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Development and validation of a UHPLC-HRMS method for the simultaneous determination of the endogenous anabolic androgenic steroids in human serum
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.ORCID iD: 0000-0002-2485-4276
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Being performance enhancing hormones, endogenous anabolic androgenic steroids (EAAS) are banned from most competitive sports by the World Anti-doping Agency (WADA).. In anti-doping control laboratories, routine assays are mainly performed on urine samples of athletes in and out of competitions. Serum constitutes a promising alternative to urine as it is less subjected to manipulation or contamination issues that may influence the method sensitivity. The simultaneous determination of EAAS and their conjugated metabolites is very challenging due to their contradicting chemical behaviors at the ionization interface of the mass spectrometer. This may prejudice their detection or limit the method sensitivity. Herein, we have addressed these challenges and developed a new method for the simultaneous determination of unconjugated, sulphate and glucuronide EAAS (Androsterone, Etiocholanolone, testosterone, epitestosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione and 17a-hydroxyprogesterone) in human serum using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The use of mass spectrometric detection in full scan mode facilitated the study of the most versatile adducts for detection and quantitation. Using solid phase extraction as sample preparation protocol prior to analysis, the method limits of quantitation ranged from 0.006 to 7.904 ng/mL and the recoveries ranged from 70.2 to 96.5 %. The method calibration was performed in untreated serum representing realistic matrix composition with correlation coeffecients ranged from 0.9859 to 0.9988. Finally, the serum- levels of the investigated steroids were determined in 4 male and 1 female human subject to provide estimates of baseline levels based on individual values.

Keywords [en]
Doping, Endogenous Anabolic androgenic steroids, Chromatography, UHPLC, Mass spectrometry, Solid phase extraction.
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-172564OAI: oai:DiVA.org:su-172564DiVA, id: diva2:1348245
Available from: 2019-09-03 Created: 2019-09-03 Last updated: 2019-09-04Bibliographically approved
In thesis
1. Analytical Methods For Sports Drugs: Challenges and Approaches
Open this publication in new window or tab >>Analytical Methods For Sports Drugs: Challenges and Approaches
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Drugs used to enhance human performance in sport competitions are prohibited by the world anti-doping association (WADA). Biological samples from athletes are continuously tested for adverse analytical findings regarding the identity and/or quantity of the banned substances. The current thesis deals with the development of new analytical methods to determine the concentrations of certain drugs used by athletes and even by regular users for therapeutic purposes. The developed methods aim to analyze the contents of these drugs in the biological matrices; plasma, serum and saliva to provide a successful approach towards either doping detection or therapeutic monitoring. β-adrenergic blockers such as propranolol and metoprolol are used in sports to relief stress and as therapeutic agents in the treatment of hypertension. Both drugs are in chiral forms and available only as racemic mixtures. The different pharmacology of each enantiomer necessitates the monitoring of each enantiomer by stereoselective analytical technique such as chiral liquid chromatography for separation and mass spectrometry for selective detection. The Endogenous anabolic androgenic steroids (EAAS) on the other hand are only notoriously used in sports to increase muscle mass and strength. A method utilizing high-resolution mass spectrometry (HRMS) coupled to ultra-high performance liquid chromatography (UHPLC) was developed for the simultaneous determination of EAAS and their conjugated metabolites to provide a better insight into the steroidal module of the athlete biological passport (ABP). Moreover, the steroidal profile was assessed in serum using the proposed method after the administration of Growth hormone injection as an approach toward the implementation of a new endocrinological module based on steroids biomarkers to hormone doping.  Biological samples contain many components that may interfere with the analytical measurements. Therefore, sample preparation methods were developed using solid phase extraction (SPE) and miniaturized techniques such as microextraction by packed sorbents (MEPS) for the purification and pre-concentration of analytes prior to LC/MS analysis.

Place, publisher, year, edition, pages
Stockholm: Department of Environmental Science and Analytical Chemistry, Stockholm University, 2019. p. 89
Keywords
Sports Drugs, Doping in Sports, Steroids, LC-MS/MS, Chiral analysis, high-resolution mass spectrometry, Sample preparation, Biological samples, solid phase extraction
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-172566 (URN)978-91-7797-835-0 (ISBN)978-91-7797-836-7 (ISBN)
Public defence
2019-10-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2019-09-25 Created: 2019-09-03 Last updated: 2019-09-13Bibliographically approved

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