Quantification of corticosteroids in human plasma by liquid chromatography thermospray mass spectrometry using stable isotope dilution

Liquid chromatography-thermospray mass spectrometry (LC-TSP-MS) using isotope dilution was investigated for quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma. Complete separation attained by a LiChroCART Supersupher reversed-phase column and elution with 0.05 M ammonium formate-tetrahydrofuran-methanol (180:53:17, v/v/v) resulted in a significantly large isotope effect of the deuterium-labeled analogs on the HPLC behavior and caused difficulty in quantification. Reduction of the isotope effect on the retention times using 0.05 M ammonium formate-acetonitrile (65:35, v/v) permitted accurate quantification of cortisol and cortisone by the isotope dilution LC-TSP-MS, although separation between cortisol and prednisone was incomplete.