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Development and application of analytical procedures for the GC-MS/MS analysis of the sulfates metabolites of anabolic androgenic steroids: The pivotal role of chemical hydrolysis.
Journal of Chromatography B ( IF 3 ) Pub Date : 2020-07-27 , DOI: 10.1016/j.jchromb.2020.122280
Michele Iannone 1 , Francesco Botrè 2 , Dayamin Martinez-Brito 1 , Roberta Matteucci 1 , Xavier de la Torre 1
Affiliation  

In this work, we present a gas-chromatography tandem mass spectrometry (GC–MS/MS) method for the identification of the sulfo-conjugate metabolites of pseudo-endogenous steroids (endogenous steroids when administered exogenously). We have preliminarily evaluated the performances of different preparations of sulfatases from Pseudomonas aeruginosa and Helix pomatia, characterized by various origins and catalytic activities, and compared the efficacy of the enzymatic hydrolysis with chemical hydrolysis, performed with a mixture of ethyl acetate, methanol, and sulphuric acid. A procedure for the selective isolation of steroid conjugates from the urine matrix has been designed and optimized, based on the “sequential” extraction of the glucuro-conjugated and of the sulfo-conjugated fractions, performed by two different direct methods, i.e. by ion paired extraction or solid-phase extraction. More specifically, the former method is based on the use of N,N-dimethylephedrinium bromide as the ion paired extraction reagent, while the latter on the use of WAX® (weak anion exchange) cartridges. The performance of the newly developed procedure has been assessed by the analysis of real urine excretion samples collected after the oral intake of a single dose of dehydroepiandrosterone (DHEA) or androstenedione (AED), measuring the concentration of epiandrosterone (EpiA) sulfate.

Our results have shown the following: (i) although the yields of chemical hydrolysis and enzymatic hydrolysis are in some cases quite similar, the former is generally preferable since it results in the quantitative cleavage of sulfate moiety; (ii) ion paired extraction has been selected as the most reliable method for direct isolation of sulfate steroids from urine matrices; (iii) EpiA sulfate allows to prolong the detectability of DHEA and AED when compared to routinely used steroidal target compounds.



中文翻译:

合成代谢雄激素类固醇的硫酸盐代谢物的GC-MS / MS分析程序的开发和应用:化学水解的关键作用。

在这项工作中,我们提出了一种气相色谱串联质谱法(GC-MS / MS),用于鉴定拟内源性类固醇(外源性类固醇)的磺基共轭代谢物。我们已经初步评估了铜绿假单胞菌Polix螺旋藻不同硫酸盐酶制剂的性能具有各种来源和催化活性,并比较了酶水解与化学水解的功效,该水解是在乙酸乙酯,甲醇和硫酸的混合物中进行的。从葡萄糖基质结合部分和磺基结合部分的“顺序”提取中,通过两种不同的直接方法(即通过离子对)进行了设计和优化,从尿液基质中选择性分离类固醇结合物的程序已被设计和优化。萃取或固相萃取。更具体地说,前一种方法基于使用N,N-二甲基麻黄溴化铵作为离子对提取试剂,而后一种方法基于使用(弱阴离子交换)柱。

我们的结果表明:(i)尽管在某些情况下化学水解和酶促水解的产率非常相似,但是前者通常是优选的,因为它导致硫酸盐部分的定量裂解;(ii)选择离子对萃取作为从尿液基质中直接分离硫酸盐类固醇的最可靠方法;(iii)与常规使用的类固醇目标化合物相比,EpiA硫酸盐可以延长DHEA和AED的检测时间。

更新日期:2020-08-04
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