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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 14  |  Issue : 4  |  Page : 209-215

A stability indicating novel analytical method for the determination of lamivudine and dolutegravir in bulk and its tablets using reverse phase high-performance liquid chromatography


1 Department of Pharmaceutical Analysis, Raghavendra Institute of Pharmaceutical Education and Research – Autonomous, Ananthapuramu, Andhra Pradesh, India
2 Department of Chemistry, Jawaharlal Nehru Technological University - OTPRI, Ananthapuramu, Andhra Pradesh, India

Date of Submission01-Apr-2022
Date of Decision04-Nov-2022
Date of Acceptance23-Nov-2022
Date of Web Publication16-Dec-2022

Correspondence Address:
K V Lalitha
Department of Pharmaceutical Analysis, Raghavendra Institute of Pharmaceutical Education and Research – Autonomous, Ananthapuramu, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ajprhc.ajprhc_38_22

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  Abstract 


Aim: A simple and sensitive analytical method was developed to simultaneously estimate lamivudine (LAM) and dolutegravir (DOL), anti-retroviral drug combination in bulk and dosage forms. Materials and Methods: Separation of analytes was done on a BEH Shield RP18 (2.1 mm × 100 mm × 1.7 mm, 5 μm Particle size) using sodium dihydrogen phosphate pH 4.9 adjusted with orthophosphoric acid: Methanol (60:40, %v/v) as mobile phase pumped at 1.0 ml/min. A photodiode array detector was used to find the detection wavelength at an isosbestic point of 292 nm while maintaining the column temperature at 30°C. With a total run period of 8 min, the mobile phase was utilized as a diluent. The International Council on Harmonization guidelines were followed in the method's validation. The method's capacity to indicate stability was confirmed by experiments on forced degradation. Results: LAM and DOL eluted at 2.88 and 3.83 min, respectively. Both the drugs exhibited excellent linearity between 105.00–315.00 and 17.50–52.50 μg/ml for LAM and DOL, respectively. The LOD and LOQ were found to be 4.51 and 15.03 μg/ml for LAM and 5.82 and 19.41 μg/ml for DOL, respectively, which are very minute concentrations. Conclusion: The method was therefore found to be quite sensitive. The proposed high-performance liquid chromatography technique was thereby sensitive, reproducible, accurate, and reliable for the measurement of LAM and DOL.

Keywords: Anti-viral, dolutegravir, lamivudine, methanol, sodium dihydrogen phosphate


How to cite this article:
Lalitha K V, Reddy J R, Nayakanti D. A stability indicating novel analytical method for the determination of lamivudine and dolutegravir in bulk and its tablets using reverse phase high-performance liquid chromatography. Asian J Pharm Res Health Care 2022;14:209-15

How to cite this URL:
Lalitha K V, Reddy J R, Nayakanti D. A stability indicating novel analytical method for the determination of lamivudine and dolutegravir in bulk and its tablets using reverse phase high-performance liquid chromatography. Asian J Pharm Res Health Care [serial online] 2022 [cited 2023 Feb 6];14:209-15. Available from: http://www.ajprhc.com/text.asp?2022/14/4/209/363939




  Introduction Top


Due to advancements in human immunodeficiency virus (HIV) medicine, patients with the HIV are living longer and providing increasingly lucrative lives.[1] To properly treat HIV, multiple medication treatment is used, which entails taking at least three or more medicines alone or in combination on a regular basis.[2] Extensive research on multidrug treatment, however, has indicated that a two-drug combination of lamivudine (LAM) and dolutegravir (DOL) efficiently manages HIV illness.[3] In order to treat HIV-1 successfully in adolescent and adult patients who have no known or suspected HIV protection, the Committee for Medicinal Products for Human Use has approved a fixed-dose combination of 300 mg LAM and 50 mg DOL.[4] Chemically, DOL [Figure 1] is a (4R,12aS)-N-( [2,4-difluorophenyl] methyl)-3, 4, 6, 8, 12, 1 2a-hexahydro-7-hydroxy-4-methyl- 6,8-dioxo-2H-pyrido[1′,2′:4,5]pyrazino [2,1-b] [1,3]oxazine-9-carboxamide, integrase strand transfer inhibitor, which prevents the integration of viral DNA into the genetic material of the host (human immune cells), inhibits HIV replication (T cells) [Figure 1]a.[5] LAM is chemically 4-amino-1-([2R,5S]-2-[hydroxyl methyl]-1, 3-oxathiolan-5-yl)-1, and 2-dihydropyrimidin-2-one.[6] The active form of LAM, LAM triphosphate (3TCTP), works as a competitive inhibitor of the enzyme reverse transcriptase, preventing DNA replication. [Figure 1] shows the chemical structures of DOL and LAM.[7]
Figure 1: Chemical structures of LAM and DOL. LAM: Lamivudine, DOL: Dolutegravir

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In the pharmaceutical industry or research, an effective analytical method is required so that a drug may be examined separately or in combination with other pharmaceuticals. A thorough review of the literature found that only a few analytical procedures, such as ultraviolet (UV) and reverse-phase high-performance liquid chromatography (RP-HPLC), have been described for estimating LAM and DOL separately.[8],[9],[10],[11],[12] The estimation of LAM, DOL, tenofovir disoproxil fumarate, butaver sulfate, or abacavir in triple combination is also possible using specific RP-HPLC methods.[13],[14],[15],[16],[17] In April 2019, the food and drug administration (FDA) approved LAM and DOL fixed-dose film-coated tablets for the treatment of HIV-1, according to official FDA announcement. To far, there has been no evidence in the literature that the RP-HPLC technique for estimating bulk and tablet LAM and DOL is stable. As a result, we have started working on developing an effective, sensitive, and cost-effective RP-HPLC methodology for determining percentage purity and assessing the stability of DOL and LAM in both bulk and tablet dosage forms. The established method's validation was carried out in accordance with the Q2 standard of the International Council on Harmonization (ICH) recommendations.[18],[19]


  Materials and Methods Top


Chromatographic conditions

Both drug standards were given by MSN laboratories in Hyderabad, India. Water, acetonitrile, and methanol were furnished by Sigma-Aldrich (LC grade). We bought a 0.22 mm membrane filter, sodium hydroxide (NaOH), hydrogen peroxide, and hydrochloric acid (HCl) of analytical grade from SD Fine Chem in Mumbai, India. Purchased from the neighborhood pharmacy market, Dovato (ViiV healthcare) contains LAM and DOL with label indications of 300 mg and 50 mg, respectively. The grade of all the chemicals utilized was analytical or LC. An BEH Shield RP18 (2.1 mm × 100 mm × 1.7 mm, 5 μm Particle size) with a photodiode array (PDA) detector and EZ Chrome Elite software were used to construct the approach. Sodium dihydrogen phosphate, pH 4.9, orthophosphoric acid, methanol (60:40,%v/v), and 1.0 ml/min pumping of a mobile phase were used to separate the two analytes. A PDA detector was used to find the detection wavelength at an isosbestic point of 292 nm while maintaining the column temperature at 30°C. With a total run length of 8 min, the mobile phase was employed as a diluent.

Preparation of phosphate buffer

Mix together 11.45 g of dipotassium hydrogen phosphate and 28.80 g of sodium dihydrogen phosphate to make 1000 ml of solution. Dilute orthophosphoric acid was used to get the buffer's pH to 4.9.

Preparation of mobile phase

400 mL of Methanol (40%) was transferred to a 1000 mL volumetric flask and added 600 mL of Phosphate buffer pH 4.9 (60%) to it, mixed thoroughly, and kept for sonication in an ultrasonic water bath for 10 min. Under vacuum filtration, the fluid was filtered through a 0.45 filter. As a diluent, the mobile phase was employed.

Preparation of working standard solutions of lamivudine

The final concentration of 450.0 μg/mL was obtained by taking 4.5 mL from the above solutions and diluting it to 10 mL with the mobile phase. To achieve this, 10.0 mg of LAM that had been accurately weighed was transferred to a 10 mL volumetric flask, dissolved in, and diluted to the mark with the diluent.

Preparation of dolutegravir standard working solutions

Ten milligram of DOL, accurately weighed, was added to a 10 mL volumetric flask, dissolved therein, and diluted to the proper volume with the diluent. From the aforementioned solutions, 0.75 mL was taken out and diluted to the proper volume with mobile phase, resulting in a final concentration of 75.0 μg/mL.

Preparation of Sample for assay determination

An amount equal to 10 mg of DOL was weighed and transferred to a 10 mL clean and dry volumetric flask after 20 LAM and DOL tablets were crushed to a powder. Then, 7 ml of diluent was added, and it was thoroughly dissolved using a sonicator. Finally, using the same solvent, the volume was brought up to par (primary formulation stock solution). A standard 10 ml flask was then filled with 0.75 mL of the excess stock solution, which was then diluted to the proper concentration using the same solvent. 75.0 μg/mL was the desired end concentration. Measured the peak regions for LAM and DOL after injecting 10 L into the HPLC system and determined the formulation's percentage assay.

The chromatographic method's validation

The created technique was approved in accordance with the ICH Guidelines, Q2 (R1), 2005.[18] Precision, accuracy, linearity, detection and quantification limitations, robustness, and forced degradation experiments were all used to verify the approach.

System suitability

To confirm system performance, parameters for system appropriateness were tested. Six further injections of the same standard preparation were used to gauge the system's accuracy. Measurements were made of all crucial factors, such as peak area, retention durations, tailing factor, peak resolution, and theoretical plate number.

Specificity

The capacity to assess whether the target analyte can be measured accurately and specifically in the presence of additional components that may be anticipated to be present in the sample is known as specificity. To assess the method's specificity, chromatograms of standard solutions, commercially available samples, and placebos were compared. The same excipients used in commercial formulations were employed to make the placebo.

Precision

Repeatability

The capacity of the suggested technique to repeat measurements over a brief time under the same analytical circumstances is referred to as repeatability. Six measurements were taken using nominal working concentrations of 450.00 μg/mL for LAM and 75.00 μg/mL for DOL to demonstrate repeatability for the current approach.

Intermediate precision

The intermediate accuracy of the suggested technology allows reproducing measurements on various days in a laboratory. On 3 consecutive days, samples were injected at their nominal working concentrations of 450.00 μg/mL for LAM and 75.00 μg/mL for DOL. For the mean peak area and the standard deviation of LAM and DOL, precision was reported as a percentage of relative standard deviation (%RSD).

Accuracy

The proposed assay procedure's accuracy is calculated and represented as a percentage of the standard analyte recovered from the sample matrix. It is customary to add a standard with a known quantity to the sample solution. Each level of analyte concentration was examined in triplicate at 50%, 100%, and 150% of the nominal concentration. The standard solutions that were put to the placebo were examined using the suggested procedure.

Linearity

If the concentration of the analyte present in the sample falls within a certain range, an analytical process can produce findings that are directly proportional. As a result, five concentrations ranging from 225.00–675.00 μg/mL for LAM to 37.50–112.50 μg/mL for DOL were used to test linearity. Each solution was filtered through a 0.45 m Millipore filter before being added in triplicate to the chromatographic apparatus. The analytical curve was assessed using a concentration versus mean area plot. The equation was created using a least-squares regression technique.

Detection and quantification limits

The slope (S) of the analytical curve based on DL = 3.3 (σ/S) and QL = 10 (σ/S) and the residual standard deviation of the regression line (σ) have been used to compute the detection and quantitative limitations for the analytical technique.

Robustness

The ability of a methodology to be unaffected by minute changes in the parameters is known as method resilience (ICH 2005). According to this study, flow rate (1.0 ± 0.2 mL/min) and change in organic content of mobile phase (±5%) were the three chromatographic parameters utilized to evaluate resilience. Three independent tests were done on the resolution between peaks parameter at fixed levels both above (+) and below (−) the nominal value.

Forced degradation studies

Stress testing must be done to explain the inherent stability properties of the active ingredient, according to the ICH Guidelines, Q1A R2, 2005 guideline,[19] which is titled stability testing of novel pharmacological substances and products. This project's objective was to use the suggested methodology to conduct stress degradation experiments on the LAM and DOL.

Acidic and alkaline hydrolysis

Transfer 0.75 mL to two pairs of 10 mL standard flasks from the primary stock solution. To one set of two 10 mL standard flasks for acidic conditions, 3 mL of 1 N HCl was added to the aforementioned solution. In a second set of 10 mL standard flasks, 3 mL of 1 N NaOH was added for alkaline degradation. The standard flask was then placed in a water bath and heated to 75°C for 4 h for acid samples and 70°C for alkaline samples, respectively. For LAM and DOL, respectively, the two sets of solutions were neutralized and diluted to a final volume of 10 mL. The resultant mixture should be cooled to room temperature. Use a 0.22 mm syringe to filter the solution before adding it to the HPLC system's vials.

Oxidative degradation

Into a 10 mL standard, pour 0.75 mL of the primary stock solution. The volume was brought up to the required level using diluents and 3 mL of 6% (w/v) hydrogen peroxide added to provide 450.00 μg/ml and 75.00 μg/ml for LAM and DOL, respectively. For 5 h, the standard flask was heated to 70°C. After filtering the outcome through a 0.22 mm syringe filter, the solution was chilled and added to the vials of the HPLC system.

Thermally induced degradation

In order to get 450.00 μg/ml and 75.00 μg/ml for LAM and DOL, respectively, 0.75 mL of main stock solution was transferred to a 10 mL standard flask, and the volume was built up to the required level using diluent. The final product was refluxed at 85°C for 4 h. The mixture was subsequently cooled to room temperature. After filtering with a 0.22 mm syringe filter, add to the vials of the HPLC system.

Photodegradation

Pipette 0.75 mL of the stock solution into a 10 mL standard flask, and then add diluent to the desired volume to get the desired concentrations of 450.00 μg/ml and 75.00 μg/ml for LAM and DOL, respectively. The samples were then put onto a Petri dish and left in a photostability chamber for 24 h at 1.2 million Lux hours of UV light and 200 Wh/m2 of UV light. Bring the finished product to room temperature. Use a 0.22 mm syringe to filter the solution before adding it to the HPLC system's vials.


  Results and Discussion Top


Method validation

According to ICH, method validation comprises putting an analytical technique through its paces to make sure it is precise, specific, and repeatable throughout the spectrum of concentrations and under the specified analytical circumstances. The created technique was approved in accordance with the ICH Guidelines, Q2 (R1), 2005.[18],[19]

System suitability study

By examining several parameters, system appropriateness was shown to be within the ICH limit. [Table 1] presents the findings.
Table 1: System suitability results

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Specificity

There was no interference during the examination of LAM and DOL, proving the method's specificity. There is no excipient interference with this approach. As a result, the LAM and DOL peaks do not intersect. LAM and DOL peaks were adequately separated, as shown in [Figure 2]a to c despite the presence of peaks for excipients, which are substances used in pharmaceutical formulations.
Figure 2: Representative chromatograms of LAM and DOL (a) Blank, (b) Standard, (c) Sample. LAM: Lamivudine, DOL: Dolutegravir

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Accuracy

The accuracy was assessed using the recovery study, and the findings are shown in [Table 2]. LAM standard recovery was 99.38%, while DOL normal recovery was 99.76%. The procedure appears to be accurate based on the findings that were achieved.
Table 2: Results of accuracy (n=3)

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Precision

The accuracy of the analysis sheds light on procedure random error. Estimates were made on the reproducibility and intermediate accuracy of LAM and DOL. The percentage RSD for LAM and DOL was 0.25 and 0.38%, respectively (reproducibility precision). Reproducibility and intermediate precision both depend critically on %RSD being <2.0%. We were able to determine the %RSD intermediate precision by analyzing sample solutions of dosage forms over 3 days in a row with six replicates each. For LAM and DOL, the coefficients of variation were 0.75% and 0.37%, respectively. This shows that the approach that was created has excellent accuracy. The findings from the study of commercially available samples are displayed in [Table 3].
Table 3: Precision results

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Linearity

Five concentrations at the level of 50%–150% (225.00, 337.50, 450.00, 562.50, and 675.00 μg/ml of LAM and 37.50, 56.25, 75.00, 93.75, and 112.50 μg/ml of DOL) were used to build the linearity. The correlation coefficient was determined to be more than 0.999 for both analytes, and the peak areas of the analytes were found to be linear in the concentration range under study. [Figure 3]a and [Figure 3]b displays the data and curve for linearity.
Figure 3: Linearity curves of (a) LAM (b) DOL. LAM: Lamivudine, DOL: Dolutegravir

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Detection and quantification limits

The standard deviation of the response and the slope of the calibration curves were used to calculate LOD and LOQ. The LOD and LOQ, which are very low values, were determined to be 4.51 and 15.03 μg/ml for LAM and 5.82 and 19.41 μg/ml for DOL, respectively.

Robustness

By analyzing the test solutions following small but intentional changes in the circumstances, such as flow rate (±0.1 ml/min) and the change in mobile composition (±5%), the resilience of the analytical approach was assessed. Throughout several iterations of the analytical circumstances, the data on system appropriateness were determined to be good. Results for system appropriateness were unaltered even when the analytical conditions were slightly altered. [Table 4] displays the robustness of LAM and DOL results.
Table 4: Robustness results

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Assay

When the suggested approach was used on tablets of LAM and DOL, the mean% assay result was 99.76% and 99.72%, respectively. The outcomes are shown in [Table 5].
Table 5: Results of assay

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Forced degradation tests were conducted to evaluate the stability-indicating capability of the proposed analytical technique. These investigations involved exposing the sample solution to various stress conditions, such as acid, base, peroxide, UV, and heat. For stress samples, assay investigations were performed against a reference standard. The created method's capacity to indicate stability is confirmed by the suggested analytical method's ability to identify the analyte even in the presence of degraded products. [Table 6] presented the findings of the stress investigations, and [Figure 4] displays the chromatograms of the LAM and DOL stress studies.
Table 6: Results of degradation studies

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Figure 4: Degradation chromatograms. LAM: Lamivudine

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  Conclusion Top


For the simultaneous measurement of LAM and DOL in tablet formulations, a straightforward, accurate, and precise RP-HPLC analytical approach was created and validated. This method's low LOD and LOQ make it possible to detect and measure this contaminant in very low quantities. Due to the fact that both peaks are clearly separated from one another and the peaks of the excipients, the approach is highly straightforward and precise, making it particularly ideal for regular quality control analytical work.

Acknowledgments

The authors are thankful to the management of Raghavendra Institute of Pharmaceutical Education and Research–Autonomous Ananthapuramu, Andhra Pradesh, India, for providing the necessary facilities to carry out this research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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