Quantitation of a de‐fluorinated analogue of Casopitant Mesylate by normal‐phase liquid chromatography/mass spectrometry

Abstract

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.

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

“…A preliminary investigation was performed by LC-MS and confirmed co-elution of casopitant mesylate with one of its defluorinated analogues [11]. Following these results, a strategy for the detection and quantification of this co-eluting impurity was put in place, by means of the evaluation of two new methods involving LC-MS and NMR spectroscopy, respectively.…”

“…In order to confirm the semi-quantitative NMR results, an alternative method for the accurate quantification of the syn 2 (6) de-fluorinated diastereoisomer in casopitant mesylate drug substance was successfully developed and validated [11]. The original reversed-phase liquid chromatography-ultraviolet detection method for routine testing of casopitant mesylate was initially considered for a possible revision.…”

“…A normal-phase LC-MS method for accurate quantification of the de-fluorinated impurity in casopitant mesylate was previously developed and validated [11].…”

Coupling normal phase liquid chromatography with electrospray ionization mass spectrometry: strategies and applications

“…22 Third, only in very rare cases does the make-up solvent consist of only solvents. 70 More commonly, a good make-up solvent contains minor additives, such as formic acid (0.1-2% v/v), 71,72 ammonia (0.15-2.5% v/v) 69 or ammonium acetate (NH 4 Ac) (2-40 mM), [73][74][75] as was discussed in Section 2. The type of additive has not usually been optimized, but can have profound effects.…”

Search for improved fluorinated stationary phases for separation of fluorine-containing pharmaceuticals from their desfluoro analogs

“…Fluorine incorporation helps to block the metabolism and increase the bioavailability and efficacy of pharmaceuticals [2][3][4][5]. The chromatographic separation of closely related species coming from the synthesis of organofluorine compounds enables research in this area, but the separation of mixtures of closely related fluorination isomers and the separation of fluorine-containing drugs from their desfluoro analogs can be challenging [6][7][8][9][10][11], owing to the close structural similarities and small differences in physical properties between desired and undesired components.…”