The medication violations in racehorses at Louisiana racetracks from 2016 to 2020

Abstract Introduction/Background The number of publications for most common drug violations in racehorses is limited. This study reports the most common medication violations in racehorses at four major racetracks in Louisiana between 2016 and 2020. Methods During this 5‐year period, 27,237 blood samples and 25,672 urine samples collected during the course of normal race meeting activities were analysed by initial screening procedure utilizing Liquid Chromatography Mass Spectrometry (LC‐MS/MS). Following initial screening, suspect samples were subject to quantitative or semi‐ quantitative confirmation analysis by LC‐MS/MS. Results The total number of violations reported was 534 (1.01% of the total number of specimens analysed). The total number of violations reported in Thoroughbred horses was 210 while the total number of violations reported in Quarter Horses was 324. The percentage of total violations was %0.59 for all the specimens analysed in Thoroughbred horses while this percentage was %1.9 for all the specimens analysed in Quarter Horses during this 5‐year period. The most frequent violations included the overages (concentrations of permitted medications equal to or exceeding the set threshold) of clenbuterol (165 violations), non‐steroidal anti‐inflammatory drugs (NSAIDs) such as phenylbutazone (73 violations), combination of phenylbutazone with flunixin (45 violations) and muscle relaxant methocarbamol (40 violations). Discussion/Conclusions The total number of violations were relatively low during 5‐year period, but wide varieties of medications with different pharmacological actions were confirmed in performance horses in Louisiana. The most frequently reported violations in Louisiana were for permitted therapeutic medications (clenbuterol, phenylbutazone, flunixin methocarbamol) with established threshold and/or withdrawal guidelines in racehorses.


INTRODUCTION
The number of test samples for drug screening by anti-doping laboratories continues to grow. Thus, the challenge faced by equine and human doping control laboratories is the increase in easily available drugs that are capable of affecting performance of equine and human athletes (Liu et al., 2011). Increased access to drugs that are now available without prescriptions through internet purchase further compounds the problem. Today, many drugs known to have a performance-enhancing effect are prohibited in racehorses by the Association of Racing Commission International (ARCI). Within these guidelines, ARCI has established five classes of prohibited substances ranging from Class 1 substances, those demonstrating the greatest potential to enhance performance, to Class 5 substances, those demonstrating the least potential for enhancing performance ( Drug Testing Standards and Practices Program, 2020;ARCI, 2021a, b). Drugs that are intended for use in horses are found in lower classes. Drugs that are not intended for use in horses are placed in higher classes, particularly, if they might affect the outcome of a race. Drugs that are recognized as legitimately useful in racehorses but could affect the outcome of a race are found in the middle or higher classes (Drug Testing Standards and Practices Program, 2021;ARCI, 2021a, b)).
The ARCI Drug Classification Scheme is based on pharmacology, drug use patterns and the appropriateness of a drug for use in the racing horse. Drugs that are known to be potent stimulants or depressants are placed in higher classes, while those that have little effect on the outcome of a race are placed in lower classes. Some considerations are also given to placement of drugs based on practical experience with their use and the nature of positive tests. Twenty-eight medications are currently approved for usage in performance horses with established thresholds and/or withdrawal guidelines by ARCI (2021b).

Instrumentation and methodology
The analytical methodologies used for the plasma and urine screening were validated as per the ISO: IEC 17025:2017 guidelines for horse racing laboratories in compliance with Association of Official Racing Chemists (AORC,2021) and ARCI standards. The identification of substances was based on the compound's chromatographic and mass spectrometric properties. Instrument mass calibration (positive and negative), cleaning of sweep cone and ion transfer tube was done before every batch run. After calibration and cleaning, a system suitability test was performed before every batch run to verify the instrument performance and its suitability for the run. The methods used were validated on the following parameters including system suitability, specificity, identification capacity, matrix effect, precision, extraction efficiency, limit of detection (LOD) and carry-over. Liquid chromatography highresolution mass spectroscopy screening analysis was performed using

RESULTS
During  (Table 1). Tables 2 and 3 (Tables 4 and 5). The percentage total violations was 0.59% for all the specimens analyzed in Thoroughbred horses while this percentage was 1.9% for all the specimens analysed in Quarter Horses during this 5-year period (Tables 4 and 5).
In the study presented here, the most common violations were reported for ARCI Class 4 medications followed by Class 3, Class 2, Class 1 and Class 5 medications (Tables 4 and 5 (Table 7).

DISCUSSION AND CONCLUSIONS
The data obtained from four major racetracks in Louisiana indicated that ARCI Class 4 drugs were the most common violations ( tion associated with respiratory disease in horses (Erichsen et al., 1994;Sasse and Hajer, 1978). While clenbuterol does provide bronchodilation, clenbuterol also has repartitioning effects on skeletal muscle that mimic the anabolic effects of androgenic/anabolic steroids Kearns et al., 2001, Reeds et al., 1986Ricks et al., 1984;Spurlocket al., 2006). World Anti-Doping Agency lists clenbuterol as a banned anabolic agent along with other beta-2 agonists.
Currently, ARCI Uniform Classification of Foreign Substances identifies clenbuterol as a Class 3 substance.
The second most frequent violations were for the overages of phenylbutazone (73 violations) and combination of phenylbutazone with flunixin (45 violations). A common practice in racetrack medicine in the USA is to administer the two NSAIDs within close proximity (24 hours apart) of each other. Phenylbutazone and flunixin meglumine are arguably two of the most commonly administered therapeutic substances in racetrack medicine. In equine medicine, phenylbutazone has continued to dominate the treatment of pain, particularly, that is associated with joint and muscle conditions (Tobin et al., 1986). Advantages of phenylbutazone in equine medicine include extensive clinical experience of efficacy and safety over both short-and long-treatment periods, and availability in parenteral and a range of oral (powder, paste and bolus) formulations (Lees and Higgins, 1985;Lees et al., 1986). Flunixin is a NSAID analgesic and anti-pyretic used in horses, cattle and pigs.
It is often formulated as the meglumine salt (Lees and Higgins, 1985).
Flunixin is recommended for the alleviation of inflammation and pain associated with musculoskeletal disorders in the horse. It is also recommended for the alleviation of visceral pain associated with colic in the horse (Knych et al., 2021;Ziegler et al., 2019). Florham Park, NJ, USA) (Rumpler et al., 2014). Compounding pharmacies advertise that they prepare methocarbamol for use in horses in various formulations including oral powders, capsules, oral paste, oral suspension in oil and a transdermal gel.
The ARCI Uniform Classification of Foreign Substances currently identifies methamphetamine as a Class 1 substance. Methamphetamine is a strong CNS stimulant mainly used as a recreational drug and less commonly as a treatment for Attention Deficit Hyperactivity Disorder and obesity (Brewer et al., 2016;Cruickshank and Dyer, 2009;Knych et al., 2019). Methamphetamine exists as two enantiomers: dextro-methamphetamine and levo-methamphetamine (Brewer et al., 2016). Dextro-methamphetamine is a much stronger central stimulant than levo-methamphetamine (Brewer et al., 2016).
There is no veterinary approved usage of methamphetamine in animals. L-methamphetamine (absent any D stereoisomer) is present in over the counter inhalers with FDA approval for the treatment of cold and flu symptoms (vicks, vapour inhaler, etc.) (Brewer et al., 2016).
There were 11 d-methamphetamine violations in 2019 from four major racetracks in Louisiana. The LSRC recognizes that certain ARCI Class 1 and 2 substances not natural to the horse may be detected in trace amounts in official samples collected after race due solely to their prevalence in nature and/or the racing environment. For this reason, the LSRC established a urinary threshold for methamphetamine at 10 ng/mL regardless of the isomer found in a particular sample in 2020.
Levamisole is an anti-helminthic drug and gained forensic interest after it was found that it was used as a cocaine adulterant. It also has  (Tobin et al., 2013). Given these circumstances, the optimal strategy is to select medications and administration routes resulting in the shortest detection times possible, and to make statistically appropriate allowance for the major unknown in this process, namely, horse-to-horse biological variability (Tobin et al., 2013). It is also important that the racetrack veterinarians and trainers should strictly follow the ARCI Controlled Therapeutic Medication Schedule for Horses in terms of dosing, route of administration, dosing interval and duration of treatment, in addition to, drug formulations approved when using therapeutic medications in racehorses to avoid medication violations. Additionally, the racetrack veterinarians and trainers should also be familiar with rules and regulations established by a Racing Commission in a particular state to avoid any medication violations.