Abstract: A modified quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction method was developed for the simultaneous determination of 42 steroid hormones in milk and milk powder by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Sample pretreatment was studied and the detection conditions were optimized to obtain a rapid and accurate method. The steroid hormones from samples were extracted with 0.1% formic acid in acetonitrile followed by a salting-out step with anhydrous MgSO4 and NaCl. After centrifugation, the supernatant was cleaned up with 50 mg of octadecylsilane (C18), 100 mg of primary secondary amine (PSA), and 300 mg of aluminum oxide (Al2O3-N) and re-dissolved with 40% formic acid acetonitrile. The separation was performed on a Waters ACQUITY UPLC? BEH C18 column (2.1 mm × 100 mm, 1.7 μm) with gradient elution using acetonitrile and water containing 0.1% formic acid as the mobile phase. The electrospray ionization mass spectrometry was operated in the positive mode using multiple reaction monitoring (MRM). The target compounds were quantified by the matrix-matched external standard method. Under the optimal conditions, the calibration curves of all the analytes were linear in the concentration range of 0.5–500 μg/L with correlation coefficients larger than 0.99. The limits of quantitation (LOQs) ranged from 0.06–1.5 μg/kg. The average recoveries for milk and milk powder at three spiked levels ranged from 70.3%–118.1% with relative standard deviations (RSDs) of 0.6%–14.7%. The method due to its simplicity, rapidity, high accuracy and high precision was suitable for the simultaneous detection of 42 steroid hormones in milk and milk powder.

Key words: QuEChERS, ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), milk, milk powder, steroid hormones