Determination of Serum Cortisol by High Performance Liquid Chromatography with Serum Direct Injection
1988 Volume 16 Issue 4 Pages 188-193
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1988 Volume 16 Issue 4 Pages 188-193
A rapid method for measuring serum cortisol has been developed by reversed phase high performance liquid chromatography (HPLC) with a column switching technique. The precolumn and analytical column used were BSA-ODS columns (20μm, TSK; 4.6 I.D. ×10mm) and (5μm, TSK;4.6 I.D.×150mm), respectively.Both columns were connectd to a high pressure switching valve (7000, Rheodyne). A serum sample (200μl) was directly injected onto the precolumn, from which serum cortisol was eluted. The eluate was transferred to the analytical column and separated, monitoring the absorbance at 245 nm. A peak corresponding to cortisol was observed at the retention time of 28.2min on the chromatogram and well separated from other glucocorticoids except for prednisolone. In the precision tests, the coefficients of variation (C.V.) for the intra-run and inter-run assays were found to be 3.48% and 2.84%, respectively. The recovery rate of cortisol was almost quantitative. The detection limit of cortisol in serum was 1μg/dl. The standard curve showed linearity in the range of 20 to 200μg/dl.
The proposed method for measuring serum cortisol requires only a small volume of the sample and no internal standard.
