Quantitative-Profiling Method of Serum Steroid Hormones by Hydroxylamine-Derivatization HPLC–MS

Abstract

A sensitive and rapid high performance liquid chromatography–mass spectrometry (HPLC–MS) method was developed and validated for simultaneous quantification of ten steroid hormones, including estrogens, androgens, progesterones, and corticosteroids four classes of steroids. The following ten steroid hormones were analyzed: progesterone, 21-deoxycortisol, estrone, 4-androstenedione, testosterone, dihydro-testosterone, androstenone, dehydroepiandrosterone, corticosterone and cortisone. Stable deuterated isotopes were used as internal standards for quantification. Sample preparation with and without derivatization were performed after liquid–liquid extraction, and the corresponding results were compared according to sensitivity and selectivity. Hydroxylamine derivatization was found to improve the ionization efficiency of the analytes for electrospray ionization MS analysis. The gradient of mobile phase and experimental parameters for HPLC separation were optimized. The lower limits of quantification were in the range of 0.05–5 ng mL −1 with wide linear range for the ten steroid hormones. The intra-day precision < 11.1% and recovery of 84.5–120% with negligible matrix effect were achieved, where within the acceptance limits of the FDA guideline. Total HPLC–MS analysis time was 6 min. This method enables simultaneous quantification of steroids in human serum. It will be helpful for the serum steroid profiling in order to understand various endocrinology diseases. Graphical Abstract

Mass Spectrometry as a Crucial Analytical Basis for Omics Sciences

“…However, preliminary derivatization is still used for this type of analysis, using a targeted modification of steroid substructures. Such approaches, including the conversion of carbonyl groups by reaction with hydroxylamine or 2-hydrazinopyridine, were proposed for the highly sensitive determination of endosteroids by HPLC/ESI-MS and UHPLC/ESI-MS [ 101 ], respectively. We can also note a preliminary modification of carbonyl groups with the introduction of a fixed charge (Girard’s reagent), carried out to detect and determine off-target steroid metabolites by a combined method of LC with high-resolution MS (LTQ-Orbitrap) [ 102 ].…”

Progress and Challenges in Quantifying Carbonyl-Metabolomic Phenomes with LC-MS/MS

“…However, the poor hydrophobicity of NH 2 OH itself has limitation to improve the retention of metabolites. Liu et al [88] quantified ten steroid hormones by hydroxylamine derivatization using HPLC-MS and a LOQ was achieved in the range of 0.05-5 ng/mL which was increased by 3.9-202.6 times after derivatization. Because hydroxylamine reacts fast and shows no contaminations to MS, Chen et al [89] developed a fully automated in-tube SPME/LC-PCD-MS method, and demonstrated rapid and accurate quantification of urinary hexanal and heptanal by simple sample preparation and avoiding LC separation of E-/Zisomers of oximes (Figure 4).…”

Determination of Ketosteroids in Human Urine Using Dispersive Liquid-Liquid Microextraction and Ultra High-Performance Liquid Chromatography-High Resolution Mass Spectrometry

“…In addition, it was possible to inject the reaction mixture into the chromatograph without additional purification steps after obtaining the derivatives. Hydroxylamine was previously used to determine steroid hormones in human biological fluids with high sensitivity [3,23,24]. Scheme 1 shows the derivatization reaction using hydroxylamine as a derivatizing agent.…”