Screening of testosterone esters in human plasma

Abstract

The detection of an intact ester of testosterone in plasma is leading towards unequivocal proof of the administration of exogenous testosterone. In the current study, a sensitive screening method for the detection of nine testosterone esters in human plasma was developed. By preparing oxime derivatives of intact testosterone esters, the sensitivity of the assay was increased. Furthermore, the method included liquid-liquid extraction (LLE) as sample clean-up, as well as online separation of the target analytes from the derivatization solution. The analysis was performed by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The method developed herein is simple and rapid, and was validated according to World Anti-Doping Agency (WADA) guidelines.

Detection of testosterone esters in blood

“…0.025% H 2 O) and all solvents (high performance liquid chromatography (HPLC) grade) were obtained from Merck (Darmstadt, Germany). 17α‐Methyltestosterone, hydroxylamine hydrochloride (≥99.0 %), pyridine (anhydrous, 99.8 %), and formic acid (analytical grade) were supplied by Sigma (St Louis, MO, USA), whereas deuterium labelled (d6) testosterone acetate was made by in‐house synthesis . An internal standard (IS) solution was prepared with methyltestosterone and d6‐testosterone acetate at a concentration level of 100 ng/mL and 50 ng/mL, respectively.…”

“…Column selection was performed by a Maylab Mistraswitch column selector (6 column selection system) (Maylab Analytical Instruments, Vienna, Austria). All analytical conditions were set as previously described for the assay …”

“…The solvents were 0.2% formic acid in water (A) and methanol containing 0.1% formic acid (B) and the eluent was diverted from the MS/MS to waste for the first 7 min following each injection. Detailed chromatographic conditions have been previously described . The mass spectrometer was equipped with a heated electrospray ionization (ESI) source and was operated in positive ionization mode with a spray voltage set at 3000 V. The capillary temperature was adjusted to 300 °C.…”

“…The detection window for the testosterone esters are depending on the half‐life of the ester, as well as the sensitivity of the analytical method. The analytical method applied in the current study was previously developed by the laboratory, and involves the detection of oxime derivatives, in order to achieve low limits of detection (LODs) …”

Highly sensitive avidin‐biotin ELISA for detection of nandrolone and testosterone in dietary supplements

“…[6][7][8][9] Some of them come from the black market and some are intentionally added to food supplements and sold as protein supplementation, vitamins, minerals, or carbohydrates, for example. In most cases, simple 17β-esters [14] are seized (e.g. Otherwise AAS pose a threat to those people who merely take dietary supplements to maintain performance and vitality.…”