Use of LC-MS/MS for the Open Detection of Steroid Metabolites Conjugated with Glucuronic Acid
- 30 April 2013
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 85 (10), 5005-5014
- https://doi.org/10.1021/ac4001749
Abstract
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.Keywords
This publication has 35 references indexed in Scilit:
- Analysis of conjugated steroid androgens: Deconjugation, derivatisation and associated issuesJournal of Pharmaceutical and Biomedical Analysis, 2009
- Conjugated steroids: analytical approaches and applicationsAnalytical and Bioanalytical Chemistry, 2008
- Factors influencing the steroid profile in doping control analysisJournal of Mass Spectrometry, 2008
- Liquid chromatographic–mass spectrometric analysis of glucuronide‐conjugated anabolic steroid metabolites: method validation and interlaboratory comparisonJournal of Mass Spectrometry, 2008
- Metabolism and excretion of anabolic steroids in doping control—New steroids and new insightsThe Journal of Steroid Biochemistry and Molecular Biology, 2006
- Optimizing the Hydrolysis of Codeine and Morphine Glucuronides in UrineTherapeutic Drug Monitoring, 2002
- Direct measurement of steroid sulfate and glucuronide conjugates with high-performance liquid chromatography-mass spectrometryJournal of Chromatography B: Biomedical Sciences and Applications, 1996
- Comparison of the Hydrolysis Rates of Morphine-3-Glucuronide and Morphine-6-Glucuronide with Acid and β-Glucuronidase*Journal of Analytical Toxicology, 1995
- Helix pomatia induced conversion of some 3β-hydroxysteroidsJournal of Steroid Biochemistry, 1984
- Hydrolysis of Pregnanediol Glucuronide by Sodium PeriodateBiochemical Journal, 1967