Identification of the long-acting erythropoiesis-stimulating agent darbepoetin alfa in human urine by liquid chromatography-tandem mass spectrometry
Identification of the long-acting erythropoiesis-stimulating agent darbepoetin alfa in human urine by liquid chromatography-tandem mass spectrometry
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DOI：
10.1007/s00216-013-6836-y
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发表时间：
2014-02-01
影响因子：
4.3
通讯作者：
Kageyama, Shinji
中科院分区：
文献类型：
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作者：
Okano, Masato;Sato, Mitsuhiko;Kageyama, Shinji
重组人促红细胞生成素（rhEPO）的滥用增加了红细胞的增殖/产生，这增强了氧运输能力，并且对人类健康和体育运动的公平性具有严重后果。对于运动药物检测，目前rhEPO的分析方法主要是凝胶电泳法，如等电聚焦-聚丙烯酰胺凝胶电泳。质谱法对于兴奋剂控制中rhEPO的可靠鉴定是必不可少的。本研究采用自下而上的方法，建立了人尿液中达贝泊苷α的高灵敏度、高通量质谱定性检测方法。该新方法涉及人尿液（10 mL）的免疫纯化，在碳酸氢铵缓冲液（pH 7.8）中用内切蛋白酶Glu-C（V8-蛋白酶）进行蛋白酶消化，以及使用带电表面混合C-18柱的超高效液相色谱法，结合电喷雾电离高灵敏度串联质谱法，以提高靶分子的选择性。从达贝泊苷α消化的特异性片段为（90）TLQLHVDKAVSGLRSLTTLLRALGAQKE（117）（V-11）。尿达贝泊苷α的检测下限为1.2 pg/mL。确认分析的检测限估计为5 pg/mL。开发的方法允许高通量确认分析，即样品制备6小时，每个样品的分析运行时间仅为10分钟;这种高通量方法大大降低了实验室的工作量。静脉注射15 μ g达依泊汀α（n = 3）后收集的人尿液中可鉴别出达依泊汀α。这种质谱方法是一种用于检测人体尿液中达贝泊苷α的创新和强大的工具，用于兴奋剂控制检测。
The misuse of recombinant human erythropoietin (rhEPO) increases the proliferation/production of erythrocytes, which enhance oxygen transport capacities, and has grave consequences with respect to human health and fairness in sports. For sports drug testing, the current analytical methods for rhEPOs are mainly gel electrophoretic methods, such as isoelectric focusing-polyacrylamide gel electrophoresis. Mass spectrometry is fundamentally necessary for the reliable identification of rhEPOs in doping control. In this study, a high-sensitivity and high-throughput mass spectrometric qualitative detection method for darbepoetin alfa in human urine was established by a bottom-up approach. The novel method involves the immunopurification of human urine (10 mL), protease digestion with endoproteinase Glu-C (V8-protease) in an ammonium bicarbonate buffer (pH 7.8) and ultra-performance liquid chromatography using a charged surface hybrid C-18 column coupled with electrospray-ionisation high-sensitivity tandem mass spectrometry for improved selectivity of the target molecules. The specific fragment digested from darbepoetin alfa was (90)TLQLHVDKAVSGLRSLTTLLRALGAQKE(117) (V-11). The lower limit of detection of urinary darbepoetin alfa was 1.2 pg/mL. The limit of detection for the confirmation analysis was estimated to be 5 pg/mL. The developed method allows high-throughput confirmation analysis, namely 6 h for sample preparation and an analytical run time of only 10 min per sample; this high-throughput method dramatically decreases the workload in the laboratory. Darbepoetin alfa could be identified in human urine collected after the intravenous administration of 15 mu g darbepoetin alfa (n = 3). This mass spectrometric method is an innovative and powerful tool for detecting darbepoetin alfa in human urine for doping control testing.