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 chromatography/ionspray-tandem mass spectrometry procedure (HPLC/MS/MS) has been developed to quantify cyclophosphamide in human urine from hospital personnel involved in drug preparation and administration of antineoplastic alkylating agents. This methodology, which includes liquid-liquid extraction with ethylacetate, requires no derivatization procedures, preventing cyclophosphamide (CP) from possible thermal and chemical decomposition 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 microL-1 to 1.9 ng microL-1 urine. This methodology was validated by the use of ifosfamide as internal standard. The assay was linear over the range 0 to 3.2 ng microL-1 urine, with a lower limit of quantification of 0.2 microL-1. The limit of detection was assessed at 0.05 ng microL-1 urine. This method is characterized by a coefficient of variation < 10%. Standard calibration curves, obtained on three different 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 different concentrations (0.5, 0.8, 3.2 ng microL-1) was always more than 85%. The extraction efficiency of cyclophosphamide from urine samples was 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 solutions was adjusted to 7.