0% READ
FULL TEXT
INTRODUCTION
Lamivudine is a reverse transcriptase that has been clinically reported to be helpful against the hepatitis B, HIV-1, and HIV-2 viruses.1 It improves liver histology stages and the development of seroconversion to antigen in beneficial hepatitis B.2 Human Immunodeficiency Virus (HIV) is a retrovirus that primarily attacks the immune system by targeting CD4 T cells. The treatment of HIV/ AIDS relies on antiretroviral therapy, which includes a combination of medicines that inhibit viral replication and reduce the viral load, thereby improving patient survival and quality of life. LMU is chemically 4-amino-1-((2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-2(1H)-pyrimidinone; it is a Nucleoside Reverse Transcriptase Inhibitor (NRTI) that interferes with the viral replication process by inhibiting the activity of the reverse transcriptase enzyme.3 Tenofovir Alafenamide is also classified as an NRTI it undergoes intracellular conversion to its active metabolite, which competitively inhibits viral reverse transcriptase, leading to unseasonable termination of viral DNA extension. TA is chemically isopropyl (2S)-2[[[(1R)-2-(6-aminopurin-9yl)-1-methyl-ethoxy]-methyl-phynoxy-phosphoryl]-amino]-propanoate used as an HIV.4 Dolutegravir is an integrase inhibitor that prevent the integration of viral DNA into the host genome. It does this by chelating magnesium ions in the active site of the HIV integrase enzyme, thereby blocking the beachfront transfer step necessary for viral replication, DTG is highly effective in rapidly lowering viral load while simultaneously boosting the number of CD4 cells in patients treated with 10, 25, or 50 mg once daily, in addition to a nucleoside reverse transcriptase inhibitor (NRTI) background. DTG is a chemically (4R,12aS)-N-[(2,4-difluorophenyl) methyl]-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido [1′,2′:4,5] pyrazino[2,1-b]1,3 oxazine-9-carboxamide. As mentioned in the literature, several approaches are practical for predicting TA, LMU and DTG individually. Among these, LMU, TA and DTG are extensively used antiretroviral agents with distinct mechanisms of action that contribute to effective HIV operation.5
LMU, DTG and TA in pharmaceutical dosage forms cannot be simultaneously estimated through a simple and rapid analytical process by using assessment of white analytical chemistry as indicated by the literature review. Furthermore, no statistical method for evaluating combinations of LMU, DTG and TA was identified through our conventional prior-art search. Multiple research studies that describe research they conducted do, however, include methodological flaws, such as the use of an ion-pair reagent in preparing the mobile phase and a run time surpassing 12 min. Additionally, photo-stable, the temperature and water conditions are not utilized for forced degradation. In the literature search, limited RP-HPLC stability-indicating assay method has been previously reported for the simultaneous quantification of all three drugs. In the present study, we discuss the development and validation of a simple, precise, accurate, and inexpensive RP-HPLC stability-indicating assay method for quantifying LMU, DTG and TA in pharmaceutical dosage forms by white analytical chemistry using green assessment tools. Based on a review of literature of all drugs are individual and combine a few methods that have been developed by [UV],6 [HPTLC],7,8[RP-HPLC],9,11 and bioanalytical.12
The objectives were to reduce or eliminate operations that generate hazardous materials, which pose serious threats to the environment and public health. The 12 principles of green chemistry, also known as "productively," were introduced by the same researchers who developed the concept of implementing green chemistry. The development of novel techniques and protocols involves carefully selecting the right solvents, materials, and technologies to regulate the safety and reactivity of chemical processes. The aim of this study is to improving method through green chemistry to reduce toxic waste through material reuse, energy conservation and process efficiency enhancements.13 Green analytical techniques have emerged as a result of this new idea; they are faster than standard methods, use fewer chemicals and solvents, and generate less waste, all of which reduce toxicity. In addition to green chemistry principles, Galuszka et al. introduced 12 green analytical principles under the acronym "significance," focusing on chemometric data use and miniaturized systems.14
White Analytical Chemistry (WAC) is an evolution of Green Analytical Chemistry (GAC), condensing its 12 principles into four key green principles that highlight the core aspects of GAC. WAC evaluates method quality by integrating analytical, practical, ecological and functional factors, using the green, red and blue color model to define a "white" (balanced) method.15,17 The red group assesses the validation characteristics of the analytical method, including sensitivity, accuracy, precision and scope. The green group focuses on GAC-related environmental safety factors, including reagent toxicity, reagent quantity and type, waste production, energy utilization and overall environmental impact. The blue group focuses on operational aspects, including cost, time efficiency, practical requirements and simplicity. A score of 100 is fully satisfactory, 75 is excellent but not universally suitable and 50 is occasionally acceptable. Averages are based on parameter scores in green, red and blue categories.18,19
MATERIALS AND METHODS
Chemicals and reagents
The Active Pharmaceutical Ingredients (API standards) sample of Lamivudine 99%, Dolutegravir 99.5% and Tenofovir alafenamide 99.3% were provided from Mylon Pharmaceuticals in Hyderabad, India, as a gift sample. A tablet Acriptega containing 300 mg Lamivudine, 300 mg Tenofovir alafenamide and 50 mg Dolutegravir, in a fixed dosage combination, was purchased from a local market in Nagpur, India.
All reagents utilized in this study were of HPLC grade and were sourced from SD Fine-Chem, Mumbai. The HPLC analytical methods were conducted using Milli-Q water, acetonitrile 99.8%, methanol 99.8% as the respective solvents. Triethyl amine 99% Extra pure, Hydrochloric acid 35 to 38% AR grade sd Fine, Sodium hydroxide 98% AR grade (SRL) Hydrogen peroxide 6% w/v Loba chemie Pvt. Ltd., ortho phosphoric acid 85% AR grade SRL.
Instrumentation
Agilent 1100 HPLC consisting of a pump G1310A, ISO auto sampler, auto injector, DAD detector (Model G13148), thermostatic column compartment connected with Chem Station software (Agilent Technologies, USA), associated with a Agilent Zorbax Eclipse C18 (250×4.6 mm, 5µ) Column. Shimadzu analytical balance (Model AY220, Japan) for accurate weighing, a Systronics digital pH meter (Model µ 361, Ahmedabad, India) for pH adjustment, and a Spectra Lab ultrasonic bath Sonicator (India).
Mobile phase preparation (Preparation of 0.05% triethylamine buffer solution)
TEA (0.05mL) was measured accurately by pipetting into 1000 mL Mili Q water and dissolved. The pH was adjusted to 3.0 with dilute with ortho phosphoric acid.
Standard solution preparation
Accurately weighed quantity of API powders equivalent to 60 mg of LMU, 60 mg of TA and 10 mg of DTG were taken into a 25 mL of volumetric flask and methanol was added to dissolve the contents, followed by the addition of methanol to make up the final volume of concentration 2400 µg/mL of LMU, 2400 µg/mL of TA and 400 µg/mL solutions respectively. Further, 0.05 mL of the stock solution was pipette out into 10 mL volumetric flask and final volume was made up with methanol to obtain final concentration of 12ppm for LMU, 12ppm for TA and 2 ppm for DTG working solution. The solution was filtered through a 0.22 µm membrane filter before analysis.
Tablet Sample preparation
Twenty tablets were weighed, crushed, and an amount of tablet powder 276.6 mg equivalent to 60 mg of LMU, 60 mg of TA and 10 mg of DTG was transferred
to 25 mL volumetric flask. Methanol was added to dissolve it, sonicated for 10 min and the final volume was made up with methanol. Further, 0.05 mL of the stock solution was pipette out into 10 mL volumetric flask and final volume was made up with methanol to obtain final concentration of 12ppm for LMU, 12ppm for TA and 2 ppm for DTG sample solution. The solution was filtered through a 0.22 µm membrane filter before analysis.
EXPERIMENTAL WORK
System suitability
Twenty microliters of the working solutions (12 µg/mL for LMU, 12 µg/mL for TA and 2 µg/mL for DTG) were injected. The system suitability parameters were determined by making six replicate injections and analyzing each solute for its retention time, tailing factor (T) and theoretical plate number (N). A chromatogram for the LMU, DTG and TA standard was illustrated in (Figure 1).
Accuracy
Recovery studies were conducted to verify the accuracy of the proposed method at concentration 24, 24 and 4 µg/mL LMU, TA and DTG respectively. Known quantity of standard solution at three distinct concentrations (80%, 100%, and 120%) was spiked and then calculated the recovery using area of chromatogram.
Precision
Six replicate injections of working solution (12, 12, and 2 µg/mL of LMU, TA, and DTG respectively) were used to determine precision at both intraday and inter-day levels.
Linearity and range (R2 Value)
Pipette out 0.05, 0.1, 0.15, 0.2 and 0.25 mL of standard stock solution of LMU, TA and DTG in 10 mL of volumetric flask separately and made up the volume to obtained the various concentrations of (12, 24, 36, 48, 60 µg/mL LMU and TA) and (2, 4, 6, 8, 10 µg/mL DTG) respectively.
Limit of detection (LOD) and Limit of quantitation (LOQ)
The least amount of analyte in a sample that can be identified is known as the Limit of Detection (LOD) for a method. The lowest analyte concentration in a sample that can be determined with a sufficient degree of precision and accuracy under the specified operating conditions of the method is known as the Limit of Quantitation (LOQ).
The standard deviation of the response (SD) and the slope of the calibration curve(s) at values near the LOD can be used to calculate LOD and LOQ using the following formulas:
LOD = 3.3 × S /SD and LOQ = 10 × S /SD
Robustness
Robustness parameter was assessed at slight and deliberate adjustments, such as changes in flow rate (0.5 and 0.9 mL/min), mobile phase composition (65% Methanol: 35% Buffer and 75% Methanol: 25% Buffer) and changes in wavelength (262 nm and 266 nm). The robustness was examined using 12 µg/mL for LMU, 12 µg/mL for TA and 2 µg/mL for DTG in triplicate.
Standard solution stability
At room temperature, the stability of the standard solution was tested every 24 and 48 hr. The system's appropriate characteristics, including theoretical plates, tailing factors, retention time and resolution were recorded.
Mobile phase stability
At ambient temperature, the stability of the mobile phase was systematically evaluated at intervals of 24 and 48 hr. The parameters including peak areas, theoretical plates, tailing factors, retention time and resolution were recorded.
Analysis of the marketed sample
The optimized method was applied to determine the purity of marketed formulation (Acriptega). The tablet sample solution was suitably diluted to obtain various concentrations of 12 µg/mL for LMU, 12 µg/mL for TA and 2 µg/mL for DTG and injected into the chromatographic system. The corresponding peak areas were determined and assay values were calculated.
% Assay = Sample Peak Area/Standard peak area * Std. concentration/Sample concentration *100
Forced degradation study
Forced degradation study was conducted according to ICH guidelines to establish the stability-indicating nature of method. Standard stock solution of LMU (2400 µg/mL), TA (2400 µg/mL) and DTG (400 µg/mL) were used. For acid hydrolysis, 3 mL of 0.1 N HCl was added to the 1 mL of the above standard stock solution LMU, TA and DTG solutions in a round bottom flask and refluxed at 70ºC for 1 hr the solution was neutralized with 0.1 N NaOH after acid hydrolysis then 0.05 mL were diluted with 10 mL methanol to obtained the desired concentration (12 µg/mL of LMU, 12 µg/mL TA and 2 µg/mL of DTG. Degraded sample were injected to the HPLC systems and compare the result with undegraded samples respectively. Similarly, alkali hydrolysis was performed using 0.1 N NaOH under the same conditions, followed by neutralization with 0.1 N HCl and subsequent analysis. Oxidative degradation, 1 mL of LMU, 1 mL of TA and 1 mL of DTG was treated with 3 mL of 3% hydrogen peroxide at RT for 1 hr then 0.05 mL of this solution was diluted with 10 mL of methanol and injected. For thermal degradation, 60 mg of LMU, 60 mg of TA and 10 mg of DTG were degraded in an oven then diluted in 25 mL of volumetric flask and make up the volume. Further 0.05 mL of above solution diluted to 10 mL of methanol and then injected. The neutral degradation, 3 mL of standard stock solution reflux with 3 mL of water for 2 hr. Then 0.05 mL of solution were diluted with 10 mL methanol and solution were injected. The degraded samples from each condition were analysed using HPLC and the chromatograms were compared with those of undegraded samples to identify and resolve any degradation peaks. The percentage of drug degraded under each condition was determined.
Statistical Analysis
The HPLC data is analyzed utilizing the Chem Station software. The outcomes were computed as the mean ± Standard Deviation (SD) to assess accuracy, while the Relative Standard Deviation (RSD) was determined to evaluate precision. Additionally, the regression coefficient was computed to ascertain the linearity parameter. AGREE software (an analytical greenness calculator version 0.5 beta) and MO-GAPI, BAGI (calculator version 1.4.3) and ASGA (version v0.37) software were used to examine the method’s greenness profile.
RESULTS AND DISCUSSION
Method development and optimization of chromatographic conditions
The approach to developing novel medicine combinations is unavoidable and always shows an advantage in treating illnesses. New techniques will always be needed to analyse the purity of the new medication combinations in the dosage form. In compliance with the updated ICH Q2(R2) guidelines.20
However, rather than formulating an analytical approach using toxic solvents, if the method has developed based on eco-friendly solvents, then the developed method can be eco-friendly and sustainable. By removing hazardous chemicals, replacing them with more environmentally friendly alternatives, and eliminating waste production, the ecologically benign analysis will be feasible. According to the Environmental, Health and Safety (EHS) system, deleterious solvents like ACN, tetrahydrofuran, should avoid in LC separations. Instead, the employment of biodegradable solvents is advocated for the purpose of drug separation within LC methodologies. This current method development has structured such a way that mitigate adverse effects to both the analyst and the environment. However, numerous additional factors have been considered apart from the solvent when analysing the mixture's drugs. When evaluating fixed-dose combinations, it is crucial to keep in mind that the method should function for an extended period without requiring further validation. To eliminate all the impediments, the present study has been conducted using RP-HPLC, which enables the analysis of chemicals at low concentrations, to establish a green and sustainable approach for the future.
The ICH Q2(R2) guidelines were followed in the validation of the developed method. The achievement of a satisfactory resolution between three drugs must be evaluated to develop a unique stability-indicating high-performance liquid chromatographic method for the quantification of LMU, TA and DTG.
To achieve good separation between all three components, different buffer pH conditions and varying proportions of solvents, such as methanol and buffer were tested. Detection was also performed at a wavelength of 264 nm.
Finally, a mixture of methanol and buffer 0.05% Triethylamine in a 70:30 ratio achieved satisfactory results using isocratic elution on a C18 column (250 mm × 4.6 mm, 5 µm) and a flow rate of 0.7 mL/min and injection volume 20µL resulting in optimal chromatographic separation, in a continuous run of up to 10 min.
Validation of the developed method
Using ICH Q2(R2) recommendations, the final optimized conditions were applied to the suggested RP-HPLC method and validated for criteria such as linearity, accuracy, and precision. Additionally, this method was used to determine the medications' % purity using the marketed formulation. Finally, various stressful conditions were used to conduct the forced deterioration study.
System Suitability
The system's suitability was tested to see how well it worked with recently made stock solutions. Comparing the Rt with those of standards allowed us to confirm the peak identities for LMU, DTG, and TA. The retention times for LMU, TA, and DTG are 3.142, 4.612, and 6.792 min, tailing factor mean are 0.621, 0.625 and 0.72 as well as theoretical plates are 5462, 5762 and 6241 respectively, as shown in (Table1). The system suitability parameters and resolution were reproducible and within the limits of the guidelines. The standard chromatograms for the three drugs are depicted in (Figure 1).
Accuracy
Accuracy (Trueness) was determined with the help of three different quality control concentration levels 80%, 100% and120%, for all the three drugs and the recovery percentage was found to be within limits as shown in Table 1. The results for the accuracy as the mean recovery of LMU, DTG and TA for 80% was found to be 100.2%, 99.81% and 99.49 respectively, for 100% was found to be 99.51%, 99.30% and 99.39% and for 120% was found to be 99.77%, 99.21% and 100.4%. Therefore, the given method shows satisfactory accuracy.
Precision
Intra-day precision for the investigated combination was assessed by calculating the percent Relative Standard Deviation (%RSD) after analyzing three freshly prepared solutions in six replicates of 12 µg/mL LMU, 12 µg/mL TA and 2 µg/mL on the same day. Intermediate precision the proposed method's inter-day accuracy was assessed by measuring six replicates over three days and calculating the percent Relative Standard Deviation (%RSD). The %RSD for intraday precision of LMU, TA and DTG was obtained as 0.3, 0.13 and 0.01 respectively and the %RSD for inter-day precision LMU, TA and DTG was obtained 0.31, 0.12 and 0.01 respectively. The %RSD of both the intraday and inter-day was less than 2%, indicating good precision of the proposed method as shown in Table 1.
Linearity and Sensitivities
Linearity can be measured at various analyte concentrations 12-60 μg/mL LMU, 12-60 μg/mL TA and, DTG 2-10 μg/mL respectively. Linear regression equations were y=123.45x-88.478 R²=0.9999 for LMU, y=211.39x-112.23 R²=0.9997 for DGT, and y=100.14x-201.99 R²=0.9994 for TA. Adequate sensitivities for the developed method concerning the revealed LOD and LOQs were achieved. LODs and LOQs were (0.12μg/mL and 0.38μg/mL), (0.21μg/mL and 0.34μg/mL) and (0.24 μg/mL and 0.73μg/mL) for LMU, DTG and TA respectively. The results of both linearity and sensitivity in were depicted in Table 1.
Robustness
At flow rate of 0.5 and 0.9 mL/min the result for LMU % RSD was 0.13 0.46, DGT for 0.06, 0.04 and TA for 0.02, 0.30 respectively. The mobile phase (65% Methanol: 35% Buffer and 75% Methanol: 25% Buffer) % RSD were 0.11, 0.03 for LMU, 0.13, 0.11 for DTG and 0.005, 0.02 for TA respectively. At wavelength 262 nm it shows % RSD 0.35, 0.001, 0.14
for LMU, DTG and TA respectively and at wavelength 266 nm it shows % RSD 0.05, 0.033, 0.05 LMU, DTG and TA respectively. The standard deviation of the peak areas was calculated for each parameter and the %RSD was found to be less than 2%. The low values of the %RSD, as shown in Table 1, of three drugs, which indicated the method's robustness.
Analysis of the marketed sample
The method has been successfully applied for the analysis of a marketed tablet. The percent of drug content in tablet of LMU, DTG and TA was found to be 100.22%, 99.99%, 99.99%, respectively as well as %RSD of LMU, DTG and TA was found to be 0.075, 0.017 and 0.175 respectively. No interference from the additives has been observed, results are shown in Table 2.
Forced Degradation Studies
Drug combinations were assessed using various stressed conditions like acid, alkali, oxidation, thermal and neutral degradation.21 Degradation chromatogram was represented in (Figure 2 a-e). The results are summarized as follows: LMU showed maximum degradation under acidic conditions (13.18%), followed by basic conditions (10.89%). The drug exhibited comparatively low degradation under oxidative (1.65%), thermal (1.69%), and neutral (2.29%) conditions. This indicates that Lamivudine is more susceptible to acid and base hydrolysis, suggesting the need for pH control during formulation and storage. DGT was found to be relatively stable under most conditions, with base-induced degradation (10.85%) being the highest. Thermal degradation (5.94%), while oxidative (2.85%), neutral (2.73%), and acidic (0.51%) conditions caused minimal degradation. These results suggest that DGT is more prone to alkaline hydrolysis but stable under acidic and oxidative environments. TA exhibited significant degradation under oxidative (17.85%) and basic (17.80%) conditions, indicating high sensitivity to oxidation and alkaline media. Moderate degradation was observed under acidic (9.50%) and thermal (2.93%) conditions, while it remained stable under neutral conditions (0.30%). Hence, protection from oxidative and basic environments is crucial for the stability of TA formulations were calculated in Table 3. The findings demonstrated that the devised approach was adequate and that there was no interaction between excipients and drugs in the commercial formulation. The results indicate that the proposed approach is suitable for the routine assessment of these drugs' combination formulations.
Whiteness and greenness profile assessment
A comprehensive comparison of published and proposed RP-HPLC methods for the concurrent stability study of LMU, DTG and TA using RGB12 scales, Mo-GAPI, AGREE, BAGI and ASGA tools is presented in Table 4 and Figure 3. The assessment is divided into three main scales: red (validation efficiency), green (environmental friendliness) and blue (cost and time efficiency). The red scale evaluates aspects like scope, sensitivity, accuracy, and precision. The green scale considers factors such as toxicity, environmental impact, waste generation, and power consumption. The blue scale examines cost efficiency, time efficiency, analytical steps and user-friendliness. Finally, a white analytical scale combines these scores to provide an overall assessment of the methods' performance. This multi-faceted approach allows for a holistic comparison of the techniques, considering their analytical performance, environmental impact, and operational efficiency.22
Red-scales profile assessment
For the study of validation efficiency of published and proposed RP-HPLC methods, four principles for Red model-based assessment (R1 to R4; see Table 4) were applied as per ICH Q2 (R1) guidelines. The published RP-HPLC methods obtained a score of 80 out of 100 for all four principles for the estimation of LMU, TA and DTG. The proposed method has obtained 10 points extra for its scope and application due to the simultaneous estimation of LMU, TA and DTG. Hence, the proposed method has obtained a score of 90 out of 100 for the validation assessment by the Red model.23,24
Green-scale profile assessment
The green-scale analytical profile assessment compares the environmental friendliness of the published and proposed RP-HPLC methods for the concurrent stability study of LMU, DTG and TA. The proposed method demonstrates superior environmental performance across multiple parameters. It significantly reduces the use of toxic organic solvents, requiring less than 30 mL compared to over 100 mL in published methods. This reduction in solvent usage not only decreases the risks of human and animal toxicity but also minimizes environmental impact. The proposed method generates less than 100 mL of toxic organic waste, which is considerably lower than the published methods. However, power consumption remains similar, with both methods consuming more than 1.0 kwh. The environmental friendliness of the proposed method is further validated by higher scores in green analytical tools: AGREE (0.77 vs. 0.60), Mo-GAPI (78 vs. 60), and AGSA (76 vs. 61). Consequently, the proposed RP-HPLC method achieved a green-scale assessment score of 76 out of 100, significantly outperforming the published methods' score of 60, thus demonstrating its superior environmental efficiency for the concurrent stability study of LMU, DTG, and TA.25
Blue-scale profile assessment
The blue-scale analytical profile assessment compares the cost and time efficiency of the published and proposed RP-HPLC methods for the concurrent stability study of LMU, DTG and TA. The proposed method demonstrates superior performance across multiple parameters. It is more cost-efficient, requiring fewer resources for concurrent stability study and bioanalysis compared to the published methods. The proposed method is also more time-efficient, as it requires less time for analysis. In terms of analytical steps, the proposed method is simpler and easier to handle, requiring fewer steps for the concurrent stability study. The proposed method is more user-friendly, as it utilizes a single chromatographic condition instead of the multiple conditions required by published methods. These improvements in efficiency and usability contribute to the proposed RP-HPLC method achieving a blue-scale assessment score of 90 out of 100, outperforming the published methods' score of 80. This higher score indicates that the proposed method is more cost-effective, time-efficient, and user-friendly for the concurrent stability study of LMU, DTG, and TA.26
White-scale profile assessment
The white-scale analytical profile assessment provides a comprehensive evaluation of the published and proposed RP-HPLC methods for concurrent stability study of LMU, DTG, and TA, integrating the red, green, and blue scale assessments. The proposed method demonstrates superior overall performance compared to the published methods. The whiteness profile score, which is an average of the red, green, and blue scale scores, is significantly higher for the proposed method at 85.33 (90 + 76 + 90)/3, compared to 73.33 (80 + 60 + 80)/3 for the published methods. This higher score reflects the proposed method's improvements in analytical efficiency, environmental friendliness, and cost-time efficiency. Notably, the proposed method achieves an 'excellent white' status with a whiteness profile score exceeding 85, while the published methods are classified as 'enough white' with a score below 75. A key factor contributing to this superior performance is the proposed method's use of a single chromatographic condition, as opposed to the multiple conditions required by the published methods. This comprehensive assessment underscores the overall superiority of the proposed RP-HPLC method for the concurrent stability study of LMU, DTG, and TA, demonstrating its potential to enhance analytical processes in pharmaceutical research and quality control.27 Green chemistry principles advocate reducing hazardous chemicals, minimizing waste, and enhancing energy efficiency. In HPLC analysis, solvent consumption is a significant environmental concern; thus, greenness assessment tools are applied to quantify and reduce the ecological impact. systematically evaluating and optimizing critical method parameters, enhancing robustness and sensitivity beyond previously reported.28,29
| Sl. No. | Validation Parameter | Observed Value | ||
|---|---|---|---|---|
| Lamivudine | Dolutegravir | Tenofovir Alafenamide | ||
| 1. | System suitability (mean) | |||
| Retention Time | 3.142 | 4.612 | 6.792 | |
| Theoretical Plate | 5462 | 5762 | 6241 | |
| c. Tailing Factor | 0.621667 | 0.625 | 0.72 | |
| 2. | Accuracy (% Recovery) | Mean Percent Recovery | ||
| 80% | 100.2 | 99.81 | 99.49 | |
| 100% | 99.51 | 99.30 | 99.39 | |
| 120% | 99.77 | 99.21 | 100.4 | |
| 3. | Precision | |||
| Intraday precision (%RSD) | 0.3 | 0.01 | 0.13 | |
| Interday precision (%RSD) | 0.31 | 0.01 | 0.12 | |
| 4. | Linearity | |||
| Concentration range (mcg mL-1) | 12-60 | 2-10 | 12-60 | |
| Y=ax+b | y = 123.45x - 88.478 | y = 100.14x - 201.99 | y = 211.39x - 112.23 | |
| R² | 0.9999 | 0.9994 | 0.9997 | |
| 5. | LOD, Limit of Detection | 0.12 µg/mL | 0.21 µg/mL | 0.24 µg/mL |
| 6. | LOQ, Limit of Quantitation | 0.36 µg/mL | 0.34 µg/mL | 0.73 µg/mL |
| 7. | Robustness | 0.13, 0.46 | 0.06, 0.04 | 0.02, 0.30 |
| Mobile phase (Methanol: Buffer (65:35) and Methanol: Buffer (75: 25) | 0.11, 0.03 | 0.13, 0.11 | 0.005, 0.02 | |
| Wavelength (262 nm and 266 nm) | 0.35, 0.05 | 0.001, 0.033 | 0.14, 0.05 |
| Lamivudine | Dolutegravir | Tenofovir alafenamide | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Sl. No. | Standard Area | Sample area | % of Drug | Standard Area | Sample area | % of Drug | Standard Area | Sample area | % of Drug |
| 1 | 1381 | 1385 | 100.28 | 305.03 | 305.02 | 99.99 | 1025.55 | 1025.51 | 99.99 |
| 2 | 1382 | 1386 | 100.28 | 305.04 | 305.01 | 99.99 | 1025.51 | 1025.53 | 100.0 |
| 3 | 1380 | 1384 | 100.28 | 305.01 | 305.04 | 100.0 | 1025.53 | 1025.52 | 99.99 |
| 4 | 1384 | 1385 | 100.07 | 305.02 | 305.03 | 100.0 | 1025.52 | 1025.51 | 99.99 |
| 5 | 1382 | 1386 | 100.28 | 305.03 | 305.01 | 99.99 | 1025.54 | 1025.53 | 99.99 |
| 6 | 1381 | 1383 | 100.14 | 305.04 | 305.02 | 99.99 | 1025.51 | 1025.54 | 100.0 |
| Mean | 1381 | 1383 | 100.22 | 305.02 | 305.03 | 99.99 | 1025.53 | 1025.51 | 99.99 |
| Std dev | 1.103 | 1.121 | 0.07 | 0.509 | 0.508 | 0.016 | 2.093 | 2.083 | 0.021 |
| %RSD | 0.08 | 0.074 | 0.075 | 0.166 | 0.161 | 0.017 | 0.206 | 0.208 | 0.175 |
| Drug substance | Degradation condition | %Drug degraded |
|---|---|---|
| Lamivudine | Acid | 13.18 |
| Base | 10.89 | |
| Oxidation | 1.65 | |
| Thermal | 1.69 | |
| Neutral | 2.29 | |
| Dolutegravir | Acid | 0.51 |
| Base | 10.85 | |
| Oxidation | 2.85 | |
| Thermal | 5.94 | |
| Neutral | 2.73 | |
| Tenofovir alafenamide | Acid | 9.50 |
| Base | 17.80 | |
| Oxidation | 17.85 | |
| Thermal | 2.93 | |
| Neutral | 0.30 |
| RGB12 multi-colored analytical chemistry scale | Reported RP-HPLC methods for concurrent stability study of Lamivudine, Dolutegravir and, Tenofovir alafenamide | Proposed RP-HPLC method for concurrent stability study of Lamivudine, Dolutegravir and, Tenofovir alafenamide |
|---|---|---|
| Red analytical scale for assessment of validation efficiency | ||
| R | No stability-indicating RP-HPLC method has been reported in the literature to date for the concurrent stability-indicating estimation of LMU, DTG, and TA. | This study develops and validates a RP-HPLC method for the simultaneous estimation of LMU, DTG, and TA in pharmaceuticals. It's a "unique, simple, successful, quick and accurate stability indicating" technique, compliant with ICH requirements. Applications include quality control, stability testing, forced degradation studies and regulatory compliance in the analysis of HIV medications. |
| R2 -Sensitivity (LOD and LOQ) | The reported individual RP-HPLC methods for stability studies of LMU, DTG, and TA exhibit sensitivity at the microgram level using separate and dedicated chromatographic conditions. | The RP-HPLC method's sensitivity is demonstrated by its Limit of Detection (LOD) and Limit of Quantification (LOQ) values. For LMU, DTG, and TA, the LOD values are 0.12, 0.11 and 0.24 μg/mL, while the LOQ values are 0.36, 0.34, and 0.73 μg/mL, respectively. These low values indicate high sensitivity, crucial for accurate pharmaceutical analysis and quality control. |
| R3 -Accuracy | The reported individual RP-HPLC method for stability study of LMU, DTG, and TA complied with the accuracy study according to ICH guidelines. However, it required more resources for the validation study. | The RP-HPLC method's accuracy was rigorously evaluated through recovery studies conducted at three distinct concentration levels: 80%, 100%, and 120%. The results demonstrated consistently high recovery percentages for all three drugs under analysis. LMU showed recovery rates of 100.2%, 99.51%, and 99.77% at the respective concentration levels. DTG exhibited recovery percentages of 99.81%, 99.30%, and 99.21%, while TA displayed 99.49%, 99.39% and 100.4% recovery. |
| R4 -Precisions | The reported individual RP-HPLC method for stability study of LMU, DTG and TA complied for precision study with ICH guidelines. But required more resources for validation study. | The precision study evaluated intraday and interday variability for LMU, DTG, and TA. Results showed low %RSD values: LMU (intraday: 0.3%, interday: 0.31%), DTG (intraday: 0.01%, interday: 0.01%), and TA (intraday: 0.13%, interday: 0.12%) . These low %RSD values indicate high precision and reproducibility of the RP-HPLC method for all three drugs. |
| Red scale assessment score | Hence, the published RP-HPLC methods have obtained red-scale assessment score of 80 out of 100 for concurrent stability study of LMU, DTG and TA. | Hence, the proposed RP-HPLC method has obtained red-scale assessment score of 90 out of 100 for concurrent stability study of LMU, DTG and TA. |
| Green analytical scale for assessment of environment friendliness using AGSA, Mo-GAPI and AGREE tools | ||
| G1 -Human and animal toxicity | The individual published RP-HPLC method for concurrent stability –indicating estimation of LMU, DTG and TA required more than 100 mL of toxic organic solvents such as methanol and acetonitrile. | The proposed RP-HPLC method for concurrent stability –indicating estimation of LMU, DTG and TA required less than 30 mL of toxic organic solvents such as methanol. |
| G2 -Environmental impacts | The acetonitrile and methanol used in reported individual RP-HPLC method for concurrent stability study of LMU, DTG and TA are hazardous to the environment. | In the proposed RP-HPLC method eco-friendly mobile phase used, Methanol and TEA buffer for stability study of LMU, DTG and TA used in is less hazardous to the environment than published methods. |
| G3 -Toxic waste generation | The individual published RP-HPLC method for concurrent stability study of LMU, DTG and TA have generated more than 100 mL toxic organic waste. | The proposed RP-HPLC method for concurrent stability study of LMU, DTG and TA is generating less than 100 mL toxic organic waste. It is always less than published methods |
| G4 -Power consumption | The power consumption of the published RP-HPLC methods is more than 1.0 KWh. | The power consumption of the proposed RP-HPLC method is more than 1.0 KWh. |
| Green analytical tools | The AGREE tool showed score of 0.60 out of 1.0 for published RP-HPLC method. | The AGREE tool showed score of 0.77 out of 1.0 for proposed RP-HPLC method. |
| Green scale assessment scores | Hence, the published RP-HPLC methods have obtained green-scale assessment score of 60 out of 100 for concurrent stability study of STM and GMP. | Hence, the proposed RP-HPLC method has obtained green-scale assessment score of 76 out of 100 for concurrent stability study of STM and GMP. |
| Blue analytical scale for assessment of cost and time efficiency using BAGI tool | ||
| B1 -Cost efficiency | The published individual RP-HPLC methods have required more resources for concurrent stability study and bioanalysis of LMU, DTG and TA. Hence, the published RP-HPLC methods are more costly than proposed RP-HPLC method. | The proposed RP-HPLC methods have required fewer resources for concurrent stability study and bioanalysis of LMU, DTG and TA in human plasma. Hence, the proposed RP-HPLC method is more economical than published RP-HPLC methods. |
| B2 -Time efficiency | The published individual RP-HPLC methods have required more time for concurrent stability study and bioanalysis of LMU, DTG and TA. Hence, the published RP-HPLC methods are more time consuming than proposed RP-HPLC method. | The proposed RP-HPLC methods have required less time for concurrent stability study and bioanalysis of LMU, DTG and TA in human plasma. Hence, the proposed RP-HPLC method is faster than published RP-HPLC methods. |
| B3 -Analytical steps | The published individual RP-HPLC methods have required more analysis steps for concurrent stability study of LMU, DTG and TA. Hence, the published RP-HPLC methods are more complex than proposed RP-HPLC method. | The proposed RP-HPLC method has required less analytical steps for concurrent stability study of LMU, DTG and TA in human plasma. Hence, the proposed RP-HPLC method is easier to handle than published RP-HPLC methods. |
| B4 -User friendliness | The proposed RP-HPLC methods for concurrent stability study of LMU, DTG and TA is less user-friendly as it required multiple chromatographic conditions to set. Hence, the proposed RP-HPLC methods are less user-friendly than published RP-HPLC methods. | The proposed RP-HPLC method for concurrent stability study of LMU, DTG and TA is more user-friendly as it required single chromatographic condition to set. Hence, the proposed RP-HPLC method is more user-friendly than published RP-HPLC methods. |
| Blue scale assessment score | Hence, the published RP-HPLC methods have obtained blue-scale assessment score of 80 out of 100 for concurrent stability study of LMU, DTG and TA | Hence, the published RP-HPLC methods have obtained blue-scale assessment score of 90 out of 100 for concurrent stability study of LMU, DTG and TA |
| White analytical scale for assessment of multicoloured analytical efficiency using RGB12 scales | ||
| Whiteness profile scored | The whiteness profile score of the published RP-HPLC methods have obtained score of 73.33 average of (80+60+80). | The whiteness profile score of the proposed RP-HPLC method has obtained score of 85.33 average of (90+76+90). |
| Whiteness status | Hence, the published individual RP-HPLC methods using multiple chromatographic conditions are enough white (whiteness profile score of less than 75) for concurrent stability study of LMU, DTG and TA | Hence, the proposed RP-HPLC methods using single chromatographic conditions is excellent white (whiteness profile score of more than 85) for concurrent stability study of LMU, DTG and TA |
CONCLUSION
This study developed and validated a simple, reliable, and stability-indicating reverse-phase HPLC method for the simultaneous estimation of LMU, DTG, and TA in pharmaceutical formulations. The method demonstrated excellent linearity (R²> 0.9999, 0.9997, and 0.9994), recoveries of LMU, DTG and TA for 80% was found to be 100.2%, 99.81% and 99.49 respectively, for 100% was found to be 99.51%, 99.30% and 99.39% and for 120% was found to be 99.77%, 99.21% and 100.4% respectively and precision (intraday %RSD: 0.3, 0.01, 0.13; interday %RSD: 0.31, 0.01, 0.12) within limit. Sensitivity was confirmed with low limits of detection (0.12, 0.24, 0.21 µg/mL) and quantification (0.36, 0.73, 0.34 µg/mL). The method complied with ICH Q2 (R2) guidelines. Forced degradation studies under acidic, alkaline, oxidative, thermal, photolytic, and hydrolytic conditions confirmed the method’s stability-indicating capability. Degradation products were well separated and did not interfere with drug peaks, establishing specificity. Environmental impact was minimized through green analytical approaches, with favorable scores from AGREE (0.77), Mo-GAPI (78), and AGSA (76). The RGB model further supported sustainability, yielding a high overall score (85.33), indicating eco-friendly performance. The method is also cost-effective compared to conventional chromatographic techniques. Overall, the proposed isocratic RP-HPLC method is simple, precise, and suitable for routine analysis of LMU, DTG, and TA in pharmaceutical laboratories. All three compounds were successfully separated using the recommended strategy, and the active contents were also calculated. The suggested plan is stability-indicating and specific. It ensures accurate quantification, effective separation under stress conditions, and supports both regulatory compliance and environmentally conscious practices
SUMMARY
The simultaneous measurement of LMU, DTG, and TA in tablet formulation was accomplished through the development and validation of a novel, simple, and environmentally friendly RP-HPLC method. Under ideal chromatographic conditions, the approach demonstrated good linearity (R2> 0.999), accuracy (≈99%), precision (%RSD < 0.5), and sensitivity, with a distinct separation. It confirmed specificity by successfully indicating stability under a range of stress settings free from interference from degradation products. With a high whiteness score (85.33), environmental assessment techniques (AGREE=0.77, Mo-GAPI=78, AGSA=76) showed its exceptional sustainability and greenness. Thus, the method is accurate, robust, cost-effective, and environmentally sustainable for routine quality control of LMU, DTG, and TA in pharmaceuticals.
