Mass spectrometry for steroids.

Abstract

This issue of the Annals includes several articles reporting analysis of steroids using mass spectrometry (MS). Many papers on the use of MS for steroid measurement and assay interference have been published recently in the Annals, reflecting a growing appreciation of the power of this physicochemical technology. Formerly, MS was primarily used for steroids in clinical research and reference centres. Combining MS with gas chromatography (GC–MS) is still a highly valued technique for profiling the range of urinary steroid metabolites in the diagnosis of adrenal and some gonadal disorders, and for the most accurate of steroid quantitative assays (e.g. reference/gold standard methods). The significant developments in MS for clinical laboratories in the last 15 years have come through the combination of liquid chromatography (LC) with tandem MS (LC–MS–MS) adopting new techniques of forming ions at their interface. Samples for LC–MS/MS ideally need minimal preparation before analysis. Protein precipitation, liquid– liquid extraction, solid phase extraction and supported liquid extraction are all used. In some cases, sample preparation can be automated or performed in 96-well plates. A precolumn may be incorporated before the LC process to improve sensitivity by holding back potential interferents and hence reduce background signal. Phenyl, C8 and C18 high-performance LC columns have undergone refinement with increasingly smaller particles being used to enhance resolution. Mobile phase constituents include water or aqueous buffers (ammonium acetate and formic acid) with organic modifiers. Ammonium fluoride is added in oestrogen analysis to improve sensitivity. The electrospray source is most widely used for steroids. Instruments vary in design to focus the sample beam, which is important when measuring low concentrations such as testosterone in saliva. The aim is to deliver the analyte to the mass spectrometer over a narrow time frame; positive ions are usually detected for steroids but for oestrogens negative ion analysis is preferred. Selected reaction ion monitoring is a two-step process to focus ions in the first sector (MS1) and then generate fragments in a collision cell, which are examined by the second sector (MS2). Steroid assays are usually stable isotope dilution methods with variable levels of deuterium incorporation in the internal standard. This increases the likelihood that the internal standard will behave in exactly the same way as the analyte throughout the procedure and therefore compensate for any effects on signal intensity through ion suppression or enhancement. The substitution of three atoms of deuterium is common but when more than three stable isotope atoms are added, the labelled steroid elutes ahead of the natural steroid thus risking independent ion suppression. The sites of incorporation may not always be appropriate for MS–MS methods depending on the fragmentation path and stabilities of charged ions. The final procedure must undergo validation for ion suppression, accuracy, recovery, imprecision, linearity, limit of quantitation and detection, specificity and stability. Some useful guidance is available to support assay development. Specificity for steroids requires knowledge of LC retention times, molecular weights and fragmentations. Owen et al., for example, tested 15 naturally occurring steroids and 12 synthetic steroids in their validation of serum concentrations of oestradiol and oestrone. LC–MS/MS methods in clinical laboratories have now been reported for nearly all steroids, and in some