5


Table 1. Experimental results obtained using LC methodologies for ondansetron impurities A, C – F in the presence of 125 µg/mL API (ondansetron).


Impurity A Impurity C Impurity D


Impurity E (imidazole) Impurity F


(2-methylimidazole) Mean


Methodology 140 ppm


Impurity A Impurity C Impurity D


Impurity E (imidazole)


Impurity F (2-


methylimidazole)


The gradient was relatively shallow, with a gradient of 2 - 16% aqueous over 6 min. The separation shows the API was well separated from all impurities, thus minimising the risk of any matrix effects.


Since the HILIC method could not be used for the quantification of unretained impurities C & D, a reversed phase method was developed to quantify the two remaining impurities. The method used a BEH C18 column (2.1 x 50 mm, 1.7 µm) maintained at 30°C along with mobile phases consisting of 0.1% (v:v) formic acid in water and 0.1% (v:v) formic acid in acetonitrile. Again, the flow rate was 0.6 mL/min and used an injection volume of 2 µL. Table 1 shows the overall results for the LC methods developed to quantify the impurities of ondansetron. All compounds showed good R2


values of >0.994 for the


calibration curves along with acceptable s/n values for the lower limits of quantitation (LLOQ). In addition, QCs run in replicates of 6 at three different concentration levels gave mean calculated concentrations within 8.7% of nominal, and all RSD values were ≤ 7.3% which shows good accuracy and precision for the methods developed.


In this analysis, because of the structural similarity between the API and impurities, there is also potential that if any in-source fragmentation occurs, it could lead to erroneous identification or quantification of compounds. In source fragmentation


occurs when a precursor compound is fragmented in the source and is then seen in Q1 at a different mass than expected. In this case, ondansetron (294 m/z) fragments in the source to form impurity D (212 m/z). Although the mass spectrometer cannot distinguish between the in-source fragmented ondansetron and native impurity D, the two compounds are separated chromatographically. The ondansetron peak in channel 212 > 184 is easily identified as ondansetron by retention time (Figure 3). Since ondansetron is not being quantified in this example, the in- source fragmentation is not problematic, however, in addition to potential matrix interferences, in- source fragmentation is another example of why a good chromatographic method where all analytes are well separated is important not just for UV but also MS methods.


HILIC RP RP


HILIC


760 ppm


Quality Control Results % Bias


2800 ppm


140 ppm


760 ppm


2800 ppm


140 ppm


% RSD


760 ppm


2800 ppm


131 749 2718 -2.6 -4.2 -2.8 1.3 1.3 1.5 164 819 2761 -4.5 1.0 -1.4 1.0 1.0 1.4 139 760 2766 -5.0 1.7 -1.5 2.3 1.4 1.5 125 669 2879 1.9 3.1 4.6 3.6 4.0 3.2


Methodology HILIC RP RP


HILIC HILIC


Calibrator Results Fit


Linear; log, log Quadratic; 1/x Quadratic; 1/x Linear; log, log Linear; log, log


R2


0.994 0.997 0.994 0.998 0.997


LLOQ s/n 3050 300 180 209 13


Supercritical Fluid Chromatography Method Development:


Supercritical fluid chromatography is a viable alternative approach to solving this analytical challenge. In SFC, the retention mechanism is most comparable to normal phase chromatography, but uses mobile phases (modifiers and additives) which are readily compatible with the source design employed for LC-MS. Supercritical fluid chromatography is known to be well suited for the retention of small polar compounds, such as imidazole and 2-methylimidazole [8]. Additionally, the low viscosity and high diffusivity of CO2


produces high efficiency


separations in relatively short run times, which can significantly increase throughput compared to traditional LC methods.


HILIC 135 811 2663 6.1 -4.5 2.2 1.5 0.7 0.8


Method development using supercritical fluid on the previously described system is analogous to LC method development, however, in place of varying the gradient and composition of aqueous and organic solvent, the gradient and composition of compressed CO2


and organic co-solvent is


varied. Methanol is the most common co- solvent used and similar to LC acidic or basic additives are often required to minimise secondary interactions and produce symmetrical peak shapes.


The final method developed for ondansetron & impurities utilised a Torus 2-PIC column (3x100 mm, 1.7 µm), maintained at 30°C (Figure 4). The co- solvent used was 0.2% ammonium hydroxide in methanol and the gradient went from 5 - 15% co-solvent over 6 minutes. The flow


Figure 3. Ondansetron in-source fragmentation, where ondansetron fragments to the same precursor mass as impurity D and thus shows up in the MRM channel. It can easily be identified by its retention time as ondansetron.


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