Profiling intact steroid sulfates and unconjugated steroids in biological fluids by liquid chromatography-tandem mass spectrometry (LC-MS-MS)

Abstract

Within the combined DFG research project "Sulfated Steroids in Reproduction" an analytical method was needed for determining sulfated and unconjugated steroids with highest specificity out of different biological matrices such as aqueous solution, cell lysate and serum. With regard to this analytical challenge, LC-MS-MS presents the technique of choice because it permits (1) analysis of the intact steroid conjugate, (2) allows for simultaneous determination of multiple analytes (profiling, targeted metabolomics approach) and (3) is independent of phenomena such as cross-reactivity. Sample work up consisted of incubation of sample with internal standards (deuterium labeled steroids) followed by solid phase extraction. Only serum samples required a protein precipitation step prior to solid phase extraction. The extract was divided in two parts: six steroid sulfates (E1S, E2S, AS, 16-OH-DHEAS, PREGS, DHEAS) were analyzed by C18aQ-ESI-MS-MS in negative ion mode and eleven unconjugated steroids (E3, 16-OH-DHEA, E1, E2, (4)A, DHEA, T, 17-OH-PREG, Prog, An, PREG) were analyzed by C18-APCI-MS-MS in positive ion mode. For steroid sulfates, we found high sensitivities with LoQ values ranging from 0.08 to 1 ng mL(-1). Unconjugated steroids showed LoQ values between 0.5 and 10 ng mL(-1). Calibration plots showed excellent linearity. Mean intra- and inter-assay CVs were 2.4% for steroid sulfates and 6.4% for unconjugated steroids. Accuracy - determined in a two-level spike experiment - showed mean relative errors of 5.9% for steroid sulfates and 6.1% for unconjugated steroids. In summary, we describe a novel LC-MS-MS procedure capable of profiling six steroid sulfates and eleven unconjugated steroids from various biological matrices.

Simultaneous quantification of cholesterol sulfate, androgen sulfates, and progestagen sulfates in human serum by LC-MS/MS

“…Mass spectrometer conditions were as reported before ( 16 ). Briefl y, the capillary temperature and the vaporizer temperatures were set to 270 and 350°C, respectively.…”

“…In 2003, Liu, Griffi ths, and Sjövall ( 15 ) provided a method to obtain a more detailed profi ling of sulfated steroids in human plasma. Recent work has focused on extending the number of sulfated steroids analyzed simultaneously in a single LC-MS analysis ( 16,17 ).…”

Free and sulfated steroids secretion in postpubertal boars (Sus scrofa domestica)

“…Measurement of free and sulfated steroids by liquid chromatography-tandem mass spectrometry A 4 , testosterone, PREGS, DHEAS, E 1 S, and E 2 S, were quantified in boar heparin plasma using isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS), as previously published (Galuska et al 2013). Briefly, the samples were mixed and equilibrated with deuterated internal standards (d 4 E2S, d 4 PREGS, d 3 T, d 7 -4A, and d 6 DHEAS).…”

High levels of oxysterol sulfates in serum of patients with steroid sulfatase deficiency

“…The sulfated steroids can be analyzed as intact molecules ( 21 ) without any need for derivatization, providing important structural information, e.g., position of the sulfate in the structure. Of note, sulfated steroids have very specifi c transitions in MS/MS experiments (21)(22)(23).…”

“…By means of non-targeted MS tools, the comparison of serum from RXLI patients with serum from The mass spectrometer was operated fi rst in negative parent ion scan mode (precursor ion scan mode) with a product mass of 96.9, a scan range of m/z 30-650, a scan time of 1 s, and collision energy of 45 V. The same parameters were applied to a product mass of 79.9. These product masses were selected taking into consideration previous studies, which showed that 96.9 and 79.9 are the most common fragmentation products of sulfated steroids, with the exception of estrogen sulfates (21)(22)(23).…”