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

BackgroundHyper–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.MethodsSaliva/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.ResultsThe E and F calibration 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).ConclusionsThis 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.