17


Table 3. Limit of quantification (LOQ), Limit of detection (LOD), relative response factor (RRF) and relative retention times (RRT) of impurities.


Impurities


Salmeterol Related Salmeterol


Salmeterol Imp. D Salmeterol Imp. G


Fluticasone Prop. Related Fluticasone Propionate Fluticasone Prop. Imp. A Fluticasone Prop. Imp. B Fluticasone Prop. Imp. C Fluticasone Prop. Imp. D Fluticasone Prop. Imp. E Fluticasone Prop. Imp. F Fluticasone Prop. Imp. G Fluticasone Prop. Imp. H Fluticasone Prop. Imp. I


RRT


LOQ (µg/ mL)


- 0.360


~ 1.08 0.150 ~ 1.97 0.190


- 0.190


~ 0.63 0.175 ~ 0.73 0.215 ~ 0.91 0.155 ~ 0.96 0.100 ~ 1.03 0.250 ~ 1.05 0.180 ~ 1.40 0.110 ~ 1.37 0.110 ~ 1.41 0.095


LOD (µg/ mL)


0.120 0.050 0.063


0.063 0.058 0.072 0.052 0.033 0.083 0.060 0.037 0.037 0.032


Table 4. Average Accuracy (recovery, %) results for SX, FP and their related substances Compounds


Salmeterol Salmeterol Imp. D Salmeterol Imp. G


Compounds


Fluticasone Propionate Fluticasone Prop. Imp. A Fluticasone Prop. Imp. B Fluticasone Prop. Imp. C Fluticasone Prop. Imp. D Fluticasone Prop. Imp. E Fluticasone Prop. Imp. F Fluticasone Prop. Imp. G Fluticasone Prop. Imp. H Fluticasone Prop. Imp. I


SX and FP peak areas was not be more than 5.0%, with the tailing factor for SX and FP peaks less than 2.0 and finally, ensuring that the column efficiency (theoretical plates) for SX and FP peaks was greater than 2000.


3. Results and Discussion 3.1. Method Optimisation 3.1.1. Column Selection


Injections with different columns were conducted to determine the conditions required to obtain the best separation of analyte peaks and placebo peaks. The optimum peak shape, retention time, tailing


94.5 95.3


96.0 108.3 95.7 104.7 RRF -


1.67 0.99


-


0.88 0.83 0.93 0.92 1.00 1.05 0.87 1.04 1.07


so hence this buffer was used. Methanol and acetonitrile were both evaluated, with methanol producing a higher retention time, higher column back pressure and more peak tailing. It was decided to progress using acetonitrile as the organic modifier. Different gradient programs of 0.1 M buffer and organic solvents were also evaluated. After many trials, based on the peak shape, peak symmetry, retention time and peak tailing, the gradient profile, using a flow rate of 2 mL/min, shown in Table 1 was developed.


3.1.3. Detection of Wavelength


SX, FP and their related substance peaks were scanned between 200-400nm wavelengths by photo-diode array detector. A wavelength of 228 nm was found to be optimum for all analyte peaks.


3.1.4. pH of the Buffer (LOQ) 30 % 100 % 120 %


98.4 98.9


(LOQ) 40 % 100 % 120 %


112.1 94.4


101.4 106.6 91.3 81.9


105.1 101.1 99.9


118.6 110.5 114.6 113.5 103.1 110.5 109.4 111.6 111.9


105.6 92.2


108.5 100.2 101.8 99.4


100.6 97.6 92.0


106.1 91.4


107.3 94.2 97.1 93.8 98.4 94.7


101.2


factor, and column efficiency were obtained with a Hypersil BDS C8 column (15 cm x 4.6 mm, 5 µm).


3.1.2. 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. The peak shape, peak symmetry, retention time and peak tailing when using 0.1 M ammonium dihydrogen phosphate were also found to be better than obtained using other buffers,


Various trials on pH of the 0.1 M ammonium phosphate buffer were made to achieve the optimum pH at which all peaks related with APIs and impurities separated well. Based on peak shape, peak tailing and theoretical plate count, suitable pH of the buffer was found as 2.9.


The optimised chromatographic conditions were selected as a gradient elution of buffer (0.1M NH2


PO4 , at pH2.9) and acetonitrile at


228 nm, 2.0 mL/min flowrate, 30º C column temperature and 20µL injection volume. A typical HPLC chromatogram (Figure 3) shows the separation of all 16 known related substances of SX and FP.


3.2. Method Validation


The developed method was validated according to the ICH Q2(R1) guideline [10] for various parameters such as specificity, precision, accuracy, linearity, robustness, limit of detection (LOD), limit of quantification (LOQ) and solution and mobile phase stability.


3.2.1. Specificity


The peak purity indices for the impurity solutions were determined with PDA detector under optimised chromatographic conditions. The FDA guidelines indicated that well separated peaks, with resolution, Rs > 2 between the peak of interest and the closest eluting peak, are reliable for the quantification [11].


Peak purity indices (purity angle < purity


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