采用LC-MS/MS检测人尿中睾内脂方法研究

LC-MS/MS Determination of Testolactone in Human Urine

目的：建立人尿中睾内脂的LC-MS/MS检测方法。方法：采用Agilent Zorbax XDB-C18柱（2.1×50mm,3.5μm）,流动相采用pH3.5甲酸铵缓冲液（A）和乙腈（B）进行梯度洗脱,离子检测方式为多反应离子检测（MRM）;离子极性为正离子,定量的离子对为m/z301→121,定性的离子对为m/z301→225。结果：测定人尿中内源性物质不干扰样品和内标,样品分析时间为7分钟,睾内脂线性范围为0.1～50μg/ml,提取回收率大约为60%,日内和日间的RSD均小于10%。结论：本方法经考察符合国际反兴奋剂机构（WADA）的要求,可以应用于常规的兴奋剂检测工作。

Objective To develop a sensitive and specific LC-MS/MS method for determination of testolactonein human urine. Methods A C18 column（2.1×50mm, 3.5μm）was used. The mobile phase was a mixture ofacetonitrile and the buffer solution（ammonium acetate-water solution adjusted with formic acid to pH 3.5）at aflow rate of 0.5ml/min. A mass spectrometer equipped with electrospray ionization source was used as a detectorand operated in the positive mode. In multiple reaction monitoring（MRM）mode, the ion transitions of m/z301 →121 and m/z 301 →225 was used to qualify and quantify the testolactone, respectively. ResultsChromatograms showed no endogenous interfering peaks with the urine blank sample. Each analysis wascompleted within 7min. The calibration was linear in the concentration range within 0.1-50μg/ml. The intra-batchand inter-batch RSD were less than 10%. The recovery rate of the extraction was about 60%. Conclusions Themethod is proved to meet the requirements of WADA and be suitable for routine screening.