35
chromatogram hence preventing interfering peaks. With the given chromatographic conditions the PDA detector was used to detect the peak purity.
For a peak to be considered pure, purity angle must be less than purity threshold. SX and FP peaks in the chromatogram were found to be pure as it is seen in Table 2. All compounds were completely separated and no drift in analyte retention time was observed.
Table 2: Purity results for SX and FP. Salmeterol Compounds Test Solution
Peak Purity Angle
Peak Purity Angle
Fluticasone Propionate
Peak Purity Angle
Peak Purity Angle
0.087 0.541 0.040 0.260 Standard solution 0.202 0.556 0.044 0.244 2.7.2. Linearity and range
Fourteen different concentrations of standard solutions were prepared to test the linearity range. The calibration curve was plotted as peak area versus concentration of the standard solutions.
The nominal concentration of test solutions for SX and FP are 0.0025 mg/mL and 0.025 mg/mL, respectively. Relative response factors were determined by preparing standard solutions at different concentration levels ranging from LOQ concentration to 0.0125 mg/mL for SX and to 0.125 mg/mL for FP.
The correlation coefficient (r) should not be lower than 0.998 to establish the criterion of linearity according to Kazakevich and LoBrutto [9]. As can be seen in the Graph 1, the correlation coefficients (r) were greater than 0.998. Therefore, it can be concluded that the analytical method was linear for this concentration range.
2.7.3. Limit of Quantification (LOQ)
LOQ values for SX and FP were determined based on signal-to-noise approach according to ICH guidelines. The results were tabulated in Table 3.
Table 3. Limit of quantification (LOQ) of SX and FP. Impurities
Salmeterol Xinafoate
Fluticasone Propionate
2.7.4. Accuracy
The accuracy of the method was determined by recovery experiments. Recovery studies were carried out with three injections at three different concentrations. Three concentration levels of 80%, 100% and 120% of the specification level of SX and FP were prepared. Three samples were prepared for each level. The experimental results (shown in Table 4) reveal that recoveries were obtained between 80% - 120% for SX and FP.
Table 4: Average Accuracy (recovery, %) results for SX and FP.
Compounds 80 % 100 % 120 % Salmeterol Xinafoate
99.5 100.2 99.2
Fluticasone Propionate
2.7.5. Precision
For the precision parameter of the method validation, system precision, repeatability and intermediate precision studies were performed. System precision studies were carried out by consecutively injecting
100.4 101.0 100.5 Compounds
Salmeterol Xinafoate
Fluticasone Propionate
LOQ (µg/mL) 0.030 0.067 Table 5: Precision results for SX and FP.
Values% System
Precision 0.36
0.18 2.7.6. Robustness
To demonstrate the robustness of the method, system suitability parameters were verified by making changes in chromatographic conditions such as change in column temperature ± 5ºC, change in mobile phase ratio ± 10 (v/v), change in flow rate ± 0.1 mL/min., change in buffer pH ± 0.1. The retention time and the difference between the results at normal conditions and modified conditions were calculated. According to these modifications excluding the change in mobile phase ratio, it can be concluded that method was robust over an acceptable working range of its HPLC operational conditions.
Repeatability 1.4 1.8
Intermediate Precision
1.0 1.2
the standard solutions for six times. Repeatability was studied by consecutively injection of six test solutions, which were prepared separately. Intermediate precision was carried out by injecting six injections of standard and sample solutions within- laboratory variations: different days, different analysts, and different equipment. The relative standard deviation and difference between two analysts were calculated. The lower RSD % values (<10.00) indicate good precision of the developed method shown in Table 5.
Graph 1: Salmeterol Linearity Results of Assay Analytical Method Validation for Cyplos 50/500 mcg Powder For Inhalation.
Graph 2: Fluticasone Propionate Linearity Results of Assay Analytical Method Validation for Cyplos 50/500 mcg Powder For Inhalation.
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