Simultaneous determination of prednisolone and Cortisol in serum by HPLC and by isotope dilution—mass spectrometry

Abstract

A reference method for the simultaneous assay of Cortisol and prednisolone by isotope dilution-mass spectrometry has been developed. A fixed amount of ²H₄-labelled Cortisol is added to a fixed amount of serum. The mixture is then extracted, converted into methoxime-trimethylsilyl derivative, and subjected to analysis by combined gas chromatography-mass spectrometry. The ions at m/e 603, m/e 605 and m/e 609 are followed through the gas chromatography. These ions correspond to the M-31 ions in the mass spectrum of methoxime-trimethylsilyl ether derivative of prednisolone, unlabelled Cortisol and ²H₄-cortisol, respectively. With the use of a standard curve, the concentration of prednisolone can be calculated from the ratio between the peak at m/e 603 and the peak at m/e 609 and the concentration of Cortisol can be calculated from the ratio between the peak at m/e 605 and the peak at m/e 609. The coefficient of variation was about 2% with respect to determination of prednisolone and about 3% with respect to determination of Cortisol. This method was compared with a routine method based on high-performance liquid chromatography (HPLC) The coefficient of variation was 2–3 % with respect to determination of Cortisol and 1–3 % with respect to determination of prednisolone. There was a good correlation between the isotope dilution method (x) and the HPLC method (y). In the assay of serum prednisolone the regression analysis gave the following equation: y = 1.02x −69 nmol/l, r = 0.99. In the assay of serum Cortisol, the regression equation was: y = 1.07x − 14 nmol/l, r = 0.95. It is concluded that the HPLC-method, used for routine determination of Cortisol and prednisolone, has an accuracy sufficient for its intended use.

Key-words:

• prednisolone
• cortisol
• mass fragmentography
• high-performance liquid chromatography
• isotope dilution