Factors affecting untargeted detection of doping agents in biological samples.

Doping control is essential for sports, and untargeted detection of doping agents (UDDA) is the holy grail for anti-doping strategies. The present study examined major factors impacting UDDA with metabolomic data processing, including the use of blank samples, signal-to-noise ratio thresholds, and the minimum chromatographic peak intensity. Contrary to data processing in metabolomics studies, both blank sample use (either blank solvent or plasma) and marking of background compounds were found to be unnecessary for UDDA in biological samples, the first such report to the authors' knowledge.

Automated identification of unknown doping agents in confiscation samples by flow-injection mass spectrometry and mass spectral library searches.

Rapid and accurate identification of unknown compounds within suspicious samples confiscated for sports doping control and law enforcement drug testing is critical, but such analyses are often conducted manually and can be time-consuming. Here, we report a methodology for automated identification of unknown substances in confiscation samples by rapid automatic flow-injection analysis on a liquid chromatography coupled to high-resolution mass spectrometry system and identifying unknown compounds with Compound Discoverer software. The developed methodology was validated by comparing the automated identification results with those obtained from manual syringe-infusion experiments and manual tandem mass spectral library searches.

High Precision Calibration Algorithm for Binocular Stereo Vision Camera using Deep Reinforcement Learning.

Camera calibration is the most important aspect of computer vision research. To address the issue of insufficient precision, therefore, a high precision calibration algorithm for binocular stereo vision camera using deep reinforcement learning is proposed. Firstly, a binocular stereo camera model is established.

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

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. Fifty-seven 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).

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

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 screening analyses in a single liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) run. In the present study, an LC-HRMS/MS method was developed and validated to detect prohibited substances in equine sports.

Identification of ex vivo catabolites of peptides with doping potential in equine plasma by HILIC-HRMS.

Bioactive peptides pose a great threat to sports integrity. The detection of these peptides is essential for enforcing their prohibition in sports. Identifying the catabolites of these peptides that are formed ex vivo in plasma may improve their detection.

A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry.

Bioactive peptides possess pharmacological effects and can be illicitly used in sports. To deter such misuse, an untargeted method using high resolution mass spectrometry (HRMS) has been developed for comprehensive detection of multitudinous exogenous peptides in equine plasma and urine. Forty-four peptides were extracted using mixed-mode solid-phase extraction (SPE) from plasma and urine, separated with a hydrophilic interaction liquid chromatography (HILIC) column, and detected on an HRMS instrument.

High-throughput doping control analysis of 28 amphetamine-type stimulants in equine plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

A hydrophilic interaction liquid chromatography-tandem mass spectrometry method (HILIC-MS/MS) was developed for the simultaneous determination of 28 amphetamine-type stimulants (ATSs) in equine plasma for doping control analysis. In this method, stimulants were recovered from equine plasma by liquid-liquid extraction (LLE) at pH 9.5 using methyl tert-butyl ether and detected on a Thermo Finnigan triple quadrupole mass spectrometer operating in positive-ion mode electrospray ionization.

Detection and confirmation of α-cobratoxin in equine plasma by solid-phase extraction and liquid chromatography coupled to mass spectrometry.

α-Cobratoxin (CTX) is a large peptide (71 amino acids) with strong analgesic effect and may be misused in sports such as horse racing. To prevent such misuse, a sensitive method is required for detection and confirmation of the toxin in equine samples. CTX was extracted from equine plasma using an optimized mixed-mode solid-phase extraction (SPE) procedure.

Comprehensive solid-phase extraction of multitudinous bioactive peptides from equine plasma and urine for doping detection.

The ability to analyze biological samples for multitudinous exogenous peptides with a single analytical method is desired for doping control in horse racing. The key to achieving this goal is the capability of extracting all target peptides from the sample matrix. In the present study, theory of mixed-mode solid-phase extraction (SPE) of peptides from plasma is described, and a generic mixed-mode SPE procedure has been developed for recovering multitudinous exogenous peptides with remarkable sequence diversity, from equine plasma and urine in a single procedure.

Validated LC-MS-MS Method for Simultaneous Analysis of 17 Barbiturates in Horse Plasma for Doping Control.

A rapid and sensitive method for simultaneous screening, quantification and confirmation of 17 barbiturates in horse plasma using liquid chromatography-tandem mass spectrometry is described. Analytes were recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether, separated on a C18 column, and analyzed in negative electrospray ionization mode. Multiple-reaction monitoring was employed for screening and quantification.

Confirmatory analysis of etanercept in equine plasma by LC-MS for doping control.

Etanercept is a protein-based medication for the treatment of human patients with rheumatoid arthritis and other autoimmune-based diseases; its pharmacological action is to inhibit and antagonize tumour necrosis factor alpha. Etanercept was rumoured to be used in horse racing in North America. To detect such use, the aim of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) method for confirmation of etanercept in equine plasma.

Identification of sample donor by 24-plex short tandem repeat in a post-race equine plasma containing dexamethasone.

Background: Animal sport such as horseracing is tainted with drug abuse as are human sports. Treatment of racehorses on race day with therapeutic medications in most cases is banned, and thus, it is essential to monitor the illicit use of drugs in the racing horse to maintain integrity of racing, ensure fair competition and protect the health, safety and welfare of the horse, jockeys and drivers. In the event of a dispute over the identity of the sample donor, if the regulator can provide evidence that the DNA genotype profile of the post-race sample matched that of the alleged donor, then the potential drug violation case might be easily resolved without legal challenges.

Sensitive hydrophilic interaction liquid chromatography/tandem mass spectrometry method for rapid detection, quantification and confirmation of cathinone-derived designer drugs for doping control in equine plasma.

Rationale: Cathinone derivatives are new amphetamine-like stimulants that can evade detection when presently available methods are used for doping control. To prevent misuse of these banned substances in racehorses, development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method became the impetus for undertaking this study.

Methods: Analytes were recovered via liquid-liquid extraction using methyl tert-butyl ether.

Detection, quantification, and identification of dermorphin in equine plasma and urine by LC-MS/MS for doping control.

Dermorphin is a unique opioid peptide that is 30-40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples.

Validated UHPLC-MS-MS method for rapid analysis of capsaicin and dihydrocapsaicin in equine plasma for doping control.

A method involving ultra high-performance liquid chromatography-tandem mass spectrometry was developed and validated for the analysis of capsaicin and dihydrocapsaicin in equine plasma. The analytes were recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether and separated on a sub-2 micron column. The mobile phase was composed of 2 mM ammonium formate and methanol.

Ex vivo spontaneous generation of 19-norandrostenedione and nandrolone detected in equine plasma and urine.

19-Norandrostenedione (NAED) and nandrolone are anabolic-androgenic steroids (AASs). Nandrolone was regarded solely as a synthetic AAS until the 1980s when trace concentrations of apparently endogenous nandrolone were detected in urine samples obtained from intact male horses (stallions). Since then, its endogenous origin has been reported in boars and bulls; endogenous NAED and nandrolone have been identified in plasma and urine samples collected from stallions.

Pharmacokinetics of intra-articular, intravenous, and intramuscular administration of triamcinolone acetonide and its effect on endogenous plasma hydrocortisone and cortisone concentrations in horses.

Objective: To compare pharmacokinetics of triamcinolone acetonide (TA) following i.v., intra-articular (i.

Efficient use of retention time for the analysis of 302 drugs in equine plasma by liquid chromatography-MS/MS with scheduled multiple reaction monitoring and instant library searching for doping control.

Multiple drug target analysis (MDTA) used in doping control is more efficient than single drug target analysis (SDTA). The number of drugs with the potential for abuse is so extensive that full coverage is not possible with SDTA. To address this problem, a liquid chromatography tandem mass spectrometric method was developed for simultaneous analysis of 302 drugs using a scheduled multiple reaction monitoring (s-MRM) algorithm.

Simultaneous separation and determination of 16 testosterone and nandrolone esters in equine plasma using ultra high performance liquid chromatography-tandem mass spectrometry for doping control.

The potential for using testosterone and nandrolone esters in racehorses to boost the biological concentrations of these steroids and enhance athletic performance is very compelling and should be seriously considered in formulating regulatory policies for doping control. In order to regulate the use of these esters in racehorses, a sensitive and validated method is needed. In this paper, we report such a method for simultaneous separation, screening, quantification and confirmation of 16 testosterone and nandrolone esters in equine plasma by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).

Sequence elucidation of an unknown cyclic peptide of high doping potential by ETD and CID tandem mass spectrometry.

Identification of an unknown substance without any information remains a daunting challenge despite advances in chemistry and mass spectrometry. However, an unknown cyclic peptide in a sample with very limited volume seized at a Pennsylvania racetrack has been successfully identified. The unknown sample was determined by accurate mass measurements to contain a small unknown peptide as the major component.

Analysis of gabapentin in equine plasma with measurement uncertainty estimation by liquid chromatography-tandem mass spectrometry.

Gabapentin (GPT) is an antiepileptic drug that was approved in 1993 for use in the management of neurotrophic pain and as an adjunctive therapy for refractory partial seizure in humans. It is also being tested in veterinary medicine as an adjunctive medication in the treatment of pain due to laminitis, neuropathic, or chronic pain. Gabapentin is readily available by prescription and even on the internet; therefore, it has the potential of being used in racehorses to mask pain.

Confirmatory analysis of continuous erythropoietin receptor activator and erythropoietin analogues in equine plasma by LC-MS for doping control.

Continuous erythropoietin receptor activator (CERA) is the third generation of recombinant human erythropoietin (rhEPO) medication that retains the effect of promoting red blood cell production but has longer duration of action in the body. CERA, rhEPO, and darbepoetin alpha (DPO) can be misused to enhance performance in both human and equine athletes. To deter such misuse, a very selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has now been developed for identification of CERA, rhEPO, and DPO in equine plasma.

Detection and confirmation of 60 anabolic and androgenic steroids in equine plasma by liquid chromatography-tandem mass spectrometry with instant library searching.

In 2008, Pennsylvania (PA) became the first State in the USA to ban and enforce the ban on the use of anabolic and androgenic steroids (AAS) in equine athletes by using plasma for analysis. To enforce the ban, a rapid and high-throughput method for analysis of 60 AAS in equine plasma was developed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analytes were recovered from plasma by liquid-liquid extraction (LLE) using methyl tert-butyl ether, separated on a reversed-phase C₁₈ column and analyzed by electrospray ionization mass spectrometry.