Instrument
The method was established by utilizing analytical grade chemicals and reagents. Dolutegravir standard gift sample was provided by Hetero Drugs Pvt. Ltd. and marketed solid dosage form (Tivicay) was procured from local pharmacy. The absorbance of the analytical solutions was determined by using double–beam Shimadzu Ultraviolet–Visible Spectrophotometer 1800. Spectral bandwidth 0.1 nm, wavelength accuracy ± 0.1 nm and a pair of 1 cm path length matched quartz cells were included in it.
Chemicals and reagents
1,10-phenanthroline reagent (0.5% w/v)
The reagent, 1,10-phenanthroline (0.5 g) was accurately weighed and dissolved in sufficient methanol (in a volumetric flask) to produce 100 mL.
Ferric chloride reagent (0.3% w/v)
Ferric chloride (0.3 g) was weighed accurately and dissolved in sufficient distilled water (in a volumetric flask) to produce 100 mL.
Dolutegravir standard stock solution
The stock solution of dolutegravir (1000.00 μg/mL) was made by solubilizing 10 mg in 10 mL of acetonitrile and water (1:1). The solution was further diluted with distilled water to get the required concentration of dolutegravir for the λmax determinations and for further analysis.
Analysis of dolutegravir using Fe3+/1,10-phenanthroline
Aliquots of 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mL of dolutegravir standard solution (1000.00 μg/mL) were progressively taken in to 10 mL volumetric flasks. To this ferric chloride solution (2 mL, 0.3% w/v), 1,10-phenanthroline solution (1 mL, 0.5% w/v) were added and shaken vigorously and lay aside for 15 min to ensure the color development through redox-coupling reaction. The volume of the volumetric flask was made up to the mark with double distilled water to accord the ultimate concentrations holding 40.00–140.00 μg/mL of dolutegravir.
Blank solutions were made by adopting identical methodology mentioned above, by omitting the corresponding analyte. Then the absorbance of the colored compound was recorded at 520.0 nm against corresponding blank. All measurements were recurrent six-fold for every concentration.
Method optimization
The analytical method was optimized for the reagent concentration (ferric chloride and 1,10-phenanthroline), time for color development and mole ratio of the reaction and the details were provided in next sections.
Method validation
The proof of the method was established based on linearity, accuracy, precision, sensitivity, and robustness according to International Council for Harmonization guidelines [28].
Linearity
The linearity was examined in pure solutions (n = 6) over the concentration span of 40.00–140.00 μg/mL for dolutegravir. Calibration curve was plotted and from that slope, intercept, and correlation coefficient were computed.
Accuracy
The accuracy of the methodology was decided by recording the recoveries of the analyte using method of standard additions. Distinct levels of standard solutions (80, 100, and 120%) of dolutegravir was spiked to pre-quantified samples and analyzed by proposed method. Each sample was prepared in triplicate at each level. The mean percentage recoveries and percentage relative standard deviation were figured out statistically.
Precision
Precision is the level of repeatability of results as reported between samples analyzed on identical day (intra-day) and samples scampered on three completely distinct days (inter day) in order to examine the intra- and inter-day variant in the method. Solutions accommodating 40.00, 80.00, and 140.00 μg/mL of dolutegravir were subjected to the present spectrophotometric method. The discrepancies in the absorbance of the analyte solutions on intra- and inter-day were deliberately expressed in percentage relative standard deviation.
Sensitivity and robustness
Sensitivity of the method was denoted by limit of detection and limit of quantification values, determined based on standard calibration curve. They were calculated using the formulae 3.3 σ/s and 10 σ/s, respectively, where “σ” is the standard deviation of the y-intercept of the regression equation and “s” is the slope of the calibration curve. Sandell’s sensitivity was calculated from the ratio of molecular weight and molar absorptivity of the dolutegravir. Further the robustness of the analytical method was established by measuring the absorbance of the colored complex by making small changes in the concentration of ferric chloride and 1,10-phenanthroline.
Assay of dolutegravir
Twenty tablets of dolutegravir (Tivicay) were weighed accurately and ground to fine powder. A quantity of powder analogous to 200 mg of dolutegravir was dissolved in acetonitrile and water (1:1), the contents were shaken thoroughly for 5 min. Then, the volume was done up to 10 mL with acetonitrile and water and screened through Whatmann’s filter paper (No. 42). To the 1 mL of above filtrate, 2 mL of ferric chloride solution (0.3% w/v), 1 mL of 1,10-phenanthroline reagent (0.5% w/v) were added and shaken vigorously. The emerged solution was diluted up to 10 mL with double distilled water and the colored chromogen was spectrophotometrically measured at 520.0 nm against the corresponding blank.