A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma
A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma
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DOI：
10.1016/j.jchromb.2009.02.006
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发表时间：
2009-04-01
影响因子：
3
通讯作者：
Decosterd, L. A.
中科院分区：
文献类型：
--
作者：
Hodel, E. M.;Zanolari, B.;Decosterd, L. A.
在治疗反应的各种决定因素中，达到足够的血液水平对治愈疟疾至关重要。为了帮助我们提高对抗疟药物的药代动力学、疗效和毒性的认识，我们开发了液相色谱-串联质谱法（LC-MS/MS）需要200 μ l血浆，用于同时测定14种抗疟药物及其代谢物，这些药物是目前疟疾一线联合治疗的成分（蒿甲醚、青蒿琥酯、双氢青蒿素、阿莫地喹、N-去乙基阿莫地喹、本芴醇、去丁基本芴醇、哌喹、咯萘啶、甲氟喹、氯喹、奎宁、乙胺嘧啶和磺胺嘧啶）。通过蛋白质沉淀、蒸发和在甲醇/甲酸铵20 mM（pH 4.0）1：1中复溶的组合纯化血浆。使用均含有0.5%甲酸的20 mM甲酸铵和乙腈的梯度洗脱，然后冲洗并重新平衡至初始溶剂组成长达21 min，获得抗疟药物的反相色谱分离。使用基质匹配校准样品进行分析物定量，通过电喷雾电离-三重四极杆质谱法，通过在正模式下的选择反应监测检测来进行。根据FDA的建议对该方法进行了验证，包括评估提取率、基质效应变异性、总体工艺效率、标准加入实验以及抗疟药在血浆中的短期和长期稳定性。在体外和疟疾患者样本中测试了在溶血存在下含内过氧化物的抗疟药的反应性。用这种方法，青蒿素的信号强度降低了约20%，在血浆中的溶血红细胞的存在下，而其衍生物基本上不受影响。该方法具有精密度（日间CV%：3.1-12.6%）和灵敏度（碱性/中性抗疟药和青蒿素衍生物的定量下限分别为0.15-3.0和0.75-5 ng/ml）。这是第一个涵盖目前使用的抗疟药的宽范围LC-MS/MS分析。与以往的方法相比，该方法在方便性（14种主要抗疟药和代谢物的单一提取程序，显著缩短了分析时间）、灵敏度、选择性和通量方面有所改进。虽然它的主要限制是设备的投资成本，但血浆样品可以在现场收集，并在-80 ℃下储存之前在4 ℃下保存长达48小时。它适用于检测受试者中药物的存在以用于筛选目的和量化治疗后的药物暴露。它可能有助于填补目前抗疟药药代动力学/药效学关系方面的知识空白，并更好地确定不同患者人群的治疗剂量范围。(C)2009爱思唯尔有限公司版权所有。
Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200 mu l of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin,amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20 mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20 mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5 ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48 h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations. (C) 2009 Elsevier B.V. All rights reserved.