Kinetic study of USP blue tetrazolium assay with methylprednisolone, hydrocortisone, and their hemisuccinate esters by high-pressure liquid chromatography.

The reactions of blue tetrazolium (I) with methylprednisolone and hydrocortisone and their hemisuccinate esters at ambient temperature under USP assay conditions in alcohol USP and in absolute alcohol were followed by high-pressure liquid chromatography (HPLC) and spectrophotometry. The disappearance of the ester and the increase and then decrease in the alcohol, as well as the formation of several reaction products with time, were observed by HPLC analysis. The reduction of I was observed spectrophotometrically. A sequential kinetic model was used to describe the overall reaction. The rate constants for the hydrolysis of the hemiester (k1), the reaction with I (k2), and the degradation of the parent steroid (k3) were determined by discrete kinetic experiments using HPLC. The following observations were made: (a) k1 is proportional to [H2O] and is the rate-limiting step, (b) k2 is about 100 times the value of k3 and (c) k2 for methylprednisolone is about the same as for hydrocortisone and appears to be independent of the concentration of water. With these rate constants, simulated time-concentration profiles for the reaction of the esters with I favorable compared with experimental data in alcohol USP and absolute alcohol. This study shows that the USP blue tetrazolium assay with these esters has potential for variability and is not stability indicating.