Recent Developments in the Direct Determination of the Ratio (Tetrahydrocortisol+alloTetrahydrocortisol)/tetrahydrocortisone in Urine

Use of on-line perfusion column purification of urine samples to get method ruggedness Glucocorticoids play an essential role in normal physiology by modulating metabolic and immune responses. They also have an important function in the pathogenesis of several diseases. The interconversion of hormonally active glucocorticoids and their inactive 11-keto forms is mediated by 11β-hydroxysteroid dehydrogenase (11β-HSD) which plays a pivotal role in the prereceptor regulation in the glucocorticoid hormone action. The ratio (tetrahydrocortisol+allo-tetrahydrocortisol)/tetrahydrocortisone is directly correlable with such enzymatic activity. Indeed, changes in the activity of 11β-HS may be important in several human diseases, such as hypertension, obesity, oligomenorrhea and hirsutism, etc.. Several methods have been proposed for the determination of that ratio. Most of them require extraction and derivatization steps and make use of gas-chromatographic techniques. The analytical methods moves from a previous report, making use of HPLC-ESI-MS-MS by Selected Reaction Monitoring (349-91 and 349-105 for tetrahydrocortisol; 347-91 and 347-149 for tetrahydrocortisone. HPLC separation was obtained on a C8 2.1x50 mm, 5 µm particle size column using a gradient of water, methanol and acetonitrile containing 0.1% formic acid. Problems arised when massive sample batches were analysed. We report here the use of a column switching system, using a Poros R1/20 2.1x30 mm perfusion column for on-line purification of the samples. Deuterated steroids were used as internal standards. The application of the original method showed a progressive degradation of the system performances as indicated by the strong reduction of the internal standard signal through the batch, up to completely lose sensitivity after some tens of matrix injections. This represents a huge complication as the accuracy and precision of the results become less reliable during the course of the analysis. Moreover the cost of the whole method is going to be strongly affected by the short life of the HPLC columns. The use of guard columns did not solve the problem as the contaminating material (probably residual proteins and peptides) is not completely trapped and partially reaches the analytical column. For instance, when analyzing a batch of 103 urine sample, in triplicate, the internal standard signal area decreased from about 20000 to 1900 counts. For this reason we decided to take advantage of the use of a switching valve and a perfusion column, able to retain the analytes, but leaving most of the high molecular weight stuff to pass to waste. This new improved method has been tested with the analysis in triplicate of a consistent number of samples