A Novel Targeted Analysis of Peripheral Steroids by Ultra-Performance Supercritical Fluid Chromatography Hyphenated to Tandem Mass Spectrometry

Abstract

Ultra-performance supercritical fluid chromatography–tandem mass spectrometry (UPSFC–MS/MS) is an alternative method for steroid analysis. Continuous development of analytical methodologies for steroid profiling is of major importance in the clinical environment to provide useful and more comprehensive data. The aim of this study was to identify and quantify a large number of endogenous steroids from the four major classes (estrogens, androgens, progestogens and corticosteroids) simultaneously within a short analytical time. This novel UPSFC–MS/MS method with electrospray in positive ionisation (ESI+) mode is robust, selective and present sufficiently high sensitivity to profile nineteen steroids in 50 µL human plasma. Under optimised conditions, nineteen different steroids were separated with high efficiency in the multiple reaction monitoring (MRM) mode. The linearity of the method was good with correlation coefficients (R2) in the range of 0.9983–0.9999 and with calibration range from 0.05–500 ng/mL in human plasma. The intraday and interday precision of the method, as RSD, was less than 15%. The accuracy of the nineteen analytes varied between 80 to 116%. Finally, the novel method was successfully applied for the determination of nineteen steroids within 5 minutes providing the possibility to use it for research as well as routine healthcare practice.

Hormonal determinants of mammographic density and density change

“…The strengths of our study are the large number of samples and the fast, sensitive and reliable UPSFC-MS/ MS method for simultaneous quantification of 17 endogenous steroids [30]. Some hormones display a circadian rhythm; we thus included the time of day of blood draw in our models.…”

“…All blood samples were collected at study entry and handled in accordance with a strict 30-h cold-chain protocol at the Karolinska Institutet high-throughput biobank. Hormones were measured in blinded peripheral blood plasma by the UPSFC-MS/MS system (Waters Corporation, Milford, USA), as described previously [30]. Sample preparation for the analysis of desulfated steroid hormones was carried out through liquid-liquid extraction with tert-butyl methyl ether (MTBE) followed by derivatisation with methoxyamine.…”

“…Data acquisition range was set to m/ z 100-600. The quantification was based on a multiple reaction monitoring (MRM) method; collision energy and cone voltage were set as described previously [30], using individual analysis of standard desulfated steroids and DHEAS (100 ng/mL). Quantification of hormones was performed using the suitable deuterated internal standards, and limit of quantification (LOQ) of desulfated steroids (0.05-0.5 ng/mL) and DHEAS (0.01 ng/ mL).…”

“…Values were missing for all hormones and across all batches. Information on LOQ and linear range for all steroinds can be found in [30]. Data was acquired, analysed and processed using the MassLynx TM4.1 software (Waters, Milford, USA).…”

Supercritical fluid chromatography – Mass spectrometry: Recent evolution and current trends

“…Considering the diversity of MS analyzers present on the market (i.e. single and triple quadrupole, time of flight, ion trap, Orbitrap and hybrid instruments), it is possible to perform qualitative and quantitative analysis at very high sensitivity [3][4][5], as well as to generate elevated MS resolution between compounds having very similar mass-to-charge values [6,7].…”