A study of validational aspects of a high speed hplc methodology

Abstract

The European Pharmacopoeia, analytical method for salicylic acid related substance determination in aspirin, has been transferred from a Phenomenex® Luna Cl 8 250 mm x 4.6 mm 5 μm column to a Phenomenex® HST column 100 mm x 3.0 mm 2.5 μm column. The influence that variations in the operational parameters of high speed HPLC have on the chromatographic properties were investigated. Parameters investigated were temperature, flow rate and injection volume. These parameters were studied on a high speed HPLC system making use of an HST column 100 mm x 3 .0 mm, 2.5 μm. Chromatograms and data obtained from each set of conditions were evaluated and statistically analysed through general linear model univariate analysis so as to gain better understanding of how various conditions affect the output/result on a high speed HPLC, and extrapolate the ideal conditions for this system. An attempt was made, to validate the developed method for specificity, linearity, accuracy and precision according to guidelines within the International Conference on Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH). Temperature was found to affect selectivity and resolution of the method. An increase in temperature was observed to reduce capacity factors of both aspirin and salicylic acid with the result of reducing selectivity. An "ideal" temperature of 15 °C, at which maximum resolution satisfying the acceptance criteria required by the system suitability was chosen. Increasing the flow rate resulted in a reduction in the analysis runtime at the expense of higher column back pressures. An "ideal" flow rate of 0.7 mL/min was established at which acceptable column back pressure resulted and a relatively short runtime was attained. A reduction in injection volume resulted in a reduction of column overloading and an increase in %RSD. An injection volume of 4 μL was chosen as the ideal injection volume for this method. The validation for the extrapolated conditions showed linearity for salicylic acid ranging from a concentration of 0.002 mg/mL to 0.015 mg/mL, LOQ was determined to be 0.0007 mg/mL and the method was found to be specific. Accuracy and precision was established at the 0.1 % composition of salicylic acid, however further improvements are required for better accuracy at 0.05% and 0.12% of salicylic acid in test solution level.