Detection of seventy-two anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry in multiplexed targeted MS2 mode and gas chromatography-tandem mass spectrometry.

Anabolic and androgenic steroids (AAS) are banned substances in both human and equine sports. They are often administered intramuscularly to horses in esterified forms for the purpose of extending their time of action. The authors' laboratory has previously reported an UHPLC/HRMS method using quadrupole-Orbitrap mass spectrometer in full scan and parallel reaction monitoring (PRM) mode for the detection of 48 AAS and/or their esters in horse hair. However, two injections were required due to the long duty cycle time. In this paper, an UHPLC/HRMS method using multiplexed targeted MS2 mode was developed and validated to improve the coverage to 65 AAS and/or their esters in a single injection. In addition, a GC/MS/MS method in selected reaction monitoring (SRM) mode was developed to screen for another seven AAS and/or their esters not adequately covered by the UHPLC/HRMS method using the same sample extract after derivatisation with pentafluoropropionic anhydride. The UHPLC/HRMS and GC/MS/MS methods in combination allowed the detection of 72 AAS and/or their esters with estimated limits of detection down to sub to low ppb levels with good interday precision. Method applicability was demonstrated by the detection of boldione and 4-androstenedione in two out-of-competition hair samples and testosterone propionate in a referee hair sample.

Screening of over 100 drugs in horse urine using automated on-line solid-phase extraction coupled to liquid chromatography-high resolution mass spectrometry for doping control.

A fast method for the direct analysis of enzyme-hydrolysed horse urine using an automated on-line solid-phase extraction (SPE) coupled to a liquid-chromatography/high resolution mass spectrometer was developed. Over 100 drugs of diverse drug classes could be simultaneously detected in horse urine at sub to low parts per billion levels. Urine sample was first hydrolysed by ß-glucuronidase to release conjugated drugs, followed by centrifugal filtration. The filtrate (1mL) was directly injected into an on-line SPE system consisting of a pre-column filter and a SPE cartridge column for the separation of analytes from matrix components. Through valves-switching, the interfering matrix components were flushed to waste, and the analytes were eluted to a C18 analytical column for refocusing and chromatographic separation. Detections were achieved by full-scan HRMS in alternating positive and negative electrospray ionisation modes within a turn-around time of 16min, inclusive of on-line sample clean-up and post-run mobile phase equilibration. No significant matrix interference was observed at the expected retention times of the targeted masses. Over 90% of the drugs studied gave estimated limits of detection (LoDs) at or below 5ng/mL, with some LoDs reaching down to 0.05ng/mL. Data-dependent acquisition (DDA) was included to provide additional product-ion scan data to substantiate the presence of detected analytes. The resulting product-ion spectra can be searched against an in-house MS/MS library for identity verification. The applicability of the method has been demonstrated by the detection of drugs in doping control samples.