LIQUID-LIQUID-EXTRACTION PROCEDURE FOR TRACE DETERMINATION OF CYCLOPHOSPHAMIDE IN HUMAN URINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY TANDEM MASS-SPECTROMETRY

A sensitive, specific and accurate high performance liquid chromatogra phy/ionspray-tandem mass spectrometry procedure (HPLC/MS/MS) has been developed to quantify cyclophosphamide in human urine from hospital pe rsonnel involved in drug preparation and administration of antineoplas tic alkylating agents. This methodology, which includes liquid-liquid extraction with ethylacetate, requires no derivatization procedures, p reventing cyclophosphamide (CP) from possible thermal and chemical dec omposition reactions. We detected the excretion of this unmetabolized alkylating drug in 50% of all the study participants. The amount of CP ranged from 0.1 ng mL(-1) to 1.9 ng mL(-1) urine. This methodology wa s validated by the use of ifosfamide as internal standard. The assay w as linear over the range 0 to 3.2 ng mL(-1) urine, with a lower limit of quantification of 0.2 ng mL(-1), The limit of detection was assesse d at 0.05 ng mL(-1) urine. This method is characterized by a coefficie nt of variation <10%, Standard calibration curves, obtained on three d ifferent days, had correlation coefficients always greater than 0,998, The intra and interday precision were within 11%, and accuracy was in the range 99-103%, The mean extracted recovery assessed at three diff erent concentrations (0.5, 0.8, 3.2 ng mL(-1)) was always more than 85 %, The extraction efficiency of cyclophosphamide from urine samples wa s also studied at six different pH values (pH 4, 5, 6, 7, 8, 10), The maximum extraction efficiency was obtained when the pH of urine soluti ons was adjusted to 7,0, (C) 1998 John Wiley & Sons, Ltd.