GC‐MS method development and validation for anabolic steroids in feed samples

Abstract

A GC-MS method for the determination of AAS used as growth promoting agents using SIM in piglet feed samples has been developed and validated, using testosterone as internal standard. The formation of volatile steroid derivatives was carried out by derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide. The optimum separation was achieved using a Zebron ZB-5 column under a gradient temperature elution, allowing the separation of steroids in 18 min. The required sample treatment process was discussed. A leaching using ACN, saponification using a binary NaOH/MgCl2 solution, and LLE using ethyl acetate were finally selected. Method validation has been carried out according to the Commission Decision 2002/657/EC criteria established for quantitative confirmatory methods. The extraction efficiencies, CCalpha and CCbeta for these compounds were in the ranges 78-98%, 10-21 and 18-35 mug/kg, respectively. The repeatability and the within-laboratory reproducibility at 1, 1.5, and 2 CCbeta concentration levels were smaller than 8.2, 7.5, and 5.8% and 12.2, 9.5, and 7.5%, respectively. Accuracy was in the 99-103% range. The robustness was evaluated using the Youden robustness test. The proposed method was applied to the analysis of steroids spiked in different kinds of animal feed samples with satisfactory results.

Microwave‐accelerated derivatization prior to GC‐MS determination of sex hormones

“…Various methods have been developed for the determination, screening, and confirmation of hormones in different types of samples, such as gas chromatography (GC) 3, 4, high‐performance liquid chromatography (HPLC) 5–7, gas chromatography‐mass spectrometry (GC‐MS) 8–20, and high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS) 21–25. GC‐MS has been a major suitable technique for the hormone analysis due to better separation and higher distinguishing power over HPLC.…”

“…However, due to the poor volatility and thermal stability of some polar compounds, derivatization step is usually required prior to GC to produce volatile products and to improve chromatographic signal. Some common derivatization reagents which can react with the hydroxyl or keto functional groups in sex hormones, such as N,O ‐ bis ‐(trimethylsilyl) trifluoroacetamide 14, 15, N ‐ tert ‐butyldimethylsilyl‐ N ‐methyltrifluoroacetamide 19, N ‐methyl‐ N ‐(trimethylsilyltrifluoroacetamide) 8, 10–13, 16, 18, 20, N,O ‐ bis ‐(trimethylsilyl)‐acetamide 26, 27, and heptafluorobutyric acid anhydride (HFBA) 8, 14, 17, have been widely used. At the same time, several common derivatization strategies for GC‐MS analysis have also been reported.…”

“…The determination of sex hormone residues in meat, kidney fat, urine, and faeces was studied 8–14, 18. As the complex constitutes including oils, fats, vitamins, minerals, proteins, carbohydrates, and preservatives (such as ethoxyquin and propionic acid) exist in feed, the determination of hormones in feed samples has always been a big challenge 5, 6, 16.…”