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

Abstract

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...

Validation of urinary excretion of cyclophosphamide as a biomarker of exposure by studying its renal clearance at high and low plasma concentrations in cancer patients

“…Thus, highly sensitive analytical methods for determination of CP in plasma and urine were developed and validated regarding LOD, precision and stability. The LODs in this study were lower compared to previous methods for determination of CP in plasma (Sadagopan et al 2001;Liu et al 2004) and urine Sannolo et al 1999;Pethran et al 2003;Kasel et al 2004). The precisions of our methods were satisfactory and the stability of CP in the samples was high.…”

A Direct Injection LC/ESI-MS/MS Analysis of Urinary Cyclophosphamide as an Anticancer Drug for Monitoring Occupational Exposure

“…4B. Although the present method is an extremely simple analytical method which does not require even deproteinization, it was possible to measure with extremely high sensitivity compared with the method reported previously [4][5][6]. The results of stability test are summarized in Table 2.…”

“…This suggested that medical staff could be exposed to these sources in situations other than drug preparation and administration. In 2004, Kasel et al [6] reported an analytical method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) to quantify the concentrations of cyclophosphamide (detection limit: 5 ng/mL) and its metabolites, N-dechloroethylcyclophosphamide, 4-ketocyclophosphamide, and carboxyphosphamide, in urine. Although the method was reliable compared to the traditional GC/MS method, it was unable to detect trace amounts of cyclophosphamide in the urine of occupationally exposed medical staff.…”

Simultaneous quantification of cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC–MS/MS)

“…However, these methods either described the quantification of CP and other metabolites in urine [2,3] or quantification of 4OHCP without CP in human plasma [4]. Sadagopan described the simultaneous quantification of CP and 4OHCP in mouse plasma [5].…”

“…Assays quantifying CP and/or 4OHCP using highperformance liquid chromatography (HPLC) with mass spectrometry (MS) detection have been described previously [2][3][4][5][6]. However, these methods either described the quantification of CP and other metabolites in urine [2,3] or quantification of 4OHCP without CP in human plasma [4].…”