ed tdc dung chdng oxy hod vd cd tac dung kich thich hormon sinh due [3], [4], [5].
Trong thanh phdn hod hpe, cdc saponin (Ginsenosid) cd gid tri ddc bift quan trpng, dupe chia lam 2 nhdm gdm: nhdm ed cdu tnic dammaran vd nhdm ed cdu tnic khung olean. Trong 2 nhdm trfn, dang chii y la nhdm saponin cd eau tnic dammaran. Vi eae saponin cua nhdm nay mang lai hoat tinh sinh hpc cua nhdn sdm.'Trong cdc Ginsenosid ed eau tnic dammaran, thi G-Rbl ddng vai trd quan trpng nhdt trong vifc ddnh gia chdt lupng cua nguyfn lifu vd cae ehe phdm ehiia nhan sdm.
Hifn nay, nhdn sdm dupe bdo chf dudi nhiiu dang khae nhau vd theo quan diem sinh dupe hpe thi hifu luc tdc dung eiing khae nhau. De ndng cao chat lugmg san xudt thude, vifc nghifn ciiu sinh kha dung va tuomg duomg sinh hpc Id vifc ldm can thift.
D I ddnh gia sinh khd dung vd xdc dinh mdt sd thdng sd dupc ddng hpc cua G-Rbl trong cac chi phdm chiia nhdn sdm tren thuc nghifm, chiing tdi tiln hanh df tdi:
'TSfghifn ciiu dinh lugmg Ginsenosid Rbi trong huylt tuomg chd bang phuang phap HPLC"
2. Thvc nghifm
2.L Nguyen vdt lipu, hod chdt
- Chdt chudn: G-Rbl (Hang WAKO - Nhdt Ban).
Chdt chudn ndi: Digoxin (Vifn Kifm nghifm thudc thdnh phd Hd Chi Minh).
- Acetonitril loai diing trong HPLC (Merck - Diie).
- Methanol loai PA.
- Cdt chiit xudt pha rdn SPE C18: Hoat hda ldn lupt bdng 2 ml methanol vd 2 ml nude.
Huylt tuomg chd : khdng chiia G-Rbl
2.2. Thiit bi , d^ng cu: Da dupe hifu chudn theo ISO/IEC 17025 vd GLP
May sdc ky ldng hifu nang eao LC lOAT (Shimadzu - Nhdt Bdn)
Cdc thilt bi va dung cu khde: Mdy ly tdm, can phdn tieh, micropipet ede loai vd cac dung eu do luomg bdng thuy tinh cd dp chinh xde cho phep.
2.3. Ddi tieffng vd phuomg phdp nghien ciru 2.3.1. Doi tugmg nghiin cicu
Tao cdc miu G Rbi trong huyft tuomg chd co ndng dp khde nhau dl khdo sat (ty if nuac/huylt tuong chd phdi nhd hom hoae bdng 5%).
2.3.2. Quy trinh xic ly mdu chiit
Ldy 1 ml huyet tuomg ehd ehiia G - R b l trpn vdri 10 pi Digoxin ndng dp 5 pg/ml. Sau do, eho thfm (ciing thf tich) dung dich methanol 60% trong nude, khuay hdn hpp trfn trong mpt phiit. Sau khi ly tdm vai tdc do 10.000 vdng/phiit trong 10 phiit, gan ldy dieh ly tam vd eho qua cdt SPE da dupe boat hda. Rira gidi bdng 2 ml nude, bd dich rua gidi, sau dd rua gidi tifp bang
1 ml methanol 90% trong nudc. Dung dich methanol sau khi rua gidi dupc ly tdm vdi tdc d6 20.000 vdng/phiit trong 10 phiit. Gan ldy dich ly tam, lpc qua mdng lpc 0,45 pm va tifm vdo cdt sdc ky.
2.3.3. Thdm dinh phuang phdp
Theo cdc chi tifu cua hudmg din thdm dinh phuong phap trong dich sinh hpc [2], [7].
3. Ket qua vd ban luan
3.L Xdy dung phieorng phdp phdn tich 3.1.1. Lua chgn npi chudn
Chpn Digoxin, theo phuang phdp cua Odani vJi cdng su [6].
3.1.2. Diiu kiin sdc ky
Sau khi khdo sat, lua chpn cac thdng sd cua qui trinh sdc ky dl kha nang tdch pic cua G-Rbl khdi cac thdnh phdn tap ehdt ed trong huylt tuomg chd la phii hop, chiing tdi tim dupc cdc difu kifn nhu sau:
- Cdt: Supelcosil C18 (150 x 4,6 mm; 5 pm);
10 Tap chi KIEM NGHIEM THUOC - So 4.2010; T|p 8.(30)
- Pha ddng: Acetonitril - dung dieh dfm NalLPO, 20 mM (35:65);
Detector: UV vdi bude sdng phat hifn la 203 nm;
Tdc dp ddng: 1 ml/phiit;
The tieh tifm: 10 pi.
3.2. Thdm dinh phirorng phdp 3.2.1. Tinh ddc hiiu
Tifm miu huylt tuomg chd khdng cd chdt phan tich (miu trdng) vd mdu thu Id miu huylt tuomg chd cd ehiia G - R b l . Tifn hdnh sdc ky theo diiu kifn da lua chpn, thu dupc sac ky dd ciia miu huylt tuomg trdng va G-Rbl trong huylt tuomg d hinh 1 va 2.
m
at
»
:J
J V ^ A A J U IHinh 1. Sdc ky do cua mdu huyit tuang trdng
G-Rbl
^X.^
I.S
i \ * % t t « «
da dupc xii ly vao hf thdng HPLC. Kit qua dupc trinh bay d bang 1.
Bdng 1. Kit qud kiim tra suphit hgp cua hi thdng sdc ky
Ldn
1 2 3 4 5 6 Trung
binli SD RSD (%)
G-Rbl Thai gian luu (phdt) 13,497 13,450 13,502 13,445 13,490 13,442 13,471 0,03 0,21
Dien tich pic (mAU.s)
6520 6532 6571 6539 6586 6541 6548,17
25,07 0,38
Digoxin Thoi gian luu (phut) 18,607 18,619 18,609 18,613 18,606 18,613 18,611 0,01 0,02
Dien tich pic (mAU.s)
1683 1684 1688 1662 1673 1664 1675,66
11,01 0,66
Dg phdn
gidi (R)
>2
Ket qua d bang 1 cho thay cac gid tri RSD cila thdi gian luu (RSD < 0,21%) va difn tich pic (RSD < 0,66%) diu cd dp lap lai tdt. Ddng thdi, dp phdn gidi (R) deu > 2. Dieu do chiing td cac diiu kifn cua hf thdng HPLC dn dinh va phu hpp de dinh luomg G-Rbl.
Trfn sac ky do (hinh 2) xuat hifn pic cua G-Rbl va cd tj^= 13,471 phiit va Digoxin cd t^= 18,611 phiit.
Tai cae thdi difm nay, tren sac ky do ciia miu huyft tuong trang (hinh 1) khdng xuat hifn pic la. Kit qua trfn cho thdy: cac difu kifn sac ky nhu da neu tren la phu hpp cho vifc tach G-Rbl ra khdi cac chat khac ciia huyft tuomg.
3.2.3. Duong chudn vd khodng tuyen tinh
Phan tich cdc mdu chudn G-Rbl trong huyet tuomg chd cd ndng dp 50 - 100 - 500 - 1.000 - 1.500 - 2.000 2.500 ng/ml va chudn ndi (Digoxin) cd ndng dp 20 pg/ml, chiing tdi thu dupc ket qua d bang 2 va hinh 3.
Bdng 2. Kit qud xdc dinh khodng tuyin tinh
Hinh 2. Sdc ky do cua G-Rbl + Digoxin trong huyit tuomg
Kft qua trfn cho thdy sac ky dd cua mdu trdng khdng cd pic tai thdi gian luu tuomg iing vdi thdi gian luu ciia pic G-Rbl diiu dd chiing td phuomg phap cd tinh dde hifu.
3.2.2. Dp phii hgrp cua hi thong sdc ky
Tifm 6 ldn miu huylt tucmg chd chiia G - R b l (ndng dp 2.000 ng/ml) vd Digoxin (ndng dp 50 ng/ml)
T9p chf KltM NGHIEM THU6C - So 4.2010; Tap 8.(30)
Nongd^
(ng/ml) 50 100 500 1000 1500 2000 2500
Dien tich pic (mA U.s) G-Rbl
1280 1654 2401 3754 5287 6332 7640
Digoxin 1603 1692 1681 1610 1654 1693 1672
Ty 1$ di$n tich pic G-Rbl/
Digoxin 0,798 0,977 1,428 2,331 3,196 3,740 4,569
Nong dg thuc (ng/ml)
56,66 97,36 463,90 945,30 1540,50 1908,80 2414,50
So vdi gid tri '^"''^
(%) 113,32 97,36 92,78 94,53 102,70
95,44 96,58 Phuomg trinh h6i quy: y = 0,0015 x + 0,7636
He s6 tuong quan tuyln tinh: r = 0,9983
11
» J -
500 1000 1500 2000 25(X) 3000 C(ng/mO
Hinh 3. Dd thi bieu dien su tuang quan tuyen tinh giiia ty le dien tich pic G-Rbl/Digoxin vd ndng do
G-Rbl trong huyet tuomg cho
3.2.4. Xdc dinh giai hgn dinh lugmg duai (LLOQ) Phdn tieh mau trang va mau chudn cd ndng dp 50 ng/ml trong huyft tuomg (ndng dp thap nhat trong khoang tuyfn tinh). Xac dinh ddp iing pic cua mau chudn. Tinh ndng dp G-Rbl trong miu chudn tu dudng chudn; tii do xac dinh dp diing bang each so sdnh vdi gia tri thuc cd trong miu. Kit qua xac dinh gid tri LLOQ cua phuomg phdp dupc trinh bay d bang 3.
Bdng 3. Ket qud xdc dinh gidi hgn dinh luang dudi
STT
1 2 3 4 5 Trung
binh SD RSD
(%)
Di^n tich pic (mAU.s) Mdu chuan
(SO ng/ml) G-Rbl
1062 1032 1045 1111 1239 1097,8
84,82
7,72
Digoxin 1740 1542 1674 1685 1653
Mdu trdng 13-14
phut 62 76 92 64 89
18-19 phut 75 86 111
85 101
Dg dung so v&i nong dg thuc
(%)
88,5 89,3 81,04 115,8 106,4 96,2
14,3
14,86
50 ng/mii uai yeu cau gim nan uum luyiig auoi cua phuang phdp. Difn tich cua mdu chuan va ndi chudn khoang 14 ldn va 18 ldn so vdi ddp iing cua miu huyft tuomg trdng cd cimg thdi gian tuang iing. Dp lap lai sau 5 ldn phdn tieh cua mdu chuan cd RSD < 15%
(7,72%), dp diing ndm frong khodng 80 -120%.
3.2.5. Xdc dinh dp chinh xdc (dp diing vd dp lap lgi) Pha cac miu LQC (100 ng/ml); MQC (1.000 ng/ml);
HQC (2.000 ng/ml). Mdi loai miu QC gdm it nhdt 5 miu dpc lap cd cung ndng dp. Tifn hanh xu ly miu va dinh lugmg theo cdc difu kifn da lira chpn. Kit qua xac dinh dp diing vd dp lap lai frong ngay vd giua cde ngay dupe trinh bay d bdng 4.
Bdng 4. Ket qud khdo sdt dd chinh xdc cua phuong phdp (n=6)
Do chinh xdc
Trong ngdy Khac ngdy
LQC (100 ng/ml) Do diing
'(%) 88,98 104,24
RSD (%) 6,91 6,70
MQC (1.000 ng/ml) Dg dung
(%) 105,84 99,97
RSD (%) 8,2 4,71
HQC (2.000 ng/ml) Dg dung
(%) 113,28
99,83 RSD
(%) 7,17 4,54
Kft qud d bdng 4 eho thay: Phuang phdp dinh lugmg cd dp diing tdt (88,98 -113,28 %), dp Ifch chuan nam frong gidi ban cho phep (± 15 %), dp lap lai ttong ngay vdi gia tri RSD nhd (6,91 8,2%), dap iing yeu cdu ve dp diing, dp chinh xac cua phuang phap phan tich frong dich sinh hpc.
3.2.6. Do on dinh cua mdu
3.2.6.1. Dp dn dinh frong qua trinh xir ly miu: Dupc tiln hanh frfn cdc mdu LQC (100 ng/ml) va HQC (2.000 ng/ml). Kit qua dupe trinh bay d bang 5.
Bdng 5. Kit qud khdo sdt dp on dinh cua mdu
Tir kft qud d bdng 3, chiing tdi thdy: Ndng dp
Thai gian
Mdu sau khi xu ly Mdu xulydi& nhift dg phong sau 8h
Mdu LQC (100 ng/ml) Dg dung
(%) 116,5
113,5 RSD
(%) 5,67
8,22
Mdu HQC (2.000 ng/ml) Dg dung
(%) 102,1
104,4 RSD
(%) 3,07
2,28
12 Tap chf KIEM NGHIEM THUOC - S6 4.2010; Tdp 8.(30)
Kit qud d bdng 5 cho thdy: sau khi xii ly 8 gid, ndng dp G - Rbi cd dp diing va dp lap lai phii hop vdi phep dinh lugmg cde chdt frong dieh sinh hpc.
3.2.6.2. Dp dn dinh ddi ngdy: Dupe tiln hanh fren cac mdu LQC va HQC. Xdc dinh nong dp G - Rbi tai cac thdi difm ban ddu vd sau 10, 20, 30 ngay bao quan d nhift dp - 35°C. Kft qua dupe trinh bay d bang 6.
Bdng 6. Kit qud nghiin cicu do on dinh ddi ngdy cua mdu (n = 6)
Giai doan
Ban dau Sau 10 ngdy Sau 20 ngdy Sau 30 ngdy
LQC (100 ng/ml) Dg diing (%)
111,5 108,3 111,7 109,5
RSD (%) 4,93 2,43 5,72 7,15
Mdu HQC (2.000 ng/ml) Bg ddng (%)
103,4 105,2 103,2 109,0
RSD(%) 4,88 4,20 4,16 3,65
Kft qua bang frfn cho thdy: ndng dp G - Rbi frong cac miu bdo qudn d nhift dp - 35°C frong thdi gian 10, 20 va 30 ngdy, khdng cd gi khac bift so vdi ndng dp G-Rbl ban ddu. Dieu dd chiing td: Ndng dp G - Rbi frong huyet tuomg chd sau 30 ngay bao quan d nhift dp - 35°C vdn dn dinh.
4. Ket luan
- Da xdy dung phuang phap dinh lugmg G - R b l frong huylt tuomg chd. Cd thi iing dung phuang phap nay de nghifn ciiu sinh kha dung cua cdc ehe phdm ed chiia nhan sam frfn thuc nghifm.
- Da tham dinh phuomg phdp dinh lugmg G - R b l frong huylt tuomg chd theo quy trinh vl thdm dinh phuang phap phan tich cac chdt frong dich sinh hpc.
Cac chi tieu ve tinh dac hieu, khoang tuyln tinh (50 - 2.500 ng/ml), gidi han dinh lugmg dudi (50 ng/ml), dp chinh xac va dp dn dinh cua mdu dat yeu cau theo quy dinh phdn tich cac chat frong dich sinh hgc.
Tai lieu tham khao
1. Do Tat Lai (2005), Nhimg cdy thuoc vd vi thuoc Viet Nam, Nha xuat ban Y hoc - Ha Noi
2. Phiing Thi Viiih(2009), Phdn tich mdu sinh hoc trong nghien ciru sinh khd dung vd tuang duomg sinh hgc. Tap chf Kilm nghiem thu6c, 1, tr. 24-26.
3. Arich S. et al (1997), Double-blind clinical effect of Korean ret ginseng power, Kisho and Linshio, vol. 13 (6), pp.
165-177.
4. Byung Hoon Han, Myung Hwan Park and Yong Nam Han (1985), Studies on the antioxidant components of Korean ginseng(V): the mechanism of antioxidant activity ofmaltol and phenolic acid, Korean Biochem, Vol.18 (4). pp. 337- 340.
5. David D. Kitts, Arosha N. Wijewickreme and Chun Hu (2000), Antioxidant properties of a North American ginseng extract. Molecular and Cellular Biochemistry, pp. 1-10.
6. Odani T, Tanizawa H and Takino Y (1983), Studies on the absorption, distribution, excretion and metabolism of ginseng saponins III. The absorption, distribution and excretion of ginsenoside Rbi in the rat. Chemical and Pharmaceutical Bulletin, 31(3), pp. 1059-1066.
7. U.S Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, "Guidance for Industry-Bioanalytical Method Validation" (May 2001).
8.
SUMMARY
An adequate and accurate HPLC for determination ofginsenoside Rbi in dog plasma has been developed and validated.
Chromatography was performed with column of Supelcosil C18 (150x4.6 mm; 5 fim), the mixture of Acetonitrile - 20 mM NaH^O^ buffer solu tion (35:65) was used as the mobile phase. The method was linear within the range of ginsenoside Rbi concentration of 50 - 2500 ng/ml. The LLOQ was 50 ng/ml. The validated method is suitable for bioavailability study ofginsenoside Rbi.
Tap chi KltM NGHI$M T H U 6 c - S6 4.2010; Tdp 8.(30) 13
