Development of a highly sensitive and selective UPLC/MS/MS method for the simultaneous determination of testosterone and 5α-dihydrotestosterone in human serum to support testosterone replacement therapy for hypogonadism
Development of a highly sensitive and selective UPLC/MS/MS method for the simultaneous determination of testosterone and 5α-dihydrotestosterone in human serum to support testosterone replacement therapy for hypogonadism
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DOI：
10.1016/j.steroids.2008.01.018
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发表时间：
2008-07-01
期刊：
影响因子：
2.7
通讯作者：
Yang, Eric
中科院分区：
文献类型：
--
作者：
Licea-Perez, Hermes;Wang, Sherry;Yang, Eric
一种高灵敏度和高选择性的定量方法，以准确地测定睾酮（Te）和5 α-二氢睾酮（DHT）在人血清中是至关重要的Te替代治疗性腺功能减退症的成功。为此，我们已经开发并验证了一种半自动化和相对高通量的方法，在96孔板格式，使用超高效液相色谱串联质谱（UPLC/MS/MS）同时测定Te和DHT在人血清中。使用甲基叔丁基醚（MTBE）通过液-液萃取从300 μ L人血清中萃取Te和DHT沿着以及内标物[（2）H-（3）]-Te和[（2）H（3）]-DHT，然后用2，3-吡啶二羧酸酐衍生化并固相萃取用于样品净化。采用2，3-吡啶二羧酸酐化学衍生的新方法，实现了双氢睾酮的质谱灵敏度和选择性。Te和DHT衍生物的内源性类固醇衍生物的基线分离，实现了使用UPLC技术上的C18固定相柱与1.7 μ m的粒径。通过标准加入实验证明了使用双重活性炭剥离的女性人血清作为替代基质制备校准标准品的有效性。该方法在Te的浓度范围为0.2-40 ng/mL和DHT的浓度范围为0.01-2 ng/mL进行了验证。验证和研究样品分析结果表明，该方法耐用、精密、准确，非常适合支持药代动力学研究，其中1天内可提取和分析约300份样品。(C)2008年爱思唯尔公司All rights reserved.
A highly sensitive and selective quantitative method to accurately determine testosterone (Te) and 5 alpha-dihydrotestosterone (DHT) in human serum is crucial to the success of Te replacement therapy for hypogonadism. To this end we have developed and validated a semi-automated and relatively high-throughput method in a 96-well plate format using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC/MS/MS) for the simultaneous determination of Te and DHT in human serum. Te and DHT along with the internal standards [(2)H-(3)]-Te and [(2)H(3)]-DHT were extracted from 300 mu L of human serum by liquid-liquid extraction using methyl tertiary-butyl ether (MTBE), followed by derivatization with 2,3-pyridinedicarboxylic anhydride and solid-phase extraction for sample clean up. A novel chemical derivatization approach using 2,3-pyridinedicarboxylic anhydride was employed to achieve the MS sensitivity and selectivity required for DHT. Baseline separation of Te and DHT derivatives from endogenous steroid derivatives was achieved using UPLC technology on a C18 stationary-phase column with 1.7 mu m particle size. The validity of using double charcoal-stripped female human serum as surrogate matrix for preparation of calibration standards was demonstrated through standard addition experiments. The method was validated over the concentration ranges of 0.2-40 ng/mL for Te and 0.01-2 ng/mL for DHT. The validation and study sample analysis results show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in 1 day. (C) 2008 Elsevier Inc. All rights reserved.