Detection of Methylphenidate in Equine Hair Using Liquid Chromatography-High-Resolution Mass Spectrometry.

Benjamin C Moeller,Amel Clifford,Luis Flores,Rick M Arthur,Mary Mosburg,Gwendolyne Alarcio

doi:10.3390/molecules26195798

Abstract

Methylphenidate is a powerful central nervous system stimulant with a high potential for abuse in horse racing. The detection of methylphenidate use is of interest to horse racing authorities for both prior to and during competition. The use of hair as an alternative sampling matrix for equine anti-doping has increased as the number of detectable compounds has expanded. Our laboratory developed a liquid chromatography–high-resolution mass spectrometry method to detect the presence of methylphenidate in submitted samples. Briefly, hair was decontaminated, cut, and pulverized prior to liquid–liquid extraction in basic conditions before introduction to the LC-MS system. Instrumental analysis was conducted using a Thermo Q Exactive mass spectrometer using parallel reaction monitoring using a stepped collision energy to obtain sufficient product ions for qualitative identification. The method was validated and limits of quantitation, linearity, matrix effects, recovery, accuracy, and precision were determined. The method has been applied to confirm the presence of methylphenidate in official samples submitted by racing authorities.

Highlights

Methylphenidate is a synthetic central nervous system stimulant frequently prescribed to treat attention deficit disorder and narcolepsy in humans [1]
Methylphenidate was first synthesized in the 1940s, approved by the US FDA in the mid-1950s, and marketed by its brand name Ritalin
Studies conducted in the 1960s clearly show that methylphenidate administration to horses at doses ranging from ≈0.1 to 1.2 mg/kg have effects on the central nervous system with increases in pulse, respiratory rate, and blood pressure observed [3]

Summary

Introduction

Methylphenidate is a synthetic central nervous system stimulant frequently prescribed to treat attention deficit disorder and narcolepsy in humans [1]. Studies conducted in the 1960s clearly show that methylphenidate administration to horses at doses ranging from ≈0.1 to 1.2 mg/kg have effects on the central nervous system with increases in pulse, respiratory rate, and blood pressure observed [3]. The absorption, distribution, metabolism, and excretion properties of methylphenidate or structurally similar agents such as ethylphenidate have not been investigated in great detail in the horse, but some studies have shown that these compounds are relatively quickly cleared and primarily excreted in urine following intramuscular, intravenous, or oral administrations [3,4,5]. The major metabolite of methylphenidate in humans is ritalinic acid. Studies with incubations of methylphenidate in equine liver microsomes confirm that

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