Development, optimization and validation of modified QuEChERS based UPLC-MS/MS for simultaneous determination of nine steroid hormones in milk powder and milk

Abstract

The simultaneous detection of a group of nine steroid hormones including six estrogens (17β-estradiol, estriol, estrone, ethinyl estradiol, diethylstilbestrol, and hexestrol), two glucocorticoids (dexamethasone, and hydrocortisone), and one progestogen (17α-hydoxyprogesterone) in milk powder and milk was performed. A modified QuEChERS method combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for analysis of the target compounds. To optimize the chromatographic conditions and QuEChERS process, the mobile phase additive, the amount of extraction solvent and the sorbent type were investigated, respectively. Under optimal conditions, the methodology applied to milk powder and milk was validated using five internal standards showing good linearity (R² ≥ 0.9955 and 0.9925), recovery (83.8–119.5% and 87.9–117.7%), intra-precision (2.9–9.5% and 2.1–12.1%), inter-precision (1.6–11.0% and 3.1–9.9%) and the limit of quantification (0.01–2.2 μg kg⁻¹ and 0.01–2.0 μg L⁻¹). Finally, the analysis of commercial milk products revealed the presence of 17α-hydoxyprogesterone, hydrocortisone, dexamethasone, 17β-estradiol, and estrone in these samples.