Determining the optimum derivatizing agents and parameters to improve the detection of anabolic-androgenic steroids

Introduction: Anabolic-androgenic steroids (AASs) typically require derivatization to increase the volatility and/or thermal stability of compounds to make the compound more amenable for gas chromatography-mass spectrometry (GC-MS) analysis. The typical method of derivatization used for AASs is trimethylation using MSTFA with the occasional addition of various catalysts, although some methods also utilize BSTFA with 1% TMCS. Typically, derivatization of AASs via the addition of MSTFA is usually carried out by heating at 60°C for 1hr. Publications have used microwave assisted derivatization (MAD) for steroids which has shown to significantly decrease long derivatization times.
Objective: To determine the yields of AASs subjected to traditional derivatization methods and microwave-assisted derivatization (MAD).

Methods: To 4 mL vials (n=5), 100 μL (10 ug/mL) of methanolic solutions of 17α-methyltestosterone, 19-norandrosterone, 2α-methyl androsterone, 6β-hydroxy metandienone, boldenone, clenbuterol, clostebol, DHEA, drostanolone, epitestosterone, fluoxymesterone, testosterone, mestanolone, mesterolone, metandienone, methylclostebol, oxandrolone, oxymetholone and diazepam (internal standard) were added. Diazepam was chosen as the internal standard as this does not undergo derivatization. Vials were then evaporated to dryness using a Genevac (DNA-23050-A00). Samples (n=5) were then reconstituted in 50 μL MSTFA, BSTFA +1% TMCS or MSTFA/NH4I/ethanethiol before being microwaved at 700W using an iGENIX microwave (Model: IG2071) for 30s, 1mins, 3mins or 5mins. Additionally, the AAS mixes were subjected to room temperature (control), and conventional heating at 37°C, 50°C, 75°C, 90°C utilizing a TECHNE Dri-Block DB-2D for 15mins, 30mins,1hr and 2hrs. After heating, the AAS mixes were analyzed using an Agilent 7820A GC (column: Zebron ZB-1 (30m x 0.25mm, 0.25μm)) coupled with a mass spectrometer 5977B MSD (Agilent Technologies Inc., UK). The average peak area ratio (APAR) was calculated using the peak area response for diazepam and each analyte, allowing for a degree of normalization between parameters, as well as accounting for any inter- and intraday differences. SPSS (version 28) was used to conduct a MANOVA to determine any statistical significance of results.

Results: MSTFA/NH4I/ethanethiol outperformed MSTFA and BSTFA +1% TMCS (p<0.05) in almost all circumstances for all analytes. The overall optimal method was found to be MSTFA/NH4I/ethanethiol incubated for 15mins at 37°C using the traditional heat block. It was found that longer incubation times resulted in lower average peak area ratios (APARs) using the traditional heating block and microwave. As microwave samples were subjected to longer heat times the coefficient of variation also increased. This was potentially due to the difference in temperature experienced by the samples due to varied positioning within the microwave, meaning each vial was exposed to different paths of microwave deflection.

Discussion: The results of this project show that AASs can be successfully derivatized using MAD, producing consistently detectable APAR comparable to traditional heating block incubation methods, in a much shorter time frame. Overall, MSTFA/NH4I/ethanethiol was shown to produce the highest APARs for the AASs derivatized. The use of MAD significantly reduces sample preparation time resulting in faster GC-MS methods for the routine detection and analysis of these analytes.