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 11β‐hydroxysteroid dehydrogenase type‐1 (11βHSD1) is altered in diseases such as obesity, inflammation and psychiatric disorders. In rodents 11βHSD1 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 11βHSD1 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 × 150 mm; 2 μm) fused core column at 25°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 ➔ 121.2, m/z 347.1 ➔ 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.