Evaluation of Some Experimental Parameters in the Determination the Ratios of Cortisol to Cortisone and their Tetrahydrometabolites in Urine

Introduction of a perfusion on-line cleanup for the elimination of proteic matter buildup onto the HPLC column Cortisol (F) plays an important role in human physiology and is a marker for the diagnosis of many pathological states. It is secreted by the adrenal glands, while cortisone is produced by 11β-hydroxysteroid dehydrogenase (11β-HSD) isoenzymes, which interconvert cortisol to hormonally inactive cortisone. Cortisol concentrations in the body are controlled by the relative activity of 11β-HSD type 1, which is responsible for the reversible conversion of cortisone to cortisol, and 11β-HSD type 2 that catalyzes the irreversible conversion of cortisol to cortisone. A correct balance between the type 1 and 2 enzymatic activities maintains the physiological level of cortisol. The ratio of either cortisol to cortisone (F/E) or that of some metabolites allows an evaluation of the activity of 11β-HSD. Sample preparation: centrifugation at 5000 rpm, enzyme hydrolysis by β-Glucuronidase/Arylsulfatase in acetate buffer 0.5 M (pH 5.1) containing IS, 1:20 (v/v) dilution with water. On-line cleanup: SPE by perfusion column Applied Biosystems POROS R1/20 2.1 x 30 mm. HPLC separation: the column (Phenomenex Luna C8 2 mm x 50 mm, 3 µm particle size), kept at 50 °C, is eluted at 250 µl/min by a two solvents gradient, where solvent A is water/methanol (50/50, v/v) with 0.1% formic acid, and solvent B is methanol/acetonitrile (50/50, v/v), containing 0.1% formic acid too. MS-MS detection: MRM, performed on an Applied Biosystems API 4000 triple quadrupole mass spectrometer, equipped with a turbo ionspray source. The main target of this work is the optimization of a previously presented method for the determination of tetrahydrocortisol (THF), allo-tetrahydrocortisol (A-THF) and tetrahydrocortisone (THE) and their ratio (THF +A-THF)/THE. In this respect we introduced the parent compounds E and F in order to get the F/E ratio as well. Moreover we checked the influence of some potentially critical parameters, such as the use of water or urine for the calibration standards, the response factor of THF and A-THF and the on-line SPE conditions. Our findings demonstrate that calibration standars can be prepared in water, as the influence of the matrix on the ratio is negligible and it is possible to use either THF or A-THF as a standard for the calibration of both. The final method was tested with some batches of samples, and it demonstrated its reliability and ruggedness. On the contrary of what observed in our previous reports, HPLC performances remain stable for hundreds of injections, without any degradation of the HPLC column, very likely due to some proteic matter buildup.