Universität zu Köln

Piper, Thomas and Thevis, Mario (2022). Addressing recent challenges in isotope ratio mass spectrometry: Development of a method applicable to 1-androstene-steroids, 6 alpha-hydroxy-androstenedione, and androstatrienedione. Drug Test. Anal., 14 (11-12). S. 1891 - 1904. HOBOKEN: WILEY. ISSN 1942-7611

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Abstract

In 2020, the confirmation of the non-endogenous origin of several pseudo-endogenous steroids by means of isotope ratio mass spectrometry (IRMS) was recommended by the World Anti-Doping Agency (WADA), in addition to previously established target analytes for IRMS in sports drug testing. To date, however, IRMS-based methods validated in accordance with current WADA regulations have not been available. Therefore, the aim of this research project was the development and validation of a method to determine the carbon isotope ratios (CIR) of all newly considered pseudo-endogenous steroids, encompassing the anabolic androgenic steroids comprising a 1-ene-core structure (5 alpha-androst-1-ene-3 beta,17 beta-diol, 5 alpha-androst-1-ene-3,17-dione [1AD], 17 beta-hydroxy-5 alpha-androst-1-en-3-one, 3 alpha-hydroxy-5 alpha-androst-1-ene-17-one [1AND], and 3 beta-hydroxy-5 alpha-androst-1-ene-17-one [1EpiAND]), as well as steroids referred to as hormone and metabolic modulators (androsta-1,4,6-triene-3,17-dione [TRD] and its main metabolite 17 beta-hydroxy-androsta-1,4,6-triene-3-one) and 6 alpha- and 6 beta-hydroxy-androst-4-ene-3,17-dione. With peak purity of target analytes being critical for IRMS analyses, a twofold high-performance liquid chromatography (HPLC)-based sample purification was employed, with all analytes being acetylated between the first and second HPLC fractionation. Using established gas chromatography/combustion/IRMS instrumentation, limits of quantification were estimated at 10 ng/ml for a 20 ml urine aliquot for all analytes, except for 1AND (20 ng/ml), and combined measurement uncertainties were estimated between 0.4 parts per thousand and 0.9 parts per thousand. For proof-of-concept, samples collected after the single oral administration of a nutritional supplement containing 1AD and 1EpiAND were analyzed as well as existing excretion study urine samples obtained after the administration of 4-androstenedione and TRD. Based on the obtained results, the developed method was considered to be fit-for-purpose.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Piper, Thomas UNSPECIFIED UNSPECIFIED UNSPECIFIED Thevis, Mario UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-666573
DOI:	10.1002/dta.3361
Journal or Publication Title:	Drug Test. Anal.
Volume:	14
Number:	11-12
Page Range:	S. 1891 - 1904
Date:	2022
Publisher:	WILEY
Place of Publication:	HOBOKEN
ISSN:	1942-7611
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language ENDOGENOUS URINARY STEROIDS; DOPING CONTROL; REFERENCE-POPULATION; C-13/C-12 RATIOS; IDENTIFICATION; FRACTIONATION; METABOLISM Multiple languages Biochemical Research Methods; Chemistry, Analytical; Pharmacology & Pharmacy Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/66657