Development and Validation of Stability-indicating assay UHPLC Method for Simultaneous analysis of Dolutegravir, Lamivudine and Tenofovir disoproxil fumarate in Bulk and Pharmaceutical Formulation

Balaji Thakare¹*, Abhilasha Mittal¹, Manoj Charde², Rahul Umbarkar², Nitin Kohle¹,

Piyush Chandra¹, Manoj Kadam¹

¹Department of Pharmaceutical Chemistry, NIMS Institute of Pharmacy, NIMS University,

Jaipur, Rajasthan 303121.

²Government College of Pharmacy, Vidhya Nagar, Karad, Dist. Satara, Karad 415603 M.S.

*Corresponding Author E-mail: balaji.thakare2@gmail.com

ABSTRACT:

A stability indicating UHPLC method has been developed to analysis of Dolutegravir (DLT), Lamivudine (LVD), and Tenofovir Disoproxil Fumarate (TDF). The separation was achieved by using UPLC BEH C18 (150mm2.1mm) with 1.7 µm particle size column at ambient temperature using solvent system in a proportion of (40:60% v/v) acetonitrile:water; pH 6.5 was adjusted with 0.1% OPA. The solvent system was filtered prior to the start of the chromatographic analysis through a 0.2μm membrane (Ultipor N₆₆ Nylon 6, 6) and sonication of it for 20min. A 10μL of fixed volume (working solution) was injected and the chromatogram was studied at a detection wavelength of 262 nm. The proposed method was validated in terms of Linearity, Accuracy, Precision, Ruggedness, Robustness and stability studies. The chromatographic analysis time was approximately less than 3 minutes with complete resolutions of DLT (Rt = 1.35min), LVD (Rt = 0.69min) and TDF (Rt = 2.36min). The method exhibited good linearity range, 3-18 μg/mL, 10-60 μg/mL and 10-60 μg/mL of DLT, LVD and TDF respectively. The % amount of DLT, LVD and TDF in marketed formulation were recorded to be 99.59 ± 0.44, 99.81 ± 0.48 and 99.47 ± 0.59 respectively. The force degradation studies were performed as per ICH guidelines under the acidic, alkali oxidative and neutral conditions for different times. Therefore the developed UHPLC method can be applied for routine qualitative and quantitative analysis of DLT, LVD and TDF in bulk and pharmaceutical formulation and validated as per theICH guidelines and could be employed for the stability studies on pharmaceutical preparation within pharmaceutical industry.

KEYWORDS: UHPLC, Dolutegravir, Lamivudine, Tenofovir, Force degradation.

1. INTRODUCTION:

Dolutegravir is a HIV-1 intergrase inhibitor that blocks the strand transfer step of the integration of the viral genome into the host cell. Chemically it is (3S,7R)-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. Methods reported in literature are HPLC,^1,2,3 HPTLC,^4,5 LC-MS/MS^6,7 and UV/Vis-Spectrophotometry.^8,9

A reverse transcriptase inhibitor and zalcitabine analog in which a sulfur atom replaces the 3' carbon of the pentose ring. It is used to treat Human Immunodeficiency Virus Type 1 (HIV-1) and hepatitis B (HBV). Lamivudine is a synthetic nucleoside analogue and is phosphorylated intracellularly to its active 5'-triphosphate metabolite, lamivudine triphosphate (L-TP). Chemically Lamivudine is 4-amino-1-[(2R, 5S)-2- (hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one. Methods reported in literature are HPLC,^10,11 HPTLC,^12,13 LC-MS/MS,^14,15 and UV/Vis-Spectrophotometry.¹⁶ Tenofovir disoproxil fumarate (a prodrug of tenofovir), marketed by Gilead Sciences under the trade name Viread, belongs to a class of antiretroviral drugs known as nucleotide analogue reverse transcriptase inhibitors (nRTIs). This drug is prescribed in combination with other drugs for the management of HIV infection as well as for Hepatitis B therapy. Chemically Tenofovir disoproxil fumarate bis({[(propan-2-yloxy)carbonyl]oxy}methyl){[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methanephosphonate. Methods reported in literature are HPLC,^17,18,19,20 HPTLC²¹, LC-MS/MS²² and UV/Vis-Spectrophotometry.^23,24

The development of pharmaceutical matrices using a combination of drugs to obtain a better clinical impact is gaining popularity to address a non - tested therapeutic gap. Antiviral therapeutic agents can help minimize Human Immunodeficiency Virus (HIV)-related morbidity, increase survival, and minimize HIV transmission. Today’s HIV therapy currently consists of three-drug, while HIV pre-exposure prophylaxis currently consists of two-drug regimens; both require once-daily dosing and long-term reduction of viral infection.²⁵For quantification of cited HIV therapeutic agents, many literature findings have been documented for analysis and quantification of EMT and TDF in the various pharmaceutical and biological matrices, including; LC-MS/MS,^26-27 HPLC,²⁸ HPTLC, spectrophotometry.²⁹ To the best of our knowledge, no stability-indicating UHPLC method has been reported for the simultaneous determination of DLT, LVD and TDF in FDC matrices so far with the application of the UHPLC system. Thus, the main objective of this study was to develop a rapid, sensitive, and stability-indicating UHPLC method to estimate and assess the DLT, LVD and TDF in pharmaceutical matrices.

2. MATERIAL AND METHOD:

DLT, LVD, and TDF solubility were examined in various solvents; methanol was considered the critical solvent for selected analytes. The wavelength of 262nm was found to have the highest absorption peak intensities in the overlain PDA spectrum of DLT, LVD, and TDF. Therefore, this wavelength was used for the measurement. To achieve the goals of developing a stability-indicating assay process, reversed-phase UHPLC was selected. UPLC BEH C₁₈ column (150 mm 2.1mm, i.d., 1.7µm particle size) was selected. The solvent system in a proportion of (40:60% v/v) acetonitrile and water; pH 6.5 was adjusted with 0.1% Orthophosphoric acid (OPA) was preferably selected for analysis. The concentrations of DLT, LVD, and TDF in the working solution were 5μg/mL, 30μg/mL, and 30 μg/mL, respectively.

3. RESULTS AND DISCUSSION:

3.1 Optimized chromatographic condition:

Consequently, an excellent resolution was noticed in the case of a solvent system in a proportion of (40:60% v/v) acetonitrile and water, as well as extensive tailing of analytes was recorded. Thus, to minimize the extensive tailing of analytes, pH of the aqueous to 6.5 with 0.1% Orthophosphoric acid. Furthermore, the samples were prepared using the solvent system to lessen the impact of the solvent system. Eventually, the solvent system comprises a proportion of (40:60% v/v) acetonitrile and water, pH 6.5±0.02 adjusted with 0.1% OPA) was exhibited remarkable symmetrical peaks shape with excellent resolution of eluents, which was also sufficient due to adequate system suitability tests. The total analysis time for quantification of DLT, LVD, and TDF was below 6 min. The Rt of DLT, LVD, and TDF were 1.356±0.013 min, 0.698±0.022 min, and 2.361±0.018 min. The optimized chromatograms of DLT, LVD, and TDF are portrayed in Figure 1.

Figure 1: Optimized chromatogram of DLT, LVD, TDF

3.2 System suitability test

System suitability indicators are most commonly used to ensure that the developed analytical method is suitable for the based courses on the day the experiment was performed and determine the quality assurance chromatographic system. The parameters of system suitability were reviewed using the concentrations 5 μg/mL, 30 μg/mL, and 30 μg/mL concentrations of DLT, LVD, and TDF (six determinations). In addition, SD and RSD % were estimated for peak area and Rt (responses). The RSD % values of responses were all within 2% of one another, indicating that the method was well-designed. In addition, the tailing factor and the number of USP plates were both found to be within acceptable limits. The system suitability test is described in Table 1.

3.3 Calibration curve:

The calibration curve for DLT, LVD, and TDF was assessed using the six working solutions. First, the same was prepared using the specific aliquots from standard stock solutions were accurately moved into the 10 mL series of a calibrated flask. Then, the volume was diluted to the mark of a calibrated flask with a solvent system to get the 3 – 18μg/mL, 10 – 60μg/mL, and 10 – 60μg/mL concentrations of DLT, LVD, and TDF, correspondingly. Next, a constant proportion of 10μL solution (for each determination) was introduced into the UHPLC system using a 100μL Hamilton Syringe (Muttenz, Switzerland), which was done repeatedly (five) for each determination. Finally, the calibration curves of peak area despite the μg/mL concentrations for DLT, LVD, and TDF were plotted and investigated, employing the equation of linear regression to develop a relationship as a calibration curve. The determination coefficient (r² 0.9968, 0.9918, and 0.9999) of the calibration curve obtained from the line indicates the excellent connection between the peak areas and the DLT, LVD, and TDF concentrations. The calibration curves are portrayed in Figure 2a,Figure 2b and Figure 2c.

Figure 2a: Calibration curve for DLT

Figure 2b: Calibration curve for LVD

Figure 2c: Calibration curve for TDF

3.4 Analysis of marketed formulation and in bulk samples:

The DLT, LVD, and TDF assay in the marketed pharmaceutical formulation was performed for Viropil^TM DLT 50 mg/ LVD 300 mg/ TDF 300 mg tablet matrix. Average weight of one tablet was precisely solubilized into 50 mL of methanol and was diluted with methanol to get the final concentrations of 6 μg/mL, 20 μg/mL, and 20 μg/mL of DLT, LVD, and TDF investigated conferring to the procedure of chromatographic conditions; The peak area was estimated for the selected peak, and the outcomes are described in Table 1.DLT, LVD, and TDF (5 mg, 30 mg, and 30 mg), precise quantities were accurately weighed and transferred in distinct 100 mL of the calibrated flasks; solubilized in methanol, and the volume was diluted to the mark of a calibrated flasks with same to obtain DLT, LVD, and TDF concentrations of 50 μg/mL, 300 μg/mL, and 300 μg/mL, respectively. Finally, the suitable volumes of this were diluted with a solvent system to get the final concentrations of 6 μg/mL, 20 μg/mL, and 20 μg/mL of DLT, LVD, and TDF that was investigated conferring to the procedure of chromatographic conditions; the peak areas of analytes were assessed, and the outcomes are described in Table 2.

Table 2: Analysis of DLT, LVD, and TDF in bulk material

Drugs	Amount taken [μg/mL]	Amount found [μg/mL] ± SD	% Amount found	% RSD [n=6]
DLT	6	6.02 ± 0.04	100.34 ± 0.78	0.77
LVD	20	20.09 ± 0.12	100.47 ± 0.60	0.59
TDF	20	20.09 ± 0.10	100.45 ± 0.52	0.52

n= number of determinations

3.5 Validation:

The ICH guideline Q2(R1) was used to evaluate the design UHPLC method for DLT, LVD, and TDF for accuracy, precision (intra- and inter-day, and repeatability), sensitivity (LOD and LOQ), robustness, specificity, and selectivity.

3.5.1 Accuracy

The accuracy of the design UHPLC method for DLT, LVD, and TDF was investigated in the context of % recovery and addressed at three distinct levels, i.e., 80%, 100%, and 120%. The % recovery was appraised by adding a known amount of DLT, LVD, and TDF standard to pre-analyzed tablet solution (DLT- 6µg/mL, LVD- 20µg/mL, and TDF- 20µg/mL). The resulting solution was eventually assessed using the designed method.

The % recovery of the designed method was investigated with the aid of the formula; Recovery (%) = A-B/C100; where, A-total concentration of DLT, LVD, and TDF; B- initial concentration of DLT, LVD, and TDF and C- concentration added of DLT, LVD, and TDF. The outcomes of the % recovery of the designed method are specified in Table 3.

Table 3: Assessment of the accuracy for the quantification of DLT, LVD and TDF using the proposed UHPLC method by ICH guideline

Initial amount added μg/mL]	Addition of standard [%]	Average % recovery ±SD^a	Grand average ± SD^b	% RSD^c
DLT
6	80	99.99 ± 0.21	99.87 ± 0.82	0.82
6	100	100.08 ± 1.28
6	120	99.53 ± 1.04
LVD
20	80	99.63 ± 0.41	100.23 ± 0.77	0.77
20	100	100.31 ± 1.30
20	120	100.75 ± 0.62
TDF
20	80	99.71 ± 1.02	100.36 ± 0.57	0.57
20	100	100.78 ± 0.61
20	120	100.59 ± 0.10

^aAverage %recovery of the three different solutions at each level concentration for each analyte.

^bAverage of % recovery of the three different levels concentration.

^cAverage of % standard deviation of all recoveries for three different concentration levels.

3.5.2 Precision:

The precision measurement of the designed UHPLC method for DLT, LVD, and TDF was examined for intra- and inter-day and repeatability precision variability. The data were analyzed as a %RSD. The three distinct concentrations 6, 9, and 12μg/mL of DLT and 20, 30, and 40μg/mL of LVD and TDF were assessed using an assay at various time frames on different days for intra-day precision, and as a result of significant study for three days in a row, as per ICH guidelines. Additionally, repeatability precision variability was assessed using six determinations of 9 μg/mL of DLT and 20μg/mL of LVD and TDF concentrations. The findings of intra- and inter-day precisions variability and outcomes of repeatability precision variability are presented in Table 4.

3.5.3 Sensitivity:

The LOD and LOQ were used to measure the sensitivity of the design UHPLC method. The LOD and LOQ were determined using the standard deviation (N) of the DLT, LVD, and TDF (n=3) outcomes, as well as the slope of the calibration curve (B). LOD = 3.3 N/B and LOQ = 10 N/B were the formulas used. Serial working dilutions of 3 - 7μg/mL of DLT and 10 – 30μg/mL of LVD and TDF ranges have been acknowledging and assessed. The designed method recorded LOD and LOQ values of 0.15 μg/mL and 0.46μg/mL for DLT, 0.48μg/mL and 1.45 μg/mL for LVD and 0.45μg/mL and 1.37μg/mL for TDF, correspondingly. As a result, the design UHPLC method was found to be extremely sensitive to the solvent system.

3.5.4 Robustness:

The robustness of the design UHPLC method was determined by attempting to make substantial changes in wavelength detection, column oven compartment temperature, and flow rate. For the peak areas of DLT, LVD, and TDF, each independent variable's influence was calculated. The selected independent variables for this analysis were varied as detection of wavelength (260 – 264 nm), the temperature of column oven compartment (25 – 35 ℃), and flow rate (0.3 – 0.5 mL/min). It was noticed that such independent variables did not affect the DLT, LVD, and TDF experiments. Therefore, investigation of robustness determination has been addressed with 15 μg/mL of DLT and 50 μg/mL of LVD and TDF concentrations. The data of robustness analysis are presented in Table 5.

Table 4: Determination of the precision assay for the quantification of DLT, LVD and TDF

Standard concentration level [μg/mL]						Intra-day						Inter-day
Standard concentration level [μg/mL]			% Amount found [n=3]				% RSD			% Amount found [n=3]			% RSD
DLT	LVD	TDF	DLT	LVD	TDF	DLT	LVD	TDF	DLT	LVD	TDF	DLT	LVD	TDF
6	20	20	100.06	100.18	99.78	0.67	0.43	0.98	100.28	100.21	99.12	0.34	0.47	0.95
9	30	30	99.23	99.61	99.58	0.19	0.57	1.32	99.32	99.57	99.62	0.45	1.04	0.55
12	40	40	99.37	99.60	99.38	1.06	0.57	0.78	98.85	99.26	99.47	0.39	0.83	0.33
					Repeatability assay variability
DLT	LVD		TDF	% Amount found [n=6]					% RSD	% Amount found [n=6]			% RSD
9	30		30	99.47 ± 0.64					0.64			99.50 ± 0.68		0.68

* n= number of determinations, %RSD= percent relative standard deviation

Table 5: Evaluation of robustness experiment for determination of DLT, LVD and TDF

Parameters		Mean Peak Area ± SD				% RSD
Parameters		DLT	LVD	TDF	DLT	LVD	TDF
Flow rate [mL/min]	0.3 - 0.5	1475425.77 ± 5591.54	781559.57 ± 8685.96	615182.49 ± 5764.65	0.37	1.10	0.93
Detection of wavelength [nm]	260 - 264	1480067.17 ± 2109.95	794616.23 ± 7782.87	614910.76 ± 4189.86	0.13	0.97	0.67
Column temp. (℃))	25 - 35	1471427.78 ± 4034.92	786143.01 ± 7115.37	609586.93 ± 5326.07	0.27	0.90	0.87

* n= number of determinations, SD= standard deviation, and %RSD= percent relative standard deviation

Table 6: Summary of data on DLT, LVD and TDF degradation under different stress conditions

Degradation conditions	Number of impurities			Rt of impurities [min]			% Degradation
	DLT	LVD	TDF	DLT	LVD	TDF	DLT	LVD	TDF
Acidic hydrolysis-1 M HCl reflux for 80°C for 12 hr	02	01	02	4.292, 4.955	0.438	2.202, 3.068	9.87	8.85	17.21
Alkaline hydrolysis- 2 M NaOH for 6 days	01	01	01	2.127	1.138	2.187	6.98	7.89	12.43
Photolysis- ≥360Wh/m² at 30°C for 10 consecutive days	01	00	00	1.923	-	-	26.14	Stable	Stable
Wet heat- Digital controlled thermostatic hot air oven at 80°C for 10 hr	00	01	01	-	1.589	2.359, 2.392	Stable	3.35	26.53
Oxidation- 6 % H₂O₂v/v for 2 days	03	02	02	0.768, 0.943, 1.356	0.825, 1.012	1.502, 2.201	12.73	10.90	5.94

3.5.5 Specificity and selectivity:

Selectivity is the method of qualitatively identifying the interest of the analyte in the context of components likely to be present in the sample matrix, as opposed to specificity, which is the process of experimentally evaluating the interest of the analyte in the context of components that can also be assumed to be present in the sample matrix. The approach proposed is very selective and precise. It was noticed that there was no other specific intervention was recorded around the Rt of DLT, LVD, and TDF; there is no significant unavoidable noise in any of the baselines.

4. Stress degradation studies:

The present UHPLC approach was used to investigate the intrinsic stability of the DLT, LVD, and TDF amid various stressor conditions. First, it was inspected for acid hydrolysis, alkaline hydrolysis, oxidation, thermal (dry heat and wet heat stress), according to the Q1A (R2) guideline of ICH references, followed by Q1B for photolysis. The stressors, their preferred concentrations, and the preparation of the samples were all based on a previously existing research experiment. As a result, the DLT, LVD, and TDF were virtually insoluble in water, so the stress studies began with the stressor dissolved in methanol. To address all of the issues, minor adjustments in the mobile phase composition and flow rate were made to resolve all the potential degradants.

Tables 6 detailed the investigational conditions and stress deterioration outcomes for DLT, LVD, and TDF, respectively. The anticipated UHPLC method's ability to analyze the DLT, LVD, and TDF without being obstructed by degradation products in all cases. The specificity of the approach is addressed by suggesting the stability-indicating capacity of the anticipated inquiry.

5. CONCLUSION:

Finally, for the simultaneous quantification of DLT, LVD, and TDF in FDC tablets, we devised a reverse-phase stability-indicating UHPLC method. Since this method was specific, sensitive, accurate, linear, exact, and repeatable, it could be used to analyze DLT, LVD, and TDF in various pharmaceutical matrices. The advantages of the proposed method over the previously reported ones are the use of advanced column packaging UPLC columns with particle sizes of less than 2 µm allowed for excellent and efficient separation of the analyte concentration from the degradation products. which ultimately savings on operating costs and revealed excellent performance and analysis of results. DLT, LVD, and TDF were successfully separated from their degradation products using optimized chromatographic conditions. The analytical approach satisfied all of the validation guidelines’ acceptance criteria and may be used to acquire stability data by simultaneously estimating DLT, LVD, and TDF in pharmaceutical matrices.

6. ACKNOWLEDGMENTS:

The authors acknowledge the Institute of Pharmacy, NIMS University, Jaipur, Rajasthan, India, for the present work.

7. COMPETING INTERESTS:

The authors declare that they have no competing interests.

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Received on 29.06.2021 Modified on 23.09.2021

Research J. Pharm. and Tech 2022; 15(9):4061-4066.

DOI: 10.52711/0974-360X.2022.00681

	Tailing factor	Theoretical plates	Resolution	Retention time [min ±SD; n=6]	Viropil^TM tablets [ % Amount found ± SD] [Label claim DLT- 50 mg, LVD-300mg and TDF- 300 mg]
DLT	1.32	9641.21	-	1.356 ± 0.013	99.59 ± 0.44
LVD	0.93	2345.81	3.56	0.698 ± 0.022	99.81 ± 0.48
TDF	1.26	11228.44	6.09	2.361 ± 0.018	99.47 ± 0.59