1. INTRODUCTION:

A class of medication called antiviral medication is used to treat the viral infections¹. Abroad-spectrum antiviral is efficient against a variety of viruses while the majority of antivirals target particular viruses². Antiviral medication are a subset of the broader of antimicrobials, which also involve antibiotic (commonly known as antibacterial), antiparasitic and antifungal medications³ Tenofovir disoproxil fumarate (TEN) is the chemical name of a compound known as 9-[(R)-2 [[bis[[(isopropoxycarbonyl)oxy]methoxy] phosphinyl] methoxy] propyl] adenine fumarate⁴ (Fig. 1a).

Based on a comprehensive literature review, various analytical techniques have been documented for the determination of these medications, both individually and in combination with other drugs, across different dosage forms. High-performance liquid chromatography (HPLC)^5,6,7, high-performance thin-layer chromatography (HPTLC)⁸, and UV spectrometry⁹ are among the analytical methods that can be utilized for this purpose. HPLC, in combination with stability-indicating methods, can provide valuable insights into the study of these medications in combination with other drugs in plasma^10,11,12.

Amoxicillin (AMX) is a chemical compound with the chemical name [2S-[2a,5a,6β(S*)]]-6-[[Amino(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid¹³. It is classified as a semi-synthetic oral penicillin and is structurally related to ampicillin (Fig. 1b).

The purpose of this work was to create a straightforward, exact, accurate, and verified UPLC method for measuring tenofovir and amoxicillin.

2. INSTRUMENTATION:

The study utilized the Agilent 1200 liquid chromatographic system, which was manufactured by Agilent Technologies in Germany. The system included an auto-sampler, a UV and Photodiode Array (PDA) detector, and an Agilent 1200 injector with a 100μl loop volume. Sample weighing was performed using the Acculab ALC 210.4 analytical balance, a Digital Micro Balance manufactured by Cole-Parmer® in the United States. The pH of the buffer was adjusted using the Chemiline CL 180μc based pH-meter[Aqua Mart, India], a pH analyzer. Also vortex, sonicator, centrifuge, syringe, Whatman filter paper no. 41, volumetric flask and porcelain mortar were used.

3. CHEMICALS AND REAGENTS:

The reference standards for Tenofovir and Amoxicillin were purchased from Cipla Pharmaceuticals, located in Mumbai, India. The combined tablet containing both medications was obtained from a local market. HPLC grade methanol, HPLC grade orthophosphoric acid (80%) from Fisher Chemicals Pvt. Ltd., Belgium, and water were also procured for use in the experiment.

4. CHROMATOGRAPHIC CONDITIONS:

The mobile phase consisted of a mixture of methanol and phosphate buffer (0.1% orthophosphoric acid in water, pH 3.5) in a ratio of 30:70 (v/v), flowing at a rate of 0.2 milliliters per minute. Kinetex 1.7μ C18 100A (2.1-mm × 50-mm) was utilized as the stationary phase. To optimize selectivity and sensitivity for both medications, a detection wavelength of 230nm was chosen for the UV-PDA detector. The UPLC system operated at a temperature of 30°C.

4.1 General procedure and construction of calibration curves:

Standard stock solutions were prepared by dissolving 10 mg of TEN and AMX separately in methanol/water (50/50, v/v) in 100mL volumetric flasks, resulting in concentrations of 100.0μg/ml for each of TEN and AMX. Working standard solutions were prepared by taking 0.1, 0.5, 1, 1.5, and 2ml of the stock solutions and diluting them up to the mark with the mobile phase in 10 mL volumetric flasks. This resulted in adjusted concentrations ranging from 1.0 to 20.0μg/ml for each of TEN and AMX. To evaluate the performance of the method, triplicate injections were made for each concentration. The samples were chromatographed under the validated LC conditions. The peak areas corresponding to the investigated drugs were recorded. Subsequently, the peak areas were plotted against the corresponding concentrations to generate calibration graphs. Regression equations were then calculated for both medications based on these calibration graphs.Top of Form

5. RESULTS AND DISCUSSION:

5.1 Optimization of chromatographic conditions:

Amoxicillin had been separated in their prepared tablets and simultaneously determined by using a straightforward, selective, simple and quick UPLC method.

Both pharmaceuticals, Tenofovir and Amoxicillin, were successfully separated on a Kinetex 1.7μ C18 100A column. The mobile phase used for the separation consisted of a mixture of methanol and phosphate buffer, with a ratio of 30:70 (v/v). The phosphate buffer was prepared by dissolving 0.1% orthophosphoric acid in water, and the pH of the mobile phase was adjusted to 3.5. This chromatographic setup allowed for the effective separation of Tenofovir and Amoxicillin, enabling the analysis and quantification of each pharmaceutical compound. In the development of LC methods, achieving appropriate resolution with satisfactory peak symmetry is of utmost importance. In this study, several reversed-phase columns were evaluated, including the Kromasil 100 column (4.6mm × 250mm, 5μm) and the Kinetex 1.7μ C18 100A column (2.1-mm × 50-mm), to determine the optimal assay parameters. After evaluation, the Kinetex 1.7μ C18 100A column was found to provide the best resolution between the two medications and was therefore selected as the working column for this investigation. Various mobile phases were examined by using different ratios of aqueous phases such as buffer (0.1M orthophosphoric acid in water) with acetonitrile, methanol with acetonitrile, and methanol with phosphate buffer (0.1% orthophosphoric acid in water, pH 3.5) in a ratio of 30:70 (v/v). Ultimately, the ideal mobile phase was determined to be a mixture of methanol and phosphate buffer solution (0.1% orthophosphoric acid in water, pH 3.5) in a ratio of 30:70 (v/v). The impact of mobile phase pH was investigated within the range of 3-4, and it was found to influence the separation efficiency. Additionally, the effect of flow rate on the chromatographic performance was studied, and it was concluded that a flow rate of 0.2mL/min yielded the best results. Overall, these findings aided in establishing the optimal assay parameters for the analysis of the two medications.

5.2 Method Validation:

The chromatographic conditions previously mentioned demonstrated a satisfactory separation between Tenofovir and Amoxicillin (Fig. 4) .This separation between two medications has acceptable run time. Tenofovir and amoxicillin have retention factors (k) of 2.28 and 2.69, respectively. Resolution (R) values for Tenofovir and Amoxicillin are 8.64 and 6.18, respectively, A resolution value of 1.5 indicates a perfect separation between two adjacent peaks, indicating distinct and well-defined chromatographic peaks^[14]. This value suggests that the developed chromatographic conditions provide an excellent separation of the target compounds. The number of theoretical plates (N) is a measure of column performance or apparent efficiency. In this case, the calculated number of theoretical plates for Tenofovir is 7203, while for Amoxicillin it is 6538. Higher numbers of theoretical plates generally indicate better column efficiency, resulting in sharper and narrower peaks. The obtained values of 7203 and 6538 for Tenofovir and Amoxicillin, respectively, suggest that the column performance is satisfactory, providing sufficient resolution and peak shape for accurate and precise analysis of the compounds.

Overall, the combination of good resolution and a high number of theoretical plates demonstrates the effectiveness and efficiency of the chromatographic system in separating Tenofovir and Amoxicillin. The method's validation was deemed successful According to the International Conference on Harmonization (ICH) ^[15] .validation items; the proposed UPLC method's linearity was assessed by examining a range of various concentrations for each drug component. It was discovered that the measured responses at the specified wavelengths exhibited a direct proportionality to the concentration of the drug.

Table 1 presents the statistical validation data for the proposed methods including correlation coefficients, concentration ranges, standard deviations of the intercept (Sa), slope (Sb), and residuals (Sy/x). The correlation coefficient values which were (r > 0.99995), percentage relative standard deviation (RSD%) of the slope values, were observed to be less than 0.5%, and the percentage y-intercept values, were observed to be less than 1%, all indicate that regression analysis exhibits good linearity. Based on signal-to-noise ratio data, the calculation of the limit of detection (LOD) and the limit of quantitation (LOQ) was performed according to the ICH guidelines. The method's sensitivity of the proposed UPLC was confirmed by the calculated limit of detection (LOD) and limit of quantitation (LOQ) values. For Tenofovir, the LOD and LOQ were determined to be 0.445μg/mL and 1.348 μg/mL, respectively, while for Amoxicillin, the LOD and LOQ values were found to be 0.2550μg/mL and 0.7729μg/mL, respectively (as shown in Table 1).

Furthermore, the recovery values obtained for both analytes were found to be good, as shown in Table 1. The recoveries indicate the accuracy and reliability of the method in quantifying the analytes. Additionally, the low percentage relative standard deviation (RSD%) of less than 1% demonstrates the high precision and repeatability of the procedures used to estimate the analytes in their bulk form, as shown in Table 3,4.

Table (1): Validation data for the determination of Tenofovir and Amoxicillin using the proposed UPLC method

Parameter	TEN	AMX
Linearity range (µg/mL)	1.0-2.0	1.0-2.0
Slope (b)	9.8377	3.9772
Intercept (a)	0.663	0.213
Correlation coefficient (r)	0.99982	0.99994
Sa	1.3279	0.3074
Sb	0.1084	0.0251
Sy/x	1.6461	0.3810
LOD	0.4454	0.2550
LOQ	1.3498	0.7729

Table 2: System suitability parameters of Tenofovir and Amoxicillin

TEN
Theoretical Preparation	1.0	5.0	10.0	15.0	20.0
Average Peaks Area	9.8835	50.1736	98.5978	150.4912	195.8910
R.S.D. (%)	0.446 %	0.537 %	0.514 %	0.478 %	0.583%
Peak Symmetry	1.113	1.106	1.109	1.118	1.110
AMX
Theoretical Preparation	1.0	5.0	10.0	15.0	20.0
Average Peaks Area	4.0016	20.3322	39.7601	60.3335	79.4722
R.S.D. (%)	0.677 %	0.509 %	0.423 %	0.616 %	0.568%
Peak Symmetry	0.982	0.974	0.977	0.988	0.971

Table 3: the accuracy parameters of Tenofovir and Amoxicillin

Tenofovir
Theoretical Preparation	5	10	15
Average Peaks Area	49.4711	97.5247	148.8533
R.S.D. (%)	0.517 %	0.533 %	0.448 %
Peak Symmetry	1.11	1.116	1.107
Amoxicillin
Theoretical Preparation	5	10	15
Average Peaks Area	20.6541	40.5491	61.5308
R.S.D. (%)	0.533 %	0.476 %	0.559 %
Peak Symmetry	0.981	0.971	0.979

Table 4: the Recovery parameters of Tenofovir and Amoxicillin

Tenofovir
Recovery	Recovered Conc. (µg/ml)				Recovery (%)
Recovery	Result 1	Result 2	Result 3	Average	Recovery (%)
Spike – Recovery at 5	5.07	5.06	5.03	5.04	100.80%
Spike – Recovery at 10	10.05	10.13	10.08	10.09	100.90%
Spike – Recovery at 15	14.85	14.92	14.87	14.88	99.20%
Amoxicillin
Recovery	Recovered Conc. (µg/ml)				Recovery (%)
Recovery	Result 1	Result 2	Result 3	Average	Recovery (%)
Spike – Recovery at 5	5.05	5.06	5.02	5.04	100.40%
Spike – Recovery at 10	9.87	9.94	9.91	9.91	99.10%
Spike – Recovery at 15	15.07	15.04	15.09	15.07	100.46%

Table 5: the system repeatability of Tenofovir and Amoxicillin

Tenofovir
Concentration 5		Day 1	Day 2	Day 3
	Peak No.	Peaks Area	Peaks Area	Peaks Area
	1	49.4711	51.8223	50.1741
	2	50.0238	50.0073	49.1282
	3	50.9182	51.1646	49.8322
	Average Peaks Area	50.1377	50.9980	49.7115
	RSD %	0.377 %	0.411 %	0.396%
Concentration 10		Day 1	Day 2	Day 3
	Peak No.	Peaks Area	Peaks Area	Peaks Area
	1	97.5247	99.1182	98.9137
	2	99.2093	100.3472	99.1093
	3	98.2172	100.6466	98.0162
	Average Peaks Area	98.3170	100.0373	98.6797
	RSD %	0.524 %	0.487 %	0.505 %
Concentration 15		Day 1	Day 2	Day 3
	Peak No.	Peaks Area	Peaks Area	Peaks Area
	1	148.8533	150.0114	147.8475
	2	150.1983	149.7264	146.2098
	3	149.0139	150.4811	148.0057
	Average Peaks Area	149.3551	150.0729	147.3543
	RSD %	0.617 %	0.583 %	0.664%
Amoxicillin
Concentration 5		Day 1	Day 2	Day 3
	Peak No.	Peaks Area	Peaks Area	Peaks Area
	1	20.6601	20.1150	21.0187
	2	21.0187	20.8377	22.0294
	3	21.9367	21.4691	22.2314
	Average Peaks Area	21.2051	20.8072	21.7597
	RSD %	0.389 %	0.443 %	0.472%
Concentration 10		Day 1	Day 2	Day 3
	Peak No.	Peaks Area	Peaks Area	Peaks Area
	1	40.3219	39.4129	41.0968
	2	41.3380	39.8337	41.4388
	3	40.0079	40.2759	40.8293
	Average Peaks Area	40.5559	39.8408	41.1216
	RSD %	0.488 %	0.532 %	0.449 %
Concentration 15		Day 1	Day 2	Day 3
	Peak No.	Peaks Area	Peaks Area	Peaks Area
	1	61.1860	59.8066	62.0364
	2	60.8444	60.0265	61.5559
	3	62.0465	59.0046	62.6862
	Average Peaks Area	61.3589	59.6125	62.0928
	RSD %	0.556 %	0.675 %	0.566%

To evaluate the within-day precision and accuracy of the described methods, three replicate determinations were performed for each concentration of Tenofovir and Amoxicillin on the same day. Similarly, to assess the between-day precision and accuracy, the same three concentrations for each drug ingredient were examined over the course of three days. Three replicate determinations were performed on each day. (Table 5). By considering both within-day and between-day variations, the overall precision and accuracy of the methods can be thoroughly assessed, providing confidence in the reliability of the analytical procedure. By making minor variations in the conditions of the experiment as flow rate which was (±0.03 mL/ min), pH which was (±0.5 unit), the robustness of the established approach in the case of UPLC was assessed. The robustness tests shows that the results were be unaffected by small variations in the method procedures. The results was very closed to the main procedures (Table 6).

Table 6: the robustness of Tenofovir and Amoxicillin

Tenofovir
Robustness	K	N	R	ά	T
Methanol
43 %	2.22	7011	8.54	7.07	0.81
45 %	2.28	7203	8.64	7.18	0.91
47 %	2.25	7138	8.51	7.05	0.82
pH
3	2.19	6994	8.59	7.12	0.86
3.5	2.28	7203	8.64	7.18	0.89
4	2.17	6104	8.62	7.10	0.83
Flow rate
0.17	2.17	6906	8.51	7.08	0.88
0.2	2.28	7203	8.64	7.18	0.91
0.23	2.19	6874	8.55	7.11	0.86
Amoxicillin
Robustness	K	N	R	ά	T
Methanol
43 %	2.66	6387	6.11	8.22	0.89
45 %	2.69	6538	6.18	8.26	0.93
47 %	2.62	6409	7.13	8.19	0.87
pH
3	2.44	6323	7.21	8.43	0.85
3.5	2.52	6509	7.30	8.50	0.90
4	2.42	6389	7.23	8.46	0.86
Flow rate
0.17	2.51	6444	7.25	8.42	0.90
0.2	2.59	6512	7.37	8.49	0.94
0.23	2.54	6419	7.33	8.42	0.88

5.3. Applications:

5.3.1 Analysis of the studied drugs in their tablets:

In this experiment, a total of twenty tablets were used. These tablets were weighed, triturated in a porcelain mortar, and thoroughly mixed. The weight average of the tablets was calculated based on the collected data. To prepare the sample solution, an accurately weighed amount of powder equivalent to one tablet of the (Amoxicillin + Tenofovir) product was taken and placed in a 100mL volumetric flask. Then, 80mL of the diluent was added to the flask. The resulting mixture was subjected to sonication for a duration of 30 minutes to ensure proper dissolution. Next, 1mL of the solution was taken and further diluted with a 100mL volumetric flask, filling it up to the mark with the diluent. This step resulted in a final solution containing 50μg/mL of Amoxicillin+Tenofovir. To ensure the clarity and purity of the prepared solution, 0.45μm membrane filters were employed for filtration. The filtration process helps remove any particulate matter or impurities, resulting in a clear and suitable solution for analysis. By following these steps, a standardized and properly prepared solution of Amoxicillin+Tenofovir with the desired concentrations was obtained, ready for further analysis and evaluation.

5.3.2 Application for spiked human plasma samples:

To assess the linearity and range in plasma, the preparation of different standard solutions was done by dilution of standard stock solution and plasma with diluent in various concentrations of (Amoxicillin and Tenofovir). The preparation of plasma samples was carried by adding 1ml plasma and 1ml of standards solutions to centrifuge tube, 3ml of methanol/water (1/1). Vortexed for 2 minutes. The next step involved centrifugation. The solution was subjected to centrifugation at a speed of 4000 revolutions per minute (rpm) for a duration of 10minutes. The separation of the supernatant layer and the estimation by UPLC was done (Fig 5).Amoxicillin (1-5-10-15-20µg/ml).Tenofovir (1-5-10-15-20 µg/ml).

By using the same conditions, the analysis of three injections from each concentration were done. To assess the linearity of the calibration curve, a linear regression analysis using the least square method was employed.(fig 6,7)

Fig. 1 Chemical structure of tenofovir (a) and Amoxicillin (b)

Fig. 2 Linearity diagram of Tenofovir

Fig. 3 Linearity diagram of Amoxicillin

Fig.4 UPLC chromatogram of Tenofovir and Amoxicillin

Fig. 5 UPLC chromatogram of Tenofovir and Amoxicillin in plasma

Fig.6 Linearity of Amoxicillin in plasma

Fig.7 Linearity of Tenofovir in plasma

6. EVALUATION OF GREENNESS METHOD:

Analytical eco-scale is a unique method that was used to assess the greenness of the proposed method in comparison to the reported UPLC method.

An effective semi-quantitative tool for assessing of any greenness of analytical technique is called the analytical eco-scale. To assess the greenness of the proposed method and the reported UPLC method, the calculation of penalty points for two key parameters can be considered: the reagent parameter and the instrumentation parameter. The calculation of the reagent parameter was performed based on the quantities, physical, environmental, and the risks for the utilized reagents. The instrumentation parameters as energy consumption, quantity of waste produced by the equipment and occupational hazards. Green analytical procedure index (GAPI) is another technique for evaluating the greenness of analytical methodology¹⁶. GAPI was observed to be more progressive tool to evaluate the greenness. The various steps in this analytical process which started from the preparation of sample to the final detection, were represented by fifteen-segment pictograms. In order to show the high, medium, or low environmental effect for every phase of the analytical process, each segment comprised specific codes for each color (green, yellow, or red)¹⁷.

GAPI was used to identify Tenofovir and Amoxicillin in their pharmaceutical formulations, the results showed that distilled water solvent (green solvent) was utilized, and the results were yellow color, demonstrating that simple preparation was managed. The pictogram graph demonstrating that the adherence of UPLC method to GAPI criteria to considerable extent to their little impact on human health and environment as shown in Table (7). For concurrent research of Tenofovir and Amoxicillin in their pharmaceutical formulations with little laboratory requirements, the excellent green UPLC method has been developed.

Table 7: Results for evaluation of greenness of the proposed approach

Analytical Eco Scale Score
Reagents	No of pictograms	Signal word	Sub-total PP
1- MeOH	3٫000	Danger=2	6٫000
2- Water	0٫000	Danger=0	0٫000
3- O-phosphoric.a	1٫000	Danger=2	2٫000
	Total PP for reagents		4٫000
Imstrument	UPLC=2		2٫000
Occupational hazard	Not emit vap and gases = 0		0٫000
Waste	1-10ml = 2		2٫000
	Total PP for instruments		4٫000
	Total PP		8٫000
	Analytical Eco-Scale total score:		92٫000
Green Analytical Procedure Index(GAPI)

CONCLUSIONS:

The research described in this paper presents a straightforward analytical method for identifying TEN and AMX in a binary combination. The technique is used to measure the amount of the examined compounds in their tablets. The proposed method can therefore be advised for routine analysis and quality control of tablets containing the two medications. By using distilled water as a green solvent, methanol, and phosphoric acid as reagents, the UPLC approach's assessment of its eco-friendliness was found to comply with GAPI requirements. As a result, it is environmentally friendly. Overall, all these characteristics allow the green UPLC technique to be properly used in the routine work in the laboratories of quality control.

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Received on 07.06.2023 Modified on 14.07.2023

Research J. Pharm. and Tech 2024; 17(1):74-80.

DOI: 10.52711/0974-360X.2024.00012