Human saliva cortisone and cortisol simultaneous analysis using reverse phase HPLC technique

Background: Hyper-hypo tension (like Cushing's syndrome, apparent mineralocorticoid excess syndrome and Addison's disease) diagnostic laboratory requires cortisol (F) analysis. The simultaneous analysis of human saliva F and cortisone (E), the inactive F metabolite, by solid phase extraction and RP-HPLC was studied. Methods: Saliva/standard samples were C18-SPE extracted, dried and resuspended. E and F were analysed by isocratic RP-HPLC (acetonitrile/ water 27/73%) and UV detection. In the morning and in the evening Salivette stimulated saliva specimens were collected from healthy volunteers. Results: The E and Fcalibration curve ranges were 11.0-110.0 and 5.5-55.0 nmol/l respectively. The LOD was 0.2 and 0.1 nmol/l for E and F respectively. The intra and inter assay CVs were respectively 2.7-6.6 and 5.6-7.0% for E and 5.8-7.0 and 11.7-13.1% for F. The E and F spiked saliva sample recovery was 99% and 88% respectively. Saliva specimen stability was validated. E and F saliva levels in healthy volunteers were significantly (p<0.001) higher at 8 a.m. compared with 11 p.m. (26.4 +/- 8.9 vs. 4.3 +/- 2.9 nmol/l for E; 11.1 +/- 4.0 vs. 2.5 +/- 1.5 nmol/l for F, respectively). Conclusions: This method is suitable for periodic analyses in a clinical biochemistry laboratory for endocrinology investigation purposes, simultaneously analysing E and F levels in a saliva specimen. (C) 2009 Elsevier B.V. All rights reserved.