Tdi lieu tham khdo 1. Dugc diin Viet Nam TV, lan xuit ban thu 4,2009.
2. The United States Pharmacopeia 32.
3. The Bristish Pharmacopoeia 2010.
4. WHO/PHARM/92.558,"Collaborating Centte for Chemical Reference Substances", 1991, p.46.
SUMMARY
A method for simultaneous determination triamcinolone acetonide, nystatine and clotrimazole in Ultracomb cream by HPLC was developed. Results of validation show the condition of HPLC analysis is suitable (the liquid chromatograph is equipped with a 254 nm detector and a 250 x4.6 mm, 5 \im, RP-8 column; pH 7.0 phosphate buffer - Methanol as mobile phase with gradient program and the flow rate is about LO ml/min). The proposed method is rapid, precise, accurate and
appropriate for the quality control of the same products.
NGHIEN COru DjNH Ll/GfNG AMPHETAMIN VA METHAMPHETAMIN TRONG MAU VA Nl/GfC TIEU BANG SAC KY LONG K H 6 | PHO
TRAN VIET HUNG
Vipn Kiem nghiem thuoc Trung uomg
PHAM QUOC CHINH Vien Phdp y Quoc gia
1. Ddt van de
Amphetamin (AM) va methamphetamin (MA) la cdc chat kich thich than kinh dang amphetamin (ATS - Amphetamm Type Stimulants). Theo thdng ke eua Co quan phdng chdng ma tuy Lien Hgp Qudc (UNODC), hifn nay tten thi gidi cd khodng 25 trifu ngudi lam dung ma tuy tdng hgp ATS ttong sd dd cd tdi 60 % thugc khu vuc Ddng vd Ddng Nam A. CJ Vift Nam ATS da lan khdp cdc tinh thdnh, ddi tugng su dung nhiiu nhit la thanh thieu nien, hgc sinh, sinh vien, lai xe dudng dai...
Tren thuc tl, cac phuang phap phan tich phdt hien cac chit ma tuy nhdm nay cdn it dugc sft dung. Do dd, de cd mdt phuang phap vdi do nhay va do chinh xac cao, chung tdi tiln hdnh nghien eftu xdy dung quy trinh xac djnh ma tiiy tdng hgp AM vd MA ttong djch sinh hgc bang sac ky ldng khdi phd (LC-MS).
2. Phinmg phdp - thuc nghifm
2.1. Nguyen vpt lipu, hoa chat vd thiet bi
Hoa chdt, thuoc thur. Chit chuin methamphetamin (vilt tdt MA) 1,0 mg/ml ttong methanol, M-009, sd Id FE022908-02, USDEA Exempt TK # 61-34; Chit ehuin amphetamin (AM) 1,0 mg/ml ttong methanol, A-007, sd Id FC122006-01A, USDEA Exempt TK # 61-05; Ngi ehuin phenylephrin (PH) (chuin ldm vifc cua Khoa Vdt ly do ludng, Vifn Kilm nghifm thudc Trung ucmg), pha dung djch 1,0 mg/ml methanol;
T9p chi KlfeM NGHI$M THU6C - S6 1.2011; Tdp 9.(31)
Methanol tinh khilt HPLC, nudc cit, acid triiluoacetic (TEA), ethylacetat, methanol PA, amoniac 13,5 M...
Thiit hi: May sdc ky long khdi phd (LC-MS) Thermo-Finnigan LCQ Advantage Max., cgt sdc ky Agilent SB-C18: 15 cm x 5 mm, 3,5 nm; Cgt chiit pha rdn AccuBOND II Evidex Cartridges, 200 mg, 3 ml (Cdt mix-mode: Trao ddi cation va pha ddo, Agilent technologies).
Mdu nghiin cim: Miu mau va miu nudc tilu khdng nhiim ma tiiy (miu ttdng) vd cac miu nudc tieu, miu mau cua ngudi nghi ngd sft dung ma tuy gfti den giam dinh tai Khoa Hda phap - Vien Phap y (^dc gia.
2.2. Phiromg phdp nghien cdu 2.2.1. Dieu kien sdc ky
- Cgt: Agilent SB-C18: 15 cm x 4,6 mm; 3,5 nm Pha ddng: MeOH TEA ttong nudc 0,05 %, ti If 90:10 (v/v)
Tdc do ddng: 0,2 ml/phut Thi tich tiem miu: 25 nl Thdi gian phdn tich: 10 phut
Detector khdi phd: Ky thudt ion hda phun difn tii ion duomg (+ESI); Khi Sheatii Gas = 20, Auxylary Gas = 6; Chi do chgn lgc ion (SIM), chay ddng thdi (3 events) lin lugt bdt manh m/z 168 ± 1 (Event 1) dl do
11
tin hieu PH, m/z 150 ± 1 (Event 2) dl do tin hieu MA va vdi manh m/z 136 ± 1 (Event 3) dl do tin hieu AM.
2.2.2. Chudn hi mdu vd dung dich thic
Mdu chudn: Cae dng chuin MA, AM ndng do 1 mg/ml pha thanh cae dung dich cd ndng do tft 0,005 ng/ml - 1 ng/ml ttong metiianol. Chuin ndi PH ndng do 1 ng/ml ttong methanol.
Cdc mdu nu&c tieu vd mdu nhiim: Sft dung mdu mau va mau nudc tilu frdng them chuin dl dugc cac dung djch nhiim cd ndng do 0,005 ng/ml; 0,025 ng/ml;
0,05 ng/ml; 0,250 ng/ml; 0,500 ng/ml va 1,000 ng/ml chuin ndi PH 0,1 ng/mJ.
Xu ly mdu: Liy 5 ml nudc tieu hoac 5 ml mau chdng ddng vao frong dng thuy tinh (16 x 100 mm).
Vdi miu mdu thi them 5 ml dung djch dem phosphat 0,1M pH 7,0. Ldc sieu am frong 10 phut, ly tam 5 phiit va liy ldp dung djch. Them 100 nl dung djch chuin ndi phenylephrin 1 ng/ml ttong methanol.
Ldm sgch vd ldm gidu mdu: Cho 3 ml methanol
chdy qua cdt, dieu chinh tde do chdy 2 ml/phut (su dung bom hut dp suit gidm). Them 3 ml dung dieh dem phosphat 0,1M pH 7,0 tdc do 2 ml/phut. Nap mdu vao cdt vdi tdc do 2 ml/phut. Rfta cdt vdi 10 ml nudc cit, tdc do 5 ml/phut. Them 5 ml dung dich HCl 0, IN tie do 5 ml/phut. Hut khd cdt 1 phut, sau dd thim udt cdt bdng 100 nl dung mdi rfta giai. Rfta gidi bdng 5 ml hon hgp ethylacetat - methanol - amoniac (80:18:2 v/v) tdc do 2 ml/phut. Them 2-3 gigt dung djch HCl IN, ldm kho dudi ddng khi Nj d 46°C. Hda can bdng 1 ml pha dong rdi tiln hanh phan tich sac ky.
3. Ket qua va ban luan 3.1. Bp dac hieu - chpn lpc
Vdi chuomg trinh sdc ky da lua chgn, sac ky dl cua AM, MA vd PH ndng do 0,05 ng/ml dugc ghi d hinh 1 cho thiy pic cua AM la 6,36 phut, cua MA la 6,33 phut va cua PH la 6,14 phut. Chi do chay 3 event cho phep tach va xft ly rieng tftng pic. Sac ky dd cua miu nudc tilu ttdng hinh 3 khdng cd pic tai cac vj tri nay
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Hinh 1. Sdc ky do cua dung dich hdn hop chudn AM, MA vd PH ndng dp 0,05 pg/ml Sdc ky do hinh 1 sau khi xft ly rieng re, dugc ghi d hinh 2
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Hinh 2. Sdc ky do xic ly riing re, AM (Event 3), MA (Event 2) vd PH (Event 1)
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Hinh 3. Sac ky do mdu nuac tieu trang 3,2. Ddnh gid phucmg phdp
3.2.1. Khodng tuyen tinh vd do lap lgi
Ket qua khao sat trong mau vk trong nuac tieu tring thi hien a bang 1, hinh 4, hinh 5.
Bdng L Duong chudn cika AMvd MA trong mdu vd nu&c tieu
Ndng dp (ng/ml)
0,005 0,025 0,050
0,250 0,500 1,000 Phuang trinh
hoi quy
Ti If difn tich pic trong mdu AM/PH
0,107209 0,718311 1,80955 (TB 6 lan tiem)
RSD = 3,45%
8,702247 18,65512 34,40751 y = 34,822x +0,1126
R^ = 0,9982
MA/PH 0,120538 1,033707 2,16676 (TB 6 Ito tiem)
RSD = 4,64%
13,62824 23,65965 46,99241 y = 47,057x^0,2478
R^ = 0,9981
Ti If difn tich pic trong nu&c tieu AM/PH
0,111535 0,708097 1,587792 (TB 6 Ito tiem)
RSD = 3,11%
7,506785 17,8124 33,6176 Y=34,044x-0,1593
R^= 0,9981
MA/PH 0,137368 1,061456 2,009264 (TB 6 Ito tiem)
RSD = 3,19%
12,9758 24,9574 46,8285 y = 47,324x +0,2278
R^ = 0,9982
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Hinh 4. (a): Tuang quan hoi quy tuyin tinh giita ti Hinh 5. (c): Tuomg quan hoi quy tuyin tinh giita ti li AM/PH vd ndng dp cua AM trong mdu li AM/PH vd ndng dp cua AM trong nu&c tiiu (b): Tuang quan hdi quy tuyin tinh giita ti li MA/PH (d): Tuang quan hoi quy tuyin tinh giita tile MA/PH
vd ndng dp ciia MA trong mdu vd ndng dp cua MA trong nu&c tieu
Tap chf KIEM NGHlf M THU6C - S6 1.2011; Tip 9.(31) 13
Nhdn xet: Kit qua thu dugc d cac bang 1 va hinh 4, hinh 5 cho thiy trong khoang ndng do da khao sat tu 0,005 |ig/ml din 1,000 jig/ml cd su tuong quan tuyen tinh chat che gifta ndng do AM va MA vdi ti sd dien tich pic AM/PH va MA/PH ti-ong mau va nudc tilu. (He sd tuong quan r > 0,99).
3.2.2. Khdo sdt dp dting ciia phucmg phdp
Kit qua khao sat do dung tren cac miu mau va nudc tieu tring them ehuin ndng do 0,05 ^g/ml dugc trinh bay tiong bang 2.
Bdng 2. Kit qud khdo sdt dp dung cua phuang phdp
Mau
1 2 3 4 5 6 TB T^ 1$ tliu h^i
Ndng df da bidt AM 0,05 fig/ml, MA 0,05 ng/ml Luang AM Am du(fc (ng/ml)
Mdu 0,04697 0,04304 0,04406 0,04634 0,04377 0,04557 0,04495 89,92 % RSD =3,48%
Nu&c tieu 0,04765 0,04659 0,04357 0,04645 0,04453 0,04323 0,04533 90,67 % RSD=3,98%
Lugrng MA dm dugrc (fig/ml) Mdu
0,04793 0,04379 0,04596 0,04645 0,04402 0,04688 0,04583 91,67 % RSD=3,56%
Nu&c tieu 0,04678 0,04428 0,04683 0,04652 0,04456 0,04704 0,04600 92,00 % RSD=2,69%
Ket qua khao sat cho thay phuong phap djnh lugng cd do dung dao ddng ti-ong khoang 89,92% din 92,00%, dieu nay la chap nhan dugc vdi mau la djch sinh hgc.
3.2.3. Khdo sdt dp Igp lgi
Ket qua sd lieu d cac bang 1, bang 2 cho thiy AM, MA trong mau va nudc tilu d ndng do 0,05 |xg/ml co do lap lai kha tot (RSD<15 %). Vdi do dao ddng RSD < 5 % trong djch smh hgc thi phuong phap nay cd do lap lai cao.
3.2.4. Xdc dinh gi&i hgn phdt hiin vdgi&i hgn dinh luang
Vdi AM, tai ndng do 0,0025 ng/ml (2,5 ng/ml) sic ky dd cho tin hieu S/N=3 (sic ky dd hinh 6), thoa man diiu kien ciia gidi ban phat hien (djnh ti'nh) theo quy djnh. Vay gidi ban djnh lugng cua AM la 0,0075 ng/ml (7,5 ng/ml; 7,5 ppb). Vdi MA, tinh dugc gidi ban djnh lugng la 0,0025 ng/ml (2,5 ng/ml; 2,5 ppb) va gidi han djnh tinh la 0,0008 ng/ml (0,8 ng/ml; 0,8 ppb, sic ky dd hmh 6). Trong diiu kien nghien eftu cua chiing tdi thi khong co su khac nhau dang kl gifta gidi ban djnh tinh va gidi ban dinh lugng tiong mau va nudc tilu.
IMA, L01>=0.8iig/ml
H U f%* tmM ^mM tata tMS UIU
AIM, LOI>=2,Sng/nil
ntA tms IMA •14 iMM I M 4 IITJ
Hinh 6. Sac ky dd ciia AM, MA khdo sdt LOD vd LOQ
14 Tap chi KIEM NGHIEM THUOC - So 1.2011; T§p 9.(31)
3.3. Ap dung quy trinh tren diphan tich mau mdu vd nudc tiiu thtrc ti
Van dung vao phan tich cac mau nudc tieu va mau thuc tl. Kit qua dugc the hien d bang 3. Hinh 7 la SKD phat hien AM va MA trong nudc tieu cua 1 ngudi su dung.
Bdng 3. Mdu thuc te gici t&i Vien Phdp y Quoc gia
TT
1 2 3 4 5
Hg ten
Nguyen Thanh H Nguyen Thi D Phiing thj O Ha Vto Q Trto Binh A
Bia chi
San La Bac Giang
Yen Bai Nghe An HaNpi
Mdu AM
(fig/ml) 0,002 0,011 0,007 (-)
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MA (ng/ml)
0,005 0,24 0,19 (-)
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Nu&c tieu AM
(ng/ml) 0,016
(-) (-)
MA (ng/ml)
0,028
; ^ ; . - ^ « &
(-) (-)
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Hinh 7. Sdc ky do mdu nu&c tieu cita Nguyen Thanh H Ky hieu (-): Am tinh vdi AM hoac MA.
Nhdn xet: Tu kit qua phan tich 7 miu mau va nudc tilu thu dugc tu 5 ddi tugng, chung tdi nhan thiy co 3 mau mau duong tinh vdi MA va AM, 1 miu nudc tilu duong tinh vdi MA va AM
4. Ket luan
- Xay dung hoan chinh phuang phap djnh tinh va djnh lugng AM va MA trong nudc tieu, mau: Xu ly mau bang phuang phap chiit pha rin, djnh tinh va djnh lugng bing sic ky long khdi phd. Phuang phap da phan tich cd do nhay cao, co tinh dac hieu, thdi gian phan tich ngin, phan tich diing va chinh xac ddi tugng cin phan tich.
- Da ap dung quy trinh phan tich dugc 7 miu giam djnh phap y trong do co 4 mau mau (3 miu duang tinh vdi MA va AM), va 3 miu nudc tilu (1 miu duong tinh vdi MA va AM).
Tai lieu tham khao
1. Anthony C. Moffat, M David Osselton, Brian Widdop (2004), Clarke's Analysis of Drugs and Poisons.
Third edition, volume 11, pages 1226-1228
2. Hendrickson H, Laurenzana E, Owens SM (2006), "Quantitative Determination of Total Methamphetamine and Active Metabolites in Rat Tissue by Liquid Chromatography With Tandem Mass Spectrometric Detection". AAPS Journal.; 8(4): E709-E717. DOI
3. Ming-Ren Fuh, Ti-Yu Wu and Tzuen-Yeuan Lin(2005), "Determination of amphetamine and methamphetamine in urine by solid phase extraction and ion-pair liquid chromatography-electrospray-tandem mass spectrometryDepartment of Chemistry", Soochow University., P.O. Box 86-72, Taipei, Taiwan
4. E.M.Thuman, M.S.Mill (1998), "Solid phase extraction". Principle and practice. Chemical analys. Volume 147
Tap chi KIEM NGHIEM THUOC - So 1.2011; Tap 9.(31) 15
