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Published on: April 25, 2016
Quantitative analysis of 11-dehydrocorticosterone and corticosterone for preclinical studies by liquid
Manu Verma1, Karen Sooy2, George Just2
1University/BHF Centre for Cardiovascular Sciences, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK.
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 microm) 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.
