Comparison of Reversed Phase High Performance Liquid Chromatography and Competitive Protein Binding Assay in the Quantification of Cortisol in Bovine Plasma

A reversed phase high performance liquid (HPL) chromato-graphic method was developed for quantification of cortisol in bovine plasma. The reliability of this method was compared to that of a competitive protein binding assay (CPBA). Samples were adjusted to > pH 12 with NaOH, extracted with methylene chloride, evaporated to dryness and reconstituted in methanol/water (55%/45%) for HPLC determination. Satisfactory separation and sensitivity were achieved using an Altex Ultrasphere C-8 column (15 cm × 4.6 mm) with isocratic elution of methanol/water (57%/43%) and UV detection (242 nm). The detection limit of the CPBA (0.16 ng) was lower than for the HPL chromatographic method (0.54 ng); however, precision was significantly better for the HPL chromatographic method than for the CPBA. Values for the CPBA were significantly higher than those obtained using HPL chromatography, probably due to the positive interference of non-specific binding of plasma components in the CPBA. The specificity of HPL chromato-graphy for cortisol was validated by mass spectrometry.