Determination of N,N',N"-triethylenthiophosphoramide in biological samples using capillary gas chromatography

A sensitive assay for the determination of N,N',N"-triethylenthiophosphoramide (thioTEPA) in microvolumes of human plasma and urine has been developed. ThioTEPA was analysed using gas chromatography with selective nitrogen-phosphorus detection, after extraction with ethyl acetate from the biological matrix. Diphenylamine is the internal standard. The limit of quantitation was 0.1 ng/ml, using only 100 microl of sample; recoveries ranged between 85 and 100% and both accuracy and precision were less than 10%. Using a flame ionisation nitrogen-phosphorus detector, the assay was not linear over the concentration range of 2-1000 ng/ml for plasma and 10-1000 ng/ml for urine. Linearity was accomplished in the range of 1-1000 ng/ml for plasma and urine when a thermionic nitrogen/phosphorous detector was used. The stability of thioTEPA in plasma proved to be satisfactory over a period of 3 months, when kept at -20 degrees C, whereas it was stable in urine for at least 1 month at -80 degrees C. ThioTEPA plasma concentrations of two patients treated with thioTEPA are presented demonstrating the applicability of the assay.