Development of Validation and Stability Indicating Method of Anti-HIV Dolutegravir Drug and its Related Impurities by Using RP-HPLC

A simple, rapid and robust reverse phase HPLC method was developed and validated for the determination of impurities in Dolutegravir drug substance. The main aim of this study is to reduce the run time of the HPLC method by developing and validating a new, less expensive HPLC method. The chromatographic separation of Dolutegravir and its related impurities is carried out by using C8 column (150 × 4.6 mm), 5 μm with 0.1% trifluoroacetic acid in water as mobile phase A, methanol as mobile phase B. The flow rate is 1.0 mL/min with gradient elution mode. The wave length for detection is 240 nm (UV detector). The developed method was validated and proved that the method was specific, accurate and precise as per ICH. The system suitability criteria found to be within the limits. The limit of detection and limit of quantification demonstrate that the method is sensitive. The linearity curve was found to be linear and the correlation coefficient obtained is not less than 0.998. The average percentage recoveries of impurities were in the range of 97 to 101%. The proposed method was found to be suitable and accurate for quantitative determination of impurities in Dolutegravir drug substance.

Impurity profiling of drug substances and products is a very critical task in the pharmaceutical industry under regulatory conditions [4][5][6][7]. The presence of unknown impurities, unwanted solvents, even at very low levels, may change the effect of drug efficiency and cause side effects. Therefore, impurity profile of drug substance and product should be carried out by using stability indicative analytical method.
High Performance Liquid chromatography is one of the effective separation analytical tools to determine and quantitate the impurities. By using HPLC, we can separate a mixture of compounds to identify and quantify into individual components. In literature survey, various quantification methods are available for the determination of Dolutegravir in combined dosage form [8][9][10][11].  Chandra Sekhar et al. separated Dolutegravir enantiomer and Diastereomer by using chiral RPLC [11]. Srinivas et al. reported the separation of Dolutegravir optical isomers by using chiral HPLC [12].
There is no literature available for the determination of impurities in Dolutegravir drug substance. In this present research work, a stability indicative HPLC method was developed for separation of impurities and degradation products by considering 0.1% as specification limit along with short run time and validated the method according to ICH guidelines [13][14][15].

OPTIMIZATION EXPERIMENTS
In the process of developing impurities separation by HPLC method, different parameters were studied which influence the separation such as using different columns, different mobile phases and column temperatures. HPLC columns used for development of method were C 18 , C 8 with different combination of mobile phases. In this development separation between impurities and Dolutegravir was not achieved. Hence we tried with Kromasil C 8 column, trifluoroacetic acid buffer in different combination of mobile phase with organic solvents acetonitrile and methanol. Finally specific method was optimized in this column. Method was finalized with satisfactory resolution among all impurities with Mobile phase A is 0.1% of trifluoroacetic acid in water and mobile phase B is methanol with gradient elution.

MATERIALS AND REAGENTS
Dolutegravir drug substance and related impurities were procured from Clearsynth laboratory. The HPLC grade methanol and acetonitrile procured from Rankem India Pvt. limited. Trifluoro acetic acid procured from Thermo scientific, India and Pure milli-Q water is used with the help of Millipore purification system (Millipore®, Milford, USA).

INSTRUMENTATION AND CHROMATOGRAPHIC CONDITIONS
WATERS HPLC, Model: 2695 equipped with 2996 photo diode array detector was used for development and method validation, with an automated sample injector. Kromasil C 8 , 150 × 4.6 mm, 5 µm column was used for the separation. 0.1% of trifluoroacetic acid in water is used as mobile phase A and methanol is used as mobile phase B. Analysis was carried out in Gradient mode with flow rate of 1.0 mL/min and injection volume was 10 µL. The column temperature was 35°C, the run time was 20 min and the gradient programme is shown in Table  1. The data was acquired at 240 nm. The output signal was monitored and integrated using Empower 2 software.

Sample solution preparation
A 25 mg of Dolutegravir was weighed, transferred to 50 mL volumetric flask, dissolved in 20 mL of diluent by sonication, made up to the mark with diluent and the resulting concentration of the solution is 0.5 mg/mL.

Impurities solution preparation (0.1%)
Stock solution-1: Each 10 mg of impurity-A, impurity-B and impurity-C were weighed and transferred to 100 mL volumetric flask, dissolved and made up to mark with diluent to give stock solution-1 (0.1 mg/mL).

Stock solution-2:
From the above solution 1 mL is transferred into a 10 mL volumetric flask, volume was made up to mark with diluent to give a solution containing 0.01 mg/mL of Impurities. It is further diluted to 2.5 mL in 50 mL of diluent to give a solution containing 0.0005 mg/mL.

System suitability solution preparation
25 mg of Dolutegravir sample is weighed and transferred into 50 mL volumetric flask, dissolved in 20 mL of diluent by sonication, 2.5 mL of impurity stock solution-2 is added and is made up to the volume with diluent solution containing concentration of 0.5 mg/mL of Dolutegravir and 0.0005 mg/mL of each impurity.

Method validation
The validation of HPLC method was carried out for the determination of Impurity-A, Impurity-B and Impurity-C in Dolutegravir drug substance as per the ICH guidelines to demonstrate that this method is stability indicative for intended use.
System suitability: The system suitability was performed for every validation parameters by injecting of system suitability solution containing 0.5 mg/mL of Dolutegravir and 0.0005 mg/mL of each impurity.
Specificity (Selectivity): The blank, system suitability solution and spiked sample solutions were prepared as per methodology. Individual impurities were prepared at 0.1 mg/mL and spike sample solutions were prepared as per specification limit and injected into HPLC system. The retention time of all peaks obtained in the resulting chromatograms were recorded. Based on the results, all impurities are eluted at different retention times and adequately resolved from each other and also from main peak. Representative chromatogram is shown in Figure 2 and experimental data is given in Table 2.  From the above chromatogram, there was no interference observed due to blank at the retention times of Dolutegravir and its related impurities. All impurities are separated with good resolution.
In order to assess the stability indicating nature of the HPLC method, Dolutegravir samples were stressed by acid, base, oxidation, heat, light (Fluorescent overall illumination with UV) and humidity. The degraded samples were analyzed by using a photodiode-array detector. The peak purity of Dolutegravir and its related impurities were passed. The forced degradation conditions are mentioned in Table 3 and the results are mentioned in Table 4. From the results, no degradation was observed when Dolutegravir sample was exposed to acid, base, hydrolysis, light, humidity and heat. Slight degradation was observed in peroxide conditions. According to the stress study, none of the degradants co-eluted with the Dolutegravir peak and its related impurities formed.

Limit of detection (LOD) and Limit of quantitation (LOQ):
The detection limit is considered as very low level of concentration of an analyte in a sample that can be detected, but not necessarily quantitated. The detection limit was determined as the lowest concentration for which the response is approximately three times greater than the baseline noise. The limit of quantitation is considered as the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method. The LOD values obtained for Dolutegravir and its impurities are listed in Table 5 and corresponding representative chromatogram is shown in Figure 3.   Based on above results for LOD, S/N ratio of each component was within the limit.
The LOQ values obtained for Dolutegravir and its impurities are listed in Table 6 and corresponding representative chromatogram is shown in Figure 4.  Based on above results for LOQ, S/N ratio of each component was within the limit.
Precision at LOQ: The precision at LOQ was performed by analyzing six replicate injections of Dolutegravir and each impurity at LOQ level. Results of peak area of Dolutegravir and each impurity were summarized in Table 7. Based on the above results, it was observed that the % RSD for the peak areas of Dolutegravir and its impurities obtained from QL level was within the acceptable limit.
Linearity: The linearity of the method was demonstrated for Dolutegravir and its related impurities by analyzing the solutions ranging from LOQ to 150% of the specification limit (Table 8). The correlation coefficient for impurity B was 0.9996, Dolutegravir was 0.9995, Impurity A was 0.9994 and Impurity C was found to be 0.9997 which indicates good linearity ( Figures  5-8).    Accuracy: The accuracy of the method was determined by using solutions containing Dolutegravir sample spiked with each impurity at LOQ, 50%, 100% and 150% of the working strength of Dolutegravir. All the solutions were prepared in triplicate and analyzed. The percentage recovery results obtained for each impurity was listed in Table 9. The percentage recovery values obtained for each impurity at LOQ level, 100% and 150% were within the acceptable limit.
System Precision: The system precision was performed by analyzing six replicate injections of standard solution at 100% of the specified limit with respect to the working strength of Dolutegravir. Results of peak area of Dolutegravir and its impurities are summarized in Table 10. The % RSD for the peak areas of Dolutegravir and its impurities obtained from six replicate injections of standard solution was within the limit.

Method Precision:
The precision of the method was determined by analyzing a sample of Dolutegravir spiked with each impurity at 100% of the specification limit (Six individual sample preparations). Data obtained is summarized in Table 11. From the above results, the % RSD for the impurities from method precision study was within the limit.

Robustness:
The chromatographic conditions were deliberately changed to evaluate the robustness of the existing method. To determine the robustness of method, system suitability solution is prepared as per methodology and injected into HPLC at different altered conditions to check the method's ability like flow rate (± 10%), column oven temperature (± 5°C) and wavelength (± 3 nm) from actual method conditions. No significant change is observed by changing flow, temperature, wavelength and system suitability also complied as per methodology. The robustness results are summarized in Table  12. From the robustness study, system suitability criteria comply with the results.

CONCLUSION
From the above experimental results it was concluded that, the newly developed method for the simultaneous estimation of related substances (i.e. Impurity A, Impurity B and Impurity C) in Dolutegravir was found to be simple, precise and accurate with high resolution and shorter retention time. The present proposed methodology makes is cost effective which can be implemented for routine analyses in pharmaceutical industry.