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), to 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 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 󠇆μm) fused core column at 25o C, with an acidified water; acetonitrile gradient over 10 minutes. Analytes were detected by multiple reaction monitoring after positive electrospray ionization (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. CONCLUSION: 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.