Quantification of steroids in human and mouse plasma using online solid phase extraction coupled to liquid chromatography tandem mass spectrometry.

Steroids are powerful signalling molecules regulating a variety of physiological processes such as cell proliferation, cell differentiation, and reproduction. Variations in steroid concentrations may hint at hormonal imbalances or metabolic disorders that allow for a diagnosis of human diseases. The measurement of steroid hormone concentrations is relevant to basic research as well as clinical sciences. For example, the systematic generation of mouse mutants has led to a need for comprehensive phenotyping. As these mouse models may bear defects that affect steroid synthesis or homeostasis, knowledge of the mouse steroid metabolome could help to identify these mutants and connect phenotypes to their molecular cause 1.Metabolic characterisation of a large human cohort recently led to the discovery of associations between phenotype, metabolome, and genotype as shown by us 2. To expand our knowledge of the metabolome to other substance classes, a high-throughput, robust, sensitive and specific steroid phenotyping method is required. For several years, immunoassays have been the standard method for measuring steroid concentrations. Recently, liquid chromatography tandem mass spectrometry methods for steroid analysis have been developed that combine the advantages of immunoassays (i.e. sensitivity) with the specificity of mass spectrometry 3. This protocol describes a high-throughput method to analyze several different steroids in plasma. By using online-SPE coupled to LC-MS/MS, six different steroids (androstenedione, testosterone, cortisone, cortisol, progesterone, 17OH-progesterone) are readily quantifiable in human plasma, while three steroids (androstenedione, testosterone, corticosterone) can be quantified in mouse plasma. The method requires a minimum of hands-on time by the experimenter while simultaneously providing concentrations of several interesting steroids