Printed in the Republic of Korea "/"-:5*$"- 4$*&/$& 5&$)/0-0(: Vol. 20, No. 2, 138-146, 2007 -$.4.4 w x q p š ³ Á Áw Á Á Á 1 g ( ), 2 ( ) w wq, 3 w w w Sensitive determination of paroxetine in canine plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) Kyu Young Chang 1, Seung Woo Kang 1,3, Sang Beom Han 3, Jeong-Rok Youm 3, Kyung Ryul Lee 1,2 G and Hee Joo Lee 1,2 1 Dept. of Drug Development Supporting Service. BioCore Co., Ltd., 108-1, Yangjea-dong, Seocho-gu, Seoul, 137-130, Korea 2 Dept. of Pharmacokinetics, Seoul Medical Science Institute, Seoul Clinical Laboratory, 7-14, Dongbinggo-dong, Yongsan-gu, Seoul, 140-809, Korea 3 College of Pharmacy, Chung-Ang University, 221, Huksuk-dong, Dongjak-gu, Seoul, 156-756, Korea (Received November 7, 2006; Accepted February 27, 2007) : x q p (LLE) wš j m v-k»(lc-ms/ms) w w w. q p ü t w v p TBME(tert-butyl methyl ether) wš d w k z, 100 µl w LC-MS/MS w. HPLC Capcell Pak UG120(2.0 150 mm, 5 µm) f w, 50% m p (ph 3, formic acid ) w š, 0.2 ml/min w. MS/MS SRM(selective reaction monitoring) q p v p, ƒƒ m/z 330 192, m/z 310 148 w 0.02~5 ng/ml (R 2 ) 0.9993 ùkü. w w 0.02 ng/ml, ü ƒ 7.67 % w š, y 92.96~102.99 % x q p w w p,, y š y w. Abstract: A simple and sensitive method for the determination of paroxetine in canine plasma was developed and validated by liquid-liquid extraction and liquid chromatography-tandem mass spectrometry (LC-/MS/MS). Fluoxetine was used as an internal standard. Paroxetine and internal standard in plasma samples were extracted using TBME (tert-butyl methyl ether). A centrifuged upper layer was then evaporated and reconstituted with Corresponding author Phone : +82-(0)2-3461-9133 Fax : +82-(0)2-3461-0590 E-mail: heejoolee0@yahoo.co.kr 138
³ Á Áw Á Á Á 139 mobile phase of 50% acetonitrile adjusted to ph 3 by formic acid. The reconstituted samples were injected into a Capcell Pak UG120 (2.0 150 mm, 5 µm) column. Using MS/MS with SRM (selective reaction monitoring) mode, the transitions (precursor to product) monitored were m/z 330 192 for paroxetine, and m/z 310 148 for internal standard. Linear detection responses were obtained for paroxetine concentration range of 0.02 5 ng/ml. A correlation coefficient of linear regression (R 2 ) was 0.9993. Detection of paroxetine in canine plasma was accurate and precise, with limit of quantification at 0.02 ng/ml. The method has been successfully applied to pharmacokinetic study of paroxetine in healthy beagle dogs. Key words : Paroxetine, LC-MS/MS, canine plasma, pharmacokinetic study q p(paroxetine) (3S-trans)-3-[(1,3-benzodioxol- 5-yl-oxy)methyl]-4-(4-fluorophenyl)piperidine yw (Fig. 1) ƒ, w xk y,, œy, z zœs, h e š. q p m k w (selective serotonin re-uptake inhibitor, SSRI), 1-4 y w z w m y yw Fig. 1. Chemical structures of (A) paroxetine, (B) fluoxetine (I.S.). k w» m y 5-9. w wg x ùkù š, yz (monoamine oxidase inhibitors, MAOIs) y» ƒ š 5-9. q p šx 3~8, x» 15~22 š, x 2, mw š. 10 q p j m v(hplc) 11-14» j m v(gc) 15-16 w. j m v q p y w š xÿ»» w dansyl chloride y w xÿ» w š. w w ƒ 5 ng/ml w w» ƒ û, ƒ ƒ š x w ƒ 0.2 ng/ml w. 12 j m v-»(lc-ms) j m v-k» (LC-MS/MS, triple quadrupole ion trap ) w x q p š š 17-21 š, š (solid phase extraction, SPE) - (liquid-liquid extraction, LLE). ù j m v-» w w (limit of quantitation, LOQ) ƒ x 0.05~5 ng/ml 100 j š. q p š w» w, - (LLE) j m v-k»(lc-ms/ms) Vol. 20, No. 2, 2007
140 LC-MS/MS w x q p š w š w û w, FDA ƒ w (validation) w. w, q p n w x wš, w x q p w, w w. x»» q p(paroxetine) ü t w v p(fluoxetine) Sigma (St. Louis, MO, USA) qt w. w m p, s, k Fisher Scientific (Springfield, NJ, USA) HPLC w w, w TBME (tert-butyl methyl ether)»k p 1 w. HPLC Thermo Finnigan Surveyor HPLC (Thermo Electron Co., San Jose, CA, USA), w autosampler CTC ANALYTICS PAL (Zwingen, Switzerland), k»(ms/ms) TSQ Quantum Ultra (Thermo Electron Co., San Jose, CA, USA) w. w MS/MS collision cell o 90 {, y (S/N ratio) j š w š. HPLC f Shiseido Capcell Pak UG120(2.0 Ü 150 mm, 5 µm, Shiseido, Tokyo, Japan) w, f y w ChemTech Korea (Seoul, Korea) stainless steel frit wx(0.5 µm) w. l e Thermo Electron Xcalibur (Ver. 1.4) w. t q p t t 50% k 1 mg/mlƒ w z, þ w œx w x q p ƒ ƒƒ 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2 5 ng/ml ƒ t x. w ü t v p 50 % k 1 mg/ mlƒ wš, 50 % k w 100 ng/mlƒ w z, Ÿw 4 o C w. t x 0.3 ml x š» ü t v p 20 µl ƒw š k yw» 10 yww.» TBME(tert-butyl methyl ether) 1.5 ml š k» 15 w. 13,000 rpm 2 w z, x þ š d þ g. d k, d» d w Áòw x»š 40 C o» g. ƒ z, 100 µl ƒw w k, 13,000 rpm 5 w. Áòw d w z, 20 µl LC-MS/MS w.» ü t vj w q p vj ƒ š w. -$.4.4 y x q p š LC-MS/MS š19w y w z ww. x w. f w Shiseido Capcell Pak UG120(2.0Ü150 mm, 5 µm, Shiseido, Tokyo, Japan) w, f y w stainless steel frit wx(0.5 µm) w. 50 % m p w z,» s ƒw ph 3.0 wš, 0.2 µm membrane filter wš, q» ƒ w w. f 40 C w o w š, 0.2 ml/min w w. w MS/MS, y ESI(electrospray ionization) kw, positive ion mode SRM(selected reaction monitoring) w. Nebulizing gas ƒ, collision gas šƒ w š,»k MS/ MS q l spray voltage 5 kv, sheath gas pressure 20, aux gas pressure 10, capillary temp. 320 o C, tube lens offset 78(I.S. 70), collision pressure 1.2 mtorr, collision energy 20(I.S. 10) w y w. q p ü t w v p (precursor ion) ƒƒ m/z 330, 310 y ([M+H] ) + w, (precursor ion) m/z 192, 148 ƒƒ l w. w x 5z ww ü x y w š, 5 x ww Analytical Science & Technology
³ Á Áw Á Á Á 141 x w. x yw 6 q p 16 mg/kg n w z, ƒ w w 70 C w x e o w š 3 kw 0.3 ml w w w. w LC-MS/MS w j m l ü t vj w q p vj wš, w w x q p w. š x q p q p ü t v p positive ion mode y ([M+H] ) + m/z 330 (Fig. 2) m/z 310 (Fig. 3) ƒ w», ƒ w. p q p [M+H] + m. w w product ion scan w, q p m/z 192, 178, 151, 135, 123, 109, 70 š, v p m/z 148.»ƒ ƒ j q p m/z 192 Fig. 2. Representative electrospray spectra of (A) Q1 mass spectrum of paroxetine, (B) product ion spectrum for the [M+H] + molecule of paroxetine. Vol. 20, No. 2, 2007
142 LC-MS/MS w x q p š Fig. 3. Representative electrospray spectra of (A) Q1 mass spectrum of fluoxetine, (B) product ion spectrum for the [M+H] + molecule of fluoxetine. v p m/z 148 w. q p v p ion transition m/z 330 192, m/z 310 148 w SRM w, ƒ ùkü. w œx œx q p ü t wì ƒw q p n w z 2 z w x x w z, LC-MS/MS w j m Fig. 4 Fig. 5 ùkü. q p vj 1.8, ü t v p vj 2.0, 2.6. MS/MS k œx q p v p vj w vj, vj k yw. œx, œx ü t ƒw 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 ng/ml t x ƒƒ w z LC-MS/MS w, x l w q p Y(q p/ü t vj ) = 0.72145 X(q p, ng/ml) + 0.00104 (R =0.9993) 2 0.02~5 ng/ml yw ùkü (Fig. 6). x q p w (LLOQ, lower limit of quantitation) j m y (S/N ratio) 10 wš 20% w š, y 80~120 % w 0.02 ng/ Analytical Science & Technology
³ Á Áw Á Á Á 143 Fig. 4. LC-MS/MS chromatograms of (A) blank canine plasma, (B) blank canine plasma spiked with paroxetine and internal standard (1 ng/ml as paroxetine). ml w. ƒ» 4ƒ (0.02, 0.2, 1 5 ng/ml) q p t x w, (C.V., %) ü 2.12~5.66 %, 3.15~7.67% ù kûš, y 92.96~102.99 % ùkû (Table 1), mw q p z 75.2~82.6 %. l x q p w LC-MS/MS w x w p,, y š. y q p w kƒ yw 6 q p (REFERENCE) x (TEST) 16 mg/ kg n w z, ƒ l x w. q p sw x -70 o C w z, w w t x w wš LC-MS/MS w. x x w q p - š Fig. 7 ùkü. Vol. 20, No. 2, 2007
144 LC-MS/MS w x q p š Fig. 5. LC-MS/MS chromatograms of canine plasma sample at 2 h after oral administration of paroxetine tablets (16 mg/kg). Fig. 6. Calibration curve of paroxetine in canine plasma (y = 0.72145 x + 0.00104, R 2 = 0.9993). Table 1. Precision and accuracy for the LC-MS/MS analysis of paroxetine in canine plasma Conc. (ng/ml) Precision (C.V., %) Intraday Interday Accuracy a (%) 0.02 5.66 4.28 102.99Û6.24 0.2 5.27 5.15 98.58Û5.23 1 4.38 7.67 93.15Û4.09 5 2.12 3.15 92.96Û1.97 a : Mean Û SD (n = 5) š x q p w 2000 HPLC (UV), x Ÿ»(FLD) w ƒ. ƒ ƒ xÿ» w w ƒ 0.2 ng/ml 12 w. w š vjƒ e ƒ w, w p w w w ƒ w. 1 10~20 y y. x t w ww» w ~ Analytical Science & Technology
³ Á Áw Á Á Á 145 Fig. 7. Mean plasma concentration-time profile of paroxetine after oral administration of paroxetine tablets (16 mg/kg). Bars represent standard deviation of the mean (n = 3). w, š HPLC-UV, HPLC-FLD ww» k ¼ ƒ w. w w» w 2000 l LC-MS LC-MS/MS w t y w w, q p w LC-MS, LC-MS/MS(triple quadrupole ion trap ) w š. Juan 17 plasma 0.5 ml LC-MS(single quadrupole) w w 5 ng/ml, Segura 18 plasma 1 ml ion trap LC-MS/MS w w 0.7 ng/ml, Zhu 19 plasma 0.5 ml triple quadrupole LC-MS/MS w w 0.2 ng/ml, Naidong 20 plasma 0.4 ml triple quadrupole LC-MS/MS w w 0.05 ng/ml, Massaroti 21 plasma 0.5 ml triple quadrupole LC-MS/ MS w w 0.2 ng/ml šw š q p w x ww. w plasma 0.3 ml w w 0.02 ng/ml» š w yw. TBME(tert-butyl methyl ether) w - (LLE) k (LC-MS/MS) w x q p w. t x l w 0.02~5 ng/ml (R 2 ) 0.9993 yw. ü 2.12~5.66 %, 3.15~7.67 %, y 92.96~102.99 % w w y š w. w,» š w g w 0.02 ng/ml¾ û q p» 22 72 ¾ x q p d w. x q p w w, wz x. š x 1. G. C. Dunbar, J. B. Cohn, L. F. Fabre, J. P. Feighner, R. Vol. 20, No. 2, 2007
146 LC-MS/MS w x q p š R. Fieve, J. Mendels and R. K. Shrivastava, Br. J. Psychiatry, 159, 394-398 (1991). 2. C. G. Gottfries, I. Karlsson and A. L. Nyth, Int. Clin. Psychopharmacol, 6, 354-359 (1992). 3. S. M. Holliday and G. L. Plosker, Drugs Aging, 3 278-299 (1993). 4. R. Lane, D. Baldwin and S. Preskom, J. Psychopharmacol, 9, 5-11 (1995). 5. D. K. Raap and L. D. Van de Kar, Life Sci, 65, 1217-1235 (1999). 6. Y. Zhang, D. K. Raap, F. Garcia, F. Serres, Q. Ma, G. Battaglia and L. D. Van de Kar, Brain Res, 855, 58-66 (2000). 7. B. Rodriguez de la Torre, J. Dreher, I. Malevany, M. Bagli, M. Kolbinger, H. Omran, B. Luderitz and M. L. Rao, Ther. Drug. Monit. 23, 435-440 (2001). 8. C. Duverneuil, G. L. de la Grandmaison, P. de Mazancourt and J. C. Alvarez, Ther. Drug. Monit. 25, 565-573 (2003). 9. K. Titier, N. Castaing, E. Scotto-Gomez, F. Pehourcq, N. Moore and M. Molimard, Ther. Drug. Monit, 25, 581-587 (2003). 10. C. Hiemke and S. Hrtter, Pharmacol. and Ther., 85, 11-28 (2000). 11. J. P. Foglia, D. Sorisio, M. Kirshner and B. G. Pollock, J. Chromatogr. B, 693, 147-151 (1997). 12. J. G. Shin, K. A. Kim, Y. R. Yoon, I. J. Cha, Y. H. Kim and S. G. Shin, J. Chromatogr. B, 713, 452-456 (1998). 13. C. Lpez-Calull and N. Dominguez, J. Chromatogr. B, 724, 393-398 (1999). 14. I. A. Zainaghi, V. L. Lanchote and R. H. C. Queiroz, Pharmacol. Res., 48, 217-221 (2003). 15. C. T. Lai, E. S. Gorden, S. H. Kennedy, A. N. Bateson, R. T. Coutts and G. B. Baker, J. Chromatogr. B, 749, 275-279 (2000). 16. H. J. Leis, W. Windischhofer and G. Fauler, J. Chromatogr. B, 779, 353-357 (2002). 17. H. Juan, Z. Zhiling and L. Huande, J. Chromatogr. B, 820, 33-39 (2005). 18. M. Segura, J. Ortuno, M. Farre, R. Pacifici, S. Pichini, J. Joglar and J. Segura, Rapid Commun. Mass Spectrom., 17, 1455-1461 (2003). 19. Z. Zhu and L. Neirinck, J Chromatogr. B Analyt Technol. Biomed. Life Sci., 780, 295-300 (2002). 20. W. Naidong and A. Eerkes, Biomed. Chromatogr., 18, 28-36 (2004). 21. P. Massaroti, N. M. Cassiano, L. F. Duarte, D.R. Campos, M. A. M. Marchioretto, G. Bernasconi, S. Calafatti, F. A. P. Barros, E. C. Meurer and J. Pedrazzoli, J. Pharm. Pharmaceut. Sci., 8, 340-347 (2005). Analytical Science & Technology