Endocrine Abstracts

Mass spectrometry based clinical metabolomics needs standardization of analytical assays to improve future inter-laboratory comparability and to become a successful new and established technology in clinical routine laboratories such as mass spectrometry based therapeutic drug monitoring (TDM) over the past 10 years. Steroid hormones are one of the most interesting endogenous metabolite class in clinical metabolomics today. Standardized determination of concentrations of steroid hormones in serum may aid in improvements in the clinical environment, e.g. IVF, diagnosis and treatment of steroid-related diseases in children and adults. The application of this assay will be focused on analysis of premenopausal women, i.e. the quantitation range of steroids is optimised for this clientele.

We will present a newly developed high throughput and standardized steroid LC–MS/MS based assay intending the quantitative determination of 16 steroids in human serum samples for clinical application. The steroid metabolites are testosterone, progesterone, cortisol, estradiol (E₂), DHEAS androstenedione, 17-OH progesterone, corticosterone, 11-deoxycortisol, estrone (E1), DHEA, 11-deoxycorticosterone, aldosterone, cortisone, etiocholanolone, androsterone. The assay includes standardized sample preparation and LC–MS/MS analysis in 96-well plate format. The sample preparation, needed to clean up and pre-concentrate the sample, is performed by a solid phase extraction (SPE) procedure in 96-well plate format. Serum (500 μl) sample volume is needed. Two different elution steps are necessary (first fraction: all steroids except DHEAS, second fraction: DHEAS) for highly pure extraction fractions resulting in minimal matrix effects and improved accuracy. As the result there are two subsequent LC–MS/MS runs with a total run time of 22 min.

Validation data of the assay for human serum will be presented including inter-laboratory comparisons demonstrating the precision, accuracy, stability, and reproducibility of the newly developed standardized assay.