Bioanalysis Research Group, IMIM, Hospital del Mar, Barcelona, Spain.
Anal Chem. 2013 May 21;85(10):5005-14. doi: 10.1021/ac4001749. Epub 2013 Apr 30.
In humans, conjugation with glucuronic acid is the most important phase II metabolic reaction of steroidal compounds. Glucuronoconjugated metabolites have been conventionally studied by using β-glucuronidase enzymes to release the phase I metabolites. It is well-known that hydrolysis with β-glucuronidase presents some limitations that may result in the underestimation of some conjugates. The aim of the present work was to develop and to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS) scan methods for the open detection of steroid glucuronides in urine samples. The mass spectrometric behavior of thirteen representative steroid glucuronides, used as model compounds, was studied. Characteristic ionization and collision induced dissociation behaviors were observed depending on the steroid glucuronide structure. Neutral loss (NL of 176, 194, 211, and 229 Da) and precursor ion (PI of m/z 141, 159, and 177, in positive mode and m/z 75, 85, and 113, in negative mode) scan methods were evaluated. The NL scan method was chosen for the open detection of glucuronoconjugated steroids due to its sensitivity and the structural information provided by this method. The application of the NL scan method to urine samples collected after testosterone (T) undecanoate administration revealed the presence of two T metabolites which remain conjugated as glucuronides after an enzymatic hydrolysis of the urine. 3α,6β-Dihydroxy-5α-androstan-17-one (6β-hydroxyandrosterone) glucuronide and 3α,6β-dihydroxy-5β-androstan-17-one (6β-hydroxyetiocholanolone) glucuronide were established as the structures for these metabolites, by comparing the structure of the steroids released after chemical hydrolysis with reference materials. An increase of 50-300-fold of these metabolites after oral administration of T undecanoate was observed, proving that their determination can be useful in the doping control field. Moreover, these results exemplify that significant information might be missed, unless direct methods for the determination of steroid glucuronides are employed.
在人类中，与葡萄糖醛酸的结合是甾族化合物最重要的第二期代谢反应。传统上，通过使用β-葡萄糖醛酸酶来释放第一期代谢物来研究葡萄糖醛酸共轭代谢物。众所周知，用β-葡萄糖醛酸酶水解存在一些限制，可能导致一些共轭物的低估。本工作的目的是开发和评估液相色谱-串联质谱（LC-MS/MS）扫描方法，用于开放检测尿液样品中的甾体葡萄糖醛酸。研究了作为模型化合物的十三种代表性甾体葡萄糖醛酸的质谱行为。观察到取决于甾体葡萄糖醛酸结构的特征离子化和碰撞诱导解离行为。中性损失（NL）为 176、194、211 和 229 Da 和前体离子（PI）为 m/z 141、159 和 177（正模式）和 m/z 75、85 和 113（负模式）的扫描方法进行了评价。NL 扫描方法由于其灵敏度和该方法提供的结构信息，被选为开放检测葡萄糖醛酸结合甾体的方法。NL 扫描方法应用于睾丸素（T）十一酸酯给药后采集的尿液样品中，揭示了两种 T 代谢物的存在，这些代谢物在尿液的酶水解后仍然作为葡萄糖醛酸结合物存在。3α,6β-二羟基-5α-雄烷-17-酮（6β-羟基雄甾酮）葡萄糖醛酸苷和 3α,6β-二羟基-5β-雄烷-17-酮（6β-羟基表雄烷酮）葡萄糖醛酸苷被确定为这些代谢物的结构，通过比较化学水解后释放的类固醇与参比物质的结构。口服 T 十一酸酯后，这些代谢物的浓度增加了 50-300 倍，证明其测定在兴奋剂控制领域可能有用。此外，这些结果表明，除非使用直接测定甾体葡萄糖醛酸的方法，否则可能会错过重要信息。
