Elsevier

Journal of Chromatography B

Volume 1040, 1 January 2017, Pages 14-21
Journal of Chromatography B

Liquid chromatography-tandem mass spectrometry assay for the quantification of niraparib and its metabolite M1 in human plasma and urine

https://doi.org/10.1016/j.jchromb.2016.11.020Get rights and content

Highlights

  • The first reported validated LC–MS/MS assay for niraparib and its metabolite M1.

  • The assay has successfully been validated in the 1–500 and 1–100 ng/mL ranges.

  • The assay was successfully used for a bioavailability study and a mass balance study.

Abstract

Niraparib (MK-4827) is a novel poly(ADP-Ribose) polymerase (PARP) inhibitor currently investigated in phase III clinical trials to treat cancers. The development of a new drug includes the characterisation of absorption, metabolism and excretion (AME) of the compound. AME studies are a requirement of regulatory agencies and for this purpose bioanalytical assays are essential. This article describes the development and validation of a bioanalytical assay for niraparib and its carboxylic acid metabolite M1 in human plasma and urine using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Sample pre-treatment involved protein precipitation for plasma and dilution of urine samples using acetonitrile-methanol (50:50, v/v). Final extracts were injected onto a SunFire C18 column and gradient elution using 20 mM ammonium acetate (mobile phase A) and formic acid:acetonitrile:methanol (0.1:50:50, v/v/v) (mobile phase B) was applied. Detection was performed on an API5500 tandem mass spectrometer operating in the positive electrospray ionisation mode applying multiple reaction monitoring (MRM). The assay was successfully validated in accordance with the Food and Drug Administration and latest European Medicines Agency guidelines on bioanalytical method validation and can therefore be applied in pharmacological clinical studies.

Introduction

Niraparib (MK-4827) is a novel poly(ADP-Ribose) polymerase (PARP) inhibitor currently evaluated in cancer patients in phase III clinical trials at a dose level of 300 mg orally once daily [1], [2]. Its mechanism of action has not been completely elucidated, but it is proposed that it lies in its ability to inhibit pathways involved in DNA repair processes [3]. Normal cellular functions, as well as everyday environmental stresses can lead to breaks in DNA. The principal DNA breaks are the single strand breaks (SSB), which are repaired through the base excision repair (BER) pathway, where the enzymes PARP-1 and PARP-2 play a vital role [4], [5]. If these SSBs go unrepaired, they can result, especially during replication, in the more serious double strand DNA breaks (DSB). These DSBs are repaired through other mechanisms, including the homologous recombination repair (HRR) pathway and the non-homologous end joining (NHEJ) [3]. It is understood that by targeting cells that have a mutation in DSB repair mechanisms, the addition of a PARP inhibitor such as niraparib can cause apoptosis. This concept is referred to as synthetic lethality: where two individual mutations on their own are not lethal, the combination of these mutations can lead to cell death [6]. In the current context, synthetic lethality refers to the impairment of DNA repair due to PARP inhibition with genetically predisposed DNA repair deficiencies, specifically the BRCA-1 and BRCA-2 mutations. At the same time, PARP inhibitors can sensitise cancerous cells to other therapies that are targeted at inhibiting DSB repair [3].

Similar to other compounds in the process of registration, it is required to conduct a mass balance study to elucidate the disposition and elimination of a compound. A mass balance study requires an appropriate bioanalytical assay to allow the quantification of the parent compound and known metabolites. Moreover, such an assay can be used to quantify the parent compound in an absolute bioavailability study. To the best of our knowledge, no such method has been described before. This article describes the validation of a bioanalytical method for both niraparib and its known carboxylic acid metabolite M1 in plasma and urine for the support of clinical studies such as the mass balance study and the absolute bioavailability study mentioned above. This method was validated in compliance with the Organisation for Economic Co-operation and Development (OECD) principles of Good Laboratory Practice (GLP) [7] and in accordance to the Food and Drug Administration (FDA) and latest European Medicines Agency (EMA) guidelines on bioanalytical method validation [8], [9].

Section snippets

Chemicals

Niraparib reference standard (Fig. 1) was supplied by Dishman (Dist. Ahmedabad, India), and its deuterated internal standard (IS) M002151 was manufactured by Merck (Kenilworth, NJ, USA). M1 reference standard (Fig. 1) was supplied by Metrics Inc (Greenville, NC, USA), and its deuterated internal standard D5-M1 was provided by GLSynthesis Inc. (Worcester, MA, USA). Acetonitrile (ACN), methanol (MeOH) and water (all Supra-Gradient grade) were purchased from Biosolve Ltd (Valkenswaard, The

Chromatography

The difference in mass between niraparib and M1 is 1 amu. The development of the chromatography mainly focused on the chromatographic separation of the two analytes. Another difficulty was the appearance of the C13-niraparib peak in the transition window of M1. It was therefore essential to obtain chromatographic separation to ensure the correct quantification of M1. Experiments were carried out using different compositions of mobile phases. Experiments showed that a mobile phase of methanol

Clinical application

Niraparib is a novel PARP inhibitor currently evaluated in clinical trials. The purpose of this method development and validation was to enable the support of pharmacological clinical studies, including a bioavailability study and a mass balance study where subjects received a single oral dose of 300 mg. In these studies blood samples were collected using K2EDTA tubes and plasma was obtained by centrifugation. The validated bioanalytical method described in this article was applied to quantify

Conclusion

Quantification methods for niraparib and its carboxylic acid M1 in plasma as well as in urine were validated according to the latest FDA and EMA guidelines [8], [9]. This method validation includes all procedures required to show that the determination of niraparib concentrations in plasma and urine is reliable for the intended application. The quantifiable range for niraparib and M1 was 1–500 ng/mL for plasma and 1–100 ng/mL for urine. Samples with concentrations above the ULOQ can be reliably

Acknowledgment

This work was financially supported by Tesaro Inc.

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