Method-specific serum cortisol responses to the adrenocorticotrophin test: comparison of gas chromatography-mass spectrometry and five automated immunoassays.

OBJECTIVE: The serum cortisol response to the adrenocorticotrophin (ACTH) test is known to vary significantly by assay, but lower reference limits (LRL) for this response have not been established by the reference gas chromatography-mass spectrometry (GC-MS) method or modern immunoassays. We aimed to compare the normal cortisol response to ACTH stimulation using GC-MS with five widely used immunoassays. DESIGN, PATIENTS AND MEASUREMENTS: An ACTH test (250 µg iv ACTH1-24 ) was undertaken in 165 healthy volunteers (age, 20-66 years; 105 women, 24 of whom were taking an oestrogen-containing oral contraceptive pill [OCP]). Serum cortisol was measured using GC-MS, Advia Centaur (Siemens), Architect (Abbott), Modular Analytics E170 (Roche), Immulite 2000 (Siemens) and Access (Beckman) automated immunoassays. The estimated LRL for the 30 min cortisol response to ACTH was derived from the 2·5th percentile of log-transformed concentrations. RESULTS: The GC-MS-measured cortisol response was normally distributed in males but not females, with no significant gender difference in baseline or post-ACTH cortisol concentration. Immunoassays were positively biased relative to GC-MS, except in samples from women on the OCP, who showed a consistent negative bias. The LRL for cortisol was method-specific [GC-MS: 420 nm; Architect: 430 nm; Centaur: 446 nm; Access 459 nm; Immulite (2000) 474 nm] and, for the E170, also gender-specific (female: 524 nm; male 574 nm). A separate LRL is necessary for women on the OCP. CONCLUSIONS: Normal cortisol responses to the ACTH test are influenced significantly by assay and oestrogen treatment. We recommend the use of separate reference limits in premenopausal women on the OCP and warn users that cortisol measurements in this subgroup are subject to assay interference.

Production and certification of four frozen human serum certified reference materials containing creatinine and electrolytes.

BACKGROUND: This paper describes the preparation, analysis and certification of four frozen human serum certified reference materials (CRMs) containing creatinine and the electrolytes calcium, lithium, magnesium, potassium and sodium. These materials have been prepared to give concentrations of these analytes that cover the currently accepted analytical range. METHODS: The analysis of the materials for certification purposes has been carried out using methodology traceable to primary standards, and which is acceptable as a reference method. The certification methods include liquid chromatography-mass spectrometry (LC-MS) with exact-matching isotope dilution calibration (EM-IDMS) for creatinine, inductively-coupled plasma optical emission spectroscopy (ICP-OES), ICP-MS and isotope-dilution inductively-coupled plasma mass spectroscopy (ID-ICP-MS) for the electrolytes. RESULTS: The uncertainties estimated for these certified values include a component from the characterization measurements, as well as contributions from possible inhomogeneity and long-term instability. The certified values have been corroborated by measurements obtained in a major UK External Quality Assessment scheme, which have, with the exception of the determination of creatinine at a particularly low concentration, given excellent agreement. CONCLUSIONS: The materials are intended for use by pathology laboratories and manufacturers of in vitro diagnostic (IVD) kits for validation of existing routine methodology to a traceable standard, which will promote harmonization between the different methods, instruments and IVD kits used in these laboratories.

Measurement of urinary free cortisol by tandem mass spectrometry and comparison with results obtained by gas chromatography-mass spectrometry and two commercial immunoassays.

BACKGROUND: Determination of urinary free cortisol (UFC) is an important adjunct for the assessment of adrenal function. In this study, we have analysed cortisol concentrations in urine samples by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and two immunoassays. The results were compared with GC-MS. The interference of cortisol ring-A metabolites in immunoassays was also assessed. METHODS: The GC-MS technique involved solvent extraction, LH-20 clean-up and derivatization. Only solid-phase extraction procedure was used for LC-MS/MS. The samples were analysed in positive electro-spray ionization mode, monitoring the transitions for cortisol and deuterated-cortisol at m/z 363.3 > 121.2 and m/z 365.3 > 122.2, respectively. Immunoassays were performed according to the manufacturer's instructions. RESULTS: When compared with GC-MS results both immunoassays (Coat-A-Count; approximately 1.9-fold, Centaur; approximately 1.6-fold) overestimated UFC concentrations. Cortisol ring-A dihydro- and tetrahydrometabolites contribute significantly to this overestimation. There was no interference by these metabolites in either GC-MS or LC-MS/MS methods. The sensitivity of the LC-MS/MS procedure was 2 nmol/L and the intra- and inter-assay variations were <5% in each quality-control sample. The comparison of the UFC results achieved by assaying the study samples with GC-MS and LC-MS/MS indicated that the agreement between the two methods was excellent (LC-MS/MS = 1.0036GC-MS - 0.0841; r2 = 0.9937). CONCLUSIONS: The interference of cortisol ring-A metabolites in immunoassays contribute to overestimation of UFC concentrations. The LC-MS/MS procedure had the sensitivity, specificity, linearity, precision and accuracy for the determination of UFC concentrations. The method is suitable for routine use provided that method-dependant reference values are established.

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 (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.