Scheuch Eberhard, Methling Karen, Bednarski Patrick J, Oswald Stefan, Siegmund Werner
Department of Clinical Pharmacology (ES, WS) of the Center of Drug Absorption and Transport (C_DAT), University Medicine, Greifswald, Germany.
Department of Pharmaceutical Chemistry (KM, PJB), University of Greifswald, Germany.
J Pharm Biomed Anal. 2015 Jan;102:377-85. doi: 10.1016/j.jpba.2014.09.010. Epub 2014 Sep 21.
The non-opiate analgesic drug flupirtine was shown in vitro to undergo hydrolysis followed by N-acetylation to form D13223, glucuronidation and conjugation with glutathione to form the stable mercapturic acid derivatives M-424 and M-466. To quantify flupirtine and its metabolites in samples obtained in a clinical study in healthy subjects selected on their genotype of NAT2, UGT1A1 and GSTP1, two LC-MS/MS methods were developed. The validation range for flupirtine and D-13223 in serum was 0.5-500 ng/ml. For urine and feces, the validation ranges for flupirtine and D-13223 were 20-5000 ng/ml and 5.0-5000 ng/ml, respectively. M-424 and M-466 could be quantified in urine between 5.0 and 5000 ng/ml. Free flupirtine and D-13223 were separated from serum, urine and feces with liquid-liquid extraction. For flupirtine and D-13223, the chromatography was performed on a XTerra C18 column isocratically with a mobile phase consisting of ammonium formate buffer (pH 3.5mM) and acetonitrile (50:50; v/v), for M-466 and M-424 a Synergi(®) Fusion-RP column was used and a linear gradient method with water/HCOOH (pH 3) and acetonitrile. The mass spectrometer operated both with electro spray ionization in positive multiple reaction monitoring mode. The developed methods fulfilled the current FDA criteria on bioanalytical method validation for accuracy (error: -16.9 to 11.2%), precision (1.2-13.4%), recovery, stability and matrix effects over the observed analytical range. Thus, the methods were suitable to quantify flupirtine absorption and metabolic disposition in man after single intravenous and oral dosing (100mg) and repeated oral administration (400mg once daily).
非阿片类镇痛药氟吡汀在体外被证明会先发生水解，然后进行N - 乙酰化形成D13223，接着进行葡萄糖醛酸化以及与谷胱甘肽结合形成稳定的硫醚氨酸衍生物M - 424和M - 466。为了对在一项针对根据NAT2、UGT1A1和GSTP1基因型挑选出的健康受试者的临床研究中所采集样本中的氟吡汀及其代谢物进行定量分析，开发了两种液相色谱 - 串联质谱（LC - MS/MS）方法。血清中氟吡汀和D - 13223的验证范围是0.5 - 500 ng/ml。对于尿液和粪便，氟吡汀和D - 13223的验证范围分别是20 - 5000 ng/ml和5.0 - 5000 ng/ml。M - 424和M - 466在尿液中的定量范围是5.0 - 5000 ng/ml。游离的氟吡汀和D - 13223通过液 - 液萃取从血清、尿液和粪便中分离出来。对于氟吡汀和D - 13223，色谱分析在XTerra C18柱上进行，采用等度洗脱，流动相由甲酸铵缓冲液（pH 3.5 mM）和乙腈（50:50；v/v）组成；对于M - 466和M - 424，则使用Synergi(®) Fusion - RP柱，并采用水/HCOOH（pH 3）和乙腈的线性梯度方法。质谱仪在正离子多反应监测模式下通过电喷雾电离运行。所开发的方法在观察到的分析范围内满足了当前美国食品药品监督管理局（FDA）关于生物分析方法验证的准确性（误差： - 16.9%至11.2%）、精密度（1.2 - 13.4%）、回收率、稳定性和基质效应的标准。因此，这些方法适用于对单次静脉注射和口服给药（100 mg）以及重复口服给药（每日一次，400 mg）后人体中氟吡汀的吸收和代谢情况进行定量分析。
