Use of high resolution/accurate mass full scan/data-dependent acquisition for targeted/non-targeted screening in equine doping control

You, Youwen; Proctor, Rachel M; Guo, Kevin; Li, Xiaoqing; Xue, Evan; Guan, Fuyu; Robinson, Mary Ann

doi:10.1039/d0ay02297g

Cited by 8 publications

(8 citation statements)

References 31 publications

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“…Stock ammonium formate (HCOONH 4 ) buffer solution comprising 1.0 mol/L (M) HCOONH 4 and 1.0 M FA (pH 3.6) and working buffer solution containing 5 mM each of HCOONH 4 and FA were made. The details about the drug standards, solvents, chemicals, reagents, and their preparations are described elsewhere . The name, chemical formula, exact mass of the protonated species, RT, and initial concentration of the 57 drugs spiked into equine plasma are listed in Table S1.…”

Section: Methodsmentioning

confidence: 99%

“…This LOD is close to that achieved using the targeted analytical method. 21 In addition, 26 of the 55 drugs had a low LOD (0.05 to 0.2 ng/mL), 16 of the drugs had a slightly higher LOD (0.5 to 2 ng/mL), and 5 drugs (piroxicam, 2-(1-hydroxyethyl)promazine sulfoxide, benzoylecgonine, theophylline, and caffeine) had a relatively high LOD (5 to 100 ng/mL). Although the LOD for caffeine was 100 ng/mL, it was actually detected at 0.1 ng/mL by the CD processing workflow but was filtered out by ROM (Table S6).…”

Section: Untargeted Detection Of Drugs Spiked Into Blankmentioning

confidence: 98%

“…The details about the drug standards, solvents, chemicals, reagents, and their preparations are described elsewhere. 21 The name, chemical formula, exact mass of the protonated species, RT, and initial concentration of the 57 drugs spiked into equine plasma are listed in Table S1. Notably, exact mass and RT of the drugs were not utilized for their detection during method development but were instead used to verify drug detection once the method was developed.…”

Section: ■ Experimental Detailsmentioning

confidence: 99%

“…The details about sample preparation and extraction are described elsewhere. 21 Extraction of race plasma samples and sample preparation for hydrophilic interaction liquid chromatography (HILIC) analysis are provided in the Supporting Information.…”

Section: ■ Experimental Detailsmentioning

confidence: 99%

“…Plasma by the Proposed Algorithms. Fifty seven drugs that were all detectable by our targeted LC−HRMS method at the initial concentration, shown in Table S1, 21 were spiked into blank plasma for the development of the UDD methodology. These drugs could be considered as unknowns for the purpose of UDD method development their exact masses and RTs were not used for their detection.…”

Section: Untargeted Detection Of Drugs Spiked Into Blankmentioning

confidence: 99%

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Novel Algorithms for Comprehensive Untargeted Detection of Doping Agents in Biological Samples

et al. 2021

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To address the limitations of current targeted analytical methods that can only detect known doping agents, a novel methodology that permits untargeted drug detection (UDD) has been developed to help in the fight against doping in sports. Fiftyseven drugs were spiked into blank equine plasma and were treated as unknowns since their exact masses and chromatographic retention times were not utilized for detection. The spiked drugs were extracted from the plasma samples and were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC−HRMS). The acquired LC−HRMS raw data files were processed using metabolomic software for compound detection and identification. For UDD with the resultant data, a mathematical model was created, and two algorithms were generated to calculate the ratio of the mean (ROM) and outlier index (OLI). Using ROM and OLI, the majority of the 57 drugs were accurately detected by name (52 of 57) or chemical formula (1 of 57). The limit of detection for the drugs was from tens of picograms to nanograms per milliliter. Xenobiotics and endogenous substances relevant to doping control were also identified using this untargeted approach following their extraction from real-world race samples, thus validating the UDD methodology. To the authors' knowledge, this is the first completely UDD methodological approach and represents significant advance toward using artificial intelligence for the detection of both known and emerging doping agents in sports.

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Section: Methodsmentioning

confidence: 99%

Section: Untargeted Detection Of Drugs Spiked Into Blankmentioning

confidence: 98%

Section: ■ Experimental Detailsmentioning

confidence: 99%

Section: ■ Experimental Detailsmentioning

confidence: 99%

Section: Untargeted Detection Of Drugs Spiked Into Blankmentioning

confidence: 99%

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Novel Algorithms for Comprehensive Untargeted Detection of Doping Agents in Biological Samples

et al. 2021

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Analytical advances in horseracing medication and doping control from 2018 to 2023

Gray,

Lubbock,

Love

et al. 2024

Drug Testing and Analysis

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The analytical approaches taken by laboratories to implement robust and efficient regulation of horseracing medication and doping control are complex and constantly evolving. Each laboratory's approach will be dictated by differences in regulatory, economic and scientific drivers specific to their local environment. However, in general, laboratories will all be undertaking developments and improvements to their screening strategies in order to meet new and emerging threats as well as provide improved service to their customers. In this paper, the published analytical advances in horseracing medication and doping control since the 22nd International Conference of Racing Analysts and Veterinarians will be reviewed. Due to the unprecedented impact of COVID‐19 on the worldwide economy, the normal 2‐year period of this review was extended to over 5 years. As such, there was considerable ground to cover, resulting in an increase in the number of relevant publications included from 107 to 307. Major trends in publications will be summarised and possible future directions highlighted. This will cover developments in the detection of ‘small’ and ‘large’ molecule drugs, sample preparation procedures and the use of alternative matrices, instrumental advances/applications, drug metabolism and pharmacokinetics, the detection and prevalence of ‘endogenous' compounds and biomarker and OMICs approaches. Particular emphasis will be given to research into the potential threat of gene doping, which is a significant area of new and continued research for many laboratories. Furthermore, developments in analytical instrumentation relevant to equine medication and doping control will be discussed.

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Development and validation of general plasma screening method for performance enhancing drugs in racehorses utilizing liquid chromatography–high‐resolution mass spectrometry (LC–HRMS)

Lomnicka,

Dubey,

Waller

et al. 2024

Drug Testing and Analysis

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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.

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Use of high resolution/accurate mass full scan/data-dependent acquisition for targeted/non-targeted screening in equine doping control

Abstract: High-resolution mass spectrometry (HRMS) is a very powerful technology for equine doping control analysis. The more recently developed hybrid type of Orbitrap-based HRMS instrument allows for both targeted and non-targeted...

Cited by 8 publications

References 31 publications

Novel Algorithms for Comprehensive Untargeted Detection of Doping Agents in Biological Samples

Novel Algorithms for Comprehensive Untargeted Detection of Doping Agents in Biological Samples

Analytical advances in horseracing medication and doping control from 2018 to 2023

Development and validation of general plasma screening method for performance enhancing drugs in racehorses utilizing liquid chromatography–high‐resolution mass spectrometry (LC–HRMS)

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