Quantification of 6 Glucocorticoids in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry: Method Development, Validation, and Assessment of Matrix Effects

Abstract

Glucocorticoids are a group of steroid hormones with immunosuppressive and anti-inflammatory properties. In this article, we report the development and the validation of a liquid chromatography tandem mass spectrometry method for the simultaneous quantification of prednisolone, prednisone, cortisol, cortisone, methylprednisolone, and dexamethasone in human plasma. Furthermore, matrix effects were assessed qualitatively and quantitatively. Plasma protein precipitation was performed with acetonitrile containing internal standards. Liquid-liquid extraction with dichloromethane and evaporation were used for cleanup and enrichment. The glucocorticoids were analyzed using reversed-phase chromatography and multiple reaction monitoring of positive ions. The mean extraction recovery was in the range 66.5%-104.8%, whereas the lower limits of quantification ranged from 1.5 to 4.0 μg/L. The intraday and interday accuracies of all the analytes were within 89.4%-116.6%, and imprecision was <15.6%. Ion suppression ranged from 15.3% to 27.3%. However, the matrix effects did not compromise the assay performance, with mean deviations in calculated concentrations of -4.8% to 2.1% between methanol and matrix. Short-term stability was acceptable for 5 of the analytes, with deviations from baseline between -3.4% and 8.7% after 24 hours at 4°C, although methylprednisolone was stable for 6 hours with a degradation of 10.2%. Deviations from baseline in controls stored at -20°C for 6 months ranged from -22.3% to 6.3%. All analytes were stable after 3 repetitive freeze-thaw cycles, with a maximum degradation of 5.5%. In terms of postpreparative stability, the analytes were stable after 24 and 48 hours at 4°C, with maximum degradation of 6.1% and 9.4%, respectively. A validated, sensitive, selective, and reproducible method for quantifying the concentrations of 6 glucocorticoids in human plasma by liquid chromatography tandem mass spectrometry is reported.