Int J Curr Pharm Res, Vol 14, Issue 5, 20-24Original Article
A NOVEL RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTIFICATION OF A POTENTIAL ANTI-DIABETIC DRUG METFORMIN HYDROCHLORIDE IN TABLET DOSAGE FORM
JAYASHREE A. HIREMATH1*, HARISH KUMAR1
*1Department of Pharmaceutical Chemistry M. S. Ramaiah University of Applied Sciences, Bengaluru 560054, Karnataka, India
Email: jaya98862@gmail.com
Received: 16 Jun 2022, Revised and Accepted: 22 Jul 2022
ABSTRACT
Objective: This study was conducted to develop a simple, economical, linear, rapid method for the assay studies of Metformin HCl by RP-HPLC method and to carry out the method validation.
Methods: A simple, robust and accurate method to carryout assay of Metformin hydrochloride tablet(500 mg) by RP-HPLC method in which the stationary phase used is Shimadzu shim-pack GIST C18 column with specification (5μm×4.6×250 mm). This method involves isocratic elution of mobile phase containing 70% buffer and 30% acetonitrile. The buffer used for analysis is Tetra-Butyl Ammonium Hydroxide (0.002%), the flow rate was maintained at 0.5 ml/min. detection was done at 232 nm. Principal peak for Metformin was observed at 3.5 min and the runtime for each injection was set to 10 min. The standard solutions of Metformin were prepared using purified water (milli-Q water) and scanned from 190 nm to 400 nm. Sharp peaks were observed in the range of 232 nm and thus, wavelength of 232 nm was selected and used throughout the validation process.
Results: Linearity graph generated was found acceptable and accurate and the graph was generated in the range of 50% to 150% concentration. The regression coefficient was found to be 0.999(acceptable range). Validation was carried out according to ICH guidelines and found to be acceptable.
Conclusion: This developed method was found to be simple, robust, economical, accurate, linear and can be used in the assay of Metformin tablet using RP-HPLC.
Keywords: Reverse phase-HPLC (RP-HPLC), Limit of detection, Limit of quantification, Method validation, System suitability, Relative standard deviation, Specificity, Linearity, Range, Precision, Intermediate precision, Accuracy, Solution stability and system suitability
© 2022 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijcpr.2022v14i5.2017 Journal homepage: https://innovareacademics.in/journals/index.php/ijcpr
INTRODUCTION
Analytical chemistry helps for the determination of quality, purity, safety of the chemicals and drugs by separating, quantifying and identifying the sample using certain methods and instruments. It helps in both quantitative as well as qualitative analysis of the sample, in qualitative analysis, it determines the purity and quality of the sample whereas, in quantitative analysis the concentration of the sample i.e., the amount of the expected content present within the sample can be found [1].
HPLC is High-Performance Liquid Chromatography is mainly used for the separation, identification and quantification of the components present in a mixture. Till 1960 liquid chromatography in which only glass columns were used and it was used to work in low pressure was developed to HPLC later with metal columns and high pressure. The basic working principle of HPLC is that it separates the constituent of the mixture based on relative affinities of the constituents for the stationary phase and mobile phase, which are used for the separation. Mainly there are two types of HPLC they are; reverse phase uses polar mobile phase and the non-polar stationary phase and the normal phase uses the non-polar mobile phase and polar stationary phase. Major applications of HPLC are analysing drugs, pollutants and synthetic polymers, isolation of components and used in industries in quality control departments to check the purity of the drug samples and chemicals [2].
Analytical method validation
Method validation provides evidence in documented form and gives high degree of assurance whether the developed method is suitable to produce a consistent result or not. ICH (International Conference on Harmonization) guidelines are issued by regulatory authorities to validate the newly developed method by using parameters such as accuracy, precision, intermediate precision(ruggedness), specificity, linearity and range, system suitability, robustness to test whether the developed method posess all the requirements [3].
Metformin HCl
Metformin HCl is an antidiabetic drug with a chemical name, 1-carbamimidamido-N,N-dimethylmethanimidamide hydrochloride falls under biguanide class of antihyperglycemic agent. Works by reducing production of hepatic glucose and also increases glucose uptake, it also helps to prevent complications related to cardiovascular system. Its also used for PCOS problems and also decreases levels of triglycerides and low-density lipoprotein cholesterol [4].
As per pharmacopeia only potentiometry and UV methods are mentioned for Metformin, as per European pharmacopeia and Indian pharmacopeia and as per literature HPTLC [5], HPLC [6, 8], UV [6, 9, 10], LC-MS/MS [11] were reported. Even in the mentioned HPLC method, there was a need for an improved and stable method.
Drug profile
Molecular structure
Fig. 1: Structure of metformin HCl
Chemical name: 1-carbamimidamido-N, N-dimethyl-methanimidamide hydrochloride
Molecular Formula: C4H12ClN5
Molecular Weight: 165.62
Appearance: White or almost white crystalline powder
State: solid
Melting point: 223-226 °C
pKa: 12.4
Category: Biguanide class of anti-hyperglycemic agent
MATERIALS AND METHODS
Instrumentation
This work describes a method development and validation of Metformin HCl by the RP-HPLC method. Used shimadzu: LC10AD HPLC and by using Lab solutions software, chromatography was performed by Shimadzu shim-pack GIST C18 column (5μm×4.6×250 mm) with the mobile phase composed of buffer solution (700 ml) and acetonitrile (300 ml). The flow rate was 0.5 ml/min, the Injection volume was 20 µl, and the detection is at 232 nm where column temperature was 15 ˚C. The retention time of Metformin HCl is 3.5 min and the run time for each injection was 10 min.
Preparation of buffer
Tetra butyl ammonium hydroxide was taken in a minute quantity i.e.,2 ml in 1000 ml of HPLC grade water, sonicated for better mixing of solutions using sonicator and pH was adjusted to 3 using ortho-phosphoric acid and filtered using a 0.45 µm filter paper.
Preparation of mobile phase
Mobile phase was prepared by mixing the buffer and acetonitrile in the ratio of 70:30
Standard and sample preparation
Preparation of standard solution: 50 mg of Metformin HCl (working standard sample) was weighed approximately and transferred to a 100 ml volumetric flask dissolved using mobile phase solution and made up to the volume using the same solution.
Preparation of reference solution (a): 10 ml of the prepared standard solution was transferred to 50 ml volumetric flask and diluted up to the mark using mobile phase.
Preparation of sample solution: Approximately about 5 equivalent tablets were crushed and 61.5 mg of the sample was weighed and transferred to a 100 ml volumetric flask and diluted upto the mark using the mobile phase sonicated to dissolve the drug contents and finally filtered and diluted further by taking 10 ml of this solution in a 50 ml volumetric flask and diluted upto the volume using mobile phase.
Validation of the developed method
Validation was done according to ICH guidelines for the determination of the developed RP-HPLC method for Metformin HCl in the tablet dosage form. Validation was carried out for the parameters like precision, intermediate precision (ruggedness), specificity, linearity and range, system suitability, and robustness.
RESULTS AND DISCUSSION
New method was developed for the assay studies of Metformin HCl tablet (500 mg); the following are conditions stabilized.
Table 1: Chromatographic conditions
| Chromatographic mode | Isocratic (70:30) Buffer: Acetonitrile |
| Detector wavelength | 232 nm |
| Flow rate | 0.5 ml/min |
| Injection volume | 20μl |
| Column | Shimadzu shim-pack GIST C18 column (5μm×4.6×250 mm). |
| Column oven | 25 °C |
| Run time | 10 min |
Specificity
Interference study
The blank, standard solution and sample solutions were prepared as described previously and injected in HPLC system. The retention time of all the corresponding peaks observed in the chromatogram were recorded. The obtained results are, as given in table 2. Based on the obtained result it is concluded that there is no interference due to the blank solution and placebo at a same retention time of the main peak of standard solution and sample solution chromatograms. The peak purity of Metformin HCl is 1.0
Table 2: Specificity studies
| Type of solution | Retention time (min) | Peak purity |
| Blank | No interference | NA |
| Placebo | No interference | NA |
| Standard | 3.5 | 1.0 |
| Sample | 3.5 | 1.0 |
Linearity
To evaluate the linearity of the method, 5 levels of concentrations 50%, 80%, 100%, 120% and 150% of Metformin HCl were taken. The correlation coefficient for Metformin HCl was found to be within the acceptable limit of not less than 0.999, which passes the linearity parameter.
Table 3: Linearity studies
| Concentration (%) | Area |
| 50 | 9677559 |
| 76 | 15483262 |
| 101 | 21162424 |
| 119 | 25130352 |
| 150 | 32649189 |
Fig. 2: Linearity graph
Range
For range, injected six replicates of lower and higher concentration levels of the linearity level and calculated the Mean and relative standard deviation. The relative standard deviation for Metformin HCl range levels was found to be within acceptable limits of not more 2.0%.
Table 4: Range of metformin HCl
| No. of injections | Retention time | Range (50) |
| 1 | 3.517 | 9656375 |
| 2 | 3.517 | 9650591 |
| 3 | 3.517 | 9653005 |
| 4 | 3.517 | 9653793 |
| 5 | 3.517 | 9654915 |
| 6 | 3.517 | 9657619 |
| Mean | 3.517 | 9654383 |
| SD | 0.000 | 2504 |
| %RSD | 0.000 | 0.026 |
Table 5: Range of metformin HCl
| No. of injections | Retention time | Range (150) |
| 1 | 3.508 | 32520257 |
| 2 | 3.500 | 32508290 |
| 3 | 3.500 | 32467103 |
| 4 | 3.500 | 32527585 |
| 5 | 3.508 | 32510855 |
| 6 | 3.508 | 32518020 |
| Mean | 3.504 | 32508625 |
| SD | 0.005 | 25565 |
| %RSD | 0.130 | 0.091 |
Precision
Repeatability
Six sample preparations were prepared and repeatability was checked. The relative standard deviation for Metformin HCl was found to be within acceptable limits of not more 2.0%.
The % Assay of Metformin HCl was within the acceptable limit. i.e. 98%-102% of label claim.
Table 6: Precision studies
| S. No. | Sample weight (mg) | Metformin HCl area | Assay (%) |
| 1 | 497.79 | 19003498 | 99.56 |
| 2 | 493.80 | 18851319 | 98.76 |
| 3 | 492.99 | 18823564 | 98.60 |
| 4 | 491.13 | 18749274 | 98.23 |
| 5 | 495.57 | 18820469 | 99.11 |
| 6 | 501.66 | 18918691 | 100.33 |
| Mean | 18869580 | 99.098 | |
| SD | 88605 | 4061 | |
| %RSD | 0.470 | 0.02 |
Intermediate precision
The intermediate precision was checked using a different instrument on a different day and compared with repeatability results.
Table 7: Intermediate precision/ruggedness studies
| S. No. | Sample weight (mg) | Metformin HCl area | Assay (%) |
| 1 | 509.48 | 18775093 | 101.90 |
| 2 | 509.17 | 18763514 | 101.83 |
| 3 | 507.34 | 18696083 | 101.47 |
| 4 | 506.31 | 18658191 | 101.26 |
| 5 | 504.34 | 18585724 | 100.87 |
| 6 | 501.62 | 18847912 | 100.32 |
| Mean | 18716818 | 100.272 | |
| SD | 93494 | 6016 | |
| %RSD | 0.499 | 0.03 |
Table 8: Comparative studies of repeatability and intermediate precision
| Parameters | Assay (%) |
| Mean assay in repeatability | 99.098 |
| Mean assay in intermediate precision | 100.272 |
| Absolute difference | 1.174 |
The % Assay of Metformin HCl was within the acceptable limit. i.e. 98%-102% of label claim.
The relative standard deviation of assay obtained from 12 sample preparations (Repeatability and Intermediate precision) is within the acceptance criteria of not more than 2.0%.
The absolute difference between the mean assay results obtained in repeatability and intermediate precision is within the acceptance criteria of not more than 2.
Based on the above results, it is concluded that the proposed method for assay by RP-HPLC is rugged.
Accuracy
The accuracy was carried at 3 levels, at 50%, 100% and 150%. The % recovery at 50%, 100% and 150% levels is within the acceptance criteria 98.0% to 102%
Based on the below-obtained recovery results as in table 8, it is concluded that the method for assay by HPLC is accurate.
Robustness
Robustness was carried out on standard solution by making the following alterations:
• By changing wavelength
• By changing the oven temperature
• By changing the flow rate
Table 9: Accuracy/recovery studies
| Levels | Test area | Std area | Potency | Mg/tablet | % recovery | Mean recovery (%) |
| 1 | 9873462 | 188444044 | 100.1 | 250.04 | 100.02 | |
| 9535003 | 188444044 | 100.1 | 253.25 | 101.30 | 100.52 | |
| 9436227 | 188444044 | 100.1 | 250.63 | 100.25 | ||
| 2 | 18897573 | 188444044 | 100.1 | 501.92 | 100.38 | |
| 18925005 | 188444044 | 100.1 | 502.65 | 100.53 | 100.43 | |
| 18896952 | 188444044 | 100.1 | 501.91 | 100.38 | ||
| 3 | 28074970 | 188444044 | 100.1 | 745.67 | 99.42 | |
| 28521507 | 188444044 | 100.1 | 757.53 | 101.00 | 100.39 | |
| 30271908 | 188444044 | 100.1 | 755.78 | 100.77 |
Table 10: Robustness studies
| Condition | Retention time (min) | Tailing factor | Theoretical plates | %RSD |
| Normal | 3.500 | 1.249 | 3081 | 0.024 |
| Flow rate (0.4 ml) | 4.4 | 1.266 | 3186 | 0.031 |
| Flow rate (0.6 ml) | 2.9 | 1.300 | 2434 | 0.046 |
| Column oven(20℃) | 3.55 | 1.255 | 2603 | 0.0 |
| Column oven(30℃) | 3.53 | 1.280 | 2910 | 0.085 |
| Wavelength (230 nm) | 3.53 | 1.308 | 2786 | 0.009 |
| Wavelength (234 nm) | 3.53 | 1.272 | 2767 | 0.070 |
Table 11: Stability studies
| Time (h) | Standard solution | Time | Sample solution | ||
| 25 ℃ | 25 ℃ | ||||
| Retention time (min) | RT | %Assay | Absolute difference with respect to initial | ||
| Initial | 3.558 | Initial | 3.558 | 99.47 | NA |
| 6.0 | 3.567 | 6.0 | 3.567 | 99.49 | 0.02 |
| 12.0 | 3.567 | 12.0 | 3.567 | 100.17 | 0.70 |
| 18.0 | 3.567 | 18.0 | 3.567 | 100.99 | 1.52 |
| 24.0 | 3.558 | 24.0 | 3.558 | 101.90 | 2.43 |
| 30.0 | 3.567 | 30.0 | 3.567 | 102.85 | 3.38 |
| 36.0 | 3.558 | 36.0 | 3.558 | 103.85 | 4.38 |
| 42.0 | 3.558 | 42.0 | 3.558 | 104.49 | 5.02 |
| 48.0 | 3.558 | 48.0 | 3.558 | 105.39 | 5.92 |
Table 12: System suitability studies
| Condition | Retention time | Tailing factor | Theoretical plate | %RSD |
| Specificity | 3.5 | 1.161 | 2285 | 0.129 |
| Linearity | 3.5 | 1.317 | 2741 | 0.026 |
| Range | 3.5 | 1.317 | 2741 | 0.026 |
| Repeatability | 3.5 | 1.249 | 3081 | |
| Intermediate precision | 3.5 | 1.202 | 2583 | 0.499 |
| Accuracy | 3.5 | 1.205 | 3113 | 0.470 |
Solution stability
The solution stability is the parameter used to verify the stability of the prepared solutions such as mobile phase, standard solution and sample solution and to check the duration of time till the prepared solutions are stable. From this analysis, we come to the conclusion that the prepared solution is stable upto 24 h
System suitability
Standard solution was prepared as per the methodology and injected into the HPLC system before starting every validation parameter. The percentage RSD for 5 replicate injections, tailing factor and theoretical plates of standard solution are considered.
The tailing factor of Metformin HCl fulfills the acceptance criteria of not more than 2.0.
The theoretical plates of Metformin HCl fulfills the acceptance criteria of more than 2000.
CONCLUSION
The proposed method describes the analytical method development and validation of the potent antidiabetic drug Metformin HCl developed method is validated according to ICH guidelines found to be accurate, precise, simple and economical, the developed method can be used for the quantification of Metformin HCl in regular analysis.
ACKNOWLEDGEMENT
It is also my privilege to thank dr. S. Bharath, dean Ramaiah university of applied sciences, department of pharmaceutical chemistry for allowing me to carry out this work and their guidance and technical advice in Analytical study and for providing all information and documents for my project.
FUNDING
Nil
AUTHORS CONTRIBUTIONS
All the authors have contributed equally.
CONFLICT OF INTERESTS
Declared none
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