Liquid chromatography–mass spectrometry assay for quantitation of ifosfamide and its N-deschloroethylated metabolites in rat microsomal medium

doi:10.1016/j.jchromb.2005.03.023

Journal of Chromatography B

Volume 820, Issue 2, 25 June 2005, Pages 251-259

https://doi.org/10.1016/j.jchromb.2005.03.023 Get rights and content

Abstract

A specific and sensitive quantitative assay has been developed using high performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) for the simultaneous quantitation of the antitumor drug ifosfamide (IFM) and its two metabolites, N²-deschloroethylifosfamide (N²-DCE-IFM) and N³-deschloroethylifosfamide (N³-DCE-IFM) in microsomal medium. The analytes and the internal standard (cyclophosphamide) were isolated by ethylacetate extraction from rat liver microsomes. They were analysed on a Nucleosil^® C18 HD column (125 mm × 4 mm, 5 μm) using a step gradient with the mobile phase (2 mM ammonium formate and methanol). The HPLC–ESI-MS method used selected ion monitoring of ions m/z 199.1 Th and m/z 261.1 Th and was validated in the concentrations ranges of 100–5000 ng/mL for IFM and 50–2500 ng/mL for its N-deschloroethylated metabolites (DCE-IFM) with good accuracy and precision (CV less than 15%). The low limits of quantitation (LLOQ) were found at 50 ng/mL for N-deschloroethylated metabolites and at 100 ng/mL for the parent drug (IFM). The method was applied for the determination of ifosfamide and its N-deschloroethylated metabolites in rat microsomal incubations.

Introduction

The bisalkylating agent ifosfamide (IFM) was introduced into clinical trials in the 1970s, but its early use was limited by severe urotoxicity consisting in haemorragic cystitis. This side effect led to the development of sodium mercaptoethanesulfonate (mesna) as a safe and effective means of regional uroprotection [1]. Further studies have demonstrated IFM activity against a wide range of tumour types, from soft tissue sarcomas to lymphomas both in adult and paediatric patients. Main adverse effects of IFM include urotoxicity, myelosuppression, nausea and vomiting, neurotoxicity and nephrotoxicity [2]. Le Cesne et al. have shown that high-dose regimen of IFM (HD-IFM) allowed the circumvention of resistance to standard-dose ifosfamide in advanced soft-tissue sarcomas, indicating that there is a dose–effect relationship [3]. Since the systematic use of adjuvant treatments such as mesna, granulocyte-macrophage colony-stimulating factor (GM-CSF) and setrons, and the increase of IFM dosages, neurotoxicity and nephrotoxicity are the limiting factors for IFM-based chemotherapy. Indeed, in some studies, up to 40% of the treated patients show neurological disorders (depending on the dose quantity and the administration mode) and 5% presents a Fanconi syndrome. These toxicities seem to be more frequent with children [4], [5].

Ifosfamide is a prodrug: metabolism is needed to obtain its active form. The initial activation reaction in IFM metabolism is mediated by the cytochrome P450 enzyme CYP3A4 (Fig. 1). The hydroxylation on the Carbon-4 of the oxazaphosphorine ring leads to 4-hydroxy-ifosfamide (4-OH-IFM), which is in equilibrium with its tautomeric form, the aldo-ifosfamide. The latter form may then either be oxidized by an aldehyde dehydrogenase (ALDH1) to carboxy-ifosfamide, an inactive metabolite, or it can spontaneously be decomposed by a retro-Michaël reaction to form acrolein and the isophosphoramide mustard (IPAM) which is the active moiety. IPAM is a bisalkylating agent. Acrolein is held responsible for urotoxicity. Up to 50% of a dose of IFM undergoes a separated oxidative N-dealkylation reaction, resulting from the loss of chloroethyl side-chains and producing N²-deschloroethylifosfamide (N²-DCE-IFM), N³-deschloroethylifosfamide (N³-DCE-IFM) and N²,N³-dideschloroethylifosfamide (N²,N³-diDCE-IFM). An equimolar quantity of chloroacetaldehyde (CAA) is formed in each of these N-dealkylation reactions [6], [7]. This metabolite is known to be responsible for both nephrotoxicity and metabolic neurotoxicity which may be associated with IFM treatment [8].

The direct quantitative determination of the oxazaphosphorines, such as IFM and cyclophosphamide (CPM), and their metabolites is difficult, because of their high polarity and their chemical and thermal properties. Thus, several analytical methods have been developed using gas chromatography (GC) or high performance liquid chromatography (HPLC) combined with different detection techniques [9]. The UV detection of oxazaphosphorine compounds is also problematic due to their poor spectral properties. After a GC separation, the most appropriate detector seems to be a nitrogen phosphorus detector (NPD). Because NPD has high selectivity and sensitivity for oxazaphosphorine compounds, as well as a small solvent peak in comparison with flame ionization detector [10]. A GC–NPD technique allowed simultaneous determination of IFM, N²-DCE-IFM and N³-DCE-IFM in plasma after liquid–liquid extraction and without derivatization [11]. Kerbusch et al. compared GC–NPD with GC-positive ion electron-impact ion-trap tandem mass spectrometry [12]. GC–NPD proved to be superior to GC–MS² in terms of sensitivity (LOQs 50 ng/mL and 250–500 ng/mL, respectively), and detection range and as well for accuracy and precision. Moreover, mass spectrometry detection has been used successfully with GC or HPLC for the oxazaphosphorine compounds determination [10]. The described GC–MS methods were used successfully for the sensitive determination of IFM in human plasma or in urine [13], but sample preparation needed derivatization before analysis and was time-consuming.

The purpose of this work was to develop a simple, sensitive and effective quantitative HPLC–ESI-MS method to study the in vitro metabolism of IFM and other oxazaphosphorines analogues in rat microsomes. In precedent works [14], we have developed synthesis of methylated IFM analogues to reduce side-chain hydroxylation which leads to toxic metabolites such as chloroacetaldehyde (CAA). The monitoring of the N²-DCE-IFM and N³-DCE-IFM formation can indirectly quantify the production of toxic and labile CAA.

A fast and effective assay is necessary to determine the concentrations of deschloroethylated metabolites (DCE-IFM) and of IFM in microsomal medium that will be useful to confirm the enzymes involved in the side-chain oxidation of IFM and the synthesized analogues. Since there is no available assay for the concomitant direct detection of IFM and its metabolites without derivatization in rat liver microsomes, the present HPLC–ESI-MS method has been developed and validated for the simultaneous determination of N²-DCE-IFM, N³-DCE-IFM and IFM.

Section snippets

Chemicals and reagents

Ifosfamide (HOLOXAN^®) (2-(2-chloroethylamino)-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) and cyclophosphamide (ENDOXAN^®) (2-[bis-(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) were obtained from Baxter SA (Maurepas, France). N²-DCE-IFM ([3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphonan-2-yl]amine) and N³-DCE-IFM (N-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphinan 2-oxide) were synthesized according to previously developed techniques [14]. Their

Optimisation of the HPLC–MS method

In terms of chromatographic conditions, methanol was chosen to get narrow and symmetric peaks. Different ammonium formate solutions (2, 5 and 10 mM in water) were tested. The best HPLC–MS response was obtained with 2 mM (pH 5.7) Allowing good ionization and stability of the studied oxazaphosphorine compounds. No interference was observed as shown on Fig. 2a.

Once mobile phase was chosen, the gradient conditions were determined to meet the following objectives: the separation with baseline return

Conclusion

The present work demonstrated the development of a HPLC–ESI-MS method using electrospray ion-trap mass spectrometry for the quantitation of IFM and their N²- and N³-deschloroethylated metabolites, which can be produced using rat microsomes. SIM mode detection allows the direct quantitation of both metabolites and CPM using CPM as internal standard. The HPLC–ESI-MS method was accurate and precise for the determination of N-deschlorethylated metabolites of IFM in the range of 50–2500 ng/mL and for

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Liquid chromatography–mass spectrometry assay for quantitation of ifosfamide and its N-deschloroethylated metabolites in rat microsomal medium

Abstract

Introduction

Section snippets

Chemicals and reagents

Optimisation of the HPLC–MS method

Conclusion

Gynecol. Oncol.

J. Chromatogr. B Biomed. Sci. Appl.

J. Chromatogr. B Biomed. Sci. Appl.

J. Chromatogr. B Biomed. Sci. Appl.

J. Chromatogr.

Biochem. Pharmacol.

J. Am. Soc. Mass Spectrom.

J. Chromatogr.

Biochem. Pharmacol.

Support Care Cancer

Clin. Pharmacokinet.

Proc. Am. Soc. Clin. Oncol.