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.
| Original language | English |
|---|---|
| Journal | Rapid communications in mass spectrometry : RCM |
| Volume | 33 |
| Issue number | 4 |
| Pages (from-to) | 371-380 |
| Number of pages | 10 |
| ISSN | 0951-4198 |
| DOIs | |
| Publication status | Published - 28.02.2019 |
Research areas and keywords
- Amino Acid Sequence
- Chromatography, High Pressure Liquid/methods
- Doping in Sports
- Humans
- Limit of Detection
- Male
- Mass Spectrometry/methods
- Mitochondrial Proteins/analysis
- Substance Abuse Detection/methods
