Quantification of cyclophosphamide and its metabolites in urine using liquid chromatography/tandem mass spectrometry

A reliable and easy to use liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantification of urinary concentrations of cyclophosphamide (CP) and its main metabolites excreted in urine, i.e. N ‐dechloroethylcyclophosphamide (DCL‐CP), 4‐ketocyclophosphamide (4KetoCP), and carboxyphosphamide (CarboxyCP). Sample preparation consisted of dilution of urine with an aqueous solution of the internal standard D 4 ‐CP and methanol, and centrifugation. LC/MS/MS detection was performed using a triple‐quadrupole mass spectrometer working in selected reaction monitoring mode. All analytes were quantified in a single run within 11.5 min. The limits of detection were 5 ng/mL for CP and 4KetoCP, 1 ng/mL for DCL‐CP, and 30 ng/mL for CarboxyCP. Quantification ranges were adjusted to the expected concentrations in 24‐h urine collections of patients treated with a polychemotherapy regimen (3–175 μg/mL for CP, 0.5–27 μg/mL for 4KetoCP and 0.17–9 μg/mL for CarboxyCP and DCL‐CP, respectively). The method was validated according to international guidelines of the ICH and the FDA. Copyright © 2004 John Wiley & Sons, Ltd.