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Analysis of endogenous steroids in urine by means of multi-immunoaffinity chromatography and isotope ratio mass spectrometry for sports drug testing.
Anal Bioanal Chem. 2019 Nov; 411(28):7563-7571.AB

Abstract

Detecting the administration of naturally occurring but synthetically derived steroids (e.g., testosterone) in routine doping controls is particularly laborious and time-consuming. Carbon isotope signatures determined by isotope ratio mass spectrometry (IRMS) have been established as the method of choice to generate confirmatory evidence in case of suspicious or atypical findings in steroid profile analyses; however, IRMS measurements require sophisticated sample preparation methods employing up to two high-performance liquid chromatography (HPLC) purification steps. Here, an alternative sample preparation approach is presented. Immunoaffinity chromatography (IAC) was employed to reduce the batch analysis time by omitting the time-consuming HPLC purification steps, while pre- and post-IAC sample handling followed published protocols. IAC exploits specific antibody-immunogen interactions, and the option of combining three immunoaffinity gels containing specific antibodies for testosterone, pregnanediol, and 11-ketoetiocholanolone into a multi-immunoaffinity sample preparation approach was assessed. Due to cross reactivities, also etiocholanolone, androsterone, 5β-androstanediol, and 5α-androstanediol were co-extracted and included in the testing protocol. The method was validated by determining precision, recovery, and carry over, and performing linear mixing models. IAC was found to be applicable to the determination of carbon isotope ratios in doping controls and the approach allowed for an accelerated sample preparation. Graphical abstract.

Authors+Show Affiliations

German Sport University Cologne, Center for Preventive Doping Research - Institute of Biochemistry, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.German Sport University Cologne, Center for Preventive Doping Research - Institute of Biochemistry, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.Analytical Laboratory, CER Groupe, Rue du Point du Jour 8, 6900, Marloie, Belgium.Analytical Laboratory, CER Groupe, Rue du Point du Jour 8, 6900, Marloie, Belgium.German Sport University Cologne, Center for Preventive Doping Research - Institute of Biochemistry, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany. thevis@dshs-koeln.de.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31641821

Citation

Putz, Marlen, et al. "Analysis of Endogenous Steroids in Urine By Means of Multi-immunoaffinity Chromatography and Isotope Ratio Mass Spectrometry for Sports Drug Testing." Analytical and Bioanalytical Chemistry, vol. 411, no. 28, 2019, pp. 7563-7571.
Putz M, Piper T, Dubois M, et al. Analysis of endogenous steroids in urine by means of multi-immunoaffinity chromatography and isotope ratio mass spectrometry for sports drug testing. Anal Bioanal Chem. 2019;411(28):7563-7571.
Putz, M., Piper, T., Dubois, M., Delahaut, P., & Thevis, M. (2019). Analysis of endogenous steroids in urine by means of multi-immunoaffinity chromatography and isotope ratio mass spectrometry for sports drug testing. Analytical and Bioanalytical Chemistry, 411(28), 7563-7571. https://doi.org/10.1007/s00216-019-02169-3
Putz M, et al. Analysis of Endogenous Steroids in Urine By Means of Multi-immunoaffinity Chromatography and Isotope Ratio Mass Spectrometry for Sports Drug Testing. Anal Bioanal Chem. 2019;411(28):7563-7571. PubMed PMID: 31641821.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Analysis of endogenous steroids in urine by means of multi-immunoaffinity chromatography and isotope ratio mass spectrometry for sports drug testing. AU - Putz,Marlen, AU - Piper,Thomas, AU - Dubois,Michel, AU - Delahaut,Philippe, AU - Thevis,Mario, Y1 - 2019/11/15/ PY - 2019/06/26/received PY - 2019/09/24/accepted PY - 2019/09/04/revised PY - 2019/10/24/pubmed PY - 2019/10/24/medline PY - 2019/10/24/entrez KW - Carbon isotope ratio KW - Compound-specific isotope analysis (CSIA) KW - Doping KW - Drug monitoring KW - Immunoaffinity purification KW - Testosterone SP - 7563 EP - 7571 JF - Analytical and bioanalytical chemistry JO - Anal Bioanal Chem VL - 411 IS - 28 N2 - Detecting the administration of naturally occurring but synthetically derived steroids (e.g., testosterone) in routine doping controls is particularly laborious and time-consuming. Carbon isotope signatures determined by isotope ratio mass spectrometry (IRMS) have been established as the method of choice to generate confirmatory evidence in case of suspicious or atypical findings in steroid profile analyses; however, IRMS measurements require sophisticated sample preparation methods employing up to two high-performance liquid chromatography (HPLC) purification steps. Here, an alternative sample preparation approach is presented. Immunoaffinity chromatography (IAC) was employed to reduce the batch analysis time by omitting the time-consuming HPLC purification steps, while pre- and post-IAC sample handling followed published protocols. IAC exploits specific antibody-immunogen interactions, and the option of combining three immunoaffinity gels containing specific antibodies for testosterone, pregnanediol, and 11-ketoetiocholanolone into a multi-immunoaffinity sample preparation approach was assessed. Due to cross reactivities, also etiocholanolone, androsterone, 5β-androstanediol, and 5α-androstanediol were co-extracted and included in the testing protocol. The method was validated by determining precision, recovery, and carry over, and performing linear mixing models. IAC was found to be applicable to the determination of carbon isotope ratios in doping controls and the approach allowed for an accelerated sample preparation. Graphical abstract. SN - 1618-2650 UR - https://www.unboundmedicine.com/medline/citation/31641821/Analysis_of_endogenous_steroids_in_urine_by_means_of_multi-immunoaffinity_chromatography_and_isotope_ratio_mass_spectrometry_for_sports_drug_testing L2 - https://dx.doi.org/10.1007/s00216-019-02169-3 DB - PRIME DP - Unbound Medicine ER -