API of Lamivudine and Dolutegravir was provided by Fortune Pharma, Hyderabad, as a gift sample. HPLC grade acetonitrile, methanol, Milli-Q, and remaining analytical grade chemicals were obtained from Merck India Limited, Mumbai, India.
Chromatographic conditions
RP-HPLC experiment is carried on WATERS 2695 with 2487 PDA detector with auto sampler, data-processing, and acquisition has done by using the Empower 2 software. Effective separation achieved by injecting 10 μL of the standard solution into Xbridge Phenyl (250 × 4.6 mm, 5 μ, 100 A0) column, using a mobile phase composed of methanol: buffer (0.1% v/v trifluoroacetic acid (TFA) in water) (85:15 v/v) at a flow rate of 0.8 mL/min, and the eluted analytes were detected at 258 nm wavelength. Ambient temperature was maintained in the injection port and in the analytical column. Mobile phase and standard and sample solution were filtered through a 0.45-μm nylon filter prior to injecting into the HPLC system.
Preparation of standard solution
Sixty milligrams of Lamivudine and 10 mg of Dolutegravir API powders were weighed and transferred into 100 mL volumetric flask, volume made with diluent (acetonitrile and water (50:50)) to 100 mL. One milliliter of above solution was transferred into 10 mL volumetric flask, again volume made with diluents to obtain concentration of 60 μg/mL, and 10 μg/mL for Lamivudine and Dolutegravir, respectively, said as 100% level concentrations.
Preparation of sample solution
An amount of tablet (DOVATO) powder (80 mg) equivalent to 60 mg of Lamivudine and 10 mg of Dolutegravir was weighed and transferred into 100 mL volumetric flask, volume made with diluents to 100 mL. One milliliter of above solution was transferred into 10 mL volumetric flask, volume made with diluents to obtain concentration of 60 μg/mL, and 10 μg/mL for Lamivudine and Dolutegravir respectively. Prior to injecting, sample solution was filtered through 0.4 μm Nylon filter.
Method validation
System suitability test
The system suitability test of the current method was carried out by injecting 100% level of working standard concentration in 6 replicates, and parameters like percentage relative standard deviation (% RSD), USP tailing factors (T), USP plate count (N), and resolution (R) were evaluated for the obtained chromatograms.
Linearity
The linearity of the method represents that the obtained test results are directly proportional to concentration. The linearity of the current method has performed by injecting the series of working standard concentrations ranges from 30 μg/ml to 90 μg/ml of Lamivudine and 5 μg/mL to 15 μg/mL of Dolutegravir into the HPLC system under optimized chromatographic conditions. Finally, linearity graph was plotted for concentration vs peak area and regression coefficient (r2) value determined.
Precision
The closeness relationship among observed responses of homogenous sample on multiple replications referred as precision. Usually, it can be done in the same day (intraday) and in different days (inter-day). Intraday and inter-day precision of the method were performed by injecting 100% level of working standard concentration for 6 times in a day and 3 times per day for three continuous days. Percentage RSD calculated for peak areas obtained.
Accuracy
The accuracy of the method was accomplished by recovery studies in which known amount sample solution spiked at three different standard concentration levels about 50, 100, and 150%, each level of solution injected in triplicate. The percentage mean recovery at three different levels of the drug solution was calculated.
Specificity
Specificity represents the ability of the method to determine or assess the intended drug in the presence of other substances without interferences. Ten microliter volume of prepared blank solution, 100% level pure working standard solution, and standard solution with placebo have been injected individually. The retention time (RT) of individual injection of standard sample solution alone and along with placebo was observed to assess any interference that has been happened with peaks of Dolutegravir and Lamivudine in obtained chromatograms.
Sensitivity
The LOD and LOQ were calculated by implementation of standard deviation method, in which the following formulae were used.
$$ \mathrm{LOD}=3\times \sigma /S $$
$$ \mathrm{LOQ}=10\times \sigma /S $$
where σ is the standard deviation of the intercept, and S is the slope of the linear curve.
Robustness
The robustness of the method was checked by slightly and deliberately changing the flow rate, mobile phase composition, and maximum absorption wavelength. It can be performed by evaluating the system suitability parameters after changing the HPLC flow rate (± 0.1 mL/min), wavelength maximum (± 2 nm), and mobile phase ratio (± 1 mL).
Forced degradation studies
In forced degradation studies, intentionally drug substance is exposed to conditions more intense than accelerated conditions. Chemical stability of the drug molecule can be depicted with forced degradation studies, which helps in successful development of stable formulation with appropriate storage conditions. ICH guidelines emphasized certain degradation conditions like acid hydrolysis, base hydrolysis, oxidation, thermal degradation, and photo stability in ICH Q1A, QIB, and Q2B guidelines.
Acidic degradation solution
Add 0.2 mL of 0.1 N HCl to 1 mL of the standard stock solution and reflux for 2 h at 70 °C, kept a side for 24 h at same temperature, and after that, cool the solution and neutralize with 0.1 N NaOH. Further dilution was done to get a solution having 60 μg/mL of Lamivudine and 10 μg/mL of Dolutegravir.
Alkali degradation solution
To the 1 mL of standard stock solution, add 0.2 mL of 0.1 N NaOH and reflux for 2 h at 70 °C, kept a side for 24 h at same temperature, and after that, cool the solution and neutralize with 0.1 N HCl and make up to 10 mL with diluent to obtain concentration of 60 μg/mL, and 10 μg/mL for Lamivudine and Dolutegravir respectively.
Oxidative degradation solution
To the 1 mL of standard stock solution, add 0.2 mL of 3% hydrogen peroxide and reflex for 2 h at 70 °C, kept a side for 24 h at same temperature, and after that, cool the solution and make up to10 mL with diluent to obtain concentration of 60 μg/mL and 10 μg/mL for Lamivudine and Dolutegravir respectively.
Thermal degradation solution
Place the 100 mL of standard stock solution in heating chamber at 80 °C/75% RH for 24 h. One microliter of above solution is diluted to 10 mL to obtain concentration of 60 μg/mL and 10 μg/mL for Lamivudine and Dolutegravir respectively.
To assess the percentage degradation, the prepared each solution was injected three times. In general, not more than 20% degradation of the drug is considered as effective and optimal acceptable value in analytical method with stability indicating.
Assay
Assay of the method can be done by injecting subsequent injections of standard and sample solutions, both having concentration about 60 μg/mL and 10 μg/mL of Lamivudine Dolutegravir respectively. The preparation of standard and sample solutions was mentioned prior the in methods section. Computing the percentage assay by using following formula.
$$ \%\mathrm{Assay}=\frac{\mathrm{AT}}{\mathrm{AS}}\times \frac{\mathrm{WS}}{\mathrm{DS}}\times \frac{\mathrm{DT}}{\mathrm{WT}}\times \frac{\mathrm{P}}{100}\times \frac{\mathrm{AVG}\ \mathrm{Wt}}{\mathrm{LableClaim}}\times 100 $$
where AT is the peak area of sample (tablet) solution, AS is the peak area of standard solution, WS is the weight of standard substance in mg to prepare standard solution, WT is the weight of sample (tablet) powder in mg to prepare standard solution, DS is the dilution factor of standard solution, DT is the dilution factor of sample solution, P is the percentage purity of standard substance, and AVG Wt is the average weight of the tablets in mg.
