Simultaneous determination of fourteen steroid hormone residues in beef samples by liquid chromatography-tandem mass spectrometry

Abstract

A liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of 14 steroid hormone residues (hydrocortisone (H), testosterone (T), dehydroepiandrosterone (DHEA), epitestosterone (EpiT), etiocholanolone (E), androsterone (A), progesterone (P), testosterone propionate (TP), pregnanediol (PD), pregnanetriol (PT), estriol (E3), α/β-estradiol (α/β-E) and estrone (E1)) in beef samples. Sample preparation and chromatographic conditions were optimized to minimize matrix effects. The beef samples were enzymatically digested with β-glucuronidase/arylsulfatase, treated by oscillation and ultrasonic-assisted extraction with acetonitrile. NaCl was added to the crude extract to induce separation of water and acetonitrile layers. After centrifugation and evaporation, the extract was further treated with ZnCl₂ to remove lipids and purified by solid phase extraction using LC-C₁₈, LC-Si and LC-NH₂ cartridges. The analytes were separated using Poroshell (glucocorticoid, androgen and progestin) and Hydrosphere (estrogen) C₁₈ columns with gradient elution using acetonitrile and water mobile phases. Finally, the eluents were qualitatively and quantitatively determined by electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode. Using a matrix matched external standard method, good linearity in response was obtained with correlation coefficients larger than 0.999. The detection limits of the method were about 0.004–0.24 μg kg⁻¹ and the quantification limits were 0.016–0.84 μg kg⁻¹. At the spike levels of 1.0, 2.0 and 4.0 μg kg⁻¹ for DHEA, E, A, PT and PD and 0.2, 0.4 and 1.0 μg kg⁻¹ for other drugs, the average recoveries of hormones were within the range of 66.4–115.2%, and the relative standard deviations (n = 6) were from 2.2% to 10.2%. The method was successfully used for the determination of 14 hormone residues in beef samples.