A rapid, simple, and robust reverse-phase HPLC method was developed and validated for the quantification of related substances in dolutegravir 10 mg dispersible tablets. This study primarily aimed to establish a new RP-HPLC method to quantify impurity B (a degradation impurity) as a related substance, following USP guidelines. Chromatographic separation was performed using a phenyl-hexyl column (250 × 4.6 mm, 5µ) with a mobile phase consisting of 45% buffer (sodium dihydrogen phosphate dihydrate and EDTA), 49% methanol, and 6% acetonitrile, adjusted to a pH of 2.5 ± 0.05 with orthophosphoric acid. The method used isocratic elution at a flow rate of 1.2 mL/min and maintained a column temperature of 35 °C, with detection at 258 nm using a PDA detector. The method was evaluated for stability, specificity, ruggedness, precision, linearity, accuracy, and robustness, according to USP standards, and showing high resolution and short retention times. All system suitability and validation parameters met the required criteria. The method showed excellent sensitivity, with LOD and LOQ values confirming its capability. Linearity was confirmed for both dolutegravir and impurity B with a correlation coefficient greater than 0.997. The recovery of the impurity was consistent and ranged from 80 to 120%. Overall, the method was found to be accurate and reliable for the determination of related substances in dolutegravir 10 mg dispersible tablets.
Introduction
Dolutegravir is an antiretroviral medication classified as an integrase inhibitor, which targets and inhibits the HIV enzyme integrase. By blocking the function of this enzyme, dolutegravir effectively disrupts the replication process of HIV, potentially reducing viral load in the body [1-4]. The US Food and Drug Administration (FDA) has approved this drug, which can be used in combination with Rilpivirine (brand name Edurant) to treat HIV. The chemical structure of dolutegravir is presented in Figure 1, and its chemical name is the sodium salt of (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1’,2’:4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide. It has a molecular formula of C20H19F2N3O5Na and a molecular weight of 419.38 g/mol.
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Figure 1. Structure of dolutegravir sodium |
Dolutegravir (DTG), marketed as Tivicay, is a widely used antiretroviral drug prescribed for the treatment of HIV/AIDS, often combined with other medications. It also plays a role in post-exposure prophylaxis to reduce the risk of HIV transmission following potential exposure. In pharmaceutical manufacturing, monitoring impurities in drug substances and final products is a critical regulatory requirement. Even the smallest amounts of contaminants or solvents can impact the drug’s therapeutic effectiveness and cause harmful side effects [5-7]. To accurately assess the impurity profile of dolutegravir, stability-indicating methods are necessary. High-performance liquid chromatography (HPLC) is an effective technique for separating, identifying, and quantifying individual components in a mixture, making it an essential tool in impurity detection. However, there is a lack of established methods in the literature for impurity analysis in dolutegravir products. This study presents a new RP-HPLC method specifically designed to quantify related substances in dolutegravir drug formulations. The method developed here is precise and reliable for determining the related impurities in dolutegravir dispersible tablets (10 mg).
Materials and Methods
Materials
The dolutegravir sodium standard and its related impurities were obtained from Jigs Chemicals, Ahmedabad. Methanol and acetonitrile, both of HPLC grade, were sourced from Rankem India Pvt. Limited. The study also employed dolutegravir dispersible tablets (10 mg) and ultrapure Milli-Q water.
Analytical method development
A new analytical approach for the detection of related substances in dolutegravir dispersible tablets (10 mg) was established following USP guidelines. A series of trials were conducted to identify the most suitable chromatographic parameters, such as varying buffer pH levels and adjusting the methanol and acetonitrile composition, using C-18, C-8, and Phenyl-Hexyl columns as stationary phases [8, 9]. The Phenyl-Hexyl column was specifically chosen due to its ability to provide distinct resolution between known and unknown degradation products. Compared to C-18 and C-8, this column demonstrated superior selectivity for aromatic compounds, attributed to its extended hexyl hydrocarbon functional group, which enhanced retention and separation. The optimized chromatographic conditions were achieved with a mobile phase comprising 45% buffer (sodium dihydrogen phosphate dihydrate and EDTA), 49% methanol, and 6% acetonitrile, adjusted to a pH of 2.5 ± 0.05 with ortho-phosphoric acid, under isocratic elution conditions. The flow rate was set at 1.2 mL/min, and the column temperature was maintained at 35 °C. The compounds were detected at 258 nm using a PDA detector. The chromatograms corresponding to the optimized method are displayed in Figure 2.
Optimized chromatographic conditions
Optimized Chromatographic Conditions are presented in Table 1.
Table 1. Optimized chromatographic conditions.
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Column details |
Kinetex® 5 µm Phenyl-Hexyl 100 Å, (250×4.6) mm. Make Phenomenex, Part No: 00G-4603-E0. |
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Flow rate |
1.2 mL/min |
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Injection volume |
20 µL |
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Column oven temperature |
35 °C |
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Autosampler temperature |
25 °C |
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Wavelength |
258 nm |
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Run time |
20 minutes |
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Wash vial |
Water: Acetonitrile (1:1) % v/v, respectively. |
Mobile phase/diluent preparations
Buffer preparation
1.56 g of Sodium dihydrogen orthophosphate dihydrate and 100 mg of EDTA were weighed and dissolved in 1000 mL of water, followed by thorough mixing. The solution was filtered through a 0.45 µm membrane filter. The pH was adjusted to 2.50 ± 0.05 using ortho-phosphoric acid, mixed well, and filtered again through a 0.45 µm nylon filter.
Mobile phase preparation
A mixture of the buffer solution, methanol, and acetonitrile was prepared in a ratio of 45:49:6 (v/v/v). The solution was sonicated for 5 minutes to remove any trapped air or gases.
0.1M hydrochloric acid solution
To prepare the hydrochloric acid solution, 8.5mL of concentrated hydrochloric acid was added to 500mL of water in a 1000mL volumetric flask. After mixing well, the solution was diluted to the final volume with water and mixed thoroughly.
Diluent preparation
A 1:1 (v/v) ratio of 0.1M hydrochloric acid and acetonitrile was mixed and sonicated for 5 minutes to degas the solution.
Blank
The diluent solution was used as the blank for this process.
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a) |
b) |
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c) |
d) |
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Figure 2. Optimized chromatograms |
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Standard preparations
Standard stock 1 solution preparation
twenty mg of dolutegravir sodium standard, corresponding to 20 mg of dolutegravir, was weighed and added to a 100 mL volumetric flask. Then, 70 mL of diluent was introduced, and the mixture was sonicated for approximately 5 minutes until fully dissolved. The flask was filled to the 100 mL mark with diluent, ensuring proper mixing.
Standard stock 2 solution preparation
A 5.0 mL portion of the first standard stock solution was further diluted to 100 mL with diluent and mixed well.
Standard solution preparation (1.0 ppm)
5.0 mL of the second standard stock solution was transferred to a 50 mL volumetric flask and diluted to the mark with diluent. The solution was mixed thoroughly.
Impurity standard preparations
Impurity B stock solution
2.0 mg of impurity B was weighed and placed in a 20 mL volumetric flask, followed by the addition of 10 mL of diluent. The solution was sonicated until completely dissolved and then diluted to the final volume with diluent.
Impurity B standard solution
0.5 mL of the impurity B stock solution was diluted to 50 mL with diluent and mixed thoroughly.
Placebo preparation
Crushed placebo powder, equivalent to five tablets, was weighed and transferred into a 100 mL volumetric flask. 50 mL of diluent was added, and the flask was sonicated for 15 minutes. After cooling, the volume was adjusted to 100 mL with diluent, mixed well, and filtered through a 0.45 µm nylon filter, discarding the initial 5 mL.
Sample preparation (ten mg)
Crushed dolutegravir dispersible tablets (10 mg, equivalent to 5 tablets) were weighed and transferred to a 100 mL volumetric flask. 60 mL of diluent was added, and the mixture was sonicated for 15 minutes. After cooling, the solution was diluted to the mark with diluent and mixed well. The solution was filtered through a 0.45 µm nylon filter, discarding the first 5 mL.
Spiked sample preparation (10 mg)
Crushed dolutegravir dispersible tablets (10 mg, equivalent to five tablets) were weighed and added to a 100 mL volumetric flask. 70 mL of diluent was added along with 1.0 mL of impurity B individual stock. The solution was sonicated for 15 minutes and allowed to cool to room temperature. The flask was filled to the 100 mL mark with diluent and mixed well. The solution was filtered through a 0.45 µm nylon filter, discarding the first 5 mL.
Method validation procedure
The analytical method for related substances in dolutegravir dispersible tablets (10 mg) is validated by assessing the following parameters:
System suitability
Before starting the analysis, it’s vital to confirm the correct operation of the analytical system. The preparation of both blank and standard solutions followed the defined testing procedure.
Specificity
Specificity in an analytical method ensures the ability to accurately identify the target analyte even in the presence of other compounds such as impurities, by-products, or matrix components. The responses obtained from these solutions are summarized in Table 2.
Filter compatibility study
In this study, a placebo solution and a spiked sample solution were analyzed using filters made from nylon or PVDF with a 0.45 μm pore size. The first 3.0 mL and 5.0 mL of the filtrates from each solution were discarded. The Dolutegravir response and the percentage differences between the filters were recorded.
Forced degradation study
A forced degradation study was conducted to ensure that dolutegravir and its impurities remained unaffected by any degradation products that might emerge during stability testing or throughout shelf life. This study helped identify degradation mechanisms (oxidative, acid hydrolysis, or neutral hydrolysis). Blank and standard solutions were prepared as part of the procedure.
Precision
The precision of the analytical method is evaluated by comparing individual test results. This is typically reflected in the relative standard deviation (RSD) or coefficient of variation, which indicates how consistent the results are over multiple tests.
System precision
To assess the performance of the analytical system, a standard solution was injected, and six separate measurements were taken. Retention times and area responses were recorded, and the relative standard deviation was calculated for each. Gradient, blank, and standard solutions were injected to verify system accuracy.
Method precision
For method precision, a single batch of dolutegravir dispersible tablets (10 mg) was tested six times. This helps confirm whether the method produces consistent results across different batches. The percentages of known and unknown degradation products were calculated, along with the total degradation.
Intermediate precision
Intermediate precision examines how external factors, such as changes in equipment, analysts, or environmental conditions, affect the results. By adjusting one variable at a time, the method’s robustness was reassessed, and results were compared to those from the original method precision test. Dolutegravir dispersible tablets were tested six times, and the percentages of specific and nonspecific degradation products were determined.
Linearity
Linearity measures the method’s ability to produce results directly proportional to the concentration of the analyte within a defined range. Dolutegravir and impurity B were linearized from the limit of quantitation (LOQ) up to 200% of the target limit. The regression analysis of slope, intercept, and correlation coefficient was performed for the area response, with concentration plotted on the X-axis and area response on the Y-axis (Table 3).
Limit of detection (LOD) and limit of quantification (LOQ)
The LOD is the lowest concentration of analyte that can be detected but not necessarily quantified, while the LOQ is the lowest concentration that can be quantified with precision and accuracy. These limits were determined using the signal-to-noise ratio method, and the area responses for both dolutegravir and impurity B were analyzed for their LOD and LOQ.
Accuracy
To evaluate the accuracy, the method was tested at three different levels in triplicate, including the LOQ concentration. Samples were prepared and injected at 50%, 100%, 150%, and 200% of the expected concentration, as well as at the LOQ. Recovery percentages for both dolutegravir and impurity B at these levels are presented in Table 4.
Range
The range of an analytical method is defined by the minimum and maximum concentration levels at which the method can accurately and linearly quantify the analyte. The analysis for dolutegravir’s linearity and impurity B’s precision was carried out, with calculations for the relative standard deviation (RSD), accuracy, and precision ranges for both substances.
Robustness
The robustness of a method assesses its reliability under small, controlled variations in testing conditions. This evaluation involved examining the effects of changes in parameters such as flow rate (± 10%), column temperature (± 5 °C), buffer pH (± 0.2 units), and the proportions of methanol and acetonitrile in the mobile phase (± 10%). The impact of these adjustments on the system’s stability was recorded.
Results and Discussion
Method validation
System suitability
To assess the system’s suitability, six injections of the standard solution were made, and the area responses were recorded. The following results were obtained: mean response of 62045.926, relative standard deviation (RSD) of 0.9%, theoretical plates of 18302, and a tailing factor of 1.04.
Data Interpretation: The data indicate that the system suitability criteria were satisfied, confirming that the system is operating within the required specifications.
Specificity
Table 2. Response of placebo, sample, and spiked sample solutions
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Sample Name |
Peak name |
RT (Min) |
RRT (about) |
Peak purity |
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Blank |
Blank |
1.941, 2.208 |
NA |
NA |
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Standard solution |
Dolutegravir |
13.420 |
NA |
999 |
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Placebo solution |
Placebo |
2.459,2.939,3.113,3.946, 4.219,7.439,9.613 |
NA |
NA |
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Sample as such -10 mg |
Impurity B |
8.846 |
0.65 |
998 |
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Dolutegravir |
13.513 |
1.00 |
1000 |
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Impurity D |
12.046 |
0.89 |
992 |
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Unknown |
4.426,5.306,10.953,12.486, 14.873 |
NA |
NA |
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Spiked sample solution-10 mg |
Impurity B |
8.804 |
0.66 |
1000 |
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Dolutegravir |
11.610 |
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1000 |
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Impurity D |
11.937 |
0.89 |
NA |
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Impurity C |
16.204 |
1.21 |
999 |
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Impurity A |
11.937 |
NA |
NA |
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Unknown |
4.424,5.277,5.817,10.884, 14.791 |
NA |
NA |
Data interpretation
From the results, it is evident that no interference occurred from the blank or placebo at the dolutegravir and impurity retention times. The purity of the key peaks satisfied the required criteria, confirming that the method is effective and specific for detecting related substances in dolutegravir dispersible tablets 10 mg.
Filter compatibility study
The area responses for the spiked and placebo solutions were recorded, and the percentage difference was determined for both unfiltered samples and the samples with 3 mL and 5 mL discarded filtrates, utilizing 0.45-micrometer Nylon and 0.45-micrometer PVDF filters.
Data interpretation
The results show that both the 0.45-micrometer Nylon and 0.45-micrometer PVDF filters are compatible for use in filtering the dolutegravir dispersible tablets 10 mg solution, with 5 mL of filtrate being discarded.
Forced degradation study
Table 3. % Assay, peak purity, and % mass balance for forced degradation
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S. No. |
Condition (sample) |
Impurities from the RS method |
% Assay |
Peak purity |
% Mass balance |
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1 |
Sample as such-I |
0.154 |
98.3 |
1000 |
- |
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2 |
Alkali stressed sample_5N NaOH_4 Hours at 80°C |
0.167 |
95.0 |
1000 |
96.7 |
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3 |
Neutral stressed sample_Water_4 Hours at 80°C |
0.470 |
98.9 |
1000 |
100.9 |
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4 |
Thermal stressed sample_105°C_1 Hour |
0.204 |
97.7 |
1000 |
99.4 |
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5 |
Peroxide stressed sample_5%v/v_H2O2_ 4 hours at 80°C |
0.275 |
98.0 |
1000 |
99.8 |
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6 |
Sample as such-II |
0.143 |
99.5 |
1000 |
- |
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7 |
Acid stressed sample_2N HCl_30 minutes at 80°C |
10.485 |
89.8 |
1000 |
100.6 |
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8 |
UV stressed Sample_16 hours at UV cabinet |
0.154 |
97.3 |
1000 |
97.8 |
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9 |
Photostability stressed sample (Visible) LUX stressed sample_1.2 million lux hours |
0.701 |
97.5 |
1000 |
98.6 |
Analysis of Data: The degradation studies, performed under a variety of conditions (acidic, alkaline, peroxide, thermal, neutral, UV, and visible light), showed that dolutegravir was most vulnerable to acid-induced stress, as indicated in Table 3. Despite this, the mass balance was consistently achieved across all conditions. This confirms the method’s stability, making it suitable for assessing related substances in dolutegravir dispersible tablets (10 mg).
Precision
System precision
Six injections of the standard solution were carried out, and the response areas were measured. The retention time and peak area averaged 11.424 and 62035.926, respectively. The relative standard deviations (RSD) for these measurements were 0.2 for retention time and 0.9 for peak area.
Data Evaluation: Based on these results, the method demonstrates consistent performance in terms of retention time and peak area, as evidenced by the low RSD, meeting the system precision criteria.
Method and intermediate precision
The total impurities, including impurity B and the largest unidentified impurity, were evaluated. The calculated mean impurity level was 0.29, with an RSD of 1.5.
Data Evaluation: These findings indicate that the method is stable and reproducible for determining related substances in dolutegravir dispersible tablets (ten mg).
Linearity
Table 4. Linearity of dolutegravir and impurity B
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Linearity level in % |
Concentration in ppm |
Peak area |
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Dolutegravir |
Impurity B |
Dolutegravir |
Impurity B |
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LOQ |
0.0510 |
0.0523 |
3555.489 |
2806.664 |
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10 |
0.1442 |
0.1544 |
8111.842 |
8337.053 |
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25 |
0.2479 |
0.2558 |
13362.503 |
13703.251 |
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50 |
0.4958 |
0.5115 |
29301.548 |
27605.915 |
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80 |
0.7933 |
0.8185 |
46274.471 |
44277.477 |
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90 |
0.8925 |
0.9208 |
52532.673 |
50506.496 |
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100 |
0.9917 |
1.0231 |
58391.380 |
56894.493 |
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120 |
1.1900 |
1.2277 |
70192.337 |
67637.253 |
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150 |
1.4875 |
1.5346 |
93564.320 |
83786.201 |
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200 |
1.9833 |
2.0462 |
116322.756 |
111442.089 |
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Dolutegravir |
Impurity B |
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Correlation coefficient ® |
0.998 |
1.000 |
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Regression coefficient (r2) |
0.997 |
1.000 |
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Slope |
60304.918 |
54718.663 |
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Intercept |
-978.064 |
-77.271 |
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% Intercept |
-1.7 |
-0.1 |
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Figure 3. Linearity plot of dolutegravir and impurity B |
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Interpretation of Data: The relationship between the concentration of dolutegravir and impurity B and their corresponding detector responses is directly proportional across the range from the limit of quantification (LOQ) to 200% of the specified limit. Both the correlation and regression coefficients exceeded 0.995 and 0.990, respectively. The plotted data (Figure 3) of concentration (ppm) against area response under the curve further supports this linearity. Additionally, the percentage intercept at the 100% level remains within the allowable ± 5.0%. These results demonstrate that the detector’s response is linearly related to the concentrations of both dolutegravir and impurity B.
Limit of detection (LOD) and limit of quantification (LOQ)
Interpretation of Data: Based on the linearity analysis, the LOD and LOQ were determined to be 2% (0.02 ppm) and 5% (0.05 ppm), respectively. This confirms that dolutegravir and impurity B are detectable and distinguishable at the LOD level. The signal-to-noise ratios for both the LOD and LOQ levels were consistent with the predefined acceptance criteria (Table 5).
Accuracy
Table 5. Accuracy results
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Level |
Area response |
mg Added (actual) |
mg recovered |
% Recovery |
Mean % recovery |
% RSD |
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Accuracy for dolutegravir standard |
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1 |
LOQ |
3615.189 |
0.0052 |
0.0062 |
119.4 |
121.7 |
1.9 |
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2 |
3330.135 |
0.0052 |
0.0057 |
121.9 |
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3 |
3585.911 |
0.0052 |
0.0061 |
123.9 |
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1 |
50% |
28926.182 |
0.0518 |
0.0513 |
99.0 |
97.9 |
1.6 |
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2 |
28819.656 |
0.0518 |
0.0511 |
98.6 |
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3 |
28091.431 |
0.0518 |
0.0498 |
96.1 |
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1 |
100% |
57965.946 |
0.1035 |
0.1028 |
99.3 |
98.7 |
0.7 |
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2 |
57678.986 |
0.1035 |
0.1023 |
98.8 |
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3 |
57150.173 |
0.1035 |
0.1014 |
98.0 |
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1 |
200% |
116172.218 |
0.2070 |
0.2060 |
99.5 |
99.4 |
0.2 |
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2 |
115749.264 |
0.2070 |
0.2053 |
99.2 |
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3 |
116275.024 |
0.2070 |
0.2062 |
99.6 |
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Accuracy for dolutegravir sample (spiked sample) |
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1 |
LOQ |
13458.061 |
0.0052 |
0.0040 |
92.0 |
92.0 |
1.4 |
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2 |
13587.952 |
0.0052 |
0.0043 |
90.7 |
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3 |
13553.000 |
0.0052 |
0.0042 |
93.3 |
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1 |
50% |
38966.179 |
0.0515 |
0.0506 |
98.3 |
98.1 |
0.2 |
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2 |
38894.463 |
0.0515 |
0.0504 |
98.0 |
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3 |
38932.228 |
0.0515 |
0.0505 |
98.1 |
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1 |
100% |
67419.720 |
0.1029 |
0.1030 |
100.1 |
99.4 |
0.6 |
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2 |
66871.834 |
0.1029 |
0.1020 |
99.1 |
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3 |
66841.316 |
0.1029 |
0.1020 |
99.1 |
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1 |
200% |
122252.274 |
0.2058 |
0.2041 |
99.2 |
99.3 |
0.2 |
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2 |
122600.746 |
0.2058 |
0.2048 |
99.5 |
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3 |
122172.356 |
0.2058 |
0.2040 |
99.1 |
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Data Analysis: Based on the data presented, it is evident that the analytical method adheres to the predefined accuracy standards outlined in the protocol. Therefore, the method is confirmed as accurate for the analysis of related substances in dolutegravir dispersible tablets ten mg.
Range
The range was determined by assessing the RSD, linearity, and accuracy at various concentration levels, including LOQ, 50%, 100%, 150%, and 200%, for both dolutegravir and impurity B.
Data Analysis: The results indicate that the method provides consistent linearity and accuracy from LOQ up to 200% of the defined specification limit for dolutegravir and impurity B.
Robustness
Robustness testing was conducted by deliberately altering conditions such as the flow rate (± 10%), column temperature (± 5 °C), pH (± 0.2 units), methanol concentration (± 10%), and acetonitrile concentration (± 10%). The system suitability metrics, including % RSD, theoretical plates, USP tailing factor, and the relative retention time of impurity B, were evaluated.
Data Analysis: The method demonstrated robustness when subjected to controlled variations in flow rate, column temperature, pH, and the concentration of methanol and acetonitrile in the mobile phase, based on the analysis of system suitability parameters.
Conclusion
The RP-HPLC method for analyzing related substances in dolutegravir dispersible tablets 10 mg has been successfully developed and validated following USP guidelines and specifications. The method demonstrated characteristics of stability, specificity, robustness, precision, linearity, accuracy, and high resolution with a short retention time. Therefore, this method is suitable for its intended application.
Acknowledgments: None
Conflict of Interest: None
Financial Support: None
Ethics Statement: None
