Validation of highly sensitive simultaneous targeted and untargeted analysis of keto-steroids by Girard P derivatization and stable isotope dilution-liquid chromatography-high resolution mass spectrometry

Abstract

A multiplexed quantitative method for the analysis of three major unconjugated steroids in human serum by stable isotope dilution liquid chromatography-high resolution mass spectrometry (LC-HRMS) was developed and validated on a Q. Exactive Plus hybrid quadrupole/Orbitrap mass spectrometer. This quantification utilized isotope dilution and Girard P derivatization on the keto-groups of testosterone (T), androstenedione (AD) and dehydroepiandrosterone (DHEA) to improve ionization efficiency using electrospray ionization. Major isomeric compounds to T and DHEA; the inactive epimer of testosterone (epiT), and the metabolite of AD, 5α-androstanedione (5α-AD) were completely resolved on a biphenyl column within an 18 min method. Inter- and intra-day method validation using LC-HRMS with qualifying product ions was performed and acceptable analytical performance was achieved. The method was further validated by comparing steroid levels from 100 μL of serum from young vs older subjects. Since this approach provides high-dimensional HRMS data, untargeted analysis by age group was performed. DHEA and T were detected among the top analytes most significantly different across the two groups after untargeted LC-HRMS analysis, as well as a number of other still unknown metabolites, indicating the potential for combined targeted/untargeted analysis in steroid analysis.

Differences in testosterone and its precursors by sex of the offspring in meconium

“…In an across-matrix comparison, standard curves constructed in water and charcoal stripped serum produced similar and parallel standard curves indicating no major matrix effects from meconium ( Figure 3 ). Inter- and intra-day validation (n=5) gave accuracy and precision below 15% CV except for AD which was below 25% due to low precision from one run (28). Lower precision for AD is in-line with previously reported findings for this analyte (31).…”

“…No certified reference material is available for any of the steroids examined here for meconium, and we did not have sufficient biospecimen to conduct comparisons in this study to RIA or other ligand binding assays for meconium. The assay used has been previously compared to the NIST Standard Reference Material (SRM) for hormones in serum and found to provide values within 5% of the reference method (28). …”

“…LC-HRMS analysis on the Girard P derivatives of T, AD, and DHEA were conducted on an Ultimate 3000 quaternary UPLC equipped with a refrigerated autosampler (6° C) and a column heater (60° C) coupled to a Thermo QExactive Plus HRMS as previously described (28). LC separations were conducted on a Phenomenex Kinetix biphenyl column (2.6 μm, 100 A, 100 x 2.1 mm).…”

“…We applied isotope dilution liquid chromatography-high resolution mass spectrometry (ID-LC-HRMS) using confirmatory tandem high resolution mass spectrometer (MS/HRMS) (28) to quantify the unconjugated androgen, testosterone (T) and the steroid precursors, androstenedione (AD) and dehydroepiandrosterone (DHEA) from human meconium. This study was designed to test the analytical performance of quantitative measurement of major unconjugated steroid hormones in meconium, and to assess the feasibility and utility of this measurement within a multi-center, longitudinal epidemiological study.…”

Susceptibility to traumatic stress sensitizes the dopaminergic response to cocaine and increases motivation for cocaine

“…Corticosterone was measured by liquid chromatography-mass spectrometry using adaptation of previously published method for keto-steroids(Frey et al, 2016). Briefly, 20 μL of serum was extracted by addition of 20 μL of (100 pg/μL in methanol as an internal standard) 2 H 4 -corticosterone, 15 μL saturated NaCl, acidification with 5 μL 1N HCl, 0.5 mL methyl-tert-butyl-ether, and 80 μL water.…”