limit,” or “screening limit”) necessary to control mis- use of legitimate therapeutic drugs. With input from stakeholders and veterinary experts, the rac- ing authorities establish the proper “limit” needed to prevent threats to racing that involve improper administration of drugs. Once the guidelines are issued, the laboratories are then responsible for se- lecting and applying the analytical method required to best verify compliance. In these cases, the labs use the fully validated quantitative methods re- viewed and approved by an external accrediting organization.


Sample Analysis is a 2-Step Procedure


Initially, all samples undergo a multi-stage solid phase extraction procedure, followed by analysis us- ing numerous screening methods. These screening methods most often employ chromatography sepa- ration coupled with mass-spectrometry detection. This combination affords simultaneous identifica- tion of thousands of compounds while providing limits of detection in the low parts-per-trillion con- centration. These extraordinary sensitivities are advantageous for detecting prohibited substances which have no therapeutic purpose in horse racing. However, if interpreted incorrectly, such as in the case of therapeutic drugs, these highly sensitive tools can result in positive findings days to weeks beyond any pharmacological effect. To avoid irrel- evant findings for therapeutic medications, compre- hensive standard operating procedures and method validation must be routine in all racing chemistry laboratories. A properly defined workflow includ- ing sample preparation, sample analysis, and data processing to meet predefined criteria are a mini- mum requirement. High-resolution accurate mass spectrometry is particularly important for biological samples that involve complex matrix (e.g., urine and blood). Full-scan approaches permit detection of unlimited numbers of analytes simultaneously. For a screen- ing method aiming at detection of both target analytes and unknowns, high-resolution instrumen- tation is a distinct advantage.


Confirmation


The use of mass spectrometry for confirmatory anal- ysis is mandatory in order to report a therapeutic excess or prohibited substance finding, in effect making this technique the gold standard for legally defensible data. The analytical technique of mass spectrometry is a measure of the mass-to-charge (m/z) ratio of ions, with the ratio of the analyte signal to the noise measured with a blank (back- ground). Contemporary mass spectrometers can operate in modes that provide very low background noise and have the ability to detect individual ions, enabling the extreme lower limits of detection (fem- tograms  1  1015 grams).


IN-DEPTH: MEDICATION AND THERAPEUTICS FOR RACEHORSES Limit of Detection and Limit of Quantitation


The limit of detection for analytical procedures is the endpoint of achievable determination by sta- tistical based measurement of the target analyte where the signal-to-noise is no less than 3:1. The limit of quantitation is the achievable determination by statistical-based measurement of the target ana- lyte where the signal-to-noise is no less than 10:1. When samples are analyzed the resultant data must be evaluated to determine whether the therapeutic medication is found and if administered within the parameters set by the method validation studies.


Method Validation


In common with human testing laboratories, the racing industry requires quantitative methods to establish the concentration of an analyte relative to a scientifically determined threshold medication. These quantitative methods must be validated using predefined acceptance criteria to characterize and document performance limitations. Experimental data must be collected to evaluate for accuracy, limit of detection, limit of quantitation, linearity, preci- sion, range, and specificity per US Food and Drug Administration–recommended guidelines. In addi- tion, it is a requirement of all quantitative methods to estimate the measurement of uncertainty for the procedure at the threshold value. Measurement of uncertainty is used when reporting the range of possible values within which the true value of the measurement lies. Combining these validation steps enhances the integrity of the analytical pro- cesses and provides assurance to stakeholders re- garding the reported findings.


3. New Emerging Trends/Threats


Proteins/Peptide A wide range of unregulated peptide-based drugs are now available through the internet with numer- ous Web sites offering an infinite array of purported performance enhancing products (e.g., proteins– growth hormones [GH-somatotrophin], AOD9604, Thymosin 4, Actovegin, Cerebrolysin, Hexarelin, Dermorphin analogues, growth hormone-releasing hormone [GHRH] analogues, GRF analogues, anti- inflammatory peptides, etc.). In general, peptides make poor drugs but they theoretically can make good doping agents. Peptides have poor bioavailability due to their low diffusion rate across cell membranes. They are rapidly degraded by endogenous proteases/pepti- dases, rapidly secreted by the kidneys and exhibit short half-lives after administration. All of these characteristics make peptides a challenge to detect in biological samples. The bulk of these products are intended to stimu-


late cellular processes or mimic the effects of endog- enous equine growth hormone, subsequently to build muscle, bone strength and speed recovery from injuries. The majority of the peptide products com-


AAEP PROCEEDINGS  Vol. 66  2020 375


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