Department of Toxicology, Faculty of Pharmacy, University of Medicine and Pharmacy Iuliu Hatieganu, no. 6 Pasteur, RO-400349 Cluj-Napoca, Romania.
Talanta. 2012 Sep 15;99:649-59. doi: 10.1016/j.talanta.2012.06.070. Epub 2012 Jun 29.
A simple, high-throughput, sensitive LC-ESI-MS/MS method is presented for the simultaneous determination of methadone (MET), flunitrazepam (FNZ) and their major metabolites, EDDP (2-ethilidene-1,5-dimethyl-3,3-diphenylpyrrolidone) and 7-aminoflunitrazepam (7-AFNZ), respectively, in human, rat and rabbit plasma. The isolation of the selected compounds involved a liquid-liquid extraction with ethyl acetate at a basic pH. Good chromatographic separation was achieved on a HSS T3 column (1.8 μm particle size), with a 3 min gradient elution using a mixture of acetonitrile with 0.1% formic acid (solvent A) and 5mM ammonium acetate (solvent B) as the mobile phase. The tandem mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with ionization of the analytes in positive mode. The assay was fully validated according to current acceptance criteria for bioanalytical methods validation. It was proved to be linear in the range of 0.5-250 ng/mL, with adequate accuracy and precision over this range. Based on accuracy and CV% values the LOQ and ULOQ values were set at 0.509 ng/mL and 2036 ng/mL for MET, 0.520 ng/mL and 2080 ng/mL for EDDP, 0.524 ng/mL and 2096 ng/mL for FNZ and 0.528 ng/mL and 2114 ng/mL for 7-AFNZ, respectively. The method was tested for potential matrix effects, without observing significant ion suppression. The investigated compounds stability was examined in plasma at room temperature and after three freeze-thaw cycles and in the final extract when maintained at 4 °C in the autosampler. Potential stability issues were observed only for FNZ at room temperature. The method was successfully applied to quantify the selected compounds in human, rat and rabbit plasma samples, after exposure to FNZ or simultaneous exposure to FNZ and MET.
本文介绍了一种简单、高通量、灵敏的 LC-ESI-MS/MS 方法，用于同时测定人、大鼠和兔血浆中的美沙酮（MET）、氟硝西泮（FNZ）及其主要代谢物，分别为 EDDP（2-乙撑基-1,5-二甲基-3,3-二苯基吡咯烷酮）和 7-氨基氟硝西泮（7-AFNZ）。所选化合物的分离涉及在碱性 pH 下用乙酸乙酯进行液液萃取。在 HSS T3 柱（1.8 μm 粒径）上实现了良好的色谱分离，使用乙腈与 0.1%甲酸（溶剂 A）和 5mM 乙酸铵（溶剂 B）的混合物作为流动相进行 3 分钟梯度洗脱。串联质谱检测采用正离子模式下分析物的多反应监测（MRM）模式进行。该测定方法按照生物分析方法验证的当前接受标准进行了全面验证。结果表明，该方法在 0.5-250ng/mL 范围内呈线性，在此范围内具有足够的准确性和精密度。基于准确度和 CV%值，将 MET 的 LOQ 和 ULOQ 值设定为 0.509ng/mL 和 2036ng/mL，EDDP 的 LOQ 和 ULOQ 值设定为 0.520ng/mL 和 2080ng/mL，FNZ 的 LOQ 和 ULOQ 值设定为 0.524ng/mL 和 2096ng/mL，7-AFNZ 的 LOQ 和 ULOQ 值设定为 0.528ng/mL 和 2114ng/mL。该方法经过潜在基质效应测试，未观察到明显的离子抑制。考察了化合物在室温下和经过三次冻融循环后的血浆中以及在自动进样器中 4°C 保存时的稳定性。仅在室温下观察到 FNZ 存在潜在的稳定性问题。该方法成功应用于测定 FNZ 暴露或同时暴露于 FNZ 和 MET 后，人、大鼠和兔血浆样品中选定化合物的含量。
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