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

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 (40mL) was added to 20mL of sample. This was vortexed for 10s followed by the addition of 100mL of internal standard in methanol. Following mixing and centrifugation, 10mL of sample was injected into an Acquity LC system coupled to a Quattro Premier tandem mass spectrometer. Serum samples (n ¼149) 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 ¼171) was analysed by the LC-MS/MS assay and a GC-MS assay. Results: Cortisol had a retention time of 0.98min and the assay had an injection-to-injection time of 2.6min per sample. Mean recovery was 99% and mean CV was 8%. The immunoassays gave comparisons of: Roche ¼1.23 � LC-MS/MS � 1.12nmol/L and Abbott ¼0.94 � LC-MS/MS þ 11.97. The comparison with GC-MS showed LC-MS/MS ¼1.11 � GCMS – 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.