The Effects of Autoclaving on the Stability of Physostigmine Salicylate in Buffer Solutions

Abstract

The effects of autoclaving on the stability of physostigmine salicylate in buffer solutions of pH 3.0, 4.0, and 5.3 were studied by reversed-phase, ion-pairing liquid chromatography. The influences of flushing the solutions with N2 and adding NaHSO3 to the solutions on the stability of physostigmine in pH 4.0 buffer solutions were also studied by this HPLC method. The anaerobic (N2 flushed) pH 3.0 buffer solutions retained 98.2% of physostigmine and the aerobic (without N2 flushing) pH 3.0 buffer solutions retained 95.4% of physostigmine after autoclaving at 123° C for 25 minutes. As the autoclaving time was reduced to 15 minutes, 99.5% of physostigmine remained in the anaerobic solutions of pH 3.0. For pH 4.0 anaerobic solutions of physostigmine salicylate, the addition of NaHSO₃ lowered the average percent of remaining physostigmine after autoclaving at 123° C for 25 minutes from 89.1% to 88.3%. But both values are significantly greater than the average percent of physostigmine remaining in pH 4.0, aerobic solutions without NaHSO₃, which is 87.5% and that in the similar solution but with NaHSC3, which is 85.7%. The anaerobic pH 5.3 buffer solutions retained 28.0% of physostigmine after autoclaving at 123°C for 15 minutes. No detectable amount of physostigmine remained in the unbuffered solutions either of aerobic or anaerobic conditions after autoclaving. Eseroline was the only decomposition product detected in the anaerobic, unbuffered solutions; both eseroline and rubreserine were detected in the aerobic, unbuffered solutions of physostigmine salicylate after autoclaving. The isocatalytic point for physostigmine at 123° C was calculated to be 2.79 from the base-catalyzed hydrolysis rate constant obtained in this study.