Abstract: Different hyphenated liquid chromatography (LC) and mass spectrometric (MS) techniques were investigated to set-up a method for the fast, direct anal. of betamethasone in hydrolyzed and non-hydrolyzed urine using large-volume sample injection. After the optimization of the LC parameters using a traditional UV detector and of the thermospray and mass spectrometric parameters by flow injection, urine samples (0.5 mL) were submitted to anal. by either LC combined with tandem mass spectrometry (MS-MS), coupled-column LC (LC-LC) combined with single quadrupole MS, and LC-LC-MS-MS. Both the three-step configurations (LC-MS-MS and LC-LC-MS) did not provide satisfactory results: loss of sensitivity was noted in the case of LC-MS-MS (likely due to reduced efficiency in the ionization of betamethasone in the thermospray owing to the presence of large amounts of matrix interference), while in the case of LC-LC-MS a high chemical noise resulting in insufficient selectivity of detection was observed On the contrary, LC-LC-MS-MS anal. proved to meet the demand of high speed of anal. (sample throughput, 4.5 h-1), selectivity, and sensitivity (LOQ, 1 ng/mL; LOD, 0.2 ng/mL). Notwithstanding the complex anal. system adopted, the developed procedure was manageable and very robust, provided that at the beginning of each anal. session the performance of the system was controlled by checking the retention time of the analytes on the first anal. column with UV detection and by optimizing vaporizer temperature of the thermospray by flow injection.

Abstract: Cortisone, hydrocortisone, prednisone, prednisolone, dexamethasone and betamethasone could be separated by HPLC on a 15-cm Hypersil (5 µm) column with methanol - 1,2-dichloroethane (3:197) as mobile phase (1.0 mL min-1). Each compound gave a response in a BAS LC4A electrochemical system, incorporating a mercury-coated gold electrode at -1.75 V, when 0.1 M tetrabutylammonium tetrafluoroborate (I) was added to the solvent as carrier electrolyte; calibration graphs were rectilinear and the sensitivity was 200 ng per injection. However, none of the steroids was retained on the column in the presence of I. The method should be applicable in flow injection analysis, or with post-column addition of electrolyte.