Liquid Chromatography Tandem Mass Spectrometry Measurements of Steroids in Biological Samples for Clinical Research Studies: Methodology and Application

Abstract

The advent of liquid chromatography tandem mass spectrometry (LC-MS/MS) coupled with soft ionization method has expanded the scope for measurement of steroids from biological matrices with higher accuracy, specificity, sensitivity and requires less sample preparation. The overall aim of this study is to develop and validate LC-MS/MS methods to measure steroids from biological samples for clinical research studies. A sensitive and specific LC-MS/MS assay was developed and validated to measure steroids from dried blood spots (DBS) samples to assess the feasibility and pharmacology of subcutaneous (sc) injection of androgen ester in healthy men using DBS for frequent sampling. The study demonstrated a sustained release of this androgen ester which suggests that sc injections of testosterone esters may prove to be pharmacologically effective. A LC-MS/MS assay was also developed to measure urinary androgens and estrogen in adolescents and subsequently to relate the changes in the urinary sex hormones over 12 months to the standard anthropometric markers of puberty. We found that the urine hormone measurements correlated cross-sectionally and longitudinally with age, anthropometry and Tanner stage. We also investigated whether first morning void urine hormones in growing adolescents require adjustments for urine dilution/concentration and, if so, whether urinary creatinine or specific gravity (SG) are better adjustments. The study demonstrates that urine steroid and LH concentrations in first morning void samples of adolescents are not significantly influenced by hydration status and may not require adjustments; however, if desired, both creatinine and SG adjustments are equally suitable. We also assessed whether commercially available luteinizing hormone (LH) immunoassays (immunochemiluminometric, ICL and immunofluorometric, IF) previously validated for human blood samples is suitable for urine samples kept at prolonged frozen (4 years). We found that both immunoassays are suitable for urinary LH measurements with ICL assay being more robust for quantitative urinary LH measurement such as for anti-doping purpose whereas the IF could be applicable for research studies where urine LH levels are compared within-study but not in absolute terms.