Simultaneous determination of Aprepitant and two metabolites in human plasma by high-performance liquid chromatography with tandem mass spectrometric detection
Simultaneous determination of Aprepitant and two metabolites in human plasma by high-performance liquid chromatography with tandem mass spectrometric detection
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DOI：
10.1016/j.jpba.2004.03.020
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发表时间：
2004-09-03
影响因子：
3.4
通讯作者：
Matuszewski, BK
中科院分区：
文献类型：
--
作者：
Chavez-Eng, CM;Constanzer, ML;Matuszewski, BK
建立了一种同时测定人血浆中阿瑞匹坦I（5-[2（R）-[1（R）-（3，5-双三氟甲基苯基）乙氧基-3（S）（4-氟苯基）吗啉-4-基]甲基]-2，4-二氢-[1，2，4]三唑-3-酮）和两种活性代谢物（II和III）的方法。该方法是基于高效液相色谱法（HPLC）与大气压化学电离串联质谱（APCI-MS-MS）检测，在正离子化模式下使用加热雾化器接口。使用液-液萃取从碱化血浆中分离分析物和内标物（IV）（图1）。有机萃取液经干燥后，在移动的相中复溶，进样至HPLC-MS/MS系统中，在窄孔柱上进行色谱分析（50 mm x 2.0 mm，3 μ m）Keystone Scientific的Prism R. P.分析柱，移动的流动相由乙腈（ACN）：含三氟乙酸的水组成，pH值调节至3（四十：60,v/v）以0.5ml/min的流速泵送。在以选择的反应监测模式操作的Sciex API 3000串联质谱仪上进行MS检测。分析物色谱分离后，使用m/z 535 --> 277、438 --> 180、452 --> 223和503 --> 259的前体-->产物离子组合分别定量I、II、III和IV。当处理1 ml血浆时，在10-5000 ng/ml（I和II）和25-5000 ng/ml（III）浓度范围内验证了该测定法。在标准曲线范围内的所有浓度下，试验精密度（表示为变异系数，CV）均小于10%，具有足够的试验准确度。进行基质效应实验，以证明在存在血浆基质的情况下，将纯标准品与分析物进行比较时，分析物的电离没有任何显著变化。该试验用于支持一项临床研究，其中健康受试者多次口服I，以研究阿瑞匹坦的药代动力学、安全性和耐受性。测定了两种最具活性的代谢产物的浓度，如果以高浓度存在，则会增加神经激肽-1（NK 1）受体占用水平，因此可能有助于阿瑞匹坦的止吐作用。(C)2004 Elsevier B. V.保留所有权利。
A method for the simultaneous determination of Aprepitant, I (5-[2(R)-[1(R)-(3,5-bistrifluoromethylphenyl)ethoxyl-3(S)(4-fluorophenyl) morpholin-4-yl]methyl]-2,4-dihydro-[1,2,4] triazol-3-one) and two active metabolites (II and III) in human plasma has been developed. The method was based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS-MS) detection in positive ionization mode using a heated nebulizer interface. The analytes and internal standard (IV) (Fig. 1) were isolated from basified plasma using liquid-liquid extraction. The organic extracts were dried, reconstituted in mobile phase and injected into the HPLC-MS/MS system.The analytes were chromatographed on a narrow bore (50 mm x 2.0 mm, 3 mum) Keystone Scientific's Prism R.P. analytical column, with mobile phase consisting of acetonitrile (ACN):water containing trifluoroacetic acid with pH adjusted to 3 (40:60, v/v) pumped at a flow rate of 0.5 ml/min. The MS-MS detection was performed on a Sciex API 3000 tandem mass spectrometer operated in selected reaction monitoring mode. The precursor --> product ion combinations of m/z 535 --> 277, 438 --> 180, 452 --> 223 and 503 --> 259 were used to quantify I, II, III, and IV, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10-5000 ng/ml for I and II and 25-5000 ng/ml for III when 1 ml of plasma was processed. The precision of the assay (expressed as coefficient of variation, CV) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. Matrix effect experiments were performed to demonstrate the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. This assay was utilized to support a clinical study where multiple oral doses of I were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of Aprepitant. Concentrations of the two most active metabolites, which if present in high concentrations would increase the neurokinin-1 (NK1) receptor occupancy level and therefore potentially contribute to the antiemetic action of Aprepitant, were determined. (C) 2004 Elsevier B.V. All rights reserved.