Andre Knoop1, Andreas Thomas1, Mario Thevis1,2. 1. Center for Preventive Doping Research, German Sport University Cologne,, Institute of Biochemistry, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany. 2. European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany.
Abstract
RATIONALE: The mitochondrial open reading frame of 12S rRNA type-c (MOTS-c) peptide was recently discovered and described to control metabolic homeostasis through AMPK activation along with AICAR accumulation. Consequently, it appears advisable to monitor the potential use of synthetic MOTS-c in sports, and a detection method suitable for sports drug testing purposes is necessary. METHODS: For the detection of MOTS-c in doping control plasma samples, a test method employing liquid chromatography and mass spectrometry (LC/MS) was developed. Following optimization, the assay was comprehensively validated and additional parameters such as the (long-term) stability and in vitro metabolism of the peptide were evaluated. In order to determine endogenous MOTS-c reference limits, the results generated by LC/MS-based detection were compared with those obtained with a commercially available enzyme-linked immunosorbent assay (ELISA). RESULTS: The LC/MS-based test method was fully validated for quantitative results interpretation according to the World Anti-Doping Agency's International Standard for Laboratories (WADA's ISL). It was found to be specific and sensitive, enabling a lower limit of detection (LLOD) for hMOTS-c in plasma at 100 pg/mL. Following optimization, animal MOTS-c analogues and four metabolites as well as two oxidation products were implemented. However, endogenous levels of a reference population of 20 healthy subjects studied by ELISA experiments (45.9-218.5 ng/mL) could not be confirmed by LC/MS. CONCLUSIONS: A mass spectrometric detection assay for MOTS-c in human plasma samples was developed and successfully validated according to WADA's ISL, providing an additional tool for future doping control purposes. Besides MOTS-c, the assay also includes four in vitro derived metabolites and two oxidation products, which might further improve the traceability of the drug. The analytical approach was compared with a commercially available ELISA, and considerable differences in measured MOTS-c levels were observed.
RATIONALE: The mitochondrial open reading frame of 12S rRNA type-c (MOTS-c) peptide was recently discovered and described to control metabolic homeostasis through AMPK activation along with AICAR accumulation. Consequently, it appears advisable to monitor the potential use of synthetic MOTS-c in sports, and a detection method suitable for sports drug testing purposes is necessary. METHODS: For the detection of MOTS-c in doping control plasma samples, a test method employing liquid chromatography and mass spectrometry (LC/MS) was developed. Following optimization, the assay was comprehensively validated and additional parameters such as the (long-term) stability and in vitro metabolism of the peptide were evaluated. In order to determine endogenous MOTS-c reference limits, the results generated by LC/MS-based detection were compared with those obtained with a commercially available enzyme-linked immunosorbent assay (ELISA). RESULTS: The LC/MS-based test method was fully validated for quantitative results interpretation according to the World Anti-Doping Agency's International Standard for Laboratories (WADA's ISL). It was found to be specific and sensitive, enabling a lower limit of detection (LLOD) for hMOTS-c in plasma at 100 pg/mL. Following optimization, animal MOTS-c analogues and four metabolites as well as two oxidation products were implemented. However, endogenous levels of a reference population of 20 healthy subjects studied by ELISA experiments (45.9-218.5 ng/mL) could not be confirmed by LC/MS. CONCLUSIONS: A mass spectrometric detection assay for MOTS-c in human plasma samples was developed and successfully validated according to WADA's ISL, providing an additional tool for future doping control purposes. Besides MOTS-c, the assay also includes four in vitro derived metabolites and two oxidation products, which might further improve the traceability of the drug. The analytical approach was compared with a commercially available ELISA, and considerable differences in measured MOTS-c levels were observed.
Authors: Joseph C Reynolds; Rochelle W Lai; Jonathan S T Woodhead; James H Joly; Cameron J Mitchell; David Cameron-Smith; Ryan Lu; Pinchas Cohen; Nicholas A Graham; Bérénice A Benayoun; Troy L Merry; Changhan Lee Journal: Nat Commun Date: 2021-01-20 Impact factor: 14.919