Development and validation of general plasma screening method for performance enhancing drugs in racehorses utilizing liquid chromatography-high-resolution mass spectrometry (LC-HRMS).
Abstract: The screening of drugs in plasma and urine often requires initial extraction (such as liquid-liquid extraction and solid-phase extraction) before the samples are submitted to instrumental analyses. These extraction procedures are often laborious and time-consuming. In this manuscript, a high-throughput automated assay based on liquid chromatography-high-resolution mass spectrometry (LC-HRMS) suitable for use as an initial testing procedure covering multiple classes of compounds prohibited in horse racing is described. The assay requires a 600-μL plasma aliquot, which is subjected to solid phase extraction (SPE) using OASIS HLB 96-well SPE with Biotage Extrahera system, evaporation, and reconstitution in a 96-well collection plate. LC-HRMS analyses were carried out on a Thermo Q-Exactive Mass spectrometer coupled with Thermo UHPLC system equipped with Thermo Accela ALS 2.4.0 autosampler linked to ACE Excel column. Drug targets were detected by retention time and accurate mass, with a mass tolerance window of 5 ppm in positive and negative ionization mode. The screening method was validated for over 300 drug targets in a 13-min run. Validation data including sensitivity, specificity, extraction recovery, and precision are presented. As the method employs full-scan mass spectrometry, unlimited number of drug targets can theoretically be incorporated into this method.
© 2024 John Wiley & Sons Ltd.
Publication Date: 2024-07-29 PubMed ID: 39075842DOI: 10.1002/dta.3774Google Scholar: Lookup
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Summary
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This study outlines the development and validation of a new, more efficient method of screening for performance enhancing drugs in racehorses using liquid chromatography-high-resolution mass spectrometry (LC-HRMS).
Objective of Research
- The primary objective of this research was to develop an efficient, high-throughput screening method for detecting a wide range of performance-enhancing drugs in racehorses. The researchers aim to enhance the efficiency and accuracy of drug detection using LC-HRMS technology, replacing laborious extraction procedures.
Methodology
- The proposed method involves using a 600-μL plasma aliquot from the horse, undergoing solid phase extraction (SPE), evaporation, and reconstitution in a 96-well collection plate.
- The LC-HRMS analyses were conducted on a Thermo Q-Exactive Mass spectrometer coupled with Thermo UHPLC system and Thermo Accela ALS 2.4.0 autosampler linked to an ACE Excel column.
- Retained drugs were detected by their retention time and accurate mass, with a mass tolerance window of 5 ppm in both positive and negative ion modes.
Validation
- The method was validated for over 300 drug targets, which were all detected within a 13-minute runtime.
- Validation metrics included sensitivity, specificity, extraction recovery, and precision, which were all found to be in acceptable ranges.
- The method’s full-scan mass spectrometry usage means that theoretically, an unlimited number of drug targets can be included in the screening procedure.
Implications
- The new LC-HRMS based screening method offers a quicker, more comprehensive way to detect a wide variety of performance-enhancing drugs in racehorses, with potential applications in other areas of animal and human drug testing.
- By providing an initial testing process that covers multiple classes of prohibited compounds, the method can help better regulate doping in horse racing and protect the integrity of the sport.
Cite This Article
APA
Lomnicka I, Dubey S, Waller P, Vora D, Dirikolu L.
(2024).
Development and validation of general plasma screening method for performance enhancing drugs in racehorses utilizing liquid chromatography-high-resolution mass spectrometry (LC-HRMS).
Drug Test Anal.
https://doi.org/10.1002/dta.3774 Publication
Researcher Affiliations
- Equine Medication Surveillance Laboratory (EMSL), Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
- Biomarkers Core Laboratory, Irving Institute for Clinical and Translational Research, Columbia University Medical Center, New York, New York, USA.
- Equine Medication Surveillance Laboratory (EMSL), Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
- Equine Medication Surveillance Laboratory (EMSL), Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
- Equine Medication Surveillance Laboratory (EMSL), Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
Grant Funding
- Louisiana State Racing Commission, New Orleans, LA 70119
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