高效液相色谱法测定人血浆中游离拉莫三嗪的血药浓度

doi:10.5246/jcps.2018.12.084

摘要/Abstract

摘要：

拉莫三嗪是广谱抗癫痫药,常用于治疗简单部分性癫痫发作、复杂部分性癫痫发作。由于其治疗窗窄,体内药代动力学个体间差异较大,故临床上对拉莫三嗪进行常规血药浓度监测来指导合理用药。个体差异和联合用药会影响蛋白结合率,进一步影响拉莫三嗪游离药物浓度。与拉莫三嗪总血药浓度相比,游离血药浓度与不良反应和治疗效果之间的关系更为密切。因此,确定血浆中拉莫三嗪游离药物浓度对于临床治疗更有意义。本文使用超滤法提取血浆中游离的拉莫三嗪,采用高效液相色谱法(HPLC)进行游离浓度测定,并通过选择性、线性、定量下限、准确度、精密度、回收率和稳定性的研究对该方法进行验证。结果发现,拉莫三嗪游离浓度在0.20–10.0 μg·mL^–1(y = 12.42×10^–2c + 5.47×10^–³,r = 0.9996), 线性关系良好,最低定量下限为0.2 μg·mL^{– 1}。该方法日内、日间精密度(RSD)均<15%, 准确度为±4.69%;游离拉莫三嗪测定方法在三种不同浓度下的提取回收率均满足生物样品分析要求,并且在不同储存条件下拉莫三嗪稳定性良好。该方法简便、快速、灵敏度高,适用于临床游离血药浓度监测。

关键词: 拉莫三嗪, 游离药物浓度, 超滤法, 高效液相色谱法, 治疗药物监测

Abstract:

Lamotrigine (LTG) is a widely used antiepileptic drug (AED) for the treatment of new-onset, as well as refractory epilepsy. Due to the narrow treatment window and large individual variability in the pharmacokinetics and pharmacodynamics of LTG, therapeutic drug monitoring (TDM) is necessary in clinical practice to guide dose adjustments. Individual differences and drug combinations can also affect protein binding rate, which further affects the unbound concentration of LTG. The unbound fraction is more closely related to adverse reactions and therapeutic efficacy than total concentration. Therefore, it may be more meaningful to determine the unbound LTG concentration in plasma than the total concentration.Unbound LTG in plasma was extracted by ultrafiltration. High-performance liquid chromatography (HPLC) was used to measure unbound LTG concentration. This method was validated by studies of its selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, recovery, and stability.The method was validated over a linear range of 0.2 to 10.0 μg·mL^–¹, and its LLOQ was 0.2 μg·mL^–¹. The method’s relative standard deviations (RSDs) for intra-day and inter-day precision were less than 15%, and its accuracy (RE) was ±4.69%. The recoveries of unbound LTG at three different concentrations satisfied the requirements for the analysis of biological samples, and no significant degradation of LTG was observed under different storage conditions.A simple HPLC method showed good performance when used to measure unbound LTG concentration. This method might be used to study the relationship between unboundLTG concentrations and its effectiveness according to TDM.

Key words: Lamotrigine, Unbound concentration, Ultrafiltration, HPLC, Therapeutic drug monitoring

中图分类号:

R969.1

Supporting:

王环鑫, 孙亚欣, 徐善森, 路童, 陈亚南, 肇丽梅. 高效液相色谱法测定人血浆中游离拉莫三嗪的血药浓度[J]. 中国药学(英文版), 2018, 27(12): 832-839.

Huanxin Wang, Yaxin Sun, Shansen Xu, Tong Lu, Yanan Chen, Limei Zhao. Development of a simple and rapid method to measure the free fraction of lamotrigine in plasma using HPLC: applications for therapeutic drug monitoring[J]. Journal of Chinese Pharmaceutical Sciences, 2018, 27(12): 832-839.

参考文献

[1] Valencia, I.; Pinol-Ripoll, G.; Khurana, D.S.; Hardison, H.H.; Kothare, S.V.; Melvin, J.J.; Marks, H.G.; Legido, A. Efficacy and safety of lamotrigine monotherapy in children and adolescents with epilepsy. Eur. J. Paediatr. Neurol. 2009, 13, 141–145.

[2] French, J.A.; Kanner, A.M.; Bautista, J. Efficacy and tolerability of the new antiepileptic drugs, II: Treatment of refractory epilepsy: report of the TTA and QSS Subcommittees of the American Academy of Neurology and the American Epilepsy Society. Epilepsia. 2004, 45, 410–423.

[3] von Wegerer, J.; Hesslinger, B.; Berger, M.; Walden, J. A calcium antagonistic effect of the new antiepileptic drug lamotrigine. Eur. Neuropsychopharmacol. 1997, 7, 77–81.

[4] Xie, X.; Lancaster, B.; Peakman, T.; Garthwaite, J. Interaction of the antiepileptic drug lamotrigine with recombinant rat brain type IIA Na+ channels and with native Na+ channels in rat hippocampal neurones. Pflugers Arch. 1995, 430, 437–446.

[5] Brzakovic, B.B.; Vezmar, K.S.; Vucicevic, K.M.; Miljkovic, B.R.; Martinovic, Z.J.; Pokrajac, M.V.; Prostran, M.S. Impact of age, weight and concomitant treatment on lamotrigine pharmacokinetics. J. Clin. Pharm. Ther. 2012, 37, 693–697.

[6] Punyawudho, B.; Ramsay, R.E.; Macias, F.M.; Rowan, A.J.; Collins, J.F.; Brundage, R.C.; Birnbaum, A.K. Population pharmacokinetics of lamotrigine in elderly patients. J. Clin. Pharmacol. 2008, 48, 455–463.

[7] Sondergaard, K.M.; Nielsen, K.A.; Dahl, M.; Wolf, P. Lamotrigine therapeutic thresholds. Seizure. 2008, 17, 391–395.

[8] Kanner, A.M.; Frey, M. Adding valproate to lamotrigine: a study of their pharmacokinetic interaction. Neurology. 2000, 55, 588–591.

[9] Fitton, A.; Goa, K.L. Lamotrigine. An update of its pharmacology and therapeutic use in epilepsy. Drugs. 1995, 50, 691–713.

[10] Sander, J.W.; Trevisol-Bittencourt, P.C.; Hart, Y.M.; Patsalos, P.N.; Shorvon, S.D. The efficacy and long-term tolerability of lamotrigine in the treatment of severe epilepsy. Epilepsy Res. 1990, 7, 226–229.

[11] Kilpatrick, E.S.; Forrest, G.; Brodie, M.J. Concentration--effect and concentration--toxicity relations with lamotrigine: a prospective study. Epilepsia. 1996, 37, 534–538.

[12] Rapeport, W.G.; Mendelow, A.D.; French, G.; MacPherson, P.; Teasdale, E.; Agnew, E.; Thompson, G.G.; Brodie, M.J. Plasma protein-binding and CSF concentrations of valproic acid in man following acute oral dosing. Br. J. Clin. Pharmacol. 1983, 16, 365–369.

[13] Lenn, N.J.; Robertson, M. Clinical utility of unbound antiepileptic drug blood levels in the management of epilepsy. Neurology. 1992, 42, 988–990.

[14] Gibbs, H.G.; Zimmerman, D.E.; Shermock, K.M.; Clarke, W.; Mirski, M.A.; Lewin, J.R. Comparison of free fraction serum valproic acid concentrations between inpatients and outpatients. Am. J. Health Syst. Pharm. 2015, 72, 121–126.

[15] Itoh, H.; Suzuki, Y.; Fujisaki, K.; Sato, Y.; Takeyama, M. Correlation between plasma ammonia level and serum trough concentration of free valproic acid in patients with epilepsy. Biol. Pharm. Bull. 2012, 35, 971–974.

[16] Suzuki, Y.; Itoh, H.; Abe, T.; Nishimura, F.; Sato, Y.; Takeyama, M. No effect of co-administered antiepileptic drugs on in-vivo protein binding parameters of valproic acid in patients with epilepsy. J. Pharm. Pharmacol. 2011, 63, 976–981.

[17] Zhang, Z.Q.; Dong, W.C.; Yang, X.L.; Zhang, J.F.; Jiang, X.H.; Jing, S.J.; Yang, H.L.; Jiang, Y. The Influence of Plasma Albumin Concentration on the Analysis Methodology of Free Valproic Acid by Ultrafiltration and Its Application to Therapeutic Drug Monitoring. Ther. Drug Monit. 2015, 37, 776–782.

[18] Dwivedi, R.; Gupta, Y.K.; Singh, M.; Joshi, R.; Tiwari, P.; Kaleekal, T.; Tripathi, M. Correlation of saliva and serum free valproic acid concentrations in persons with epilepsy. Seizure. 2015, 25, 187–190.

[19] March, C.; Blanke, RV. Determination of free valproic acid concentrations using the Amicon Micropartition MPS-1 Ultrafiltration System. Ther. Drug Monit. 1985, 7, 115–120.

[20] Tsiropoulos, I.; Kristensen, O.; Klitgaard, N.A. Saliva and serum concentration of lamotrigine in patients with epilepsy. Ther. Drug Monit. 2000, 22, 517–521.

[21] FDA. Guidance for Industry Bioanalytical Method Validation [website] 2013. Available at:https://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm368107.pdf. Accessed 15 March, 2017.

[22] XU, S.S.; Z, T.; Z, J.K. Determination of concentrations of lamotrigine, oxcarbazepine and its active metabolite in human serum by HPLC method. Chin. J. Clin. Phar-macol. 2015, 12, 1176–1179.

[23] de Moraes, N.V.; Lauretti, G.R.; Napolitano, M.N.; Santos, N.R.; Godoy, A.L.; Lanchote, V.L. Enantioselective analysis of unbound tramadol, O-desmethyltramadol and N-desmethyltramadol in plasma by ultrafiltration and LC-MS/MS: application to clinical pharmacokinetics. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2012, 880, 140–147.

[24] Metsu, D.; Seraissol, P.; Delobel, P.; Cinq-Frais, C.; Cuzin, L.; Izopet, J.; Chatelut, E.; Gandia, P. Is the unbound concentration of atazanavir of interest in therapeutic drug monitoring? Fundam Clin. Pharmacol. 2017, 31, 245–253.

[25] Berthoin, K.; Ampe, E.; Tulkens, P.M.; Carryn, S. Correlation between free and total vancomycin serum concentrations in patients treated for Gram-positive infections. Int. J. Antimicrob. Agents. 2009, 34, 555–560.

[26] McMillin, G.A.; Juenke, J.; Dasgupta, A. Effect of ultrafiltrate volume on determination of free phenytoin concentration. Ther. Drug Monit. 2005, 27, 630–633.

[27] Gidal, B.E.; Sheth, R.; Parnell, J.; Maloney, K.; Sale, M. Evaluation of VPA dose and concentration effects on lamotrigine pharmacokinetics: implications for conversion to lamotrigine monotherapy. Epilepsy Res. 2003, 57, 85–93.

[28] Brodie, M.J.; Yuen, A.W. Lamotrigine substitution study: evidence for synergism with sodium valproate? 105 Study Group. Epilepsy Res. 1997, 26, 423–432.

[29] Magdalou, J.; Herber, R.; Bidault, R.; Siest, G. In vitro N-glucuronidation of a novel antiepileptic drug, lamotrigine, by human liver microsomes. J. Pharmacol. Exp. Ther. 1992, 260, 1166–1173.

[30] Weintraub, D.; Buchsbaum, R.; Resor, S.J.; Hirsch, L.J. Effect of antiepileptic drug comedication on lamotrigine clearance. Arch. Neurol. 2005, 62, 1432–1436.

[31] Almeida, A.M.; Falcao, A.C.; Sales, F.; Baldeiras, I.; Rocha, M.J.; Caramona, M.M. Lamotrigine pharmacokinetic evaluation in epileptic patients submitted to VEEG monitoring. Eur. J. Clin. Pharmacol. 2006, 62, 737–742.

[32] Kanner, A.M.; Frey, M. Adding valproate to lamotrigine: a study of their pharmacokinetic interaction. Neurology. 2000, 55, 588–591.