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 mg/mL for CP, 0.5-27 mg/mL for 4KetoCP and 0.17-9 mg/mL for CarboxyCP and DCL-CP, respectively). The N-lost derivative cyclophosphamide (CP) is widely used as an antineoplastic drug and is included in many polychemotherapy regimens. CP is a prodrug and requires oxidative biotransformation to evolve its cytotoxic activity. It is subject to a pronounced metabolism in the human body, leading to active and inactive metabolites. A hydroxylation in ring position 4 represents the activating step in CP metabolism. The product 4-hydroxycyclophosphamide (4-OHCP) is in equilibrium with its ring-opened tautomer aldophosphamide (AldoCP). In humans, this first step is mediated predominantly by the cytochrome P450 2B6 (CYP2B6) enzyme, and also by members of the CYP2C subfamily and CYP3A4. [1][2][3][4] Most of the intermediate product AldoCP is subject to a spontaneous, non-enzymatic b-elimination to release the active alkylating species phosphoramide mustard and the urotoxic acrolein. Apart from the renal elimination of unchanged drug, partially competitive steps occur leading to inactive metabolites. A fraction of the parent compound is inactivated to N-dechloroethylcyclophosphamide (DCL-CP) by sidechain oxidation mediated by CYP3A4.3,5 In turn, 4-OHCP and AldoCP are detoxified by oxidation to carboxyphosphamide (CarboxyCP) and 4-ketocyclophosphamide (4KetoCP), which are excreted in urine.During the last 25 years, several methods for the determination of CP and its metabolites have been described.6-10 Gas chromatography represents a very sensitive method for the detection of the parent compound, 9,10 DCL-CP 5 and 4KetoCP in biological fluids. In addition, HPLC methods for the determination of CP concentrations in vivo have also been used. 11,12 However, these methods are not very selective or sensitive because of the low wavelength necessary for UV detection (200 nm), and they include complex extraction procedures. 6,[13][14][15] For the more reactive compounds, assays are available which require a complex derivatization before quantification in order to stabilize the analytes. 16 -18 During the last few y...