Development of a generic steroid platform, using adipose tissue as a model system

Sex steroid analysis using RPC18 (UPLC and/or fused core) has been investigated. For an 1mmx150 mm  Acquity BEH C18 column using a methanol-water gradient at 80 µL/min and full scan Orbitrap-MS detection an LOD for T and Adion is obtained of ca 20 pg on column, whereas for Adiol, DHEA and DHT this is 30 pg. DHEAS, E1 and E2 can be detected in negative ESI at a 30 pg level. Sensitivity can be improved up to 40 times using either nano-LC-MS or chemical derivatisation of the sex steroids using the LC-MS as described above. Chemical derivatisation results in detection limits of 0.5 pg on column for Girard-P derivatised T and Adion and 1 pg on column for DHEA and E1 using a LC-MS method as described above. Similarly, picolinic acid derivatisation resulted in LOD values of ca 5 pg for T, DHT, DHEA, E1 and E2. Sample enrichment and cleanup have been studied. Best results are obtained using Folch LLE combined with aminosilica cleanup resulting in recoveries of 71% and higher for sex steroids spiked in plasma.
The combined methods for cleanup and analysis using LC-ESI-MS/MS will allow sensitive and accurate quantitation of a range of steroids at levels in the order of 10 - 20 pg mL-1 plasma. This is competitive or better when compared with recently published methods describing the simultaneous analysis of a multiple steroids (e.g. Ceglarek ea Clin.Chim.Acta 401(2009)114). The sensitivity of the current approach is well within range for the analysis of steroids in plasma and adipose tissue. The developed methods will be validated using a larger panel of steroids and sterols. The resulting system will be applied for the analysis of these compounds in biological samples and allows the monitoring of sterols and steroids in tissue and plasma.