Abstract
RationaleThe 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.MethodsMass 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.ResultsReliable 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.ConclusionsThis 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.
Highlights
Glucocorticoids are essential for the regulation of metabolism, the stress response and inflammation
Glucocorticoid action is controlled at two levels: first by the hypothalamicpituitary axis, a negative feedback loop which determines the levels of circulating glucocorticoids and secondly by metabolism of glucocorticoids in the tissues
The mass spectrometer was operated in positive ion electrospray ionisation (ESI) mode using a TurboIonSpray source and data collected in unit resolution (0.7 m/z units full width at half maximum)
Summary
Glucocorticoids are essential for the regulation of metabolism, the stress response and inflammation. The best documented example of tissue metabolism of glucocorticoids is 11 -hydroxysteroid dehydrogenase 1 (11 HSD1) [1] In rodents this enzyme converts inert glucocorticoid 11dehydrocorticosterone (11-DHC) to the active form, corticosterone (CORT). The activity of 11 HSD1 is altered in several disease states including obesity, inflammation and psychiatric disorders [2] The importance of this enzyme has been revealed by murine models of global or tissue-specific disruption [3,4] or over-expression [5] of the enzyme. Li et al developed a 6-minute LC/MS method for CORT analysis (LOQ of 1 ng/mL), but not 11DHC, in mouse plasma [11]. A 14-minute steroid profiling human plasma method includes CORT with an LOQ of 0.5 ng/mL, but not 11-DHC [13]. To date there has not been a validated method focussing only on CORT and 11-DHC in murine plasma
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