A preliminary study for the method transfer of acetylsalicylic acid from HPLC to RRLC

Abstract

High performance liquid chromatography (HPLC) has long been a fundamental tool in pharmaceutical analysis. However the need for faster and more cost effective analysis has led to the introduction of Rapid Resolution Liquid Chromatography (RRLC) and hence, pharmaceutical companies are opting to transfer their methods from the conventional HPLC to the faster RRLC. RRLC operates under higher pressures and uses stationary phases of smaller particle size. This project focused on transferring the European Pharmacopeia method for aspirin, which is set on HPLC, to the RRLC using the standard column, Phenomenex Luna Cl 8 250 x 4.6mrn with a particle size of 5µm. The performance of both instruments was investigated by varying the flow rate, injection volume and temperature. The output was statistically compared using various statistical tests mainly regression, t-tests and ANOVA. Furthermore, the concentrations of the standards were also altered to check which combination of injection volume and concentration obtained the highest efficiency for the RRLC. For both HPLC and RRLC, increasing the temperature offered many advantages that included higher efficiency, reduced retention times, shorter analysis run time and less consumption of mobile phase. However it also resulted in certain disadvantages including small capacity factors and resolution as well as the possibility of sample degradation. Increasing the flow rate gave shorter retention times and hence shorter analysis times which is cost effective in terms of time, money and mobile phase consumption. When the flow rate was increased on a column of 5µm particle size, the efficiency decreased drastically. On increasing the injection volume, it was noticed that eventually peaks broadened, the plate number decreased and the resolution worsened. When the concentration of the standards were doubled and halved it was noticed that the best efficiency, resolution and asymmetry were achieved at the standard concentration. This was more prominently seen for the RRLC than HPLC. The results of the study indicated that it is important to keep the column dimensions in mind when monitoring the effect of chromatographic parameters since columns with different dimensions and particle sizes affect the chromatogram in a distinct way. It was also concluded that by using the large column, the benefits of the RRLC were not fully exploited. Hence new RRLC methods should be developed on columns of smaller dimensions and particle sizes. In the case of the European Pharmacopoeia method for aspirin the difference between the two instruments was determined to be statistically significant yet not large enough to require a revalidation when utilising a RRLC for HPLC methods.