Background The analysis of steroids in biological matrices is challenging. One can apply immunoassay as well as gas and liquid chromatography with various types of detection, depending on the available equipment and the experience of the analyst. The question is how the methods are interchangeable between themselves. Doubts were reported having compared immunoassays and chromatography-mass spectrometry, but there are scarce data on chromatographic methods with detection types other than mass spectrometry. Methods Here, we present the detailed comparison of two liquid chromatographic methods for the determination of free urinary cortisol and cortisone: one with fluorescence detection (high-performance liquid chromatography [HPLC-FLD]) and the other with tandem mass spectrometry (HPLC-MS/MS). The comparison was made with 199 human urine samples. The data analysis included Passing–Bablok and Deming regression, Bland–Altman test, Wilcoxon test, mountain plot and Lin’s concordance correlation coefficient. Results The validation data indicated that both methods met the requirements of the European Medicines Agency. However, the statistical analysis revealed the systematic bias between the two assays. The Passing–Bablok and the Deming tests showed that the HPLC-FLD method overestimated results for cortisol and underestimated measurements for cortisone. The Bland–Altman analysis estimated the mean differences between the methods: 18.8 nmol/L for cortisol and −16.9 nmol/L for cortisone measurement. Conclusions Both methods’ results led to the same conclusion in observational studies, but the techniques are not interchangeable. The literature data, the observations from the clinical setting and our experience clearly indicate that the future of steroid measurements will belong to chromatography coupled with mass spectrometry.
Development and validation of two analytical methods for urea determination in compound feed, including pet food, and yeast using high-performance liquid chromatography coupled with fluorescence detection and tandem mass spectrometry