6 May / June 2017
Figure 4. Chromatogram showing ondansetron (API) and related impurities A, C, D, E and F separated using SFC. A Torus 2-PIC column (3 x 100 mm, 1.7 µm) was used with 0.2% NH4OH in methanol as co-solvent and make-up flow.
rate was 1 mL/min, and the injection volume used was 2 µL. Because the relative amount of organic co-solvent is low (a gradient of 5 - 15% at 1 mL/min is equal to 50 - 150 µL/min of organic solvent reaching the MS probe), a make-up solvent was teed in post column to aid ionisation. The make-up solvent also consisted of 0.2% ammonium hydroxide in methanol and was added at 0.5 mL/min.
Table 2 shows the overall results for the single SFC method developed to quantify the impurities of ondansetron. All compounds showed good R2
values of >/= 0.998 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 6.1% of nominal, and all RSD values were ≤ 4.0% which shows good accuracy and precision of the method developed.
Conclusions: The development of a high sensitivity
method for the analysis of impurities of ondansetron was challenging due to a number of factors including retention of small polar impurities, required detection levels, potential matrix interferences, and in-source fragmentation. With liquid chromatography, it was necessary to develop two methods: a HILIC method for the quantitation of highly polar impurities imidazole and 2-methylimidazole, and a reversed phase method for quantitation of the less polar impurities. However, using SFC, it was possible to analyse all five impurities in a single method. Both the UHPLC and SFC methodologies were amenable with MS detection, which facilitated detection at the levels required for potential mutagenic impurities set forth by ICH M7. In addition, all methods developed for the quantification of PMIs of ondansetron met the general requirements of an accurate and precise method. Finally, any possibility for matrix effects or negative effects due to in-source fragmentation was eliminated by adequate separation of the peaks of interest from the main API peak.
Table 2. Experimental results obtained using SFC methodology for ondansetron impurities A, C-F in the presence of 125µg/mL API (ondansetron).
Fit
Impurity A Impurity C Impurity D
Impurity E (imidazole) Impurity F
(2-methylimidazole) Mean
140 ppm
Impurity A Impurity C Impurity D
Impurity E (imidazole) Impurity F
(2-methylimidazole)
760 ppm
2800 ppm
Quality Control Results % Bias
140 ppm
760 ppm
2800 ppm
140 ppm
131 749 2718 -2.6 -4.2 -2.8 1.3 164 819 2761 -4.5 1.0 -1.4 1.0 139 760 2766 -5.0 1.7 -1.5 2.3 125 669 2879 1.9
3.1 4.6 135 811 2663 6.1 -4.5 2.2
3.6 1.5
% RSD
760 ppm
1.3 1.0 1.4 4.0 0.7
2800 ppm
1.5 1.4 1.5 3.2 0.8
Linear; 1/x Linear; 1/x Linear; 1/x
Quadratic; 1/x Linear; 1/x
Calibrator Results R2
0.998 0.999 0.999 0.999 0.998
LLOQ s/n 2500 2000 2000 15 75
As stated previously, SFC shares the same selectivity as normal phase LC, thus providing a high degree of orthogonality to RPLC when utilising polar stationary phases. However, the flexibility of SFC also allows the use of conventional RP stationary phases, such as C18, yielding similar retention characteristics to RPLC when hydrophobic stationary phases are used. Combining the miscibility of CO2
with both
polar and non-polar organic solvents, SFC is a technique widely applicable to a diverse range of compounds. SFC is especially useful for separating mixtures containing polar compounds, as in the impurity example outlined above, and is also ideally suited for positional isomers, stereoisomers, diastereomers and chiral compounds. Finally, SFC is compatible with many popular detection techniques such as photodiode array, evaporative light scattering, and mass detection, making it a beneficial addition to any analytical laboratory.
References:
1. Reddy, A.V.B., Jaafar, J., Umar, K., Majid, Z.A., Aris, A. B., Talib, J., Madhavi, G., J. Sep. Sci. 2015, 38, 764-779.
2. Official Monographs, USP 38 NF33. United States Pharmacopeia and National Formulary (USP 38-NF 33) Baltimore, MD: United Book Press, Inc.; 2015. p. 4639.
3. International Conference on Harmonization M7 (ICH M7), Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk. Jun 2014.
4. Baumann, M., Baxendale, I.R., Ley, S.V. and Nikbin, N. (2011) An overview of the key routes to the best selling 5-membered ring heterocyclic pharmaceuticals. Beilstein Journal of Organic Chemistry 7, 442-495.
5. NTP Technical Report on the Toxicity Studies of 2- and 4-Methylimidazole. U.S. Department of Health and Human Services Public Health Service National Institutes of Health.
6. Highlights of Prescribing Information.
http://www.accessdata.fda.gov/drugsatfda_ docs/label/2014/020007s046lbl.pdf
7. Benvenuti, M., Burgess, J.A., Analysis of 2- and 4-methylimidazole in beverages using Alliance HPLC with Mass Detection. http://
www.waters.com/webassets/cms/library/ docs/720005200en.pdf
8. Simeone, J. et al, Exploiting the Orthogonality of the ACQUITY UPC2 System to Develop an Impurities Method for Ondansetron.
http://www.waters.com/ webassets/cms/library/docs/720005732en.pdf
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