Solid-phase analytical derivatization as a tool for the quantification of steroid hormones in human urine with HPLC-Q-ToF detection

A possibility of application of solid-phase analytical derivatization (SPAD) for the quantification of seven steroid hormones (testosterone, dihydrotestosterone, cortisone, cortisol, progesterone, 11α-hydroxyprogesterone, and estrone) in human urine was evaluated. To prepare urine samples for instrumental analysis, SPAD with hydroxylamine was applied after enzymatic hydrolysis of the sample. To achieve high recovery values, extraction and derivatization conditions were optimized. Cartridges packed with end-capped octadecylsilyl silica sorbent provided optimum extraction of target analytes, while the reaction with hydroxylamine in the cartridge was found as a simple and efficient way for the chemical derivatization of steroids. The obtained derivatives were detected by using reversed-phase ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. The proposed procedure was validated and applied to the analysis of real urine samples to prove the applicability of the proposed method for the routine analysis.