Detection and quantification of 12 anabolic steroids and analogs in human whole blood and 20 in hair using LC-HRMS/MS: application to real cases.

Abstract

We developed and validated a method to detect and quantify 12 anabolic steroids in blood (androstenedione, dihydrotestosterone, boldenone, epitestosterone, mesterolone, methandienone, nandrolone, stanozolol, norandrostenedione, tamoxifene, testosterone, trenbolone) and eight more in hair samples (nandrolone phenylpropionate, nandrolone decanoate, testosterone propionate, testosterone benzoate, testosterone cypionate, testosterone decanoate, testosterone phenylpropionate, testosterone undecanoate) using liquid chromatography coupled to high-resolution mass spectrometry. This method used a benchtop Orbitrap mass spectrometer operating with an APCI probe under positive ionization mode. Analysis was realized in full scan experiment with a nominal resolving power of 140,000. After addition of the internal standard (testosterone-D3) and incubation in phosphate buffer pH=5 for hair, 200μL of blood and 30mg of hair samples were extracted with heptane. LOQ and LOD were determined at 5 and 1ngmL-1 in whole blood and 10 to 100pgmg-1 and 2 to 20pgmg-1 in hair according to the compounds, respectively. The method was linear in the 5-1000ngmL-1 range in whole blood and between 10 or 100pgmg-1 and 1000pgmg-1 in hair with correlation coefficients >0.99, and intra- and inter-day accuracy and precision were <14.8% for all compounds except for some esters in hairs (<19.9%) probably due to an important matrix effect for these compounds. This sensitive and specific method to detect anabolic steroids has been successfully applied to two real cases, for which various anabolic steroids in whole blood, urine, and hair were identified and quantified.