A new validated stability-indicating RP-HPLC method for simultaneous quantification of dolutegravir and lamivudine in bulk and pharmaceutical dosage form

A fresh selective, rapid, accurate, precise and RP-HPLC stability-indicating method was developed and validated for the quantitative simultaneous determination of dolutegravir and lamivudine in the bulk as well as pharmaceutical dosage form. A chromatographic separation was done by using Inertsil ODS 3V (250 × 4.6 mm, 5 μm) column and mobile phase composed of phosphate buffer, pH 3.0:acetonitrile:methanol (50:20:30% v/v/v) with flow rate of 1.0 mL/min, and the detection of eluents was carried out at a wavelength of 257 nm utilizing a PDA detector. The drugs, dolutegravir and lamivudine, were subjected to varied conditions like base hydrolysis, acid hydrolysis, oxidation, thermal, photochemical and UV. The suggested method was analysed statistically and validated to fulfil requirements of International Conference on Harmonisation (ICH) and the validation covered accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), robustness, ruggedness and specificity. The retention time of dolutegravir and lamivudine were observed to be 6.36 and 2.16 min, respectively. The method was found to be linear within the range of 14.98 to 91.25 μg/mL for lamivudine and 2.54 to 15.35 μg/mL for dolutegravir. The percentage recoveries (accuracy) for dolutegravir and lamivudine were in the range of 98.35 to 102.14% and 98.01 to 101.5%. The computed relative variance (%RSD) was within the suitable criterion of less than 2.0. The suggested method was set to be precise as well as stability-indicating since no interfering degradant peaks and excipients were evident. All the peaks of degradation were successfully resolved by the use of the developed analytical method with altered retention times. Results obtained were analysed statistically and found to be acceptable in line with the ICH guidelines. Hence, such method is often employed successfully for routine analysis of active analytes in the bulk as well as pharmaceutical dosage form. It is going to be extended to review for its estimation in plasma and other biological fluids and may even be employed for quality control stability sample estimation and in cleaning method analysis during cleaning validation.

Dolutegravir and lamivudine (Dovato), manufactured by GSK Healthcare, may be a combination antiretroviral agent approved by the FDA as an entire regimen for HIV-1 infection treatment in case of adults. It is a fixeddose combination product consisting of dolutegravir 50 mg and lamivudine 300 mg. The recommended dose of the drug is once daily per oral with or without food in case of adults [3]. Dolutegravir, being an integrase strand transfer inhibitor, blocks replication of HIV by the amalgamation prevention of viral DNA into the host human immune T cells' genetic material [4]. In the pharmaceutical industry, an effective analytical method is needed to analyse a drug individually or simultaneously in combination with other drugs. Various methods of analysis were employed for quantitative determination of individual or multi-component combination assay of nucleoside polymerase inhibitor (NPI) in pharmaceutical dosage forms. Various HPLC [5][6][7][8][9][10][11][12][13][14], LC/MS/MS [15][16][17][18][19], HPTLC [20,21], UV [22][23][24] and UPLC [25] assay methods were described within the literature regarding the estimation of lamivudine, abacavir, and a few other anti-retroviral drugs individually as well as in combination with other drugs. On the converse based on the review of literature, no official method for the stability-indicating simultaneous estimation of dolutegravir and lamivudine by RP-HPLC in the bulk and tablet dosage form was performed so far. Hence, there is an undertaken effort for research that has been made to develop and validate a novel and simple analytical method for the stability-indicating simultaneous estimation of dolutegravir and lamivudine in the bulk and pharmaceutical dosage form. The proposed novel method is able to separate all the active analytes present in the pharmaceutical dosage form and validated as per the guidelines of ICH (Q2 specification) [26].

Materials
Dolutegravir and lamivudine were received as gift samples from Mylan Laboratories Limited, Hyderabad, India. The marketed pharmaceutical tablets of Dovato consisting of dolutegravir 50 mg and lamivudine 300 mg, respectively (manufactured by GSK Healthcare), were procured from the pharmacy. HPLC grade methanol, acetonitrile, Milli-Q water, orthophosphoric acid and analytical grade chemicals of sodium hydroxide, potassium dihydrogen phosphate, hydrogen peroxide and hydrochloric acid were purchased from Merck India Limited, Mumbai, India.
Instrumentation HPLC-Waters alliance (Model-2695) was used to perform chromatographic separation which consists of a column oven, an in-built auto sampler and 2996 PDA detector. The information was obtained through the use of Empower-3-software. The column utilised was Inertsil ODS 250 × 4.6 mm, 5 μm. Meltronics sonicator was used to enhance the solubility of the drugs. For pH  adjustment of the solution, Elico pH meter was employed. Sartorius balance was employed for weighing the samples.

Optimised chromatographic conditions
A chromatographic separation was achieved by using Inertsil ODS 3 V, 5 μm particle size, 250 × 4.6 mm column as a stationary phase and mobile phase composed of phosphate buffer, pH 3.0:acetonitrile:methanol (50:20: 30% v/v/v) with a flow rate of 1.0 mL/min, and the eluents were detected at a wavelength of 257 nm utilising PDA detector. The HPLC system was operated at 30°C and therefore the runtime was 10 min. Mode of separation was isocratic.
Phosphate buffer solution, pH 3.0 preparation 1.36 g of accurately weighed potassium dihydrogen orthophosphate was dissolved in 900 mL of HPLC grade water and the pH of the solution was adjusted to 3.0 with the addition of dilute phosphoric acid. Further, the volume was adjusted to 1000 mL with water and the prepared solution was filtered through 0.45 μm membrane filter and degassed to sonicate.

Preparation of mobile phase
Phosphate buffer solution pH 3.0, acetonitrile and methanol were taken within the ratio of 50:20:30% v/v/v.

Preparation of diluent
The prepared mobile phase was used as diluent.

Standard solution preparation
Ten milligrammes of each dolutegravir and lamivudine working standards was weighed accurately and transferred into an individual 10-mL clean and dry volumetric flasks. Half volume of methanol was added and then the

Preparation of sample solution
Twenty tablets were weighed and the average weight of each tablet was calculated. Then a quantity of powder equivalent to one tablet was weighed and transferred into a volumetric flask (100 mL), followed by addition of 3/4th volume of diluent and sonicated for half-hour. Further, the volume was made up with the diluent and filtered through 0.45 μm filter. 0.5 mL of the above solution was transferred into a 25-mL volumetric flask, and the volume was made up to the mark with the diluent and mixed up well to get a sample solution containing 10 μg/mL of dolutegravir and 60 μg/mL of lamivudine.

Method validation
The developed method was validated according to the ICH guidelines with reference to accuracy, precision, system suitability, specificity, linearity, limit of quantification, limit of detection and forced degradation studies [26].

System suitability
The system suitability was checked by the use of 6 replicate injections of freshly made standard solution. The perceived RSD values were within suitable limits (≤ 2.0%). Theoretical plates, resolution and tailing factor of dolutegravir and lamivudine were determined and originated to be well within the suitable limits.

Linearity
The linearity was performed for the prepared standard solutions of dolutegravir and lamivudine at various concentration levels, i.e. within a range of 14.98 to 91.25 μg/ mL for lamivudine and 2.54 to 15.35 μg/mL for dolutegravir. Each measurement was administered in triplicate. The linearity was proven by multivariate analysis by least square method.

Precision
The precision of the method was demonstrated by intraday and inter-day analysis. The concentration used for the precision studies was assumed as 100% (10 μg/mL for dolutegravir and 60 μg/mL for lamivudine). To review the intra-day precision, the analysis of drugs was repeated sixfold on the similar day, and for inter-day precision, the analysis of drugs was made for six   consecutive days for a concentration of 10 μg/mL for dolutegravir and 60 μg/mL for lamivudine.

Accuracy
For the determination of accuracy in the sample preparation method of the standard pattern, additions were done for the measurement of the recovery of drugs. A quantity of sample was taken and the standard drug was added at the levels of 50, 100 and 150%. The results obtained were analysed and observed to be within bounds.

Ruggedness
The ruggedness of the method was confirmed by analysing the sample by various analysts on various instruments on various days.

Robustness
The robustness was determined by varying three parameters from the optimised conditions of chromatography like mobile phase composition (± 5%), making small changes in the flow rate (± 0.1 mL/min) and column temperature (± 5°C).

Limit of detection and limit of quantification (LOD and LOQ)
LOD and LOQ were computed with the use of waters Empower software for signal-to-noise ratio method.

Specificity
The specificity of the method was carried out by the injection of a blank solution (i.e. without any sample), followed by injection of 10 μL drug solution into the column under the optimised conditions of chromatography, in order to demonstrate the separation of two drugs

Forced degradation studies
Such studies were carried out in order to demonstrate the optimised stability-indicating method and to find out if the method is able to quantify active pharmaceutical ingredient (API) accurately in the presence of degradation products that might be formed during various kinds of degradations pertained to drug sample. ICH guidelines (Q1A, Q1B, Q2B) highlight particular degradation environments such as base, acid, oxidation, photo and thermal stability.

Development of RP-HPLC method
Suitable method was selected based on the character of the sample (neutral or ionic molecule), its relative molecular mass and solubility. Various conditions of chromatography were applied for the analysis of dolutegravir and lamivudine in both the bulk and pharmaceutical dosage form. The result of the analysis was within the suitable limit (not less than 2.0). Figs 3, 4 and 5 represent the chromatograms of placebo, blank and sample, respectively. This is often a sign of the specificity of developed RP-HPLC method.

System suitability
It was decided by making six replicate injections from the freshly made standard solutions. The RSD values were observed to be within the generally acceptable limit (≤ 2.0%). Theoretical plates, resolution and tailing factor of dolutegravir and lamivudine were determined and located to be within the suitable limits as shown in Table 1.

Linearity, limit of detection and limit of quantification
The test for linearity was performed by making standard solutions of dolutegravir and lamivudine at various concentration levels, i.e. in a range of 14.98 to 91.25 μg/mL for lamivudine and 2.54 to 15.35 μg/mL for dolutegravir. Each measurement was administered in triplicate. The linearity was proven by multivariate analysis by least square method. The results show that a linear relationship exists between peak area and concentration of drugs within the given range as shown in Table 2 and Figs. 6 and 7. LOD and LOQ were observed to be 0.40 and 0.88 μg/ mL, respectively, for lamivudine and 0.04 and 0.11 μg/ mL, respectively, for dolutegravir. Signal-to-noise ratio values for LOD and LOQ were found to be 3.3 and 10.2 for lamivudine and 3.1 and 9.7 for dolutegravir and observed all the obtained results were found to be within acceptance criteria according to the guidelines of ICH and the results obtained were as shown in Table 3.

Recovery, precision and accuracy
Within-day precision and accuracy of the method were analyzed from replicate analysis (n = 6). Precision of the method was finalised by intra-day and inter-day analysis. The percentage relative variance (%RSD) was computed and was within the suitable criterion of < 2.0. Accuracy was represented as the percentage of the respective active analyte recovery and also plotted linearity graph (Figs. 8 and 9) between the typical amounts added and the average amount recovered for all the active analytes and located to be linear for all the concentration levels (Tables 4, 5 and 6).

Ruggedness
The ruggedness of the method was confirmed by the analysis of sample by various analysts on various instruments on various days. The results in Table 7 show that there were no significant changes within the chromatograms, which means that the developed RP-HPLC method is rugged in nature.

Robustness
The robustness was determined by varying three parameters from the optimised conditions of chromatography like mobile phase composition (± 5%), making small changes in flow rate (± 0.1 mL/min) and column temperature (± 5°C). It had been observed that small changes in such operating parameters did not cause drastic changes of the retention time of the peak of interest, tailing factor (NMT 2.0), plate count (NLT 2000), resolution (NLT 2.0) and %RSD for six replicate injections (NMT 2.0) which were found to be within the acceptance criteria. The results show that the developed method is robust as shown in Tables 8 and 9.

Forced degradation studies
In the case of forced degradation studies, aliquots of stock solutions were treated separately with 1 mL of 2N sodium hydroxide (alkaline stability), 1 mL of 2N hydrochloric acid (acid stability), 1 mL of 20% hydrogen peroxide (oxidative degradation), photo stability degradation (exposure of drug at 200 watt h/m 2 ), exposure of sample drug solution at 105°C for 6 h (dry heat degradation) and neutral degradation (refluxing with water at 60°C for 6 h). The stability of such samples was cross-checked with a fresh sample on the particular day of the study. No interference was  exhibited in the HPLC chromatograms of the degraded products at the respective analyte peaks. Hence, the individual analyte peak purity values found to be within the suitable limits; hence, the method was specific and stability-indicating (Tables 10 and 11).

Discussion
The major aim for the development of the chromatographic method was to urge a reliable technique for the quantification of dolutegravir and lamivudine from the bulk and pharmaceutical dosage form that can be employed also for degradation products. Various conditions of chromatography were applied for the analysis of dolutegravir and lamivudine in both the bulk and pharmaceutical dosage form. The results of analysis obtained was within the suitable limit (not but 2.0). The calibration curves were found to be linear over the concentration range of 14.98 to 91.25 μg/mL for lamivudine and 2.54 to 15.35 μg/mL for dolutegravir. The samples were analysed at 257 nm, the injection volume was 10 μL and the separation was done by utilising Inertsil ODS-3 V, (5 μm, 250 × 4.6 mm) column. The tailing factor and retention times were computed. The retention time of dolutegravir and lamivudine were found to be 2.169 and 6.367 min, respectively. The proposed column was selected which gave a pointy and symmetrical peak with 1.34 tailing factor and theoretical plates of 7643 for lamivudine and 1.12 tailing factor and theoretical plates of 55924 for dolutegravir. The resolution between dolutegravir and lamivudine is 22.51. The linearity of the method was confirmed statistically. The RSD values obtained for the accuracy and precision studies were but 2.0% which proved that the developed analytical method was accurate and precise. It was concluded from the forced degradation studies that all the degradant peaks exhibited during the degradation were well resolved from major drugs, i.e. dolutegravir and lamivudine, and therefore the peak purity was passed, i.e. purity angle was less than purity threshold as per waters Empower-3 software. Therefore, the method is established to be stability-indicating.

Conclusion
The developed chromatographic method is simple, accurate and selective as well as proved to be stabilityindicating for the simultaneous estimation of dolutegravir and lamivudine in the bulk and pharmaceutical dosage form. Preparation of sample is straightforward, the analysis time is short and therefore the elution is by isocratic method. To our present knowledge, no       attempts have yet been made to estimate this multidrug mixture by the stability-indicating analytical procedure. All the active ingredients were profitably resolved with good resolution and quantified. Hence, the suggested validated stability-indicating method was successfully employed to work out dolutegravir and lamivudine in the bulk and pharmaceutical dosage form. It is going to be extended to review for its estimation in plasma and other biological fluids and may even be employed for quality control stability sample estimation and in cleaning method analysis during cleaning validation.