A Novel Eco-Friendly Approach using the Concept of Hydrotropy for Analytical Estimation of Tiopronin by HPLC used to Treat Cystenuria
Harsh D Adhvaryu*, Vandana B Patel
Department of Quality Assurance, PIPR, Parul University, Vadodara, Gujarat, India.
*Corresponding Author E-mail: harshadhvaryu@gmail.com
ABSTRACT:
Introduction: In the pharmaceuticals industry, generally it is required to have aqueous solutions of a variety of insoluble drugs. To further increase aqueous solubility can be considered an innovation for increasing the efficacy and reducing adverse effects for certain drugs. In quantitative analysis of poorly water-soluble drugs there are requirements of various organic solvents. Hydrotropic materials can be a gateway to preclude the use of organic solvents and develop specific, precise, accurate and stability indicating methods for drug analysis. Objective: To select a suitable hydrotropic agent for Tiopronin and optimize its concentration of hydrotrope by developing suitable HPLC method followed by method validation of an developed analytical method by HPLC as per ICH Q2(R1). Methodology: The method was developed using 0.03M Trisodium Citrate in water as the mobile phase and a Kromasil C18 (250mm*4.6mm) 5µm column, Flow rate- 1.0mL/min, and wavelength- 235 nm. Results: The linearity range of 10-150% met the correlation coefficient > 0.998. The %RSD for method precision was < 2.0%. The recovery range of 50-150% met 98-102%. Conclusion: The analytical procedure was successfully developed and validated for specificity, Method precision, linearity, accuracy, and system suitability. Further applied for routine testing for the estimation of Tiopronin by HPLC. Additionally, the comparative greenness of the developed analytical method was superior to other literature methods.
KEYWORDS: Tiopronin, HPLC, Hydrotropy, Analytical Estimation, Greenness, ICH Q2(R1).
1. INTRODUCTION:
In pharmaceutical industry, solubility plays a vital phenomenon for efficacy of poorly water-soluble drugs. Aqueous solubility is a critical factor for bioavailability. Hydrotropes, which enhance the solubility of poorly soluble compounds, offer a green alternative to the traditional use of organic solvents. The present study focuses on developing and validating an eco-friendly, specific, precise and analytical method using the concept of hydrotropy for the estimation of Tiopronin without the use of organic solvents and a comparative study was provided for greenness of development method compared to literature method1-5.
2. OBJECTIVE:
Current research paper includes hydrotropic method development for analytical estimation of Tiopronin and comparative greenness of developed method with literature method.
3. METHODOLOGY:
3.1 Optimized Chromatographic Conditions6-10
· Mobile phase: 0.03M Trisodium Citrate in water (Organic Hydrotrope)
· Column: Kromasil C18 (250mm * 4.6mm) 5µm
· Flow: 1.0 mL/min
· Temperature: 25°C
· Injection Volume: 10µL
· Wavelength: 235nm
· Sample Concentration: 100 ppm
3.2 Buffer Preparation:
Take 900ml of Milli Q water, weigh and transfer 7.7g of Trisodium Citrate in water and dissolve further make up volume to 1000 mL with Milli-Q water.
3.3 Mobile Phase Preparation:
Use the buffer as the mobile phase.
3.4 Standard Solution Preparation:
Weigh 100mg of Tiopronin standard and transfer into a 100mL volumetric flask, add 70mL of diluent and further sonicate to dissolve. Make up volume with diluent and mix. Pipette out 5mL of the standard stock solution and transfer to 50mL volumetric flask and dilute with diluent upto the mark.
3.5 Sample Solution Preparation:
Weigh 100 mg of Tiopronin standard and transfer into a 100 mL volumetric flask, add 70mL of diluent and further sonicate to dissolve. Make up volume with diluent and mix. Pipette out 5mL of the standard stock solution and transfer to 50mL volumetric flask and dilute with diluent upto the mark.
The developed method is validated as per ICH Q2R1 with below listed parameters11-15.
· System suitability
· Specificity/selectivity
· Linearity
· Accuracy
· Method precision
3.6 Preparation of Solution:
Linearity Solution preparation16-18
Prepare a stock solution containing 1000µg/mL of Tiopronin. Prepare a series of linearity solutions as shown in Table 1 and inject into the chromatography.
Table 1: Linearity Study Data (The table presents the % linearity levels, the volume of stock solution added, dilution, and resulting concentration in µg/mL.)
|
S. No. |
% Linearity |
Linearity Stock Solution (mL) |
Dilution |
Concentration (µg/mL) |
|
1 |
10 |
10 |
100 |
10 |
|
2 |
25 |
5 |
200 |
25 |
|
2 |
50 |
5 |
100 |
50.0 |
|
2 |
100 |
5 |
50 |
100.0 |
|
2 |
150 |
15 |
100 |
150.0 |
Accuracy solution preparation19-20
Table 2: Accuracy Solutions Preparation (This table presents the accuracy levels, the amount of API spiked, dilution, and the resulting concentration in µg/mL.)
|
S. No. |
Accuracy Level (%) |
Stock of API Spiked (mg) |
Dilution (mL) |
Volume |
Dilution (mL) |
Concentration*(µg/mL) |
|
1 |
50 |
50 |
100 |
5.0 |
50 |
50.0 |
|
2 |
100 |
100 |
100 |
5.0 |
50 |
100.0 |
|
3 |
150 |
150 |
100 |
5.0 |
50 |
150.0 |
*Prepare 3 sets of each accuracy level of 50%, 100% and 150%.
Method Precision21-22
Sample Preparation:
Weigh 100mg of Tiopronin standard and transfer into a 100mL volumetric flask, add 70mL of diluent and further sonicate to dissolve. Make up volume with diluent and mix. Pipette out 5mL of the standard stock solution and transfer to 50mL volumetric flask and dilute with diluent upto the mark.
Prepare other 5 sets as per above procedure.
4. RESULTS AND DISCUSSION:
4.1 System Suitability:
Figure 1: Standard chromatogram:
Acceptance Criteria:
· Tailing factor of standard solution-1 should be NMT 2.0.
· Theoretical plate of standard solution-1 should be NLT 2000.
· %RSD for5 replicate injections of standard should be NMT 3.0%.
Table 3: System suitability details
|
Sr. No |
System suitability |
Observation |
Acceptance criteria |
|
1 |
Retention Time (min) |
3.2 |
- |
|
2 |
Theoretical Plates |
4522 |
NLT 2000 |
|
3 |
Tailing Factor |
1.8 |
NMT 2.0 |
|
4 |
Mean Area |
546379 |
- |
|
5 |
% RSD |
1.36 |
NMT 3.0% |
CONCLUSION:
All System suitability parameters meets acceptance criteria. Hence, above method can be considered suitable for indented use.
4.2 Specificity/Selectivity:
Figure 2: Blank chromatogram
Figure 3: Standard chromatogram
Figure 4: Sample chromatogram
Acceptance Criteria:
There should not be any interference at the RT of the Tiopronin peak.
CONCLUSION:
Considering no interference of blank at the RT of Tiopronin in standard and sample. Hence it can be concluded that test method is specific for estimation of Tiopronin.
4.3 Linearity:
Table 4: Linearity Results
|
Linearity Range |
Concentration (ppm) |
Area |
|
10% |
10.0 |
58945 |
|
25% |
25.1 |
144430 |
|
50% |
50.1 |
270113 |
|
100% |
100.2 |
535962 |
|
150% |
150.3 |
873382 |
|
Correlation Coefficient |
0.998 |
|
|
Slope |
10450 |
|
|
Intercept |
60311 |
|
Figure 5: Linearity Graph
Acceptance Criteria:
Coefficient of determination (r2) should be not less than 0.998.
CONCLUSION:
From the above results, Coefficient of correlation (r) meets the pre-defined acceptance criteria. Hence method can be concluded as Linear over the range of 10-150%.
4.4 Accuracy:
Table 5: Accuracy Results
|
Levels |
Actual Concentration (ppm) |
Obtained Concentration (ppm) |
% Recovery |
Mean % recovery |
% RSD |
|
50 % |
50.2 |
49.53 |
98.7 |
99.5 |
0.87 |
|
53.7 |
53.4 |
99.4 |
|||
|
50.5 |
50.7 |
100.4 |
|||
|
100 % |
98 |
96.3 |
98.3 |
98.3 |
0.17 |
|
99.8 |
98.3 |
98.5 |
|||
|
94.1 |
92.4 |
98.1 |
|||
|
150% |
158.2 |
160.2 |
101.2 |
101.2 |
0.42 |
|
166.8 |
168.0 |
100.7 |
|||
|
157.5 |
160.0 |
101.6 |
Acceptance Criteria:
% Recovery of Tiopronin at all levels should be between 98.0 to 102.0.
CONCLUSION:
From the results it is concluded that the method can be considered as accurate in the range of 50% to 150%.
4.5 Method Precision:
Table 6: Results of Method Precision
|
Injection no. |
%Assay |
|
1 |
98.3 |
|
2 |
98.6 |
|
3 |
101.9 |
|
4 |
99.4 |
|
5 |
101.4 |
|
6 |
101.3 |
|
Mean |
100.1 |
|
SD |
1.57 |
|
% RSD |
1.57 |
Acceptance Criteria:
RSD of six preparations should not be more than 2.0%
CONCLUSION:
Based on above results, % RSD of assay results of Tiopronin meets the pre-defined acceptance criteria. Hence method can be considered as Precise.
5. COMPARISON OF METHODS:
Table 7: Comparison of Developed method and Literature method
|
Criteria |
Literature Method (Organic Solvent-Based) |
Developed Method (Hydrotropy-Based) |
|
Environmental Impact |
Higher, due to organic solvents (e.g., acetonitrile) |
Lower, as it uses water-based systems and non-toxic hydrotropes |
|
Safety |
Risks from toxic, flammable organic solvents |
Safer, as water and hydrotropes are typically non-toxic |
|
Sustainability |
Less sustainable due to high solvent use and waste disposal |
More sustainable with water-based solutions and biodegradable agents |
|
Cost |
Higher due to solvent costs and waste disposal |
Potentially lower due to cheaper hydrotropes and less waste disposal |
|
Efficiency |
Well-established, high precision, but more hazardous |
May require more optimization, but can achieve similar efficiency |
|
Waste Management |
Complex and costly (organic solvent disposal) |
Easier and cheaper (water-based waste) |
6. CONCLUSION:
The proposed method was found to be specific, precise,novel, rapid, accurate, green, and economical for the estimation of Tiopronin by HPLC using the concept of hydrotropy. This method provides a more sustainable and safer alternative compared to traditional organic solvent-based methods. It can be applied effectively for routine testing and has a lower environmental impact.
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Received on 18.03.2025 Revised on 14.07.2025 Accepted on 04.09.2025 Published on 20.05.2026 Available online from May 25, 2026 Research J. Pharmacy and Technology. 2026;19(5):2009-2013. DOI: 10.52711/0974-360X.2026.00287 © RJPT All right reserved
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