4 May / June 2017 Results and Discussion:


The USP monograph for ondansetron hydrochloride [2], which is an HPLC- UV method, includes identification and quantification of 5 related organic impurities (Figure 1), including two process impurities [4] E and F, which are imidazole and 2-methylimidazole respectively. Studies done by the National Toxicology Program (NTP) on 2-methylimidazole show exposure- related increases of micronucleated normochromatic erythrocytes in peripheral blood samples of male and female mice, which is an indicator of chromosomal damage. Additionally, the amount of damage increases with increasing duration of exposure [5]. In light of this information, 2-methylimidazole can be considered to be a potentially mutagenic impurity, and due to its closely related structure for this example imidazole will also be considered a PMI.


Due to the dangers posed by PMIs, the maximum acceptable daily intake for PMIs is set to specific levels according to ICH M7: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk guidelines [3]. Depending on the treatment it is prescribed for, ondansetron may be taken for more than 30 non-consecutive days, thus the allowable PMI daily intake limit is 20 µg/ day per ICH M7. The highest possible daily dosing for ondansetron is 48 mg/day [6], which means that PMIs are allowable at 417 ppm with reference to the API, which is five times lower than the USP monograph limit of 0.2% (2000 ppm). For this reason, the use of a higher sensitivity detector, specifically a tandem quadrupole MS, was employed to provide detection levels much lower than can be achieved by UV. To mimic a true test sample, all calibrators and QCs were prepared in diluent containing 0.125 mg/ mL of the ondansetron API. When using MS detection, it is important to separate all impurity peaks from the main API peak to avoid any potential matrix effects, i.e. signal suppression or enhancement, due to the presence of API at such a high concentration which may affect the ionisation process within the MS source. Additionally, the original USP monograph utilises ion-pairing reagents to facilitate retention of the polar impurities E & F, however, the use of ion-pairing reagents with MS is known to cause signal suppression so they are often avoided. Therefore, a new higher sensitivity methodology was developed for the detection of 5 organic impurities of ondansetron utilising MS detection.


Liquid Chromatography Method Development:


For the reversed phase method development, numerous variables were evaluated to facilitate retention of the polar impurities and overall separation of the API and all 5 impurities. Multiple column chemistries were examined (including CSH fluoro-phenyl, BEH amide, HSS T3 and HSS cyano chemistries), along with various mobile phases over a range of pH. The column chemistries were chosen because of their ability to increase retention for polar compounds; however, under generic RPLC conditions of formic acid in water/ acetonitrile, both polar impurities E & F were unretained under all conditions tested. Figure 2a shows an example chromatogram generated on an HSS T3 column. The same general trend was seen for all column and mobile phase combinations tested. For these reasons, it was determined that an alternate approach was required, specifically HILIC.


HILIC chromatography is a technique that uses hydrophilic stationary phases with typical reversed phase mobile phases, with the notable difference that the aqueous mobile phase is the strong solvent which facilitates elution. The separation mode is based on a combination of partitioning, ion-exchange and hydrogen bonding with a layer of water on the surface of the particles. Common method development strategies for HILIC chromatography include screening different columns and changing the buffer and additive concentration of


the mobile phases. For this example, an existing method for 2-methylimidazole using a CORTECS HILIC column along with ammonium formate in the mobile phase was evaluated [7]. Under the prescribed HILIC conditions, impurities E & F were well retained, however, impurities C & D were not retained (data not shown). An alternate mobile phase combination was examined, specifically the use of ammonium acetate and acetic acid as the buffering system. Under these conditions, there was still no retention for impurities C & D, however, there was better separation of the API from impurities A, E and F (Figure 2b). The same gradient profile was used for both mobile phase combinations with initial LC conditions at 98% organic solvent containing either formic or acetic acid at 0.1% by volume. Because acetic acid is a weaker acid, the pH of the starting conditions and the subsequent gradient will be slightly higher for the acetic acid mobile phase combination. The use of the higher pH mobile phases resulted in increased retention of all compounds and an alternate selectivity which ultimately provided a better separation.


The final HILIC method developed was used for the quantification of impurities A, E, and F (Figure 2b). It utilised a CORTECS UPLC HILIC column (2.1 x 100 mm, 1.6 µm) maintained at 30°C. The mobile phases consisted of 0.1% (v:v) acetic acid in acetonitrile and 10mM ammonium acetate at pH 4. The flow rate was 0.6 mL/ min and used an injection volume of 2 µL.


Figure 2. a) Chromatogram showing ondansetron (API) and related impurities A, C, D, E and F separated un- der generic reversed phase LC conditions. An ACQUITY HSS T3 column (2.1 x 100 mm, 1.7 µm) was used with a linear gradient over 4 minutes going from 2 - 50% B where mobile phase A = 0.1% formic acid in water and mobile phase B = 0.1% formic acid in acetonitrile. b) Chromatogram showing ondansetron (API) and related impurities A, C, D, E and F separated under HILIC conditions. A CORTECS HILIC column (2.1 x 100 mm, 1.6 µm) was used with a linear gradient over 6 minutes going from 2 - 16% B where mobile phase A = 0.1% acetic acid in acetonitrile and mobile phase B = 10mM ammonium acetate in water at pH 4.


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