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Validation of high-performance liquid chromatography–tandem mass spectrometry assays for the quantification of eribulin (E7389) in various biological matrices

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Abstract

This paper presents specific and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) assays for the quantification of the novel anticancer agent eribulin in human plasma, whole blood, urine and faeces. These assays, developed to support clinical pharmacological studies with the drug, quantify eribulin concentration ranges of 0.2–100 ng/mL for plasma, 0.5–100 ng/mL for whole blood and urine and 0.1–25 μg/g for faeces, using sample volumes of 500 μL or 250 μg (faeces). Samples were prepared with liquid–liquid extraction, separated on a C18 column with gradient elution and analysed with a triple quadrupole MS, in positive ion mode. A structural analogue of eribulin was used as internal standard for the quantification. The assays were linear with correlation coefficients (r2) of 0.99 and better, whereby the deviation from nominal concentrations ranged from −8.2 to 8.9% with CV values of maximally 14.2%. Stability assessments demonstrated that eribulin is stable at −20 °C in plasma, whole blood, urine and faeces for at least 38, 4, 10.5 and 5 months, respectively. In conclusion, the validation results show that the assays are specific and accurate and can therefore adequately be applied to support clinical studies of eribulin.

Introduction

Eribulin mesylate (E7389) (Fig. 1A) is a nontaxane microtubule dynamics inhibitor with a distinct mode of action. While it is still being investigated in clinical trials, eribulin has recently been approved by the United States Food and Drug Administration (FDA) for treatment of patients with metastatic breast cancer, who have previously received at least two chemotherapeutic regimens, including an anthracycline and a taxane [1].

Like most anti-mitotic drugs, eribulin affects the microtubule dynamics, resulting in a cell cycle block, leading to apoptosis [2]. Unlike other tubulin-targeted agents, eribulin only inhibits the growth and not the shortening of microtubules and it induces the formation of tubulin aggregates [3]. These distinct modes of action may contribute to the results of phase II studies wherein eribulin shows activity in patients who had received previous therapy with taxanes and anthracyclines [4].

To support clinical pharmacological studies of eribulin, and especially mass balance studies, it was essential to develop and validate quantitative bioanalytical assays of eribulin in plasma, whole blood, urine and faeces. The quantification of eribulin in human plasma and urine described by Desjardins et al. [5] served as a starting point for the development of the plasma assay in this paper. As our attempts to reproduce their method resulted in an insufficient separation between eribulin and the internal standard, we further optimized the chromatographic conditions. Additionally, we present methods for quantification of eribulin in whole blood and faeces, for which thus far no methods have been published. The described validations were performed according to the FDA guidelines for bioanalytical method validation [6], [7].

Section snippets

Chemicals and reagents

Eribulin methanesulfonic acid salt and its internal standard ER-076349 (Fig. 1B) were provided by Eisai Co., Ltd, Japan. Methanol (Supra-Gradient grade), ethanol absolute (HPLC-grade) and acetonitrile (ACN, Supra-Gradient grade) were obtained from Biosolve Ltd, Valkenswaard, The Netherlands. Ethyl acetate (LiChrosolv), sodium hydroxide (NaOH) (>99%) and formic acid (98%) were purchased from Merck, Darmstadt, Germany. Water (distilled) used for sample preparation originated from B. Braun

Sample pretreatment

The apolar nature of eribulin theoretically makes it a good substrate for LLE with an organic solvent. Ethyl acetate only or in combination with methanol and ethanol (90:5:5, v/v/v) and tert-butyl methyl ether were tested. Extraction with the ethyl acetate, methanol and ethanol combination resulted in clean extracts and reproducible recoveries and was chosen as pretreatment method.

Because whole blood study samples collected from patients are stored frozen, and thereby haemolysed, it was decided

Conclusion

For the quantification of eribulin in human plasma, whole blood, urine and faeces, sensitive and accurate LC–MS/MS assays are presented. Using sample volumes of 500 μL of plasma, whole blood and urine and 250 μg faeces, linear ranges from 0.2 to 100 ng/mL for plasma, 0.5 to 100 ng/mL for whole blood and urine and 0.1 to 25 μg/g for faeces were validated. The assays have successfully been used to support clinical studies. Especially in mass balance studies they can play a major role, making it

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