Authors: Ferdinand Haller, Cornelia Prehn & Jerzy Adamski
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.
A. Sampling of blood
B. Preparation of precipitation agent containing internal standards
C. Sample preparation
D. Chromatographic separation of samples
E. MS/MS analysis
For preparation of 100 samples: hands-on time 2-3 h For measurement of 100 samples: about 8.5 h (no hands-on time, performed by autosampler and LC-MS/MS system)
Chromatography solvents and the precipitation agent should be prepared fresh for each run. Pipetting steps before addition of internal standards are critical and should be performed as exactly as possible.
Typical results for human and mouse plasma are shown in Figures 1 and 2, respectively.
Figure 1 : Representative chromatogram of measured steroids in human plasma (including internal standards)
Figure 2: Representative chromatogram of measured steroids in mouse plasma (including internal standards)
Table 1: Gradient for the separation of steroids: the 6 port switching valve was set to the inject position after 1 min. After 3.5 min the switching valve was switched back to the load position. Below MRM parameters for tandem mass spectrometry are shown.
Ferdinand Haller, Cornelia Prehn & Jerzy Adamski, Institute for Experimental Genetics, Helmholtz Zentrum Muenchen
Source: Protocol Exchange (2010) doi:10.1038/nprot.2010.22. Originally published online 16 February 2010.