Chapter 2.2 - Steroid determination—Sample preparation

Abstract

The analysis of steroids of biological importance to humans requires methods for the determination of the very low concentrations of steroids found in body fluids and sometimes in biological tissues or formed during experimental studies in vitro or in vivo. This book primarily addresses studies in humans, but much fundamental research is conducted in animals. Steroid hormones are carried in the bloodstream bound to plasma proteins with specific and nonspecific properties. In general, the concentrations of steroids in blood serum or plasma are determined as the total, after the bound steroid is displaced from binding proteins and combined with the free steroid. The concentrations of free steroids are not usually determined but methods are available after equilibrium dialysis. Steroid determination was advanced considerably by immunoassay as the analysis of steroids in a single sample extract of almost any biological sample could be achieved. After a chromatographic separation several steroids could be determined by separate immunoassays. Steroid immunoassays were amenable to automation. However, the poor specificity of immunoassays has driven the move to using chromatography combined with mass spectrometry for steroid measurements. Early methods were based on determination of single steroids but there is a greater need for simultaneous determination of several steroids in one analysis which has been called profiling or recently the metabolome. The first hurdle in a bioanalysis is to extract the analyte(s) of interest from a highly complex matrix into a simple medium for analysis by instrumental techniques. Sample preparation often requires, by far, the most time and effort of analysts. Ideally it should be easy to use, rapid, high throughput, robust and reasonably priced.