INTRODUCTION:

Lamivudine is a cytosine analogue with potent activity against human immune deficiency and hepatitis B viruses through inhibition of reversed transcriptase activity. Dolutegravir is a novel transfer inhibitor active against human immune deficiency virus¹. The drug is active against HIV-1 and also has some in-vitro activity against HIV-2. Extensive research on multiple drug therapy revealed that a two-drug regimen consisting of lamivudine and dolutegravir controls the HIV disease effectively.

Hence, the committee for medicinal products for human use recommended a fixed dose combination containing 300 mg of lamivudine and 50 mg of dolutegravir for the effective treatment of HIV-1 in adolescent and adult patients with no known or suspected protection from the integrase inhibitors.

Extensive literature search revealed that few analytical methods such as UV methods^2-5 and RP-HPLC methods^6-8 were reported for estimation of lamivudine and dolutegravir individually. Further, there are some RP-HPLC methods available for simultaneous estimation of lamivudine and dolutegravir^9-13 as well in combination with other drugs^14-18. Till date, no rapid stability indicating RP-HPLC method has not been reported in literature for the simultaneous estimation of lamivudine and dolutegravir in bulk and tablet dosage form. There is a need of sensitive and rapid analytical method for quality control of these drugs^19-30. Hence the present investigation was done to reduce the retention time and increase the sensitivity of RP-HPLC method for simultaneous analysis of lamivudine and dolutegravir.

MATERIALS AND METHODS:

Instrumentation:

The Alliance Waters HPLC (2695) with a PDA detector and connected to Empower 2 software. The chromatographic column Std discovery C18 (150 x 4.6 mm, 5 µ) at 30 ⁰C, auto sample injector (10 µL) was used for the analysis of drugs. Analytical balance (Shimadzu AUX 220D), Digital pH meter (Elico, LI-120), Ultra sonicator (Spincotech, RK 106), Hot air oven (bio-techniques, India) were utilized for this work.

Chemicals and Reagents:

The reference standards of lamivudine and dolutegravir were obtained from Hetero Laboratories, Hyderabad, India. Marketed formulation (DOVATO) with label claim 50 mg of dolutegravir and 300 mg of lamivudine per tablet was procured from local pharmacy. All solvents used for mobile phase were of HPLC grade and obtained from Merck, Mumbai, India. Premixed mobile phase was used as diluent throughout the study.

Chromatographic conditions:

Chromatographic separation of analytes was achieved at ambient temperature on Std discovery C18 (150 x 4.6 mm, 5 µ) column with isocratic elution. The mobile phase consisting of acetonitrile: phosphate buffer, pH 5.0 (40:60 % v/v) at flow rate of 1.0 mL/min. Injection volume was 10 µL and detection at 260 nm in PDA detector.

Preparation of standard solution:

In order to prepare binary standard stock solution, lamivudine (75 mg) and dolutegravir (12.5 mg) were weighed, decamped into 100 mL volumetric flask and dissolved in methanol by sonication. Volume was contrived up to the mark with methanol and the flask was shaken well. Further 1 mL of this solution was diluted to 10 mL with diluent so as get binary working standard solution concentration of 75 µg/mL and 12.5 µg/mL for lamivudine and dolutegravir, respectively.

Method validation:

The validation parameters like linearity, precision, accuracy, system suitability, sensitivity and robustness were premeditated in accordance to ICH Q2(R1) guidelines³¹.

System suitability: The efficiency of optimized method was monitored by system suitability test. It was carried out via injection of freshly prepared working standard solution into HPLC under optimized conditions for six times. The chromatographic responses studied were retention time, resolution, theoretical plate number, peak area, and tailing factor of analyte peaks. Linearity: Aliquots of 0.25, 0.5, 0.75, 1.0, 1.25, 1.5 mL were withdrawn from mixed standard stock and diluted to 10 mL with mobile phase such that the final concentration of lamivudine and dolutegravir in the range of 18.75 - 112.5 µg/mL and 3.125 - 18.75 µg/mL was obtained respectively. Calibration curve was constructed by plotting the peak area against the concentration of drug.

Precision: Repeatability of proposed method was verified by analyzing 6 replicate injections of freshly prepared working standard solution of lamivudine (75 µg/mL) and dolutegravir (12.5 µg/mL) in mobile phase on the same day. Intermediate precision was performed by analyzing replicates of same concentration solution prepared in three consecutive days. The peak area of the analytes was determined and %RSD was calculated.

Accuracy: Accuracy of the method was determined by recovery of standard that spiked to target concentration of sample at three levels (50, 100, 150%). The % recovery of drug was calculated by measuring the peak area of chromatogram.

Specificity: The Specificity of method was established by comparison the chromatogram of blank (mobile phase) and placebo solutions with test solution (analytes in mobile phase). The placebo solution comprises all the commonly used excipients for manufacturing of tablet dosage form.

Sensitivity: Limit of detection (LOD) and limit of quantification (LOQ) were premeditated from the regression analysis data of linearity studies (LOD = 3.3 σ/s, LOQ = 10 σ/s; where, σ-standard deviation 󠄎of 󠄎response, s-slope 󠄎of 󠄎calibration 󠄎curve).

Robustness: In order to assess the robustness of the method, deliberate changes in method parameters were made. The variations in the parameters include pH (± 0.2), organic phase (± 2 %) and flow rate (± 0.1 mL/min) of mobile phase. The % RSD of theoretical plate number and retention time of chromatogram obtained for every variation was calculated.

Assay of marketed formulation:

Twenty tablets of marketed formulation (DOVATO), each containing 300 mg of lamivudine and 50 mg of dolutegravir were taken, average weight was determined and crushed in to fine powder. An accurately weighed quantity of powder equivalent to 75 mg of lamivudine and 12.5 mg of dolutegravir was transferred to volumetric flask of 100 mL capacity containing methanol and sonicated for 15 min. The flask was shaken and volume was made up to the mark with methanol. The above solution was filtered through whattmann filter paper (No: 41). From the filtrate 1 mL was transferred into 10 mL volumetric flask and the volume was made up to the mark with mobile phase. The amount of lamivudine and dolutegravir present in sample solution was determined.

Forced degradation studies:

Stress studies were executed on lamivudine and dolutegravir standards under acid, base, oxidative, thermal and UV light conditions as per ICH guidelines³². Acid degradation was carried out with 5 ml of mixed stock solution, 5 ml of 1 N HCl was added and kept at 60 °C for 30 min, then neutralized with 1 N NaOH and diluted with mobile phase. Similarly, 1 N NaOH for base degradation and hydrogen peroxide (20 %) for oxidative conditions were used. Dry heat degradation (80 °C, 24 hrs.) and light degradation (UV Chamber, 200-Watt hours/m2, 24 hrs.) in photo stability chamber were performed on selected drugs in solid state, dissolved in methanol and diluted with mobile phase. These solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.

RESULT:

Method development and optimization

Chromatographic conditions for simultaneous determination of lamivudine and dolutegravir were optimized as mobile phase consists of acetonitrile: phosphate buffer, Ph 5.0 (40:60 % v/v) at flow rate of 1.0 Ml/min gave efficient chromatographic separation of analytes on std discovery C18 (150 x 4.6 mm, 5µ) column with isocratic elution. Injection volume was 10 µL and UV detection at 260 nm. The chromatogram of standard solution of lamivudine and dolutegravir under optimized method conditions was illustrated in Figure 1. Lamivudine and dolutegravir were eluted at 2.37 min and 2.97 min respectively with good resolution (>2.0). Theoretical plate count (>2000) and tailing factor (<2.0) were satisfactory.

Figure 1. HPLC chromatogram under optimized conditions

Method validation:

System suitability:

System suitability criteria may include factors such as plate count, tailing, retention and resolution. The tests were performed by collecting data from five replicate injections of standard drug solution. System suitability parameters for the proposed method were publicized in Table 1 and found that all parameters were within the limits

Linearity:

Linearity of method was determined using six different concentrations of drugs and solutions were injected in a triplicate. The method showed linearity in the range of 18.75 - 112.5 µg/mL for lamivudine and 3.125 - 18.75 µg/mL for dolutegravir. The linear regression equation for lamivudine was Y = 39406 X + 13679, with a correlation coefficient of 0.999, and for dolutegravir it was Y = 38925 X + 6160, with a correlation coefficient of 0.999.

Precision:

The precision of the developed method was assessed in terms of repeatability by analysing 6 replicate injections of mixed standard solution having concentrations of 12.5 μg/ml and 75 μg/ml of dolutegravir and lamivudine respectively. The % RSD values of the results corresponding to the peak area and retention time were calculated. The data obtained for intra-day and inter-day precision studies were given in the Table 1. The intra-day and inter-day chromatograms are similar, in terms of retention times and peak areas. No significant difference (between intra-day and inter-day precision) is observed. The % RSD values for intra-day and inter-day study were less than 2.0, endorsed the good repeatability of proposed method.

Accuracy:

The proposed method when used for extraction and subsequent estimation of dolutegravir and lamivudine from pharmaceutical formulation, after spiking with additional standard drug at three different levels (50%, 100 %, 150 %) to the sample solution containing 6.25 μg/ml and 37.5 μg/ml of dolutegravir and lamivudine respectively. Three chromatograms of each spiked concentrations are recorded for comparison and found that the retention times remained same, but peak areas proportionally changed. The data are compiled and recorded in Table 1. The data indicated good recovery of three drugs, with % RSD less than 2, suggesting accuracy of proposed method.

Specificity:

The chromatograms of blank and placebo indicated the absence of peaks. The excipients used in the formulation did not interfere with drug peaks. However, the formulation and standard chromatograms gave characteristic peaks for dolutegravir and lamivudine. Thus, the specificity of proposed analytical method was ensured.

Sensitivity:

Sensitivity of method was premeditated using LOD and LOQ. These values were calculated from calibration plot. The LOD values of dolutegravir and lamivudine were found to be 0.05 and 0.27 µg/mL. The LOQ values were found to be 0.15 and 0.82 µg/mL, respectively for dolutegravir and lamivudine. These values indicate the adequate sensitivity of proposed method.

Robustness:

Deliberate changes in optimized method parameters such as pH, flow rate and organic phase (%) were made in order to assess the robustness of the method and results were represented in Table 2. The % RSD was less than 2 for all variables, which point towards the robust method.

Table 1: Results of method validation

Parameter	Lamivudine			Dolutegravir
Precision
Repeatability	Peak area	SD	% RSD	Peak area	SD	% RSD
Intra-day	2932951	7062	0.24	467681	2104	0.45
Inter-day	2558614	6728	0.26	432404	2481	0.57
Accuracy
Recovery level	Amount Spiked (µg/ml)	% Recovery	% RSD	Amount Spiked (µg/ml)	% Recovery	% RSD
50%	18.75	99.41	0.56	3.125	100.31	0.71
100%	37.5	100.41	0.54	6.25	100.76	0.70
150%	56.25	100.68	0.55	9.375	100.01	0.41
System suitability
Retention time	2.375			2.968
Peak area	2957684			494162
Tailing factor	1.32			1.27
Theoretical plates	6270			7068
Capacity factor (K’)	1.3			1.9
Selectivity factor (α)	1.5
Resolution	4.3

SD- standard deviation; RSD- Relative standard deviation (n=6)

Table 2: Robustness data of optimized method

Factor	Change condition	Lamivudine (%RSD)		Dolutegravir (%RSD)
Factor	Change condition	Rt	Tp	Rt	Tp
Flow rate (mL/min)	0.9	1.24	0.61	1.42	0.63
	1.0	0.86	0.48	0.91	0.57
	1.1	1.17	0.56	1.34	0.66
Organic phase (%)	28	0.23	0.13	0.41	0.35
	30	0.19	0.13	0.39	0.31
	32	0.36	0.12	0.43	0.37
Buffer pH	4.8	0.91	0.26	0.87	0.33
	5.0	0.72	0.22	0.77	0.28
	5.2	0.86	0.29	0.79	0.35

RSD- Relative standard deviation (n=3)

Application of proposed method:

The proposed method was evaluated by the assay of commercially available tablet dosage form (DOVATO) containing 50 mg of dolutegravir and 300 mg of lamivudine. Sample and standard chromatograms showed identical analytical peaks at their retention times and indicated that selected drugs are clearly separated and showed no interfering peaks, due to excipients (Figure 2). Hence, the proposed method is selective for determination of title analytes. The assay results obtained were compared with the corresponding labeled amounts and the % assay of dolutegravir and lamivudine was found to be 100.36 % and 100.17 %, respectively. The % RSD was less than 2, which indicated the accuracy of the proposed method.

The developed RP-HPLC method was applied to study the behavior of drugs, under various stress conditions such as acidic, alkaline, oxidative, photolytic and thermal. The chromatograms of samples (subjected to various stress conditions) exhibited well-separated peaks of the analytes and degradation products at different retention times. Data of degradation studies was reported in Table 3. It was observed that both drugs were stable under neutral hydrolysis conditions. Significant degradation of both drugs was observed upon exposure to acid, base and oxidative conditions.

Figure 2: Chromatogram of sample solution (DOVATO tablets)

Table 3: Stability study data of lamivudine and dolutegravir

Degradation Condition	% Drug Degraded
Degradation Condition	Lamivudine	Dolutegravir
Acid (1 N HCl, 60 °C)	6.38	6.80
Base (1 N NaOH, 60 °C)	5.18	5.34
Oxidation (20% H₂O_2,)	3.72	3.30
Neutral (H₂O, 60 °C)	1.58	1.97
Photolytic (UV chamber)	1.14	1.17
Thermal (80 °C)	0.68	1.08

DISCUSSION:

In order to achieve HPLC separation of lamivudine and dolutegravir, initial trials were performed with the objective to select adequate and optimum chromatographic conditions. Parameters, such as mobile phase and their proportions, different columns were carefully studied. Choice of retention time, tailing, theoretical plates, and run time were the major tasks while developing the method. In order to get sharp peak and baseline separation of the component, required number of trials were made. To affect the best separation of the title analytes under isocratic conditions, probable combinations of buffers with variable pH values and organic solvents were tested on different columns. All validation parameters include system suitability studies, linearity, accuracy, precision and robustness are obeyed as per ICH Q2 (R1) guidelines.

CONCLUSION:

The developed stability indicating RP-HPLC method was sensitive and specific for simultaneous estimation of dolutegravir and lamivudine. The proposed method was found to be superior to literature methods due to its stability-indicating nature. The proposed method used low organic mobile phase (40 %) and it has rapid analysis time (run time 5.0 min), hence this method reduces the organic solvent consumption. The developed method showed excellent resolution and theoretical plate count. Retention times, selectivity and capacity factors are appropriate. The method is stability-indicating assay and maximum recoveries of drugs are obtained. The method was effectively validated in terms of system suitability, precision, linearity, range, accuracy, LOD, LOQ and robustness and stress indicating studies in agreement with ICH guidelines. Hence the method can be productively used for assay and stability studies of combined market formulation of dolutegravir and lamivudine.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

LIST OF ABBREVIATIONS:

HIV- Human Immunodeficiency Virus

ICH- International Conference on Harmonization

LOD- Limit of Detection

LOQ-Limit of Quantification

PDA- Photo Diode Array

RP-HPLC – Reverse Phase High Performance Liquid Chromatography

Rt – Retention time

RSD- Relative Standard Deviation

Tp-Theoretical plate count

UV – Ultra Violet

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Received on 26.09.2021 Modified on 28.11.2021

Asian J. Pharm. Ana. 2022; 12(2):105-110.

DOI: 10.52711/2231-5675.2022.00019