Analysis of cyclophosphamide and five metabolites from human plasma using liquid chromatography–mass spectrometry and gas chromatography–nitrogen–phosphorus detection

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Abstract

An assay method for the quantification of cyclophosphamide (CY) and five metabolites from human plasma is presented. The procedure is adapted to the chemical properties of the compounds of interest: non-polar compounds are extracted into methylene chloride, concentrated and analyzed by GC–NPD after derivatization, and the remaining aqueous fraction is deproteinated with acetonitrile–methanol prior to separation via reversed-phase HPLC and detection using atmospheric pressure ionization (API)-MS. Standard curves are linear over the required range and reproducible over five months. Plasma concentration–time profiles of CY and metabolites from a patient receiving CY by intravenous infusion (60 mg/kg, once a day for two days) are presented.

Introduction

Cyclophosphamide (CY) is a phosphoramide form of nitrogen mustard commonly used in cancer treatment. In addition, its toxicity to lymphocytes has made it an important immunosuppresent agent in hematopoietic stem cell transplant therapies and, more recently, in treatment of inflammatory conditions such as systemic lupus erythematosis and rheumatoid arthritis, etc. [1]. Dose escalation of CY is limited by myelosuppression, cardiotoxicity, hemorrhagic cystitis and venoocclusive disease (VOD) of the liver. The parent drug itself is not cytotoxic. It must be activated, primarily by cytochrome P450 2C9 and 3A4 in the liver, to “transport intermediates” 4-hydroxycyclophosphamide (HCY) [2] and, possibly, 3-hydroxypropylphosphoramide mustard (HPPM) [3] which enter target cells and degrade chemically/enzymatically to produce the ultimate DNA alkylator-phosphoramide mustard (PM). There are a variety of alternative metabolic pathways for CY and HCY which result in deschloroethylcyclophosphamide (DCCY), carboxyethylphosphoramide mustard (CEPM), and 4-oxocyclophosphamide (KetoCY) all of which show very low cytotoxocity. A partial scheme of CY metabolism is shown in Fig. 1.

Attaining therapeutic versus toxic outcomes in patients treated with CY requires an understanding of the individual variation in both activation and detoxifying pathways [4], [5] of the drug, which can be obtained through prospective studies analyzing all the compounds shown in Fig. 1. There are many analytical procedures for CY. Most use a gas chromatographic separation of derivatized CY with either mass spectrometry (MS) or nitrogen–phosphorus detection (NPD). The other compounds provide more of a challenge; they are UV transparent, highly polar and demonstrate varying degrees of instability. Over the years, a variety of methods have been developed using thin-layer chromatography (TLC) [6], [7], [8], colorimetry [9], [10], high-performance liquid chromatography (HPLC) [11], gas chromatography (GC)–NPD or GC–MS [3], [12], [13], [14], [15], and direct insertion probe MS analysis for some or all of the compounds of interest. GC has emerged as the method of choice for analysis of these compounds. However, the multiplicity of products resulting from derivitization [14], poor extraction efficiency [12], and the instability of the individual metabolites [15] have all served to diminish the utility of this approach. These problems are compounded in high dose therapy, where concentrations of cyclophosphamide may be up to a thousand times those of the metabolites.

We have recently developed a method for HCY analysis using HPLC with UV detection after bedside stabilization as the p-nitrophenylhydrazone [5]. Herein we describe methodology which allows the non-polar (and generally more stable) components to be extracted from plasma, derivatized, and analyzed by GC using either NPD or MS. The extracted aqueous phase is then deproteinated and analyzed for the more polar compounds by LC–MS using deuterated internal standards. Detection limits obtained for all compounds are in the low to sub-micromolar range and all analyses can be performed using a single 0.5-ml plasma sample.

Section snippets

Materials

CY was obtained from Sigma as the monohydrate; DCCY, CEPM and PM were the generous gift of ASTA Medica (Frankfurt, Germany); HPPM was synthesized in the laboratory by the method of Takamizawa et al. [16]. β-Tetradeuterocarboxyethyl phosphoramide mustard (D4-CEPM) and β-tetradeutero phosphoramide mustard (D4-PM) were synthesized in the laboratory by the methods of Takamizawa et al. [17] and Grigg and Jarman [18], respectively, using β-D4-mustard prepared by the method of Jardine et al. [15].

Results and discussion

The synthesized compounds were all >95% chemically pure by NMR analysis. The isotopically labeled internal standards were prepared with four deuteriums on the β (halogenated) carbons of the nitrogen mustard moiety. The deuteriums in this site are very stable (pKa∼20) and exchange is impossible without total fragmentation of the molecule.

The extraction procedure described is based on earlier work by other investigators [12], [15] that shows efficient, pH independent, extraction for CY and DCCY.

Acknowledgements

This work was supported in part by NIH Grants CA 18029 and GM 32165.

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