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Development and Comparison of Quantitative Methods Using Orthogonal Chromatographic Techniques for the Analysis of Potential Mutagenic Impurities
by Jennifer Simeone, Paula Hong and Patricia R. McConville Waters Corporation, Milford, MA
There are many steps during the manufacturing process of an active pharmaceutical ingredient (API) where impurities can be introduced, whether as reagents, byproducts, intermediates, etc [1]. Some of these impurities may be mutagenic, or have the potential to interact with DNA and ultimately cause carcinogenicity. Methodologies associated with monitoring API purity levels are often HPLC-UV based [2], which frequently do not provide the sensitivity levels needed to detect potentially mutagenic impurities (PMIs) at the levels required by regulatory agencies [3]. For example, ondansetron is a pharmaceutical used in the prevention of nausea and vomiting and may contain one potential mutagenic impurity, 2-methylimidazole, as well as a second impurity very closely related in structure, imidazole.
Similar to 2-methylimidazole and imidazole, many mutagenic impurities are small, highly polar compounds that are poorly retained under typical reversed phase liquid chromatography (RPLC) conditions. Alternate forms of chromatography, such as hydrophilic interaction chromatography (HILIC), or the use of ion-pairing reagents can be employed, but these often result in tedious method development or non-MS friendly mobile phases. Supercritical fluid chromatography (SFC) is known to be orthogonal to RPLC, and employs reagents which are suitable for MS detection. In this study, methods for the analysis of ondansetron and five organic impurities were developed using both liquid and supercritical fluid chromatographic methods. Both chromatographic techniques generated high sensitivity methods that met the required limits of detection and both techniques showed good accuracy and reproducibility.
Experimental:
Ammonium acetate (≥99%), ammonium formate (≥99.99%), formic acid (~98%), acetic acid (≥99.7%) and ammonium hydroxide (28.0-30.0%) were purchased from Sigma-Aldrich (St Louis, MO). Acetonitrile and methanol were Optima Grade and purchased from Fisher Chemical (Fair Lawn, NJ). Ondansetron hydrochloride and ondansetron impurities A, C, and
D were purchased from the United States Pharmacopeia (Frederick, MD). Impurity E (imidazole) and impurity F (2-methylimidazole) were purchased from Sigma-Aldrich.
All Ultra High Performance Liquid Chromatography (UHPLC) studies were conducted on a Waters ACQUITY I-Class system and all SFC studies were conducted on a Waters ACQUITY Ultra Performance Convergence Chromatography (UPC2
)
system which utilises compressed or supercritical CO2
. Both systems were
connected to a Waters Xevo TQ-S micro tandem quadrupole mass spectrometer. MS source conditions were optimised separately for UHPLC and SFC experiments.
Calibrator and quality control (QC) samples for impurities A, C, D, E and F were prepared in diluent containing 0.125 mg/ mL API (ondansetron) in methanol (SFC/ HILIC) or water (RPLC) at the following concentrations: calibrators at 15, 20, 25, 50, 75, 100, 125, 200, 300, and 500 ng/mL and QCs at 17.5, 95, and 350 ng/mL. This is equivalent to impurity A, C, D, E, and F concentrations of 120, 160, 200, 400, 600, 800, 1000, 1600, 2400, and 4000 ppm for the calibrators and 140, 760, and 2800 ppm for the QCs, where ppm is in reference to the API.
Figure 1. Structures of ondansetron and related impurities A, C-F.
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