Development of a rapid assay for the analysis of serum cortisol and its implementation into a routine service laboratory.

ABSTRACT

BACKGROUND: LC-MS/MS is rapidly becoming the technology of choice for measuring steroid hormones. We have developed a rapid LC-MS/MS assay for the routine analysis of serum cortisol. We have used this assay to investigate the effects of gender and exogenous steroid interference on the immunoassay measurement of serum cortisol. METHODS: Zinc sulphate (40 µL) was added to 20 µL of sample. This was vortexed for 10 s followed by the addition of 100 µL of internal standard in methanol. Following mixing and centrifugation, 10 µL of sample was injected into an Acquity LC system coupled to a Quattro Premier tandem mass spectrometer. Serum samples (n = 149) were analysed by LC-MS/MS and two commercial immunoassays. Results were then compared for all samples and for gender differences. A further set of serum samples (n = 171) was analysed by the LC-MS/MS assay and a GC-MS assay. RESULTS: Cortisol had a retention time of 0.98 min and the assay had an injection-to-injection time of 2.6 min per sample. Mean recovery was 99% and mean CV was 8%. The immunoassays gave comparisons of Roche = 1.23 × LC-MS/MS -1.12 nmol/L and Abbott = 0.94 × LC-MS/MS + 11.97. The comparison with GC-MS showed LC-MS/MS = 1.11 × GC-MS - 22.90. DISCUSSION: We have developed an LC-MS/MS assay for serum cortisol analysis that is suitable for routine clinical use and has been in use in our laboratory for 12 months. The availability of this assay will give more reliable results in patients receiving exogenous steroid therapy.