34 February / March 2020 2.4.3. Test solution from blister
Approximately 13.0 mg Cyplos 50/500 mcg Powder for Inhalation (containing 0.05 mg SX and 0.5 mg FP) was weighed in a 20 mL amber colour volumetric flask and made up to volume with dilution solution and dissolved by sonication in an ultrasonic bath for 15 minutes. The solution was then filtered through a 0.45 µm filter into a sample vial. (CSalmeterol
= 0.0025 mg/ mL, C Fluticasone propionate = 0.025 mg/ mL) 2.5. System Suitability
The relative standard deviations for SX and FP peak areas of six replicate injections of standard solution should not be more than 2.0%.
2.6. Optimisation of the chromatographic conditions
Optimising the reverse phase HPLC parameters, several chromatographic conditions were tested in order to achieve a suitable peak resolution and peak shape for SX and FP.
2.6.1. Column Selection
Injections on to different columns types were conducted to achieve the best separation for the analyte peaks and other interfering blank and placebo peaks. The optimum peak shape, retention time, tailing factor, and column efficiency was achieved using a Hypersil BDS C18 column (15 cm x 4.6 mm, 5 µm).
2.6.2. Mobile Phase Composition
Different compositions of mobile phase were tested to obtain sufficient selectivity and retention time for the analyte peaks. With ammonium dihydrogen phosphate buffer, high sensitivity and selectivity were achieved when compared with other buffers. Based on peak shape, symmetry, retention time and peak tailing, pH 3.0 Potassium dihydrogen phosphate was selected as the buffer preparation to be used. Different gradient programs of pH 3.0 Potassium dihydrogen phosphate buffer and organic solvents were conducted and according to experiments with acetonitrile and methanol, higher retention time, higher column pressure and higher peak tailing were observed with methanol. Hence, acetonitrile was selected as the organic modifier. After many trials, based on the peak shape, peak symmetry, retention time and peak tailing a 1.5 mL/min flow rate was selected.
2.6.3. Detection Wavelength
SX, FP and their related substance peaks were scanned between 200 nm – 400 nm by photo-diode array detector. The maximum absorption of SX and FP was determined at 210 nm and 239 nm. Therefore, to obtain the maximum absorbance in one chromatogram a wavelength gradient was applied.
2.6.4. Buffer pH
Various trials on the pH of the Potassium dihydrogen phosphate buffer were made to achieve the optimum pH at which all API
peaks are well separated. For the optimum peak shape and peak tailing, a buffer pH of 3.0 was selected.
The chromatographic conditions were optimised with a mobile phase of pH 3.0 Potassium dihydrogen phosphate buffer and acetonitrile mixture at gradient wavelength with a flow rate of 1.5 mL/min at 40ºC column temperature and 40 µL injection volume. The typical HPLC chromatogram (Figure 1) shows a satisfactory separation of SX and FP.
2.7. Method Validation
The validation of the developed method was performed in accordance with the ICH Q2 (R1) guideline [8] for the following parameters: specificity, linearity, limit of quantitation (LOQ), accuracy, precision, robustness, and solution and mobile phase stability.
2.7.1. Specificity
Specificity is the ability of the substance to be analysed to be precisely determined in the presence of the matrix effect and additives ensuring the identity of the analyte(s) of interest.
Blank solution, placebo solution, standard solution and assay test solution were analysed and peaks from each of the solutions were determined for SX and FP. Moreover, the impurities in Table 1 were injected to the system in order to determine their relative retention times in the
Figure 1: HPLC chromatogram showing the separation of SX and FP standard mixtures.
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