Validated stability indicating HPLC method to quantify lamivudine and dolutegravir in tablets

Dovato tablets (lamivudine and dolutegravir combination) are full therapy regimen for the Type 1 human immunode iciency virus (HIV-1) infection in adults without history of antiretroviral therapy. The aim of present research focused on development of validated stability indicatingmethod for the quanti ication of lamivudine and dolutegravir in combined dosage form.The separation was achieved on a Cosmicsil C18 column. The mobile phase consisted of 0.1% orthophosphoric acid (pH 3.5) – acetonitrile (50:50, v/v). Photodiode array detector was used to detect the analytes at 258 nm. The method performancewas validated in compliancewith the recommendations of the International Conference on Harmonization.The method was validated with selectivity, limit of detection (0.262μg/ml for lamivudine and 0.238μg/ml for dolutegravir), linearity (150-450 μg/ml for lamivudine and 25-75 μg/ml for dolutegravir), limit of quanti ication (0.874 μg/ml for lamivudine and 0.793 μg/ml for dolutegravir), accuracy (percent recovery was nearer to 100%), precision (percent relative standard deviationwas less than 2.0%) and robustness (system suitability values are within limits). The stability indicating method was performed by under the various stress conditions. Degradants did not interfere with lamivudine and dolutegravir detection. The developed method can suggest that quanti ication of lamivudine and dolutegravir in quality control of analytical laboratories.

Acquired immunode iciency syndrome, human immunode iciency virus, Dovato tablets, Lamivudine, Dolutegravir, Analysis ABSTRACT Dovato tablets (lamivudine and dolutegravir combination) are full therapy regimen for the Type 1 human immunode iciency virus (HIV-1) infection in adults without history of antiretroviral therapy. The aim of present research focused on development of validated stability indicating method for the quanti ication of lamivudine and dolutegravir in combined dosage form.The separation was achieved on a Cosmicsil C18 column. The mobile phase consisted of 0.1% orthophosphoric acid (pH 3.5) -acetonitrile (50:50, v/v). Photodiode array detector was used to detect the analytes at 258 nm. The method performance was validated in compliance with the recommendations of the International Conference on Harmonization.The method was validated with selectivity, limit of detection (0.262 µg/ml for lamivudine and 0.238 µg/ml for dolutegravir), linearity (150-450 µg/ml for lamivudine and 25-75 µg/ml for dolutegravir), limit of quanti ication (0.874 µg/ml for lamivudine and 0.793 µg/ml for dolutegravir), accuracy (percent recovery was nearer to 100%), precision (percent relative standard deviation was less than 2.0%) and robustness (system suitability values are within limits). The stability indicating method was performed by under the various stress conditions. Degradants did not interfere with lamivudine and dolutegravir detection. The developed method can suggest that quanti ication of lamivudine and dolutegravir in quality control of analytical laboratories.

INTRODUCTION
Acquired immunode iciency syndrome is a human immunode iciency virus (HIV)-induced medical condition. Presently, HIV infection is very danger and can readily be called a curse on mankind (Sauter and Kirchhoff, 2019). Shortly after the of icial recognition of HIV patients in the USA, HIV-1 was recognized as the causative organism (Sharp and Hahn, 2011). In Africa, HIV-2 was irst recorded in 1985 and is signi icantly distinct from HIV-1 (Sharp and Hahn, 2010). In developing nations, mortality and morbidity rates associated with HIV infections were high.
In April 2019, the U.S. Food and Drug Administration endorsed Dovato tablets as a full therapy regimen for Type 1 human immunode iciency virus (HIV-1) infection in adults without history of antiretroviral therapy (U.S. Food and Drug Administration., 2019). Davato tablets were labeling with to300 mg of lamivudine (LMD) and 50 mg of dolutegravir (DTG). DTG is an integrase inhibitor group of human immunode iciency viral drug (Min et al., 2010), (Dow and Bartlett, 2014). Integrase inhibitor blocks an enzyme called integrase present in human immunode iciency virus and stop multiplication of human immunode iciency virus (Snedecor et al., 2019).
It operates by lowering blood levels of human immunode iciency virus and hepatitis B (Quercia et al., 2018). LMD does not cure HIV, but the possibility of developing acquired immunode iciency syndrome and human immunode iciency virus related diseases such as severe infections or cancer may be reduced (Budamakuntla et al., 2014).
From the literature reveals that few HPLC methods (Devanna et al., 2017), (Dhanwate et al., 2019) and only one HPTLC (Dhanve et al., 2018) method was developed for the analysis of pharmaceutical formulations containing LMD and DTG. There is no stability indicating HPLC method was found for the quanti ication of LMD and DTG simultaneously. The International Conference on Harmonization guideline says that forced degradation study was designed to recognize the probable degradation products that further assist in determining the molecule's inherent stability and developing pathways for the degradation (Mistry and Mishra, 2018). Based on the importance of stress testing, a stability indicating method using HPLC technique was developed and validated to quantify LMD and DTG simultaneously in bulk and marketed formulation.

Standards and tablet dosage forms of LMD and DTG
Standards of LMD and DTG were obtained from Rainbow Pharma Training Labs, Hyderabad, India. Dovato tablets (ViiV Healthcare, US) labeled with 300 mg of LMD and 50 mg of DTG were bought from the local pharmacy market.

Chemicals and solvents
Analytical grade chemicals and HPLC grade solvents have been used. Hydrochloric acid, peroxide, sodium hydroxide and orthophosphoric acid were purchased from Sd Fine Chemicals Ltd., Mumbai, India. Methanol and water are from Merck India Ltd (Mumbai, India) and Milli-Q ® system (Millipore, USA), respectively.

Instrumentation and conditions
Liquid chromatographic simultaneous assay of LMD and DTG were performed with a Waters alliance liquid chromatography system equipped with an autosampler and Waters photodiode array detector. Analysis of chromatography data was done using the Empower2 version software. Chromatography separations of LMD and DTG were performed in a Cosmicsil C18 column (5 µm, 4.6 × 250 mm) with using 50% of 0.1% phosphoric acid (pH 3.5) and 50% of acetonitrile as components of mobile phase. The low rate was ixed at 1.0 ml/min, the volume of injection was 10 µl, the analytes (LMD and DTG) were monitored at 258 nm, and analysis was completed to 6 min.

Standard solutions of LMD and DTG
LMD (3000 µg/ml) and DTG (500 µg/ml) standard solution was prepared with mobile phase by using 25 ml standard lask. To construct calibration graphs, LMD and DTG calibration solutions in the range of 150-450 µg/ml and 25-75 µg/ml concentration, respectively were individually prepared in a set of 10 ml standard lasks using the stock solution (LMD 3000 µg/ml and DTG 500 µg/ml). To validate the method, 300 µg/ml of LMD and 50 µg/ml of DTG working standards were prepared from the standard stock solution.

LMD and DTG calibration graphs
10 µl of each standard dilution was injected to the HPLC system under speci ied chromatographic conditions. To plot the calibration curves were peak area Vs concentration and then calculate the correlation coef icient.

Assay of LMD and DTG in tablets
Ten tablets were triturate to the ine powder. Appropriately weighed tablet powder was equivalent to 300 mg of LMD and 50 mg of DTG was transferred into a 100 ml volumetric lask, and dissolved in mobile phase, the solution was make up with diluent up to the mark. The solution was iltered via a 0.45 µm size membrane ilter. Further respected dilutions were prepared from the above standard solution. Chromatography development was performed using conditions indicated in the "Instrumentation and Conditions" section. The peak areas of LMD and DTG were calculated and then used to analyze the content of LMD and DTG in the samples using the earlier computed calibration graphs or regression equations.

Stress testing
As indicated by International Conference on Harmonization guideline, stress test includes studying the susceptibility of LMD and DTG (Thota et al., 2018), (Ravichandran et al., 2010) standard solutions were placed under various stress conditions such as acid, base, peroxide, thermal and photolytic conditions.
Stress test was carried out by adding 10 ml of reagent to 10 ml of tablet sample (3000 µg/ml for LMD and 500 µg/ml for DTG). 0.1 N sodium hydroxide, 0.1N hydrochloric acid, 30 % peroxide   and deionized water were employed as reagents for base, acid, oxidative and neutral stress, respectively. At room temperature, the solutions were sonicated for 30 min, followed by diluted to 100 ml of mobile phase.
The thermal and photolytic stress were carried out by subjecting the tablet powder (proportional to 300 mg of LMD and 50 mg of DTG) to 105 o C for 6 hand to sun light for 24 h, respectively. After the stress time, thermal and photolytic stressed samples were transferred to the 100 ml volumetric lask, dissolved in mobile phase and diluted with the same solvent. The solution was iltered via a 0.45 µm size membrane ilter. 300 µg/ml LMD and 50 µg/ml DTG standard concentrations were prepared from the stock solution by using the mobile phase.
10 µl of the stressed sample was injected to the HPLC system. Chromatography development was performed using conditions indicated in the "Instrumentation and Conditions" section. The peak areas of both analytes were calculated and then used to analyze the percent assay and percent degradation of studied analytes.

Development of method
Different reversed phase columns such as Supelco C18, Sunsil C8, Inertsil C18 and Cosmicsil C18 were evaluated. Two solvent combinations such as phosphate buffer: methanol and orthophosphoric acid: acetonitrile of different pH, low rate and proportions were evaluated to optimize the conditions such as tailing factor, theoretical plates, resolution and retention times of LMD and DTG. Finally Cosmicsil C18 reverse phase column with mobile phase consisting of orthophosphoric acid (pH 3.5): acetonitrile (50:50, v/v). 1.0 ml/min low rate was provided the good separation of LMD and DTG. The analytes showed retention times were 1.799 min (LMD) and 3.035 min (DTG). The optimized chromatogram was shown in Figure 1.

Validation
Method validation was performed to the according to International Conference on Harmonization guideline to guarantee that the method is appropriate for intended use (Sharma et al., 2018).

Selectivity
Analysis of mobile phase blank and placebo solution was done to evaluate the selectivity of the proposed method. The chromatograms of mobile phase blank and placebo solution showed no peak (Figure 2). The proposed method is therefore selective for ana-lyzing the studied drug combination.

Linearity
Linearity was determined for LMD and DTG using standard solutions with concentrations 150-450 µg/ml of LMD and 25-75 µg/ml of DTG, injected to the HPLC system to construct calibration curves.

Limit of detection (LD) and quanti ication (LQ)
Based on the calibration curves, LD and LQ were determined as 3.3 SD/m and 10 SD/m respectively. SD is standard deviation of intercept and m is slope of regression line. Lower LD and LQ values (Table 1) con irm the adequate sensitivity of the method developed.

Precision
Precision was inspected by injecting the standard solution of LMD (300 µg/ml) and DTG (50 µg/ml) six times in to the HPLC system on the same day. The mean peak area was 5525181 and 1543574 for LMD and DTG, respectively. The results, percent relative standard deviation of peak areas, were within acceptable conditions (%RSD <2).

Accuracy
For accuracy, the tablet formulation was spiked with known quantities of LMD and DTG at 50%, 100% and 150% concentration levels and evaluated using the proposed method. Percentage recoveries of added LMD and DTG have been evaluated thrice and the indings are given as mean recovery. Recovery values (Table 2) for LMD and DTG ranged from 98.88-99.81% and 99.03-100.28%, respectively. The results con irm the adequate accuracy of the method developed.

Robustness
By changing of few chromatographic conditions, then observe the system suitability parameters such as tailing factor, resolution and theoretical plates were evaluated and veri ied the method. The conditions veri ied are Flow rate (1.0 ± 0.1 ml/min), pH (3.5± 0.1), Ratio of acetonitrile in mobile phase (50±5%), Column temperature (25 ± 2 o C), Wavelength (258 ± 2 nm). The robustness data was shown in (Table 3).

Speci icity / stability indicating nature / degradation study
Different stress conditions were applied such as acid hydrolysis, neutral hydrolysis, base hydrolysis, oxidative degradation, thermal degradation and photolytic degradation. The percent degradation and percent assay of LMD and DTG were depicted in Table 4. In all cases, the method was capable of separating degradation products from the LMD and   (Table 4).

CONCLUSIONS
Stability indicating HPLC method was developed to evaluate LMD and DTG in combined dosage form. The chromatographic separation was t achieved by using the column Cosmicsil C18. The complete analytical run time was 6 minutes. The method suggested showed good recovery in the presence of tablet excipients. The method was also precise, sensitive, selective and robust. Degradation data was suggested to develop HPLC method was speci ic and stability indicating. This method can be employed to quantify LMD and DTG in quality control test for analytical laboratories.