• Measurement of salivary cortisol with liquid chromatography-tandem mass spectrometry in patients undergoing dynamic endocrine testing.

      Perogamvros, Ilias; Owen, Laura J; Keevil, Brian G; Brabant, Georg E; Trainer, Peter J; Department of Endocrinology, Christie Hospital, Manchester, UK. (2009-03-19)
      Objective: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) eliminates cross-reactivity, which is a major limitation of immunoassays used for the measurement of salivary cortisol (SalC). We aimed to evaluate the potential of SalC measured by LC-MS/MS in patients undergoing assessment of the HPA axis. Design and patients: Cross-sectional study of 78 patients admitted for routine testing in a specialized endocrine unit. Measurements: Matched serum and saliva samples were collected from 68 patients who had a Short Synacthen Test (SST, 250 mcg IM) and 10 patients who had an Insulin Tolerance Test (ITT, insulin 0.15 U/kg IV). Serum cortisol (SerC) was measured with an automated immunoassay and SalC with LC-MS/MS. Adequate SerC responses were >500 nmol/l. Results: In all patients with adequate responses the relative increase in SalC was significantly higher than in SerC [6.4(0.3-26.1) vs 1.0(0.3-4.9), P<0.0001)]. The SerC-SalC relationship was better explained by an exponential rather than a linear model (R(2)=0.83 vs R(2)=0.65, both P<0.0001). Based on 59 patients with adequate SerC responses to an SST, an adequate SalC response was defined as 8.3 nmol/l. 7 patients following an SST and 3 patients following an ITT showed inadequate responses in both SerC and SalC, but two patients with CBG deficiency showed a low SerC with normal SalC. Conclusions: We have shown an excellent diagnostic sensitivity and specificity of LC-MS/MS SalC in the assessment of the HPA axis and superiority over SerC when CBG levels are altered. The exponential relationship between SerC and SalC supports the concept of CBG binding capacity saturation.
    • Simultaneous measurement of cortisol and cortisone in human saliva using liquid chromatography-tandem mass spectrometry: application in basal and stimulated conditions.

      Perogamvros, Ilias; Owen, Laura J; Newell-Price, John; Ray, David W; Trainer, Peter J; Keevil, Brian G; Department of Endocrinology, The Christie, Manchester, UK. ilias.perogamvros@nhs.net (2009-11-01)
      Immunoassays used for the measurement of salivary cortisol are limited by variable interference from cortisone. Salivary cortisone is a consequence of the salivary glands expressing 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) which converts cortisol to cortisone. We report a combined salivary cortisol and cortisone (SalF and SalE respectively) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to address the cortisone cross-reactivity in cortisol immunoassays and as a tool to study 11beta-HSD2 activity. The method was linear up to 400 nmol/L for SalF and 200 nmol/L for SalE and the lower limits of quantitation were 0.39 nmol/L (SalF) and 0.78 nmol/L (SalE). No evidence of ion suppression was found and precision, accuracy and recovery were within internationally accepted limits. No interference was identified from 13 structurally related steroids. SalF, SalE and SalF/SalE were significantly greater in the morning than at bed-time and following stimulation of the adrenal glands. As serum cortisol increased, an exponential rise was observed in SalF and a linear increase in SalE which reached a plateau at higher SalF concentrations. We have developed a novel, robust LC-MS/MS assay for the combined measurement of SalF and SalE. Our results confirm the 11beta-HSD2 activity of the salivary glands resulting in high SalE concentrations and the enzyme saturation at high substrate concentrations. This method can be used as a simple, non-invasive and highly specific tool to assess the value of salivary cortisol as a surrogate for free serum cortisol and as a potential novel way to assess 11beta-HSD2 activity.