Development of a stability indicating HPLC method for the Pheroid™ delivery system
Van den Berg, Elaine
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Stability plays an important role in the development of a new drug product. High Performance Liquid Chromatography (HPLC) is considered a stability indicating method of analysis. It is widely used in the pharmaceutical industry for the quantification of small organic molecules during stability testing. Previous stability studies conducted on Pheroid™-based drug products, experienced problems with the generation of reliable data by means of HPLC analysis. With these studies it was concluded that the inconclusive results could either be attributed to the stability of the delivery system itself and the compatibility of the active pharmaceutical ingredients (API's) with the delivery system, or to the usage of unsuitable HPLC methods. The aims of this study were to: i. determine if the Pheroid™ delivery system changes significantly over time at accelerated storage conditions and how these changes influence the HPLC analysis, ii. determine the effect of the anti-oxidant tert-butylhydroquinone (TBHQ) on the stability and HPLC analysis of the Pheroid™ delivery system, and iii. to suggest a suitable approach for the analysis of Pheroid™-based drug products. Pheroid™ microsponges, containing no API's, were prepared and stored for a period of three months at 5°C, 25°C+60%RH, 30°C+65%RH and 40°C+75%RH. Two of the four Pheroid™ formulations contained an extra anti-oxidant, namely TBHQ. Monthly HPLC analyses were done using existing methods for mefloquine and artesunate. In addition to HPLC analysis, particle size analysis and Confocal Laser Scanning Microscopy (CLSM) were undertaken to support the HPLC results and provide information concerning the overall stability of the Pheroid™ delivery system. After the completion of the above analyses, experiments were carried out to determine whether adjustments to some of the key chromatographic parameters could improve the separation of Pheroid™-based samples. The parameters that were subjected to change included the organic solvent, isocratic versus gradient separation, pH and detection wavelength. Two pro-Pheroid vesicles formulations were prepared and stored for a three month period at 40°C+75%RH only. No API was added to the one formulation while the other contained 2 mg/ml of mefloquine hydrochloride. Results obtained indicated that the Pheroid™ formulations changed after exposure to elevated temperature and humidity. The number of detectable peaks increased, longer run times became necessary and solubility in the sample solvent (methanol) decreased. Solubility of the Pheroid™ formulations in methanol was preserved to some extent by the presence of TBHQ. Physical signs of instability like discolouration and creaming were noted for TBHQ-containing formulations. TBHQ also seemed to have influenced the particle sizes, particle size distributions and structure of the Pheroid™ microsponges. With adjustments made to the HPLC method it was found that: i. the sample solvent is incompatible with the HPLC system, ii. very hydrophobic compounds are present in the Pheroid™-based samples, iii. acetontrile and methanol are unsuitable for both gradient and isocratic separation of Pheroid™-based samples, iv. more Pheroid™ components absorb at shorter wavelengths, and v. small changes in the pH values usually implemented do not influence the retention and selectivity of the Pheroid™ components. The Pheroid™ delivery system proved to be too complex and reversed hydrophobic for phase HPLC analysis. Preparation of the sample by only diluting the Pheroid™ formulations with pure methanol was not optimal. These samples introduced compounds to the column of which some caused interferences with the analyte peak while others were difficult to elute from the column. To continue using HPLC for the analysis of Pheroid™-based drug products, it is therefore recommended that attention should be given to the development of a more appropriate sample preparation procedure, like solid phase extraction or liquid-liquid extraction, one that will eliminate the effects of the Pheroid™ components. Physical instabilities noticed with the addition of TBHQ, suggest that there should also be attended to the compatibility and stability of each of the components in the Pheroid™ delivery system during formulation development.
- ETD@PUK