Determination of N,N',N"-triethylenethiophosphoramide and its active metabolite N,N',N"-triethylenephosphoramide in plasma and urine using capillary gas chromatography.

A sensitive assay for the determination of N,N',N"-triethylenethiophosphoramide (thioTEPA) and its metabolite N,N',N"-triethylenephosphoramide (TEPA) in micro-volumes human plasma and urine has been developed. ThioTEPA and TEPA were analysed using gas chromatography with selective nitrogen-phosphorus detection or mass spectrometry after extraction with a mixture of 1-propanol-chloroform from the biological matrix. Diphenylamine was used as internal standard. The limit of detection was 1.5 ng/ml for thioTEPA and 2.5 ng/ml for TEPA, using 100 microl of biological sample; recoveries ranged between 70 and 90% and both accuracy and precision were less than 10%. Linearity was accomplished in the range of 10-1000 ng/ml for plasma and 100-10000 ng/ml for urine using thermionic nitrogen-phosphorus detection. With mass spectrometry a linear range of 100-25000 ng/ml TEPA in plasma or urine was obtained. For thioTEPA a second-order polynomial function describes the relationship between the analyte concentration in the range of 500-25000 ng/ml and detection response. TEPA proved to be stable in plasma and urine for at least 10 weeks at -80 degrees C. ThioTEPA and TEPA plasma concentrations of two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples.