Thăm dò điều kiện định lượng đồng thời paracetamol và ibuprofen trong huyết tương bằng phương pháp SKLHNC

• N g h i e n CLFU - K y thuat<br /> <br /> journal of biologial chemistry, vol 237, N 4, April 1962,<br /> 2<br /> 10. Mala B. Rao, Aparna M. Tanksale, Mohini S.<br /> USA, (1961). Ghatge, and Vasanti V. Deshpande, " Sources of<br /> 6. Barrett, Alan J; Buttle, David J.; Rich, Daniel H, proteases ", Microbiology and Molecular Biology<br /> Pharmaceutical composition of purified chymopapain, Reviews, (1998).<br /> Patent No. 5468480, (1995). 11. Americal Chemical Society, Washington, DC,<br /> 7. Dando PM et al., unoglobin E antibodies to Reagent Chemicals ACS Specification, Volume II, 2-<br /> papaya proteinases and their relevance to 26, (1993).<br /> chemonucleolysis, Spine. Department of 12. Kapsalis AA, Stern IJ, Bornstein I. The fate of<br /> Biochemistry, Strangeways Research Laboratory, chymopapain injected for therapy of intervertebral<br /> Cambridge, UK, 1;20(9):981-5, (1995). disc disease. J Lab Clin Med, 1974 Apr; 83(4):532-<br /> 8. Rubens Monti et al. Biological Faculty, Estadual 540.<br /> Paulista university, Brasil, Purification of papain from 13. Bradford DS, Cooper KM, Oegema TR., Jr<br /> fresh latex of Carica papaya, Brazilian Archives of Chymopapain, chemonucleolysis, and nucleus<br /> Biology and Technology, v.43, n.5, p. 501-507, (2000). pulposus regeneration, J Bone Joint Surg Am, 1983,<br /> 9. Sidney P. Colowick, Nathan O.Kaplan, Dec; 65(9): 1220-1231.<br /> Methods in Ezymology, vol II, vol XIX, McCollum-Pratt 14. Staley Zucker et al, The proteolytic activities<br /> Institute, the Jonh Hopkins university, Baltimore, of chymopapain, papain and papaya proteinase III.,<br /> Maryland, (2008). Biochim. Biophys, Acta 828,196-204, (1985).<br /> <br /> <br /> <br /> T h a m d o d i e u k i e n d j n h h r o n g d o n g t h d i<br /> <br /> <br /> p a r a c e t a m o l v a i b u p r o f e n t r o n g h u y e t t i r o n g<br /> <br /> <br /> b S n g p h i r o n g p h a p S K L H N C<br /> <br /> VTnh Djnh, Le Ngoc Thanh True*<br /> Khoa Duqc, Dgi hoc Y Duqc TP. HCM<br /> <br /> Dat v i n d i - Xay dung va thim dinh qui trinh djnh lugng<br /> Hien nay co nhieu che pham la nhQ'ng "che ding thdi paracetamol va ibuprofen trong huyit<br /> pham tuong duong dugc hpc" (pharmaceutical tuang bing SKLHNC.<br /> equivalence / pharmaceutical alternative) ' . Viec<br /> 4|<br /> V a t lieu va p h i f c n g p h a p<br /> kiem tra chat lupng chu yeu dua tren su kiem - T i t ca h6a chit va dung m6i su dung la<br /> tra cac tinh chat ly hoa theo tieu chuan djnh sin. loai tinh khiit phan tich, loai dung cho SKLHNC.<br /> Viec danh gia in vitro nay khong phan anh thuc - P va I chuin lam viec dupe cung cip tu<br /> su hieu qua tri lieu cua mpt thuic trong co thi vi Vien Kiem nghiem TP. HCM. Vien nen Y<br /> sinh kha dung cua mpt thuic phu thupc vao rlt<br /> (Pymexpharm, so lot: 010605, so d§ng ki: VNA-<br /> nhieu yiu t i . Hien nay viec nghien cuu ve tuang<br /> 1971-04) va vien nen doi chiiu R (luu hanh thj<br /> duang sinh hpc chi dua tren hudng dan thu<br /> trudng, so lot: 403671, so dang ki: VNB-0525-<br /> tuang duang sinh hpc cua ASEAN la chu yeu<br /> [2], D i gop phin chuan bj cho cong tac nghien 00) chua 325 mg P va 200 mg I. miu tring su<br /> cu'u nay, chung toi thuc hien de tai: "Thim dd dung la huyet tuong dupe cung cap tu Vien<br /> diiu kien dinh luong ddng thdi paracetamol va truyen mau va huyet hpc TP. HCM.<br /> ibuprofen trong huyit tuong bing phuong phap - Djnh lugng P & I:<br /> SKLHNC" voi muc tieu: Chuan bi dung dich chuan<br /> - Tham do dieu kien dinh lupng chi pham Dung djch chuin P: h6a tan chinh xac 22 mg<br /> chua paracetamol va ibuprofen trong huyit tuong P trong 10 ml acetonitril.<br /> nhim tao so sd du lieu cho viec thu tuong duong Dung dich chuin I: h6a tan chinh xac 13,4<br /> sinh hpc cua nhung chi phim nay. mg I trong 10 ml acetonitril.<br /> <br /> 42 TAP CHI DUOC HQC - 10/2010 (SO 414 NAM 50)<br /> • N g h i e n CUHJ - Ky thuat<br /> <br /> Dung djch chuan trong huyet tuong: hoa tan giiy lpc 0,45 urn de dugc dung dich mau thu<br /> 500 pi dung djch chuan P va 500 ul dung djch trong huyit tuong dung cho phan tich.<br /> chuan I trong 9 ml huyet tuong, rung tren may Dieu kien phan tich SKLHNC: im<br /> <br /> <br /> <br /> 13c vortex de dugc dung djch co n6ng dp 110 - Cot Waters Symmetry C18 (150 x 4,6 mm,<br /> ug/ml cua P va 67 ug/ml cua I. Ket tua protein 3,5 urn), cot bao ve (2 x 4,6 mm, 5 p.m).<br /> bing each them 1ml acetonitril vao 500 ul dung - Pha dpng AcCN - H P0 0,1% (55:45, v/v).<br /> 3 4<br /> <br /> djch huyet tuong nay trong ong eppendorf, lie - Tic dp dong: 1 ml/min; Phat hien: X = 220 nm.<br /> tren vortex trong 1 phut, ly tam 6ng eppendorf<br /> nay a tdc dp 13.000 rpm trong 10 phut, loai bo Ket qua<br /> tua qua mang lpc 0,45 urn. Djch lpc dugc dung Xac djnh tinh tuang thich cua he thing<br /> sic k y l5]<br /> <br /> <br /> de pha dung djch huyet tuong chuin cua P co<br /> Chay sic ki dung djch chuin P (27,5 ug/ml)<br /> nong dp 3,44 ug/ml; 6,88 ug/ml; 13,75 ug/ml;<br /> va I (16,75 ug/ml) trong huyet tuang vgi 6 lin<br /> 27,5 ug/ml; 55 ug/ml va dung djch chuin cua I<br /> bam lien tiep. Kit qua thu dugc cho thiy cac<br /> cd ning dp 2,09 ug/ml; 4,19 ug/ml; 8,38 ug/ml;<br /> dieu kien trong phuang phap bao dam tuong<br /> 16,75 ug/ml; 33,5 ug/ml bing each pha loang thich he thing (xem bang 1 va 2):<br /> vgi miu tring huyit tuong. Bang 1: Gia tri thong sd sic ki irng vdi dinh<br /> Chuan bj mau thir cua paracetamol chuin trong pha dong<br /> Tinh khoi lugng trung binh vien tu 20 vien W r<br /> STT Dien tich N„<br /> nen Y va R, nghiln tit ca thanh bpt, can chinh<br /> e<br /> <br /> <br /> 1 3,11 1389487,54 1,22 1,31 3674,49<br /> xac mpt lupng bpt tuong duang 1/10 khoi<br /> lugng trung binh vien cua Y va R. Chuyen khoi 2 3,10 1363670,25 1,21 1,32 3750,70<br /> lugng bpt nay vao binh djnh muc 50 ml, hoa tan 3 3,10 1365813,88 1,21 1,32 3646,91<br /> vgi 20 ml ethanol 96 % va lam diy bing dung 4 3,10 1376045,33 1,21 1,32 3618,30<br /> dich H P0 0,1%. Lpc, loai bo 20 ml djch lpc diu.<br /> 3 4<br /> 5 3,10 1379716,77 1,21 1,32 3747,75<br /> Djch lpc sau khi dupe pha loang vdi huyit tuang<br /> 6 3,11 1389487,54 1,22 1,31 3674,49<br /> theo ti l§ 1:9, lie tren may lie vortex trong 1 phut.<br /> Cho 500 ul djch huyit tuong nay vao 1 ml Xm 3,11 1377370,22 1,22 1,32 3685,44<br /> acetonitril, lie tren may lie vortex trong 1 phut, ly SD 0,00 11154,90 0,00 0,00 53,62<br /> tam a tic dp 13.000 rpm trong 10 phut, lpc qua RSD(%) 0,14 0,81 0,26 0,37 1,45<br /> civa"ibuprofen chuin trong pha ddng<br /> Bang 2: Gia trj thong so sic ki irng voi dinh<br /> STT te D/?n tich k' R T N„ e<br /> <br /> 1 12,93 368.489,25 8,24 27,56 1,16 10.657,23<br /> 2 12,93 367.317,78 8,24 27,43 1,16 10.574,60<br /> 3 12,93 365.494,23 8,24 27,29 1,16 10.604,30<br /> 4 12,97 370.296,47 8,27 27,23 1,18 10.366,12<br /> 5 12,99 360.603,24 8,28 27,43 1,16 10.650,25<br /> 6 12,95 362.871,30 8,25 27,41 1,15 10.608,58<br /> Xm 12,95 365.845,38 8,25 27,39 1,16 10.576,85<br /> SD 0,03 3.617,15 0,02 0,11 0,01 107,72<br /> RSD(%) 0,19 0,99 0,22 0,42 0,81 1,02<br /> Djnh lirgng d i n g thai P va I trong huyit<br /> tuong<br /> Nhiiu qui trinh djnh lugng rieng re P va I<br /> trong huyit tuong da cong b i . Dua tren ca<br /> [1,3]<br /> <br /> <br /> <br /> sa nay, de tai da tham do viec djnh lugng ding<br /> thai P va I trong huyit tuong vgi nhiiu pha<br /> dpng khac nhau phii hgp cua acetonitril<br /> va/hoac methanol vdi nhQ'ng dung dich acid c6<br /> Hinh 1: Sic ki dd HPLC cua paracetamol va pH khac nhau. Viec tach hai chit nay dat dugc<br /> ibuprofen chuin d 220nm vdi pha dpng methanol - acid acetic 1/80<br /> <br /> TAP CHI DUQC HQC - 10/2010 (SO 414 NAM 50) 43<br /> • N g h i e n CLFU - Ky thuat<br /> <br /> (15:85), nhung thai gian phan tich doi vdi I con Dp chinh xac<br /> keo dai (10 phut). Ngugc lai vdi pha dpng 6" nong dp phan tich 26,3 pg.ml" cua P va<br /> 1<br /> <br /> <br /> acetoniltril - 0,1 % H P0 (90:10), dinh cua P<br /> 3 4<br /> 16,2 ug.mr cua I, chuin bj tren 6 miu, dp chinh<br /> 1<br /> <br /> <br /> khong dat dugc su can d6i (tailing factor). Cu6i<br /> xac dat dupe dap ung cho yeu ciu phan tich<br /> cung vdi pha dpng acetoniltril - 0,1 % H P0<br /> trong huyit tuang (RSD yeu ciu d ning dp<br /> 3 4<br /> <br /> (55:45) da dat dugc sic ky do thoa man cac<br /> thong s6 phan tich. phan tich 100 ppm la 5,3 % theo AOAC).<br /> Bang 2: Do chinh xac sau 6 lan tiem mSu cua dung dich thu trong huyit tuong<br /> STT Dien tich Ham luong P 1vien (mg) Dien tich Ham luong 11 vien (mg)<br /> 1 1.042.479 325,43 n=6 260.987 200,44 n=6<br /> 2 1.046.626 327,64 Xm = 325,08 259.310 199,71 Xm = 200,92<br /> 3 1.043.142 330,29 SD = 3,71 261.662 203,83 SD = 1,465<br /> 4 1.051.306 328,64 RSD= 1,12% 260.388 200,26 RSD = 0,73%<br /> 5 1.044.939 326,65 e = 3,05 (P = 0,95) 261.001 200,73 e = 1,21 (P = 0,95)<br /> 6 1.075.925 335,87 p = 325,08 ± 3,05 261.164 200,57 p = 200,92 ± 1,21<br /> Tinh tuyen tinh va gioi han phat hien Ket luan<br /> Thu nghiem tren 2 lin tiem mlu lap lai cho Nhung kit qua nay cho thay cd the dinh<br /> thiy co su tuyln tinh v l dien tich dinh vdi nong lugng ding thdi P va I trong chi phim va trong<br /> dp trong khoang 0,86 - 110 pg.ml" doi vdi P va<br /> 1<br /> <br /> huyit tuang bing SKLHNC. Phuang phap dat<br /> 0,52 - 67 ug.mr dii vdi I.<br /> 1<br /> dugc dp dung, dp chinh xac, tinh chuyen biet vd<br /> Gidi han djnh lugng la 0,23 pg.ml" cho P va<br /> 1<br /> dp lap lai. Qui trinh trich ly miu thuc hien nhanh<br /> 0,26 ug.ml' cho I.<br /> 1<br /> va don gian. Qui trinh cd gia trj tham khao de<br /> Dp dung tilp tuc nghien cuu v i sinh kha dung cung nhu<br /> Chuin bi dung dich thu trong huyit tuong cd tinh tuang duang sinh hpc cua nhQ'ng chi<br /> phim cd chua dong thdi hai hoat chit nay.<br /> ndng dp 26,3 ug.mr cua P va 16,2 u.g.ml" cua<br /> 1 1<br /> <br /> <br /> <br /> I. Them chit chuin P va I vdi tl Id 80 %, 100 %, Summary<br /> 120 % so vdi ham lupng cua P va I trong dung A reversed-phase high performance liquid<br /> dich thu neu trdn. Thuc hien phan tich theo qui chromatography (HPLC) method for the<br /> trinh neu trdn, kit qua cho dp dung dat dupe simultaneous measurement of paracetamol (P)<br /> 90,7 % cua P va 90,6 % cua I. and ibuprofene (I) in human plasma was<br /> Bang 3: Dp dung cua mau thu huyit tuong developed and validated. Observation of solvent<br /> them chuin cua P to precipitate of protein showed that the suitable<br /> ratio of acetoniltril : protein is 2:1. The<br /> Lugng Lugng<br /> Tile supernatant after centrifugation is injected<br /> them Dien tich tim thay Do hoi<br /> them phuc(%) directly into the chromatographic system. These<br /> (ug/ml) (ug/ml)<br /> compounds were completely separated by<br /> 80% 22,0 931.346 20,91 95,04 isocratic elution with acetonitril - H P0 0.1%<br /> 3 4<br /> <br /> 100% 27,5 1.087.220 24,45 88,92 (55:45, v/v) and detected with PDA detector at<br /> 120% 33,0 1.400.121 29,11 88,21 247 nm (for P) and at 220 nm (for I).<br /> Trung binh 90,72 Quantification was performed from calibration<br /> Bang 4: Do dung cua miu thu huyit tuong curve generated from P and I working standards<br /> them chuin cua 1 (HCMC IDQC, lot 09040804, content 99.17%<br /> and lot 26020603, content 99.69%, respectively)<br /> Lupng Lugng<br /> Ti le them them Dien tich tim thay Dp hoi spiked in blank human plasma (HCMC Hospital<br /> (ug/ml) phuc (%) of hematology & Blood tranfusion). The<br /> (ug/ml)<br /> calibration range of the method was 0.86 - 110<br /> 80% 12 218.675 11,38 94,82 p:g mf for P and 0.52 - 67 pig mf for I. Mean<br /> 1 1<br /> <br /> <br /> 100% 15 241.046 13,52 90,17 accuracies for P and I were 90.7 %, and 90.6 %,<br /> 120% \S 304.438 15,64 86,88 respectively. Mean precisions (% RSD) for P<br /> Trung binh 90,62 were 1.12 % and for I were 0.73 %. The lowest<br /> <br /> 44 TAP CHI DUQC HQC - 10/2010 (SO 414 NAM 50)<br />  N g h i e n CLFU - K y thuat<br /> <br /> limit of quantification was 0.23 pg mf and 0.26<br /> 1<br /> 2. European Agency for the evaluation of<br /> ug mf for P and I, respectively. This validated<br /> 1 medicinal products, Asean guidelines for the conduct<br /> of bioavaibility and bioequivalence studies (2001).<br /> assay is followed-up for use in pharmacokinetic<br /> 3. Demotes - Mainard F., Vincon G., Jarry C,<br /> and bioequivalence studies. Albin H.,Plasma determination of paracetamol using<br /> Key words: paracetamol; ibuprofene; human high performance liquid chromatography. Application<br /> plasma; HPLC. to a pharmacokinetic study, Ann. Biol. CH. (1984).,<br /> Tai lieu t h a m khao 42(1), 9-13<br /> 1. Ali A., Kazmi S., Plakogiannis F.M., High-pressure 4. Drugs information for the Professional Health<br /> liquid chromatographic determination of ibuprofen in Care, 22 ed., Thomson Micromedex, 7-382 (2003).<br /> nd<br /> <br /> <br /> <br /> <br /> plasma, J. Pharm. Sci, 70(8), 5-944 (1981). 5. USP 30 NF, 1088 (2007, electronic version).<br /> <br /> <br /> <br /> K h a o sat m i f c d o n h i e m n a m m o c<br /> <br /> <br /> 6" p h o n g l a m v i e c c o m a y l a n h<br /> <br /> <br /> t a i t h a n h p h 6 H 6 C h i M i n h<br /> <br /> Nguyen Van Thanh, Le Thj Ngoc Hue<br /> Khoa Duoc, Dgi hoc Y Duqc TP. Ho Chi Minh<br /> <br /> Oat van de dieu kien lam vide ngay cang dugc cai thien. Do<br /> Theo bao cao cua To chuc Y te The gioi vao dd tai cac thanh phd, da so cac van phdng diu<br /> nam 2005 cho biet chat lugng khong khi trong dugc trang bi may lanh. Nhung da tao ra nghjch<br /> nha va cao 6c van phong tren toan the gicVi dang ly la de tilt kiem dien, tranh i n , tranh khdi bui<br /> sut giam nghiem trong. Cong dong cac nha ben ngodi, nhung nai ndy tang cugng ddng kin<br /> khoa hpc quoc te lo ling ve van de 6 nhiim cua, may lanh khdng dugc ve sinh thudng<br /> khong khi d nhung m6i truong khep kin. Cac xuyen, tao dieu kien thuan Igi cho nim moc<br /> nha nghien cuu My va chau Au cho thiy con phdt trien va gay ra mdi trudng bit Igi cho sue<br /> ngugi d cac nude cdng nghiep danh hon 90% khoe. Ngoai ra, theo cac bdo cao d cac benh<br /> thdi gian cua hp trong nha. Nhung noi dupe vien cho biit hien nay ti Id benh dudng hd hip<br /> quan tam nhieu nhit la nhung khu vue ma con ngay cang gia tang trong dd cd the ke nguyen<br /> nguoi d thai gian dai nhu nha d, trudng hpc va nhan do nim moc.<br /> nai lam viec Hon thi nua, tinh trang suy giam miin djch<br /> Cac cong trinh nghien cuu cho thiy nim se cd nguy co cao trong viec nhiim nim moc<br /> mic trong nha gay hai din sue khoe con ngudi nhu AIDS, lao, tieu dudng, ciy ghep co quan,<br /> nhu dau diu, chong mat, chay mui, hit hoi, ho, ca phiu thudt...Vdi tac hai cua nim moc doi vdi<br /> viem phoi, kich ung mit, dau khdp, anh hudng sue khoe con ngudi, do dd d nude ngoai da cd<br /> thin kinh, giam sue de khang .... Ngoai ra mpt quy djnh gidi han muc dp nhiim nim mdc trong<br /> so nim mic con co kha nang sinh dpc t i nhu: 1 m khdng khi nha d. Tuy nhien nude ta chua<br /> 3<br /> <br /> <br /> <br /> <br /> Aspergillus fumigatus, A. flavus, A. niger, cd quy djnh nay va vi vay, cung chua cd nhung<br /> Penicillium sp., Fusarium sp., Rhizopus bien phap xu ly can thiet.<br /> sp...Cac dpc t i nim gay ung thu nhu aflatoxin, De tim hieu tinh hinh thuc t i lam can cu cho<br /> ochratoxin, trichothecen...' . Nim moc khong<br /> 21<br /> nhung de xuit tiep theo, de tai thuc hien vdi cac<br /> nhung gay thiet hai v i sue khoe cho con ngudi muc tieu:<br /> ma cdn gay thidt hai rat Idn ve kinh t i . - Khao sat muc dp nhiim nim mdc trong<br /> Trong tinh hinh hien nay d Viet Nam, chit khdng khi cua 20 phdng Idm viec may lanh cua<br /> lupng cupc song ngdy cang dugc nang cao, trudng hpc. Pham vi chpn lua la trudng hpc vi<br /> <br /> TAP CHI DUQC HQC - 10/2010 (SO 414 NAM 50) 45<br />