Analysis of methyloxime derivatives of intact esters of testosterone and boldenone in equine plasma using ultra high performance liquid chromatography tandem mass spectrometry

Abstract

Analysis of equine plasma samples to detect the abuse of anabolic steroids can be complicated when the parent steroid is endogenous to the animal. Anabolic steroids are usually administered intramuscularly as synthetic esters and therefore detection of the exogenous esters provides unequivocal proof of illegal administration. An ultra high performance liquid chromatography tandem mass spectrometric (UPLC-MSMS) method for the analysis of esters of testosterone (propionate, phenylpropionate, isocaproate, and decanoate) and boldenone (undecylenate) in equine plasma has been developed. Esters were extracted from equine plasma using a mixture of hexane and ethyl acetate and treated with methoxyamine hydrochloride to form methyloxime derivatives. Metenolone enanthate was used as an internal standard. After chromatographic separation, the derivatized steroid esters were quantified using selected reaction monitoring (SRM). The limit of detection for all of the steroid esters, based on a signal to noise ratio (S/N) of 3:1, was 1-3 pg/mL. The lower limit of quantification (LLOQ) for the all of the steroid esters was 5 pg/mL when 2 mL of plasma was extracted. Recovery of the steroid esters was 85-97% for all esters except for testosterone decanoate which was recovered at 62%. The intra-day coefficient of variation (CV) for the analysis of plasma quality control (QC) samples was less than 9.2% at 40 pg/mL and less than 6.0% at 400 pg/mL. The developed assay was used to successfully confirm the presence of intact testosterone esters in equine plasma samples following intramuscular injection of Durateston® (mixed testosterone esters).

Detection of prohibited substances in equine hair by ultra‐high performance liquid chromatography–triple quadrupole mass spectrometry – application to doping control samples

“…11,15,16,19 Recently (Table 1). 25 Method robustness and specificity was assessed by analysing 10 individual samples spiked at 3 times the estimated LOD of each analyte and analysing the same samples as blanks (unspiked). These LODs are equivalent to and in most cases lower than those reported in previous methods.…”

Screening of testosterone esters in human plasma

“…[26] This is most likely due to E and Z geometries caused by a restricted rotation around the carbon-nitrogen double bond. [26] This is most likely due to E and Z geometries caused by a restricted rotation around the carbon-nitrogen double bond.…”

“…Some reports can be found in the literature on the analysis of such esters in human and animal blood plasma using mass spectrometry (MS), [23][24][25][26][27][28] in addition to several methods for detecting steroid esters in hair. [28][29][30][31][32][33][34][35][36] Occasionally, derivatization prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was employed.…”

“…[28][29][30][31][32][33][34][35][36] Occasionally, derivatization prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was employed. [25,26] In this study, we have attempted to improve the methodology to allow a more sensitive analysis of human plasma samples by the inclusion of a derivatization step using hydroxylamine prior to LC-MS/MS analysis. Thus, a sensitive screening method for nine synthetic testosterone esters in human plasma was developed.…”

Monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration: An efficient and innovative strategy to screen for nandrolone abuse in entire male horses

“…[7] This steroid has never been reported as endogenous in female or gelded male horses, however, and, in these cases nandrolone administration is unambiguously indicated by the simple detection of nandrolone or its known equine metabolites. [10,11] Alternative strategies such as steroid esters analysis in hair [12][13][14] and plasma [15][16][17] or isotopic deviation measurement (δ [13] C) using a gas chromatography isotopic ratio mass spectrometry (GC-C-IRMS) technique [18][19][20] have therefore been investigated in equine but also in bovine aiming at detecting nandrolone abuse. Therefore a simple qualitative and quantitative determination of its presence in entire male horses is insufficient to prove any corresponding abuse.…”