A rapid, accurate and robust UHPLC–MS/MS method for quantitative determination of BMS-927711, a CGRP receptor antagonist, in plasma in support of non-clinical toxicokinetic studies

Highlights

• A robust assay was validated to quantify BMS-927711, a drug candidate, in plasma.

• A simplified method optimization strategy was used to develop this robust assay.

• An N-carbamoyl glucuronide metabolite was evaluated to minimize its conversion risk.

• Excellent assay robustness, precision and accuracy were achieved.

• It has been applied to non-clinical toxicokinetic studies in different species.

Abstract

BMS-927711 is a calcitonin gene-related peptide (CGRP) receptor antagonist that is being developed for the treatment of migraine. A rapid, accurate and robust assay was developed and validated for the quantitation of BMS-927711 in rat, monkey, rabbit and mouse plasma using ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS). A simplified method screening strategy was utilized that included a liquid–liquid extraction (LLE) methodology and eleven LC columns (ten sub-2 μm UHPLC columns and one 2.6 μm HPLC column) for screening with emphasis on the removal of phospholipids, avoidance of metabolite interference and ruggedness of LC conditions. A stable isotope labeled [¹³C₂, D₄]-BMS-927711 was used as the internal standard, and 50 μL of plasma samples were used for extraction by automated LLE with methyl tert-butyl ether (MTBE) in 96-well format. Chromatographic separation was achieved with an isocratic elution and a gradient column wash on a Waters Acuity UPLC^® BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with run time of 3.7 min. Positive electrospray ionization was performed using selected reaction monitoring (SRM) with transitions of m/z 535 > 256 for BMS-927711 and m/z 541 > 256 for [¹³C₂, D₄]-BMS-927711. The standard curve, which ranged from 3.00 to 3000 ng/mL for BMS-927711, was fitted to a 1/x² weighted linear regression model. The intra-assay precision was within 5.2% CV, inter-assay precision was within 5.9% CV, and the assay accuracy was within ±5.2% deviation (%Dev) of the nominal values in all the species. The stability of an N-carbamoyl glucuronide metabolite was carefully investigated, and the conversion of this metabolite to BMS-927711 was minimal and manageable without a stabilization procedure. The method was successfully applied to multiple non-clinical toxicokinetic studies in different species in support of the investigative new drug (IND) filing.

Introduction

Migraine is a neurological disorder characterized by recurrent moderate to severe headaches [1], which affects 17% of women and 6% of men in the United States [2]. Currently, 5-HT1B/1D agonists (triptans) are used as the standard of care for treating acute migraine; however, patients treated with triptans often do not receive complete and consistent pain relief, with a headache recurrence rate of 30–40% in treated patients [3], [4]. In addition, due to their vascoconstrictive side effects, triptans are generally not suitable for patients with cardiovascular diseases and hypertension. As a result, new treatments with improved efficacy and safety profiles are essential for many migraine patients. Calcitonin gene-related peptide (CGRP) is a potent vasodilator that has been shown to play a role in migraine pathophysiology and suggests that CGRP receptor antagonists could be an effective treatment for migraine patients [5], [6]. As a new and selective CGRP receptor antagonist, BMS-927711 (Fig. 1A) is being developed for the treatment of acute migraine [7]. In this manuscript, we report the development and validation of an UHPLC–MS/MS method for the quantification of BMS-927711 in rat, monkey, rabbit and mouse plasma in support of this new drug's development.

In regulated LC–MS/MS bioanalysis, the development of a robust assay is very desirable because it will help to ensure data quality and reduce the number of repeated analyses due to failed analytical runs. In addition, robust assays are easy to transfer from one laboratory to another in today's increasing demand for outsourcing bioanalytical projects to contract research organizations (CROs). A robust assay (or a high performance assay) is expected to have excellent LC peak shape, highly reproducible retention time and signal response, low carryover, and a rugged UHPLC column (high resolution, wide pH range and long life). Previously, a systematic method screening and optimization strategy was routinely applied in our laboratory during method development to achieve optimized mass spectrometry, chromatography, and sample extraction conditions [8], [9], [10]. Such a comprehensive screening strategy usually included all sample extraction options including solid phase extraction (SPE), liquid–liquid extraction (LLE), and protein precipitation (PPT) with about 20 conditions in total for evaluation [8], [9], [10]. In each extraction condition, the extraction recovery and matrix effect of each analyte were determined at two concentrations (low and high concentrations). The purpose of extraction method screening and optimization was mainly to develop an LC–MS/MS method with a high extraction recovery and low matrix effect in order to improve the detection sensitivity of analyte(s) at the level of lower limit of detection (LLOQ). Recently, with the emergence of UHPLC technology and highly sensitive tandem mass spectrometers [11], [12], as well as the increased application of stable isotope labeled internal standards, it is possible to quantify most analytes in biological samples at a very low concentration without the need for a very high extraction recovery as long as the recovery remains constant over time and concentration. As a result, it is possible to simplify the method screening and optimization process aimed at assay performance rather than achieving a higher extraction recovery.

In general, a robust LC–MS/MS assay results from the cleanness of the extracted samples, absence of metabolite interference and a rugged chromatography condition. LLE-based extraction method has been reported to give very clean extracts as indicated by the absence of endogenous peaks interfering with the SRM quantitation of the analyte even at the level of LLOQ [8], [10], [13]. In general, the selectivity, in terms of removing interference from phospholipids, achieved by LLE for the extraction of drugs and metabolites was much better than protein precipitation (PPT), and as good as or better than that obtained with SPE for several reported cases [8]. SPE-based extraction in a 96-well format has been demonstrated to be an effective method in achieving a higher extraction recovery for analytes with a variety of chemotypes; however, occasionally observed lot-to-lot variation with SPE cartridges or plates could be potential concerns for achieving run-to-run assay reproducibility [8]. With the maximal removal of phospholipids and decent LLE recovery for most of the small-molecule drugs and metabolites, several organic solvents (e.g., n-butyl chloride and MTBE) and solvent combinations (e.g., hexane/ethyl acetate and hexane/2-methyl-1-butanol) commonly used in LLE are considered to be some of the most effective solvents for LLE [8], [13]. It was reported that the relative amounts of a lyso phosphatidylcholine (C16:0 lyso-PC) in the LLE plasma extracts obtained using n-butyl chloride, MTBE and ethyl acetate (as LLE solvents) were less than 0.1, 1 and 15% as compared with a PPT extract [8]. The amounts of C16:0 lyso-PC in the n-butyl chloride and MTBE LLE extracts were significantly smaller than that seen in the SPE extracts under the conditions evaluated [8]. To simplify the assay screening and optimization procedure, we screened only LLE as the extraction method with two solvents (n-butyl chloride and MTBE) under three pH conditions (acidic, neutral, and basic) aimed at achieving cleanness of the extracted samples, since the assay sensitivity is not an issue for BMS-927711. A generic extraction method optimization strategy we used is shown in Fig. 2A, in which Step a was simplified to include only LLE option for BMS-927711 method screening. Instead of determining the absolute extraction recovery at two concentrations (low and high concentrations), plasma samples spiked with one concentration of BMS-927711 at lower limit of quantitation (LLOQ) were used for sensitivity evaluation after extraction using the LLE conditions mentioned above (no determination of the extraction recovery was required for initial optimization; data not shown).

Recently, the chromatography separation and assay selectivity in bioanalysis have been significantly improved by using UHPLC technology [8], [13]. As a result, considerable time can be saved in LC method optimization. A generic UHPLC method optimization strategy we used is shown in Fig. 2B, in which Step b was simplified to include only three mobile phase systems for BMS-927711 method screening. The detailed UHPLC columns and conditions used for the screening are shown in Table 1. Analyte stability and assay specificity (due to metabolite conversion/interference and phospholipids) were also included for evaluation using incurred samples during method development and optimization (as shown in Fig. 2B, Tests 2 and 3). One carbamoyl glucuronide metabolite of BMS-927711 (N-glucuronide, Fig. 1C) and several other metabolites in incurred rat plasma samples were carefully evaluated. The objective of LC method optimization was to separate these metabolites from BMS-927711 during method optimization. The presence of this N-glucuronide in the plasma sample poses a significant bioanalytical challenge for two reasons: (1) its potential conversion to BMS-927711 during storage or sample extraction that could result in overestimation of BMS-927711 (stability issue); and (2) its conversion to BMS-927711 in the MS source that could potentially interfere with the quantification of BMS-927711 if co-elution occurs (specificity issue). By using extraction method optimization (Fig. 2A) and LC condition optimization (Fig. 2B), bioanalytical risks due to stability and specificity issues (such as N-glucuronide) could be eliminated during validation and sample analysis. Our simplified approach has led to the development of a robust assay for the analysis of BMS-927711 in rat, monkey, rabbit and mouse plasma. The method utilized stable-isotope labeled [¹³C₂, D₄]-BMS-927711 as the internal standard and automated LLE in 96-well format to clean up the plasma samples. The validated method has been successfully used to analyze thousands of plasma samples in support of non-clinical toxicokinetic studies conducted in different species with BMS-927711.