Simultaneous determination of N,N',N"-triethylenethiophosphoramide, cyclophosphamide and some of their metabolites in plasma using capillary gas chromatography.

Abstract

A sensitive assay for the simultaneous determination of N,N',N"-triethylenethiophosphoramide (thioTEPA), its metabolite N,N',N"-triethylenephosphoramide (TEPA), cyclophosphamide (CP) and its metabolite 2-dechloroethylcyclophosphamide (2-DCE-CP) in plasma has been developed and validated. The analytes were determined using gas chromatography with nitrogen/phosphorus selective detection after liquid-liquid extraction with chloroform using 100 microl of plasma. Diphenylamine (for TEPA, thioTEPA and 2-DCE-CP) and imipramine (for CP) were used as internal standards. The limits of quantitation for thioTEPA, TEPA, CP and 2-DCE-CP were 5, 5, 50 and 250 ng/ml, respectively. Linear calibration curves were observed over two decades of concentration. Accuracy, within-day and between-day precision were less than 13% for all analytes. Stability of the analytes proved to be satisfactory for at least 1 month, stored at -70 degrees C. Analysis of samples obtained from patients receiving cyclophosphamide, thioTEPA and carboplatin in a high-dose regimen demonstrated the applicability of the assay.