Detection, identification and quantification of a new de-fluorinated impurity in casopitant mesylate drug substance during late phase development: an analytical challenge involving a multidisciplinary approach.

During late phase development of the selective NK1 receptor antagonist casopitant mesylate, a de-fluorinated impurity was discovered and quantified by an orthogonal analytical approach, using NMR and LC-MS. A dedicated (19)F NMR method was initially developed for first line identification and semi-quantification of the impurity. Subsequently, a more accurate quantification was achieved by means of a selective normal-phase LC-MS method, which was fully validated. The results obtained on the development batches of the drug substance were used by the project team to set up a suitable control strategy and ultimately to ensure patient safety and the progression of the project.

Quantitation of a de-fluorinated analogue of casopitant mesylate by normal-phase liquid chromatography/mass spectrometry.

The introduction of Quality by Design (QbD) in Drug Development has resulted in a greater emphasis on chemical process understanding, in particular on the origin and fate of impurities. Therefore, the identification and quantitation of low level impurities in new Active Pharmaceutical Ingredients (APIs) play a crucial role in project progression and this has created a greater need for sensitive and selective analytical methodology. Consequently, scientists are constantly challenged to look for new applications of traditional analytical techniques. In this context a normal-phase liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method was developed to determine the amount of a de-fluorinated analogue impurity in Casopitant Mesylate, a new API under development in GlaxoSmithKline, Verona. Normal-phase LC provided the selectivity needed between our target analyte and Casopitant, while a single quadrupole mass spectrometer was used to ensure the sensitivity needed to detect the impurity at <0.05%w/w. Standard solutions and samples were prepared in heptane/ethanol (50:50, v/v) containing 1% of 2 M NH(3) in ethanol; the mobile phase consisted of heptane/ethanol (95:5, v/v) with isocratic elution (flow rate: 1.0 mL/min, total run time: 23 min). To allow the formation of ions in solutions under normal-phase (apolar) conditions, a post-column infusion of a solution of 0.1% v/v of formic acid in methanol was applied (flow rate: 200 microL/min). The analysis was carried out in positive ion mode, monitoring the impurity by single ion monitoring (SIM). The method was fully validated and its applicability was demonstrated by the analysis of real-life samples. This work is an example of the need for selective and accurate methodology during the development of a new chemical entity in order to develop an appropriate control strategy for impurities to ultimately ensure patient safety.

A multi-technique approach using LC-NMR, LC-MS, semi-preparative HPLC, HR-NMR and HR-MS for the isolation and characterization of low-level unknown impurities in GW876008, a novel corticotropin-release factor 1 antagonist.

A multi-technique approach was applied in order to fully characterize four low-level unknown impurities of GW876008, a novel CRF(1) receptor antagonist. Liquid chromatography (LC)-NMR spectroscopy was used in combination with LC-MS to obtain detailed information regarding the structure of the two major impurities present in batches of GW876008 and observed in the first synthetic scale-up for preclinical use. Two additional impurities were unexpectedly found at greater levels in a large scale synthesis for clinical use and their structure was elucidated by means of high resolution (HR)-MS and HR-NMR, after a small scale preparative HPLC purification step. This structural information was useful in terms of shedding light on the typical impurity profile of this new chemical entity with the aim to support the early development package for Phase I clinical studies.