Clinical Pharmacology Department, Hospital Universitario de la Princesa, Instituto Teófilo Hernando, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria la Princesa (IP), Spain.
Talanta. 2019 Jun 1;198:159-168. doi: 10.1016/j.talanta.2019.01.112. Epub 2019 Feb 1.
A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated in human plasma for the simultaneous determination of aripiprazole (ARI) and its metabolite dehydro-aripiprazole (DARI); olanzapine (OLA), risperidone (RIS), paliperidone (PAL), quetiapine (QUE), clozapine (CLO) and caffeine (CAF). CAF is included to the method because it can have an influence on drug metabolism due to competitive inhibition. The above mentioned compounds and their isotope-labeled internal standards were extracted from 200 µL human plasma samples by both, effective phospholipids-eliminating three-step microelution-solid-phase extraction (µ-SPE) and protein precipitation (PPT) for comparison. A combination of formic acid (0.2%)-acetonitrile (pH 3.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 mL/min. Run time lasted 6 min, followed by a re-equilibration time of 3 min. All analytes were monitored by mass spectrometric detection operating in multiple reaction monitoring mode and the method was validated covering the corresponding therapeutic ranges: 0.18-120 ng/mL for ARI, 0.25-80 ng/mL for DARI, 1.00-100 ng/mL for OLA, 0.70-60 ng/mL for RIS, 0.20-30 ng/mL for PAL, 0.50-160 ng/mL for QUE, 0.50-1000 ng/mL for CLO, and finally 1200-3700 ng/mL for CAF. The method was validated based on the recommendations of regulatory agencies through tests of precision, accuracy, extraction recovery, identity confirmation, trueness, matrix effect, process efficiency, stability, selectivity, linearity and carry-over effect fulfilling the guideline requirements. Our µ-SPE method results in the elimination of more than 99% of early eluting and more than 92% of late-eluting phospholipids compared to PPT. Additionally, the method was successfully applied for quantifying ARI and OLA plasma concentrations from healthy volunteers.
建立并验证了一种人血浆中阿立哌唑（ARI）及其代谢物脱氢阿立哌唑（DARI）、奥氮平（OLA）、利培酮（RIS）、帕利哌酮（PAL）、喹硫平（QUE）、氯氮平（CLO）和咖啡因（CAF）的同时测定的灵敏、简便的液相色谱-串联质谱法。CAF 被纳入方法中，因为它可能通过竞争性抑制对药物代谢产生影响。上述化合物及其同位素标记的内标通过有效去除磷脂的三步微萃取-固相萃取（µ-SPE）和蛋白沉淀（PPT）两种方法从 200 µL 人血浆样品中提取，然后进行比较。采用甲酸（0.2%）-乙腈（pH 3.0；65:35，v/v）作为流动相，在 0.6 mL/min 的流速下进行梯度洗脱。洗脱时间为 6 min，随后再平衡 3 min。所有分析物均采用质谱检测，以多反应监测模式进行监测，方法验证涵盖相应的治疗范围：ARI 为 0.18-120ng/mL，DARI 为 0.25-80ng/mL，OLA 为 1.00-100ng/mL，RIS 为 0.70-60ng/mL，PAL 为 0.20-30ng/mL，QUE 为 0.50-160ng/mL，CLO 为 0.50-1000ng/mL，CAF 为 1200-3700ng/mL。该方法是根据监管机构的建议，通过精密度、准确度、提取回收率、确证试验、准确度、基质效应、过程效率、稳定性、选择性、线性和携带效应等测试进行验证的，满足指南要求。与 PPT 相比，我们的 µ-SPE 方法可去除 99%以上的早期洗脱磷脂和 92%以上的晚期洗脱磷脂。此外，该方法还成功应用于定量分析健康志愿者的血浆中阿立哌唑和奥氮平浓度。
