Development and Validation of RP-UPLC Method for Simultaneous Determination of Lamivudine and Dolutegravir in Combined Dosage Forms

Lamivudine

Lamivudine is a reverse transcriptase inhibitor used to treat HIV and hepatitis B infections. Lamivudine is a synthetic nucleoside analogue and is phosphorylated intracellularly to its active 5'-triphosphate metabolite, lamivudine   triphosphate (L-TP). This nucleoside analogue is incorporated into viral DNA by HIV reverse transcriptase and HBV polymerase, resulting in DNA chain termination.[1-2]

• IUPAC name is 4-amino-1- [(2R,5S)-2 (hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2- dihydropyrimidin-2-one.
• Molecular Formula is C8H11N3O3S.
• Molecular weight is 229.2.

Dolutegravir

Dolutegravir is an antiviral agent used for the treatment of HIV-1 infections in combination with other antiretroviral agents. Dolutegravir is an HIV-1 antiviral agent.[3-4] It inhibits HIV integrase by binding to the active site and blocking the strand transfer step of retroviral DNA integration in the host cell. The strand transfer step is essential in the HIV replication cycle and results in the inhibition of viral activity. Dolutegravir has a mean EC50 value of 0.5 nM (0.21 ng/mL) to 2.1 nM (0.85 ng/mL) in peripheral blood mononuclear cells (PBMCs) and MT-4 cells.

• IUPAC name is N-[(2,4- difluorophenyl)methyl]-11-hydroxy-7-methyl-9,12- dioxo-4-oxa-1,8-diazatricyclo [8.4.0.0^{3,8}] tetradeca-10, 13-diene-13-carboxamide.
• Molecular Formula is C20H19F2N3O5.
• Molecular weight is 419.3.

Figure 1: Structure of Lamivudine.

Figure 2: Structure of Dolutegravir.

A literature survey conveyed that, limited methods are available for simultaneous estimation of Lamivudine and Dolutegravir. Various HPLC, [5,6,7,8,9,10,11,12,13,14] LC/ MS/ MS, [15,16,17,18,19] HPTLC, [20,21] UV, [22,23,24]  and UPLC, [25] assay methods were described within the literature regarding the estimation of lamivudine, abacavir, and a few other anti-retroviral drugs individually as well as in combination with other drugs. In view of the demand for an appropriate, cost-effective RP-UPLC method for routine analysis of Lamivudine and Dolutegravir synchronized evaluation of in pharmaceutical dose type. Attempts were made to establish easy, precise, accurate as well as cost-efficient logical method for the estimate of Lamivudine and Dolutegravir. The recommended approach will be validated according to ICH guidelines. The objective of the recommended work is to establish a brand-new, simple, delicate, exact and economical logical method as well as recognition for the Synchronized evaluation of Lamivudine and Dolutegravir in pharmaceutical dose kind by utilizing RP-UPLC. To verify the established method based on ICH standards for the desired analytical application.

MATERIALS AND METHODS

Chemicals and Reagents: Lamivudine and Dolutegravir pure drugs (API), Combination Lamivudine and Dolutegravir oral tablets (Dovato), Distilled water, Acetonitrile, Phosphate buffer, Methanol, Potassium dehydrogenate ortho phosphate buffer, Ortho-phosphoric acid. All the above chemicals and solvents are from Rankem.

Diluent: Based up on the solubility of the drugs, diluent was selected, Methanol and Water taken in the ratio of 50:50 as diluent.

Preparation of Standard stock solutions: Accurately weighed 75 mg of Lamivudine, 12.5 mg of Dolutegravir and transferred to individual 50 mL volumetric flasks separately. 3/4th of diluents was added to both of these flasks and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution 1 and 2. (1500 µg/mL of Lamivudine and 250 µg/mL of Dolutegravir).

Preparation of Standard working solutions (100% solution): 1mL from each stock solution was pipetted out and taken into a 10mL volumetric flask and made up with diluent. (150 µg/mL Lamivudine of and 25 µg/mL of Dolutegravir).

Preparation of Sample stock solutions: 10 tablets were weighed and was transferred into a 100 mL volumetric flask, 50 mL of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (3000 µg/mL of Lamivudine and 500 µg/mL of Dolutegravir).

Preparation of Sample working solutions (100% solution): 0.5 mL of filtered sample stock solution was transferred to 10 mL volumetric flask and made up with diluent. (150 µg/mL of Lamivudine and 25 µg/mL of Dolutegravir).

Preparation of buffer

0.01N Na2HPO4 Buffer: Accurately weighed 1.41 gm of sodium dihyrogen Ortho phosphate in a 1000 mL of Volumetric flask add about 900 mL of milli-Q water added and degas to sonicate and finally make up the volume with water.

Procedure

Inject the samples by changing the chromatographic conditions and record the chromatograms, note the conditions of proper peak elution for performing validation parameters as per ICH guidelines.

RESULTS AND DISCUSSION

Method

The developed chromatographic method was validated for system suitability, linearity accuracy, precision, ruggedness and robustness as per ICH guidelines.

System suitability parameters: To evaluate system suitability parameters such as retention time, tailing factor and USP theoretical plate count, the mobile phase was allowed to flow through the column at a flow rate of 0.3 ml/min to equilibrate the column at ambient temperature. Chromatographic separation was achieved by injecting a volume of 1 μL of standard into HSS C18 (2.6 x 50 mm, 1.6 µm), the mobile phase of composition 70% 0.01N Na2HPO4: 30% Methanol was allowed to flow through the column at a flow rate of 0.3 ml per minute. Retention time, tailing factor and USP theoretical plate count of the developed method are shown in table 1.

Table 1: System suitability parameters.

Table 2: Assay results for Lamivudine.

Table 3: Assay results for Dolutegravir.

Figure 3: Standard chromatogram.

Figure 4: Sample chromatogram.

Figure 5: Blank chromatogram

Validation of Analytical method

Linearity: The linearity study was performed for the concentration of 0 µg/ml to 225 µg/ml and 0 µg/ml to 37.5 µg/ml level. Each level was injected into chromatographic system. The area of each level was used for calculation of correlation coefficient. Inject each level into the chromatographic system and measure the peak area. Plot a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient. The results are shown in table 4.

Table 4: Results of linearity for Lamivudine& Dolutegravir.

Figure 6: Linearity graph for Lamivudine.

Figure 6: Linearity graph for Dolutegravir.

Accuracy studies: The accuracy was determined by help of recovery study. The recovery method carried out at three level 50%, 100%, 150% and 50%, 100%, 150% Inject  the  standard  solutions  into  chromatographic system. Calculate the Amount found and Amount added for Lamivudine and Dolutegravir and calculate the individual recovery and mean recovery values. The results are shown in table 5,6.

Table 5: Showing accuracy results for Lamivudine.

Table 6: Showing accuracy results for Dolutegravir.

Precision Studies: Precision was calculated from Coefficient of variance for six replicate injections of the standard. The standard solution was injected for six times and measured the area for all six Injections in HPLC. The %RSD for the area of six replicate injections was found. The results are shown in table 7.

Table 7: Precision results for Lamivudine and Dolutegravir.

Ruggedness: To evaluate the intermediate precision of the method, Precision was performed on different day. The standard solution was injected for six times and measured the area for all six injections in UPLC. The %RSD for the area of six replicate injections was found. The results are shown in table 8.

Table 8: Ruggedness results of Lamivudine and Dolutegravir.

Robustness: As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method. The flow rate was varied at 0.2 ml/min to 0.4 ml/min. The results are shown in table 9.

Table 9: Robustness results of Dolutegravir by RP-UPLC.