Studies of the Stability of a Substituted Phosphorothioate Using an Improved High Performance Liquid Chromatography Assay Coupled with Electrochemical Detection

Abstract

A sensitive, reproducible and rapid HPLC assay was developed for preformulation and stability studies of a potential radioprotector, S-2-(3-methylaminopropylamino) ethyl-phosphorothioate, WR-3689. This method is based on the elution of WR3689 by high performance ion-pair reverse phase liquid chromatography using an electrochemical detector with an oxidation potential set at + 0.19 v. The detector response was found to be linear over the concentration range of 0.02–20 μg/mL with an absolute limit of quantitation of 170 pg. The coefficient of variation ranged from 0.75–6.52% for the interday reproducibility. This HPLC assay was employed to determine the stability of WR-3689 at various pH (2, 4, 7, 10 and 12) and temperatures (22 °C, 35 °C, 43 °C and 100 °C). Results of the stability studies indicated that WR-3689 is more stable in basic than in acidic aqueous solutions. For example, the half-life of WR-3689 at 45 °C was 13.8 days at pH 12 as compared to 12.7 h at pH 4. With regard to the effect of temperature, the half-life of this compound increased from 19.2 h at 43 °C to 41.8 days at 22 °C. In conclusion, the excellent reproducibility and stability indicating capacity of this assay makes it a useful method for formulation studies. The high sensitivity and specificity of this assay make it useful for pharmacokinetic studies.