1P.E. Society’s College of Pharmacy and Research Centre Phaltan.
2Siddhant College of Pharmacy Pune.
3Loknete Shri Dadapatil Pharate College of pharmacy Mandavgan Pharata, Pune.
Existing study has a strong, precise and cost-effective contrast phase UV Spectrophotometry method for the quantitative estimate of Tenofovir Disoproxil Fumarate (TDF) in bulk and pharmaceutical dose forms. Tenofovir Disoproxil Fumarate, nucleotide reverse transcript blocker, HIV -1 and chronic hepatitis B are widely used in the treatment of infection. Due to its therapeutic importance, the development of a reliable analytical method is required for regular quality control. Chromatographic analysis was performed using nucleosyl C18K column lum, which includes methanol and water with a mobile phase, with a ratio of 70:30 V/V at 0.8 mL/min, and at 260 nm. Retention time for TDF was found to be 6.0 ± 0.03 minutes. This method was approved in accordance with the ICH Q2 (R1) guide, excellent linear (2–10 µg/ml, R² = 0.994), precision (recovery achievement 97.73–100.56%), precision (%RSD <2%), (%RSD = 2.18). System qualification parameters such as theoretical plates (9625 ± 234) and the tailoring factor (1.20 ± 0.336) were within acceptable limits The developed RP-HPLC method, in both bulk drugs and pharmaceutical formulation, proves to be simple, specific, and reliable for regular analysis of Tenofovir Disoproxil Fumarate.
Analytical chemistry plays a critical role in the pharmaceutical industry by providing precise, accurate, and reliable strategies for the distinguishing proof, quantification, and characterization of drug substances and details. Among the instrumental techniques accessible, UV Spectrophotometry has gained prominence due to its high resolution, sensitivity, and reproducibility. Tenofovir Disoproxil Fumarate (TDF) is an antiretroviral agent belonging to the nucleotide reverse transcriptase inhibitors (NtRTIs), used in the management of HIV-1 infection and persistent hepatitis B. It may be a prodrug of tenofovir, which is converted into its active diphosphate form in vivo. The drug exerts its pharmacological impact by repressing viral invert transcriptase and ending the viral DNA chain. Since of its therapeutic importance and widespread use, it is essential to develop and validate analytical strategies for its routine quality control. UV Spectrophotometry is themost commonly employed chromatographic technique in pharmaceutical analysis. It offers preferences such as faster division, high resolution, ease of mechanization, and the ability to handle both polar and non-polar compounds. UV Spectrophotometry methods are particularly suitable for soundness t hinks about and measure of dynamic pharmaceutical ingredients (APIs) in nearness of excipients or debasement products.The explanatory strategy advancement includes precise assessment of different chromaographic parameters counting selection of column, portable stage composition, stream rate, discovery wavelength, and infusion volume. Each of these parameters is optimized to achieve maximum resolution, symmetry, and negligible run time. Once developed, the method is approved agreeing to the Worldwide Committee for Harmonization (ICH) Q2 (R1) rules to guarantee unwavering quality and reproducibility. Validation parameters such as specificity, linearity, precision, exactness, limit of detection (LOD), restrain of quantitation (LOQ), strength, and framework reasonableness are essential to affirm the method’s reasonableness for its aiming purpose. Although a few UV Spectrophotometry strategies have been reported for TDF in combination with other antiretroviral operators, there's restricted writing accessible on its standalone determination in bulk and measurement shapes employing straight forward and prudent approach. Subsequently, the display consider is centered on developing and validating a straightforward, vigorous, and exact RP-HPLC strategy for the estimation of Tenofovir Disoproxil Fumarate in bulk and its pharmaceutical measurement forms.
MATERIAL AND METHOD
Method Development by using UV Spectrophotometer
Selection of Solvent
The solubility of Tenofovir Disoproxil Fumarate was determined by adding some amount of drug in the 5ml of solvents (Methanol) at room temperature.
UV Spectrum of Tenofovir Disoproxil Fumarate was carried out in methanol by dissolving 10mg of Tenofovir Disoproxil Fumarate in 100ml methanol to urge concentration of 100µg/ml and further dilution were made with methanol. A arrangement of 10 µg/ml was kept in quartz cell of path length 10mm.The UV spectrum was recorded utilizing double beam UV visible spectrophotometer in the wavelength run of 200-400nm utilizing methanol as blank and λmax was decided. The maximum absorbance was found to be at 260nm.
A stock arrangement of Tenofovir Disoproxil Fumarate was prepared by dissolving 10mg of Tenofovir Disoproxil Fumarate in methanol, final volume was made with same dissolvable to obtain standard stock arrangement of known concentration (100µg/ml) Then 0.2ml aliquot from stock arrangement exchanged in to 10ml volumetric flask, diluted with methanol. The final volume was made up to the mark with methanol to obtain arrangement 2 µg/ml. The further dilution were made in the concentration run of 2 to 10 µg/ml from standard stock arrangement in to 10ml volumetric carafe and volume was made with methanol up to the mark.
The UV absorbance of these arrangements were measured by UV obvious spectrophotometer at 260nm. From this absorbance standard curve was plotted. Its incline and relapse coefficient were calculated.
RESULT AND DISCUSSION
Test arrangement was prepared and scanned over the range of 200-400nm. The spectra were obtained. It was observed that the sedate appeared significant absorbance at 260nm.
Fig 1.1: UV spectrum of TDF
The linearity (relationship between absorbance and concentration) was determined by analyzing three or five replicates over the concentration run of 2-10µg/ml for Tenofovir Disoproxil Fumarate API. Correlation coefficient was found to be 0.994.
Table 1.1: Linearity study of Tenofovir Disoproxil Fumarate
|
Sr. No. |
Concentration(µg/ml) |
Absorbance |
Estimate conc. |
% Recovery |
|
1 |
2 |
0.059 |
2.5 |
80 |
|
2 |
4 |
0.094 |
4.09 |
97.79 |
|
3 |
6 |
0.134 |
5.90 |
101.69 |
|
4 |
8 |
0.188 |
8.36 |
95.69 |
|
5 |
10 |
0.232 |
10.36 |
96.52 |
|
|
Mean |
0.1414 |
|
|
|
|
SD |
0.0069 |
|
|
|
|
%RSD |
4.28 |
|
|
2) Accuracy
Exactness was assessed by determination of the recovery of the method by standard addition method at 3 different concentration level 80%, 100%, and 120%. % RSD was found to be 1.023, 0.7784 and 1.3601 separately.
Table 1.2: Accuracy studies of Tenofovir Disoproxil Fumarate
|
Sr. No |
% Level |
Absorbance |
% Recovery |
|
1 |
80 |
0.236 |
100.56 |
|
|
80 |
0.232 |
|
|
|
80 |
0.236 |
|
|
2 |
100 |
0.289 |
97.73 |
|
|
100 |
0.289 |
|
|
|
100 |
0.294 |
|
|
3 |
120 |
0.368 |
98.13 |
|
|
120 |
0.367 |
|
|
|
120 |
0.368 |
|
|
Mean |
0.2346 |
0.2966 |
0.3676 |
|
SD |
0.0024 |
0.002309 |
0.005 |
|
%RSD |
1.023 |
0.7784 |
1.3601 |
3) Precision
The intra-day and inter-day precision study of Tenofovir Disoproxil Fumarate was carried out. Were %RSD found to be 1.07 and 1.53 respectively.
Table 1.3: Intra-day and Inter-day precision studies of Tenofovir Disoproxil Fumarate
|
1 |
2 |
0.055 |
0.039 |
86.58 |
97.02 |
|
2 |
4 |
0.098 |
0.096 |
93.67 |
95.69 |
|
3 |
6 |
0.132 |
0.134 |
86.05 |
84.74 |
|
4 |
8 |
0.181 |
0.176 |
99.50 |
102.43 |
|
5 |
10 |
0.231 |
0.221 |
96.99 |
101.41 |
|
6 |
12 |
0.236 |
0.231 |
99.70 |
97.82 |
|
|
Mean |
0.1394 |
0.1332 |
|
|
|
|
SD |
0.0014 |
0.002 |
|
|
|
|
%RSD |
1.07 |
1.53 |
|
|
4) Robustness
Tenofovir Disoproxil Fumarate under condition during which slight change in wavelength and % RSD was found to be 2.18.
Table 1.4: Robustness studies of Tenofovir Disoproxil Fumarate
|
Sr.No |
Conc.(µg/ml) |
Wavelength(nm) |
Absorbance |
%Recovery |
|
1 |
10 |
262 |
0.229 |
97.84 |
|
|
10 |
262 |
0.229 |
97.84 |
|
|
10 |
262 |
0.230 |
97.37 |
|
2 |
10 |
258 |
0.229 |
97.84 |
|
|
10 |
258 |
0.228 |
98.23 |
|
|
10 |
258 |
0.229 |
97.84 |
|
|
Mean |
0.229 |
0.229 |
|
|
|
SD |
0.0057 |
0.0057 |
|
|
|
%RSD |
2.18 |
2.18 |
|
5) Limit of Detection
LOD was calculated for TDF. Were LOD was found to be 0.952
LOD is calculated from the formula:
LOD= 3.3 ?/s
Where
?= standard deviation of peak area of drug for the lowest conc. in the range s= slope of the calibration curve
Table 1.5: Limit of Detection of Tenofovir Disoproxil Fumarate
|
Sr. No |
Conc. (µg/ml) |
Absorbance |
% Recovery |
|
1 |
4 |
0.098 |
93.67 |
|
2 |
4 |
0.094 |
97.79 |
|
3 |
4 |
0.098 |
93.67 |
|
|
Mean |
0.096 |
|
|
|
SD |
0.002 |
|
|
|
%RSD |
2.08 |
|
Fig 1.4: Limit of Detection of Tenofovir Disoproxil Fumarate
6) Limit Of Quantitation
LOQ was calculated for TDF. Were LOQ was found to be 1.2571
LOQ is calculated from the formula:
LOQ= 10 ?/s
Where
?= standard deviation of peak area of drug for the lowest conc. in the range s= slope of the calibration curve
Table 1.6: Limit of Quantitation of Tenofovir Disoproxil Fumarate
|
Sr. No |
Conc. (µg/ml) |
Absorbance |
% Recovery |
|
1 |
2 |
0.055 |
86.58 |
|
2 |
2 |
0.059 |
80 |
|
3 |
2 |
0.071 |
65.78 |
|
|
Mean |
0.061 |
|
|
|
SD |
0.008 |
|
|
|
%RSD |
13.11 |
|
Fig 1.5: Limit of Quantitation of Tenofovir Disoproxil Fumarate
7) Assay
The analysis was performed as stated under the procedure, repeated five times. A 10µg/ml sample solution was analyzed by UV and the absorption was recorded. The concentration and % purity were determined by the equation of linearness
Table 1.7: Assay of Tenofovir Disoproxil Fumarate formulation
|
Sr. No |
Label Claim |
Amount found (mg) |
% Assay |
|
1 |
10 |
9.75 |
97.59 |
|
2 |
10 |
10.01 |
100.31 |
|
3 |
10 |
9.82 |
98.23 |
|
4 |
10 |
9.67 |
96.79 |
|
5 |
10 |
9.89 |
98.92 |
|
|
Mean |
9.83 |
98.37 |
|
|
SD |
0.13 |
|
|
|
%RSD |
1.32 |
|
Fig: 1.6 Assay spectrums for Tenofovir Disoproxil Fumarate formulation
The column was balanced with the mobile phase. The drug's working standard solution (10µg/ml) was injected into the HPLC system. Retention time for the drug was seen: TDF 6.0 ± 0.03 minutes.
Table 1.8: System suitability parameter of Tenofovir Disoproxil Fumarate
|
Property |
Response |
|
Retention time (tR) |
6.0±0.03min |
|
Theoretical plate (N) |
9625±234 |
|
Tailing factor (T) |
1.20±0.336 |
Fig 1.7 : System suitability parameter of Tenofovir Disoproxil Fumarat
Table 1.9 Summary of chromatographic parameter selected
|
Sr.No |
Parameter |
Condition used for analysis |
|
1 |
Column |
Nucleosil C18 |
|
2 |
Mobile phase |
Methanol: water (70:30 v/v) |
|
3 |
Flow rate |
0.8ml/min |
|
4 |
Detection wavelength |
260nm |
|
5 |
Injection volume |
20µL |
|
6 |
Column temperature |
300c |
CONCLUSION
A straightforward exact and temperate RP HPLC strategy was effectively created and approved for the estimation of Tenofovir Disoproxil Fumarate (TDF) in bulk and pharmaceutical details.The strategy utilized a versatile stage of methanol and water (70:30 v/v), with location at 260 nm, and illustrated a sharp and well-resolved top with a maintenance time of around 6.0minutes. Approval parameters, counting linearity, exactness, accuracy, vigor, LOD, and LOQ, were inside satisfactory limits as per ICH Q2 (R1) rules. The strategy shown fabulous reproducibility and specificity without impedances from excipients or pollutions. Hence, the created RP-HPLC strategy can be dependably utilized for schedule quality control examination of TDF in pharmaceutical businesses, guaranteeing consistency and compliance with administrative guidelines.
ACKNOWLEDGMENT
First and foremost, I would like to express my honest thanks to my guide and mentor, Prof. M. S. Rode, for their invaluable guidance, encouragement and constant support during this research work. His expertise and insight was important in the successful completion of this thesis. I expand my heartfelt thanks to the department's principal and head department SVPM’S College of Pharmacy, Malegaon BkI Tal- Baramati, Dist- Pune to provide the necessary facilities and a favorable environment to complete my research work. I am also grateful to laboratory staff and fellow researchers for their kind of cooperation and support during experimental work.
Last but not at least, I want to thank my family and friends for their constant support, patience and inspiration during my academic journey.
Conflict Of Interest
All authors have declared no conflict of interest.
REFERENCES
Zite Sonal Bapu*, Toge Sonali Vitthal, Handal Shubham Laxman, Analytical Method Development and Validation of Tenofovir Disoproxil Fumarate in Bulk and Pharmaceutical Formulation by UV Spectrophotometry, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 7, 1122-1132. https://doi.org/10.5281/zenodo.15838179
10.5281/zenodo.15838179
