Quantitative analysis of 11-dehydrocorticosterone and corticosterone for preclinical studies by liquid chromatography/triple quadrupole mass spectrometry

Rationale The activity of the glucocorticoid activating enzyme 11 beta-hydroxysteroid dehydrogenase type-1 (11 beta HSD1) is altered in diseases such as obesity, inflammation and psychiatric disorders. In rodents 11 beta HSD1 converts inert 11-dehydrocorticosterone (11-DHC) into the active form, corticosterone (CORT). A sensitive, specific liquid chromatography/tandem mass spectrometry method was sought to simultaneously quantify total 11-DHC and total and free CORT in murine plasma for simple assessment of 11 beta HSD1 activity in murine models. Methods Mass spectrometry parameters were optimised and a method for the chromatographic separation of CORT and 11-DHC was developed. Murine plasma was prepared by 10:1 chloroform liquid-liquid extraction (LLE) for analysis. Limits of quantitation (LOQs), linearity and other method criteria were assessed, according to bioanalytical method validation guidelines. Results Reliable separation of 11-DHC and CORT was achieved using an ACE Excel 2 C18-AR (2.1 x 150 mm; 2 mu m) fused core column at 25 degrees C, with an acidified water/acetonitrile gradient over 10 min. Analytes were detected by multiple reaction monitoring after positive electrospray ionisation (m/z 345.1.1 & x2794; 121.2, m/z 347.1 & x2794; 121.1 for 11-DHC and CORT, respectively). The LOQs were 0.25 and 0.20 ng/mL for 11-DHC and CORT, respectively. Conclusions This LC/MS method is suitable for the reliable analysis of 11-DHC and CORT following simple LLE of murine plasma, bringing preclinical analysis in line with recommendations for clinical endocrinology and biochemistry.