DETERMINATION OF PCMX CONJUGATED METABOLITES IN BLOOD-PLASMA AND URINE USING ENZYMATIC-HYDROLYSIS AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

An enzymatic-high performance liquid chromatography method with electrochemical detection was developed for the quantitation of p-chloro-m-xylenol conjugated metabolites in blood plasma and urine. The method is based on enzymatic hydrolysis of PCMX conjugates and extraction with benzene. The HPLC system consisted of a C18 reverse-phase column and a 65% methanol:35% ammonium carbonate 0.05% solution as a mobile phase. An electrochemical detector/glassy carbon electrode set at a potential of +0.9V versus Ag/AgCl/3M NaCl was used to monitor the drug. The recovery of PCMX after enzymatic hydrolysis from blood plasma samples and urine samples is approximately 98%. The limit of quantitation for plasma and urine is 5 ng/ml. The coefficient of variation is 5.1% over the range of 10-1000 ng/ml of plasma, and 4.8% over the range of 50-10000 ng/ml of urine. Data are presented to illustrate the practicality of this method for evaluation of PCMX and its conjugated metabolites in blood plasma and urine, after single intravenous administration of 200 mg dose of PCMX to a mongrel dog.