Determination of corticosteroids in tissue samples by liquid chromatography–tandem mass spectrometry

Abstract

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of corticosterone and 11-dehydrocorticosterone (11-DHC) levels in KKA y mouse liver and adipose tissue, and hydrocortisone and cortisone levels in human adipose tissue has been developed. The corticosteroids were extracted from liver tissue with methanol/water and ethyl acetate for adipose tissue samples. Corticosterone and 11-DHC were separated with a methanol gradient and hydrocortisone and cortisone with an acetonitrile gradient containing trifluoroacetic acid on a reversed-phase column within 15 min. The corticosteroids were detected after electrospray ionization in positive mode with multiple reaction monitoring (MRM). The limits of quantification (LOQ) were estimated to be 15 nmol/kg liver and 1.6 nmol/kg adipose tissue for corticosterone and 5.4 nmol/kg liver and 0.92 nmol/kg adipose tissue for 11-DHC. The LOQ was estimated to be 0.2 nmol/kg adipose tissue for hydrocortisone and 0.4 nmol/kg adipose tissue for cortisone. The limits of detection (LOD) at 3 times S/N were estimated to be 0.07 nmol/kg adipose tissue for hydrocortisone 0.1 nmol/kg adipose tissue for cortisone. The variation of endogenous levels in KKA y mouse from different animals (CV%) was high with mean liver tissue levels of 117 ± 25 (S.D.) nmol/kg for corticosterone and 62 ± 19 (S.D.) nmol/kg for 11-DHC ( n = 5) and adipose tissue levels of 39 ± 20 (S.D.) nmol/kg for corticosterone and 2.4 ± 0.9 (S.D.) nmol/kg for 11-DHC ( n = 9). Endogenous levels in human biopsy samples from adipose tissue were 12 ± 7.0 (S.D.) nmol/kg for hydrocortisone and 3.0 ± 1.6 (S.D.) nmol/kg for cortisone ( n = 16). The new LC–MS/MS methods showed sufficient sensitivity and selectivity for determination of endogenous levels of corticosteroids in both KKA y mouse liver and adipose tissue samples and human adipose tissue samples. The selectivity of the methods was verified by analysis of two different product-ions from each analyte.