Simultaneous determination of capecitabine and its metabolites by HPLC and mass spectrometry for preclinical and clinical studies

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Abstract

A reverse-phase high-performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of capecitabine, its intermediate metabolites (DFCR, DFUR) and the active metabolite 5-fluorouracil in mouse plasma, liver and human xenograft tumours. The method was also cross-validated in human plasma and human tumour for clinical application. The method has greater sensitivity than previously published methods with an equivalent accuracy and precision. It uses less biological material (plasma, tissue) and should therefore be applicable to biopsies in patients treated with capecitabine.

Introduction

Capecitabine (N4-pentoxycarbonyl-5′-deoxy-5-fluorocytidine, Xeloda®), is a fluoropyrimidine carbamate, which is converted in liver and tumour to the active agent 5-fluorouracil (5-FU). It is used in the chemotherapeutic treatment of patients with breast and colon cancer. Carboxylesterases (EC 3.1.1.1) located in the liver in human and in the plasma and liver in rodents convert capecitabine to 5′deoxy-5-fluorocytidine (DFCR). DFCR is then converted by cytidine deaminase (EC 3.5.4.5) both in liver and tumour to 5′deoxy-5-fluorouridine (DFUR). The formation of 5-FU from DFUR is catabolised by thymidine phosphorylase (EC 2.4.2.4), and preferential expression in tumours has been reported previously both in animal models [1] and in patients [2]. 5-FU is the active metabolite: its inhibition of thymidylate synthase (EC 2.1.1.45) and incorporation into nucleic acids are responsible for the cytotoxic activity. Extensive pharmacokinetic studies have been performed on capecitabine and its metabolites [3], [4] based on phases II and III trials [4], [5], [6]. Marked inter-patient variability was observed during these studies, although pharmacokinetic parameters were not predictive of either toxicity or response to treatment [4]. Animal models have also been used to evaluate capecitabine efficacy in different xenograft models [1], [7]. A physiologically-based pharmacokinetic model was developed and shown to predict accurately plasma concentrations of capecitabine and its metabolites [8].

As a single agent, capecitabine is generally used in the clinic using a twice daily administration schedule for 14 out of 21 days, but it is possible that other schedules of administration may be more beneficial for some patients. The duration of treatment may also be important. Finally, early markers of response/progression could be beneficial for patient management.

Performing preclinical studies in rodents allows the development of alternative schedules of administration linked to the determination of drug concentration in different organs. They may also be used to identify a better surrogate tissue to predict toxicity/response. However, one of their main pitfalls is the small quantities of biological material that can be recovered for pharmacokinetic studies, hence the interest in developing analytical methods using small volumes of blood and small quantities of tissue. This would also be beneficial in clinical studies when serial sampling is required. Finally, if an analytical method is validated for both rodent and human tissue, the comparison of preclinical and clinical data is facilitated.

Several HPLC methods have been developed over the recent years to study capecitabine and its metabolites. The difference in polarity between capecitabine and the active metabolite 5-fluorouracil has so far prevented the simultaneous analysis of both compounds by HPLC: Reigner et al. in the original method analysed independently capecitabine, DFCR and DFUR by HPLC and UV detection and 5-FU using gas chromatography [9]. Subsequently, an MS–MS method was developed by the same team [10] but using a different sample extraction and chromatography conditions for capecitabine, DFCR, DFUR on one system and 5-FU and FBAL on another. More recently, a LC–MS technique has been developed for capecitabine, DFCR, DFUR but is not suitable for 5-FU determination [11]. Zufia et al. set up a method using UV detection that allows the simultaneous detection of capecitabine, DFUR, 5-FU and dihydro-5-FU in plasma [12]. Finally, Siethoff et al. were able to determine the plasma concentration of both capecitabine and its different metabolites but using a column switching and MS–MS detection [13].

We propose a new HPLC method using small volumes of biological material, validated in plasma and tissues (liver, tumour) from rodents and humans, which allows the simultaneous quantification of capecitabine and its metabolites with a single HPLC system coupled to mass spectrometry detection. The method is fully validated for both preclinical and clinical studies and can therefore be the basis for further preclinical and clinical studies with capecitabine.

Section snippets

Chemicals and solutions

Capecitabine (batch # 26954-190A-MIL), 5′deoxy-5-fluorocytidine (DFCR) (batch # Ro 0218782-000-003), 5′deoxy-5-fluorouridine (DFUR) (batch # Ro 0219738-000-02), and 5-fluorouracil (5-FU) were provided by Hoffmann-La Roche, Basel, Switzerland. Ammonium acetate was from Sigma (Sigma, Gillingham, UK). Formic acid was from BDH (BDH, Poole, UK). HPLC grade acetonitrile was from Rathburn (Walkerburn, UK) or BDH.

Plasma and tissues from mouse and human

Human plasma was obtained from the Scottish National Blood Transfusion Service. Human

Results and discussion

The method was validated in terms of limits of quantification recovery, specificity, sensitivity, precision and accuracy, and stability.

Conclusion

Several HPLC methods have been developed in the last few years for the quantification of capecitabine and/or its metabolites, but each has had associated limitations. In contrast to previous methods, the present method was validated in plasma, tumour and liver from both mouse and human origin. It can therefore be used both for preclinical and clinical studies. It uses small quantities of biological material (50 μl of plasma, 50 mg of tumour tissue) while previous methods used 250–500 μl of plasma

Acknowledgements

We are grateful to Hoffmann- La Roche for providing capecitabine, DFCR, DFUR and 5-FU and to the Edinburgh Wellcome Trust Clinical Research Facility for access to equipment.

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