metanil yellow in chromatogram of reference preparation. The entire method was validated about the specificity linear range, LOD, LOQ, and validation results proved that this method was suitable for determination of metanil yellow illegally presented in lipstick and nail polish, and having good accuracy. The validated method was used to
test 6 samples of lipstick and 8 samples of nail polish purchased in the market, and I nail polish sample was detected to contain metanil yellow at 28.33 fig/g.
Bl/CrC DAU NGHIEN ClTU Dl/OC DONG HOC OUCfNG TIEM BAP VA TIEM TINH MACH CUA ASLEM TREN CHO
NGUYEN HOANG ANH, DAO KIM CHI Trudng Dai hoc Dirac Hd Npi 1. Dat van de
TA MANH HUNG, LE THI THU HUYEN, PHUNG THI VINH Vien Kiim nghiem thuoc Trung irang
Thudc tiem Aslem (Glycyl Funtumin hydroclorid 0,3 mg/ml), mot thudc cd tac dung kich thich miin dich khdng dac hieu, dung bd trg cho benh nhan ung thu sau phau thuat va trong cac trudng hgp suy giam miin dich khac. Chi phim do Cdng ty cd phin Dugc pham Vinh Phiic san xuat va da dugc Cue Quan ly Dugc Viet Nam cip phep luu hanh tren thi trudng (sd dang ky VD-0105-06). Mac du vay den nay, cac nghien ciiu vl dugc ddng hgc (DDH) cua chi phim nay vln chua dugc tien hanh. Nghien ciiu nay nham muc tieu xac dinh cac thdng sd DDH so cap ciia thudc tren chd thi nghiem khi tiem bap va tiem tinh mach, tao ca sd cho viec triin khai cac nghien ciiu DDH tilp theo tren ngudi tinh nguyen.
2. Nguyen vat lieu va phirong phap nghien cuu 2.1. Nguyen vat lieu, thiet bi, dung cu
2.1.1. Thudc nghien ciru
Thudc tiem Aslem (dng tiem chiia Glycyl Funtumin hydroclorid 0,3 mg/ml) dugc san xuit bdi Cdng ty cd phan Dugc pham Vmh Phiic.
Sd Id: 030609; Ngay san xuit: 06/2009; Han dung:
06/2012
2.1.2. Dpng vdt thi nghiem
Chd khde manh (ca hai gidng), can nang trung binh 10 ± 0,5 kg. Chd dugc nudi dn dinh trong 7 ngay trudc khi tien hanh thi nghiem.
2.1.3. Chdt chudn
+ Glycyl funtumin hydroclorid (GF): Chit chuin phdng thi nghiem; ham lugng: 99,0 % (nguyen trang).
-I- Salbutamol sulfat (SAL): Chit chuan phdng thi nghiem; ham lugng 99,62 %; do im 0,11 %; su dung lam chuan ndi trong phucmg phap phan tich.
2.1.4. Dung moi, hda chdt: Dat tieu chuin tinh khilt dung cho HPLC va LC/MS.
2.1.5. Cdc thiit bi phdn tich: Dugc chuin hda, dap ling yeu ciu cua ISO/IEC 17025 - 2005 va GLP bao gdm:
+ Thiet bi phan tich: May sac ky ldng khdi pho TSQ Quantum Ultra (Thermo Scientific).
-I- Thiet bi dung cu khac: Can phan tich, may ly tam, thiet bi bdc hai dung mdi, buret tu ddng, cac dung cu thiiy tinh, binh dinh miic, pipet cd do chinh xac phu hgp.
2.2. Thiet ke nghien ciiru [2]
Thiet ke nghien ciiu hai giai doan, hai phac dd (giai doan mdt tiem bip cho 6 chd, giai doan hai tiem tinh mach cho 6 chd). Thdi gian nghi giiia hai giai doan thu thudc la 4 ngay. Lilu thu: lieu dan 0,6 mg/con.
Liy 3 ml mau true tiep tii tinh mach cd cac chd thi nghiem tai cac thdi diem ngay trudc khi tiem thuoc va trong vdng 3 gid sau khi tiem thudc. Cac thdi diim lay mau nhu sau:
+ Tiem tinh mach: 2, 4, 8, 10, 12, 15, 20, 30,45, 60, 90, 120, 150 va 180 phiit sau khi tiem thudc.
+ Tiem bip: 2, 4, 6, 8, 10, 15, 20, 30, 45, 60, 90, 120, 150 va 180 phiit sau khi tiem thudc.
Mau mau sau khi lay dugc chuyin vao cac ong nghiem chiia chit chdng ddng Na-EDTA, ly tam voi tdc do 4000 vdng/phiit x 10 phiit. Sau khi ly tam, tach liy phan huylt tuong va bao quan trong tu lanh sau -35 ± S^'C trong vdng 30 ngay.
2.3. Dinh lirong Glycyl Funtumin trong huyit tucmg Xac dinh ndng do GF ti-ong huylt tuang (HT) cho thi nghiem bang phuong phap sic ky long khdi pho (LC/MS-MS) [1]. Tdm tit diiu kien sic ky, khdi ph6 va xu ly mau huylt tuang nhu sau:
14 Tap chi KIEM NGHIEM THU6c - So 2.2011; Tap 9.(32)
2.3.1. Diiu kien khoi phd di dinh luang GF vd chudn npi (SAL)
Thong $6 The cua nguon phun
Ap suat khi nitrogen cap cho ngudn ion Nhiet dp mao quan
Dien the thau kinh hoi tu ion Ion ban dau
The phan manh ion Ion tao thanh
GF 3400 V
30psi 360°C 118V m/z = 375
32 V m/z =121
SAL 3200 V
35psi 360°C 125 V m/z = 240
18V m/z =148
2.3.2. Dieu kien sdc ky long
• Cdt Luna CN 250 x 4,6 mm; 5 pm. Nhiet do cdt 50°C
• Pha ddng: Methanol - acid formic 0,5 % (60:40). Gradient tdc do ddng.
• Thi tich tiem miu: 25 pi.
2.3.3. Quy trinh xir ly mdu huyit tuang chiet tdch GF:
MIU HT chd dl ra ddng d nhiet do phdng. Liy 1 ml miu HT, them 100 pi dung djch ndi chuin SAL (ndng do khoang 20 ng/ml). Kiim hda bing 500 pi dung dich amoniac 0,1M. Chiit bing hon hgp dung mdi diethylether va cloroform (ty le thich hgp). Ly tam tach liy ldp dung mdi. Bdc hoi dung mdi dudi ddng khi nitrogen, thu cin. Hda tan cin trong 0,5 ml dung mdi thich hgp va tiem.sic ky. Xac dinh ndng do GF trong miu HT chd dua tren cac mau chuan chiia GF phan tich trong cung diiu kien.
Phuang phap LC/MS-MS dinh lugng GF trong miu HT da dugc thim dinh theo qui dinh thim dinh phuang phap phan tich dich sinh hgc ciia US - FDA [5]. Ket qua thim dinh phuang phap theo hudng dan ciia US- FDA, dugc trinh bay d bang 1.
Bdng 1. Kit qud thdm dinh phuang phdp LC/MS-MS dinh luang GF trong HT cho STT
1 2 3 4 5
Chi tieu Giai han dinh lucmg duai Khoang tuyen tinh
Do diing trong ngay - khac ngay
Do chinh xac trong ngay - khac ngay (CV%) Ty le thu hii cua GF (%)
Kit qua 0,545 ng/ml 0,5 -53,3 ng/ml; r > 0,995
92,8% - 106,4%
4,0% - 9,9%
90,7% - 94,0%
2.4. Xac ^nh cdc thong sd DDH cua GF[3], [4]
Tu kit qua xac dinh ndng do GF trong HT, xay dung dudng cong bilu dien su biin thien ndng do GF theo thdi gian v^ xac dinh cac thdng sd DDH so cap ciia thudc tren mdi chd thi nghiem.
3. Ket qu^ thirc nghiem
Qua trinh lay mau khdng gap su cd bit Igi nao. Cac miu mau dugc liy theo thdi gian du kiln. Sau khi kit thiic hai giai doan liy miu, tien hanh phan tich xac dinh ndng do GF trong huylt tuang chd bing phuang phap
T^p chi KltM NGHIfM THUOC - S6 2.2011; TSp 9.(32) 15
LC/MS-MS da dugc xay dimg va thim dinh. Ndng do GF trong HT chd sau khi tiem bip, tiem tmh mach, lilu dan (0,6 mg/con), dugc trinh bay d bang 2 va bang 3. Sic ky dd mdt sd miu tring, miu chuin, mau huylt tuang chd trudc va sau khi tiem thudc dugc trinh bay d hinh 1,2. Ehidng cong trung binh ndng do GF trong huyet tuang chd sau khi tiem lilu dan (0,6 mg/con) dugc trinh bay d hinh 3.
Bdng 2. Nong dp Glycyl Funtumin (ng/ml) trong HTcho sau khi tiem tinh mach lieu dem 0,6 mg
T.gian(phut) 10 12 15 20 30 45 60 90 120 ISO
Choi 12,2 7,1 3,0 2,7 2,5 2,6 1,7 1,9 1,3 0,8 0,8 0,7 0,6 0,6
Cho 2 14,5 9,4 8,6 5,7 5,6 4,1 3,4 2,1 2,3 1,9 1,3 1,1 0,9 0,6
Cho 3 22,5 15,5 9,0 5,9 4,0 2,6 1,9 1,4 1,2 1,0 0,8 0,7 0,6 0,5
Cho 4 30,2 15,5 13,4 10,1 6,2 4,7 3,2 2,5 1,9 1,5 0,9 0,8 0,5 0,5
Cho 5 15,0 9,0 3,7 3,4 2,8 2,8 2,4 2,5 2,3 2,1 1,4 0,9 0,8 0,6
Cho 6 15,1 13,1 7,3 5,6 4,1 4,1 3,5 2,7 2,3 1,5 1,0 1,0 0,8 0,6
Trung binh 18,2 11,6 7,5 5,6 4,2 3,5 2,7 2,2 1,9 1,5 1,0 0,9 0,7 0,6
SD 6,8 3,6 3,8 2,6 1,5 0,9 0,8 0,5 0,5 0,5 0,3 0,2 0,2 0,0
Bang 3. Nong dp,
T.gian(phut) C h o i Cho 2 Cho 3 Cho 4 Ch6 5 Ch6 6 IVung binh
SD
2 4,6 5,7 1,6 3,0 2,8 5,3 3,8 1,6
4 4,1 3,0 5,6 10,2
4,5 9,0 6,1 2,9
Glycyl
6 4,1 2,8 5,2 7,9 3,5 10,0 5,6 2,8
Funtumin (ng/ml) trong HTcho sau khi tiem bdp lieu dan 0,6 mg
8 3,8 2,7 3,9 6,2 3,7 10,2
5,1 2,8
10 2,9 2,4 3,7 5,9 3,3 8,0 4,4 2,2
15 3,2 2,2 3,2 4,5 2,3 6,2 3,6 1,5
20 2,3 2,3 3,0 4,7 2,1 5,8 3,4 1,5
30 2,0 1,8 2,0 3,2 2,3 3,8 2,5 0,8
45 1,5 1,8 1,9 3,1 1,6 2,9 2,1 0,7
60
1,2 1,1 1,7 2,7 1,2 2,5 1,7 0,7
90 1,3 0,8 0,9 1,9 0,8 1,4 1,2 0,4
120 0,7 0,7 0,8 1,3 0,6 0,8 0,8 0,3
150 0,6 0,6 0,7 1,1 0,4*
0,5 0,7 0,2
m
0,6 0,6 0,6 0,9 0,3*
0,4*
0,6 0,2
• gid tri nong dp ngoai suy, dimg de tham khao
RT: 0.00 -
1000
0 -
Intensity CD
u - 500-
0 0
7.74
0.98
0.98
2 4.71
3.80 1 5.87
4.71
3.80 ; ;
f [
5.56 ^-^^
4 6 Time (min)
NL: 1.74E3 TIC MS BUNK
NL: 1.74E3 TICR + cESISRM ms2 240.300 [147.700-148.700]
MS BLANK NL: 6.82E2 TICR + cESISRM mS2 375.000 [120.500-121.5001 MS BLANK
RT: 0.00-6.74
40000 20000 0
JiOOO:
0 4000O;
20000^
0" - 0
RT: 5.28 NL: 4.04E4 AA: 660949 TIC MS Genesis S3 RT:4.13
AA:248005 ;
RT:4.13 AA:248444
RT: 5.28
NL: 1.92E4
TICR + c E S i S R M m ^ 240.300
[147.700-148.7001 MS Genesis S3
NL: 4.04E4
AA: 650096x10 F: + c ESI SRM mS
2 4 6 Time (min)
375.000
[120.500-121.5001 MS Genesis S3
Hinh 1. Sdc ky do mdu HT trdng (trdi) vd mdu HT them chudn GF, ndng do 5 ng/ml (phdi) TIC: Sac ky dl tong ion (Total Ion Chromatography);
ESI SRM 240 [147,7-148,8]: Sic ky dl lira chon ion m/z = 240 ^ 148 (SAL);
ESI SRM 375 [120,5-121,5]: Sic ky dl lua chon ion m/z = 375-»121 (GF).
16 Tap chi KIEM NGHIEM THU6C - S6 2.2011; Tap 9.(32)
RT: 0.00 -
1000
0
Intensity
u 500
0 7.74
0.96
0.98
0
4.71
3-80 5.87
4.71
3.80
5.56 5-8^
2 4 6 Time (min)
NL; 1.74E3 TIC MS BLANK
NL:1.74E3 TICF: + cESISRM ms2 240.300 [147.700-148.700]
MS BLANK NL: 6.82E2 TIC F: + c ESI SRM ms2 375.000 [120.500-121.500]
MS BLANK
RT: 0.00-6.7^
40000 20000
0 —
flOOO
c 0 — 40000 20000 0 0
RT: 5.28 AA: 660949 RT:4.13 AA: 248005
RT:4.13 AA:248444
RT: 5.28 AA: 650096
2 4 6 Time (min)
NL: 4.04E4 TIC MS Genesis 3-I.V-10min
NL: 1.92E4
TIC F: + c ESI SRM ms2 240.300 [147.700-148.700]
MS Genesis3-I.V-10min
NL: 4.04E4
TIC F: + c ESI SRM ms2 375.000 [120.500-121.500]
MS Genesis3-I.V-10min
Hinh 2. Sdc ky do mdu HTcho trudc khi tiem (trdi) vd 10 phiit sau khi tiem tinh mach (phdi) TIC: Sac ky do tong ion (Total Ion Chromatography);
ESI SRM 240 [147,7-148,8]: Sic ky d6 lira chon ion m/z = 240 ^ 148 (SAL);
ESI SRM 375 [120,5-121,5]: SSc ky d6 lua chon ion m/z = 375 ^ 121 (GF).
Dudng cong trung binh ndng do thdi gian (n=6)
| - « - T i 6 m b l p
120.0 150.0 180.0 Thd>i gian (phut)
Hinh 3. Dudng cong trung binh nong do GF trong HTcho theo thdi gian sau khi tiem bdp vd
tiem tinh mach lieu dan 0,6 mg/con.
4. Ban luan va ket lu|n
Ket qua nghien ctiu tren 06 cho cho thay, sau khi tiem bap, GF hap thu rat nhanh vao tuan hoan chung (thai gian dat nong do cue dai trung binh khoang 4 phiit). Nong do cue dai trong huyet tuofng cho sau khi tiem bap lieu dom 0,6 mg/con trung binh khoang 6,1 ng/ml (n = 6; SD = 2,9). Nong do cue dai khi tiem tinh mach lieu don 0,6 mg/con trung binh la 18,2 ng/ml (n = 6; SD = 6,8). Sinh kha dung tuyet doi duong tiem bap trung binh khoang 91%. Ket qua nghien cuu ban dau tren cho cung cap cac thong tin dinh huong cho cac nghien cuu tiep theo, nghien cuu DDH cua Glycyl funtumin tren nguoi tinh nguyen.
Tai lieu tham khao
1. Ta Manh Hung, Le Thi Thu Huyen, Phimg Thi Vinh (2010), Nghien cuu xdy dung vd thdm dinh phuang phdp dinh luang Glycyl Funtumin trong huyet tuang cho bdng LC-MS/MS, Tap chi Dugc hoc s6 416,12/2010, trang 48-52.
2. Andes D. Craig WA (2002), Animal model pharmacokinetics and pharmacodynamics: a critical review, Int J Antimicrob Agents, Vol. 19 (issue 4), p. 261-268.
3. H.vande Waterbeemd, H.Lennemas and RArtursson (2003), Drug Bioavailability, Estimation of Solubility, Permeability, Absorption and Bioavailability, First Edition, WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim Published House.
4. Sunil S Jambhekar, Philip J Breen (2009), Basic pharmacokinetics. First Edition, Pubhshed by the Pharmaceutical Press - British.
5. US Food and Drug Administration (May 2001), Guidance for industry, Bioanalytical method vahdation.
SUMMARY
A pharmacokinetic study of Glycyl Funtumin after IM and IV administration in dogs was operated. The concentration of GF in dog plasma were determined by validated LC-MS/MS method. At the single dose of0.6mg, peak concentrations were: 6.1 ± 2.9 ng/ml and 18.2 ± 6.8 ng/ml respectively to IM and IV administration. Mean absolute bioavailability of IM route was 91%. These results will orientate away for further research in human.
Tap chi KIEM NGHI$M THU6C - S6 2.2011; T?ip 9.(32) 17
