18 May / June 2017 Table 5. Precision results for SX, FP and their related substances. Compounds
Salmeterol Imp. D Salmeterol Imp. G
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
RSD Values (%) System Precision Repeatability 1.65
1.22 0.32 0.53 0.32 0.48 0.40 0.31 0.24 0.21 0.49
threshold) were found as no additional peaks were co-eluting with the impurities. Baseline resolution was achieved for all investigated compounds. Complete and clear separation of all compounds was observed and there were no observable interferences in the chromatogram. The required resolution (Rs > 2) was achieved for all peaks, visibly confirmed in Figure 4.
3.2.2. Linearity and range
Six series of standard solutions were selected to assess 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 were 0.5 mg/mL and 5 mg/mL, respectively. Relative response factors were determined by preparing standard solutions for each impurity at different concentration levels ranging from LOQ concentration to 0.006 mg/mL for SX and its related substances, also to 0.06 mg/mL for FP and its related substances.
The y-intercepts were close to zero with their confidence intervals containing the origin. CDER recommends establishing the criterion of linearity at a level of the correlation coefficient r not lower than 0.999 [13]. As can be seen in Table 2, the correlation coefficients (r) are greater than 0.999, the acceptance threshold suggested for linearity of procedures for the determination of impurity content in bulk drug [12].
3.2.3. Limit of Detection (LOD), Limit of Quantification (LOQ) and Reporting Threshold
LOQ and LOD values for all 11 known related substances were determined based
3.2.4. Accuracy
The accuracy of the method was determined by recovery experiments. Recovery studies were carried out with six injections and four different concentrations. Known amounts for each related impurities corresponding to three concentration levels of LOQ, 30%, 100% and 120% of the specification level were spiked into sample solution. Three samples were prepared for each level. The experimental results (shown in Table 4) revealed that recoveries were obtained between 80% - 120% for all the investigated related compounds.
3.2.5. Precision
System precision studies were carried out by consecutively injecting the standard solutions for six times. Repeatability was studied by consecutively injection of six spiked test solution which are 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 (<5.00) indicate good precision of the developed method (Table 5).
2.15 4.52 0.64 1.75 2.75 2.16 2.10 1.72 1.69 1.48 2.26
Intermediate Precision
2.39 3.50 1.23 1.74 0.59 0.88 0.75 0.66 0.72 0.72 0.62
on signal-to-noise approach according to ICH guidelines. The results are tabulated in Table 3.
Reporting threshold was calculated based on the maximum daily intake of the drug and ICH Q3B(R2) [13]. According to ICH Q3B(R2), reporting threshold for maximum daily doses less than 1 g is given as 0.1%. Impurities found above the reporting threshold are reported.
3.2.6. Robustness
To demonstrate the robustness of the method, system suitability parameters was verified by making changes in chromatographic conditions, i.e. change in column temperature ± 2ºC, change in organic composition of mobile phase ± 2%, using different lots of columns. The retention time and the difference between the results at normal conditions and modified conditions were calculated. The spiked sample was injected and the resolution among all components was monitored. These experiments determined that the method is sensitive to changes in organic composition of mobile phase; however for all of the other parameters investigated the assay is robust
3.2.7. Stability
The stability of mobile phase, standard and sample solutions were carried out by keeping the solutions for 5 days and observing for changes in the area and the retention of the peaks, which were then compared with the pattern of the chromatogram of freshly prepared solutions. Relative difference (%) of known impurities was calculated based on the values of initial conditions, with the target being less than 5%. According to the stability study, the standard solution is stable for 118 hours and test solution is stable for 58 hours at 15ºC tray temperature, with the mobile phase being stable for 5 days in ambient conditions.
4. Conclusion
The proposed RP-HPLC impurity method for SX based impurities (D and G) and FP based impurities (A, B, C, D, E, F, G, H, I) was found accurate, precise, robust, specific and selective in the result of the performed validation studies. The method was validated as per ICH guidelines and it can be used for routine analysis of simultaneous quantification of 11 related substance of salmeterol xinafoate and fluticasone propionate in inhalation powder.
References
1. A. Samir, H. Salem, M. Abdelkawy, Simultaneous determination of salmeterol xinafoate and fluticasone propionate in bulk powder and Seritide®
Diskus using high
performance liquid chromatographic and spectrophotometric method, Pharmaceut. Anal. Acta. 50 (2012) 21–126.
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