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 volume was made up to the mark with diluent. 0.6 mL of lamivudine standard stock solution and 0.1 mL of dolutegravir standard stock solution were transferred into a 10-mL volumetric flask and the volume was made up to the mark with the diluent to get a standard solution containing 60 μg/mL of lamivudine and 10 μg/mL of dolutegravir.
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 intra-day 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 dolutegravir and lamivudine from impurities present, if any.
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.