Int J Curr Pharm Res, Vol 9, Issue 5, 134-139Original Article
PROTEIN BINDING STUDY OF FELODIPINE USING VALIDATED LIQUID CHROMATOGRAPHIC METHOD
SWARNA VIJITHA*, P. RAJAVEL, K. UDAYASREE
Department of Pharmaceutical Analysis, Sree Vidyanikethan College of Pharmacy, Sree Sainath Nagar, Chandragiri (Mandal), Tirupati, Chittoor (Dist), A. P.
Email: vijitha.mayu@gmail.com
Received: 22 May 2017, Revised and Accepted: 22 Jul 2017
ABSTRACT
Objective: The aim of present study was to develop and validate a new simple, easy, selective, precise, accurate reverse phase high-performance liquid chromatography for the estimation of felodipine in bulk and pharmaceutical dosage form.
Methods: The separation was carried on HPLC system consisting C18 column (150 mm ×4.6 nm, 5 µm) at room temperature coupled with a phenomenixcolumn silica with flow rate 1 ml/min. The mobile phase used was methanol: acetonitrile in the ratio of 50: 50. The drug was detected using UV-visible detector at the wavelength of 230 nm and run time was 10 min.
Results: The retention time was 3.138 min. Linearity was observed in the concentration range of 5-25μg/ml. The accuracy of the method was assessed by percentage recovery studies at three different levels at 80%, 100% and 120% of its working concentration. The percentage recovery of felodipine in the developed method was found to be in the ranges of from 99.81-100.00% that indicates the good accuracy of the method. The percentage % RSD of precision was found to be less than 2%. The method was validated as per ICH guidelines. The developed method was employed in in vitro protein binding studies using semi permeable membrane and performed by plotting calibration curve (peak area vs concentration) the % drug release of felodipine was calculated.
Conclusion: The proposed method was found to be simple, precise, accurate and consistent. The validated parameters are statistically validated for linearity, precision and limit of detection, limit of quantification, robustness, ruggedness were concluded.
Keywords: Felodipine, RP-HPLC, ICH guidelines, %RSD
© 2017 The Authors. Published Published Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2017v9i5.22157
INTRODUCTION
Felodipine is a second-generation calcium channel blocker and commonly used antihypertensive agent. Felodipine belongs to the dihydropyridine group of calcium channel blocker (with amlodipine, isradipine, and nifedipine) and is used primarily in the therapy of hypertension. Like other calcium channel blockers, felodipine acts by blocking the influx of calcium ions into the vascular smooth muscle of voltage-gated sensitive channel cells by relaxing L-type calcium channel in their inactive conformation. By preventing the influx of calcium in smooth muscle cells. Felodipine chemically, ethyl methyl-4(2, 3-chlorophenyl)-1, 4-dihydro-2, 6-dimethyl-5methyl ester, is a calcium agonist widely used in the treatment of hypertension, heart failure, and angina pectoris.
The molecular of felodipine is C18H19C12NO4 and molecular weight 384.254g/mol. It is a white or light yellow, crystalline powder, practically insoluble in water, freely soluble in acetone, acetonitrile, ethanol, methanol, with melting point 145 °C [1]. Felodipine is also used to treat mild to moderate critical hypertension [3]. Felodipinestructure was shown in fig. 1.
In literature, very few RP–HPLC [4-8], UV-Spectrophotometric [9] and HPTLC [10], methods have been reported for determination of felodipine in bulk drugs and pharmaceutical formulations and also in combination with other drugs. Hence an attempt has been made to develop a new assay method with less retention time for protein binding study of felodipine.
MATERIALS AND METHODS
Chemicals and reagents
Felodipine gift sample was procured from Madras pharmaceutical private limited. The com mercial tablet dosage form was obtained from local market labelled felogard (5 mg felodipine). HPLC methanol, acetonitrile and water were obtained from MerckspecialtiesPvt. Chennai, India. All the chemicals and reagents were analytical grade.
HPLC instruments and chromatographic conditions
The RP-HPLC method development and partial validation studies are performed using Shimadzu SPD 20ALC20Asystem. The column used for separation of the analyte is a phenomenex C18 column (150 mm ×4.6 mm 5 µm) isocratic elution with methanol: acetonitrile as mobile phase (50:50). The sample was injected through Rhenodyne injector mode. The data was analyzed and saved in spinochrome software. The flow rate of the mobile phase is 1.0 ml/min.
Preparation of mobile phase
The mobile phase was a mixture of methanol: acetonitrile, 50:50v/v solution. The mobile phase was filtered by using 0.45 µm membrane filter and sonicated for 15 min prior to use. The mobile phase is also used as diluent.
Preparation of standard stock solution
Weighed accurately about 50 mg of felodipine and transferred into 50 ml volumetric flask and dissolved in 20 ml of diluent. The final volume made up to the mark with diluent to get 1000 µg/ml concentration. From the above stock solution (1000 µg/ml) 1 ml was pipetted out and transferred into a 10 ml clean and dry volumetric flask and diluted with solvent to get 100 µg/ml solution.
Selection of wavelength
From the stock solution (100µg/ml). Further dilutions were made to get 10 µg/ml solution was scanned in UV-visible spectrophotometer in the range of 200-400 nm, where the mixture of methanol (50%) and acetonitrile (50%) was used as a blank. The wavelength of maximum absorbance (λmax) of felodipine was found at 230 nm. The spectrum was shown in fig. 2.
Preparation of sample solution for assay
Twenty tablets of felodipine were weighed and the average weight was calculated. Tablets were finely powdered and the weight equivalent to 50 mg (1.57g) was transferred into a 50 ml volumetric flask 25 ml of diluent was added and sonicated for 20 min. The final volume was made up to the mark with diluent to get 1000 µg/ml solution. The above solution was filtered by using Whatman filter paper 42. From the above solution (1000 µg/ml), 1 ml was transferred into 10 ml volumetric flask and made up to the mark with diluent to get 100 µg/ml solution. Further dilution was made by pipetting out 1 ml of 100 µg/ml solution into 10 ml volumetric flask and made up to the mark with diluent to get 10 µg/ml solution. This solution was injected under chromatographic conditions and the peak area was calculated.
Method development
The HPLC procedure optimized with a view to developing a method for Assay. The column used for separation of analyte was phenomenex C18 column (150 mm × 4.6 mm 5 µm) using isocratic elution with methanol and acetonitrile as mobile phase (50:50). The sample was injected through ryanodine injector mode. The data was acquired, stored and analyzed in spinchrome software. The flow rate of mobile was 1.0 ml/min at room temperature. The retention time of both standard and sample felodipine was 3.140 min. The peak shape of felodipine was found to be symmetrical.
Validation of the developed method
The developed method was validated as per ICH guidelines for the following parameters.
Linearity
The linearity of a method is its ability to obtain test results that are directly proportional to the sample concentration over a given range. The linearity was performed in the concentration range of 5 to 25μg/ml (5, 10, 15, 20, and 25). Then the five standard solutions of felodipine were injected into HPLC system at 230 nm by using diluent. The peak area and concentration were plotted to get a standard calibration curve were shown in fig. 3.
Precision
The closeness of agreement between a series of measurements. Multiple sampling of homogeneous samples under a prescribed condition. The precision of the method was demonstrated by method precision, system precision, intraday precision, interday precision.
System precision
The system precision was performed by injecting six replicate injections of the standard solution into HPLC system. The peak area and RSD were calculated, were found to be not more than 2%.
Method precision
The method precision performed by preparing six replicate sample preparations as per testing procedure and injected into HPLC system. The peak area and RSD were calculated, were found to be not more than 2%.
Intraday precision
Intraday precision performed by preparing six replicate sample preparations as per testing procedure and injected into HPLC system. The peak area and RSD were calculated were found to be not more than 2%.
Interday precision
Interday precision performed by preparing six replicate sample preparations as per testing procedure and injected into HPLC system.
Accuracy
The accuracy of an analytical method is the closeness of results obtained by that method to the true value for the sample. It is expressed as recovery (%) and determined by the standard addition method. Samples were spiked with 80%, 100%, and 120% of the standard and analyzed. The experiment performed in triplicate. Recovery (%) and RSD (%) were calculated for each concentration.
Limit of detection (LOD)
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantified as an exact value. The detection limit (LOD) may be expressed as
LOD = 3.3 σ/S
Where,
σ = the standard deviation of the response
S = the slope of the calibration curve
Limit of quantification (LOQ)
LOQ is defined as the lowest concentration of the substance (analyte) in a sample that can be estimated quantitatively with acceptable precision, accuracy and reliability by a given method under stated experimental conditions. Quantification limit (LOQ) may be expressed as
LOQ = 10 σ/S
Where,
σ = the standard deviation of the response
S = the slope of the calibration curve
Robustness
The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. It is concluded that the method is robust as it is found that the %RSD is less than 2. The results are shown in table 3.
Ruggedness
Ruggedness is defined as the reproducibility of results when the method is performed under actual use conditions. This includes different analysts, laboratories, columns, instruments, the source of reagents, chemicals, solvents etc. Method ruggedness may not be known when a method is first developed, but insight is obtained during subsequent use of that method. The results are shown in table 4.
Protein binding studies
Calibration curve
The calibration curve was performed in the concentration range of 10 to 60μg/ml. Then the six standard solutions of felodipine were injected into HPLC system at 230 nm by using diluent. The peak area and concentration were plotted to get a standard calibration curve was shown in fig. 6.
Preparation of bovine serum albumin
Bovine serum albumin solution was prepared by dissolving the 300 mg of bovine serum albumin in 25 ml of distilled water.
Preparation of stock solution
Weighed accurately about 50 mg of felodipine was transferred into clean and dried 50 ml volumetric flask. To that 25 ml of diluent was added and sonicated for 5 min and the final volume was made up to the mark with diluent to obtain 1000µg/ml. From the above solution, 100µg/ml concentration was prepared by transferring 1 ml into 10 ml volumetric flask and made up to the mark with diluent.
Preparation of semi permeable membrane
An egg shell was kept in concentrated HCL solution for about five minutes. The outer layer gets dissolved leaving membrane. The membrane was removed and washed with distilled water.
Procedure
Both sides open ended cylinder was taken and semi permeable membrane was tied to one end. 20 ml of bovine serum albumin and 20 ml of 100µg/ml felodipine solution was placed inside the semi permeable membrane. The cylinder was kept inside the beaker containing mobile phase.5 ml of solution was collected at different intervals (10-60 min) from the beaker and the same volume was replaced with mobile phase to maintain sink conditions. Then the solution was injected into HPLC system. From the chromatogram peak area was noted and percentage drug release was calculated. Results are shown in table 5.
RESULTS AND DISCUSSION
The current RP-HPLC technique is most accurate, reliable and precise method of analysis for the quantitative estimation of felodipine in bulk drugs and its pharmaceutical dosage form. The current method system suitability was accounted by measuring tailing factor, plate count for the peak of felodipine from standard solution and percentage RSD of the area for the peak of felodipine from six replicate injections of standard solution. The obtained values are 0.9 as tailing factor (should not be more than 2.0), 15500 as plate count (should not be less than 2000), and percentage RSD of the area as 0.5 (should not be not more than 2.0). The typical felodipine retention time in current developed method was about 3.140. All Variations resulted with the very good percentage RSD of assay i.e. less than 2.0%.
Linearity and range
The calibration curve showed good linearity in the range of 5–25µg/ml for felodipine API with correlation coefficient (R2) value of 0.994 (fig. 1). A typical calibration curve has the regression equation y = 149.9+153.2 for felodipine.
Precision
Method precision
Method precision was determined by performing sample under the test concentration showed % RSD less than 2 concerning % assay of the drug which indicate that the method developed is method precise by the test of repeatability and hence can be understood that the method gives consistently reproducible results shown in table 4.
System precision
Six consecutive injections of the sample at working concentration showed % RSD less than 2 concerning % assay of the drug which indicates that the method developed is system precise by the standard of repeatability and hence can be understood that the method gives consistently reproducible results shown in table 4.
Intraday precision
Intraday precision studies were performed by injecting a standard solution of 10μg/ml at several intervals i.e., 1, 3 and 5 h within a day and the relative Standard deviation was calculated. The results are shown in table 4.
Interday precision
The Interday precision of the proposed method was determined by analysing the corresponding responses on different days for 10 µg/ml felodipine standard solution. The relative standard deviation was calculated. The results are shown in table 4.
Limit of detection and limit of quantification
Method sensitivity was estimated in terms of limit detection and limit of quantification by using formula. The detection of LOD and LOQ is based on the standard response of felodipine by plotting separate calibration curve.
LOD=3.3×150.123/165.7=2.9 µg/ml
LOQ=10×150.123/165.7=9.0 µg/ml
Where s is the slope of calibration plot, σ is standard deviation calculated using values of y-intercept of the regression equation.
Accuracy
The mean recovery of the method determined by standard addition method and found to be 100%. The values of % recovery and % RSD, listed in table 3 indicate that the method is accurate.
Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters. For the proposed method robustness was checked by making small deliberate changes in wavelength i. e,±0.2 nm. After making these small changes showed thevery slight difference in peak area for estimation of felodipine. The values are given in table 2 point out method is robust.
Ruggedness
It is the reproducibility of test results under operating condition from analyst to analyst, column to column variations. The ruggedness of the method was performed by comparing the results between two analysts, columns in the same laboratory and results given table 3.
Protein binding studies
Protein binding studies were performed based on the calibration curve (concentration vs peak area) and then calculating the percentage drug release.
Fig. 1: Structure of felodipine.
Fig. 2: UV spectrum of felodipine
Fig. 3: Calibration curves of felodipine
Fig. 4: Typical chromatogram of standard felodipine
Fig. 5: Typical chromatogram of sample felodipine
Fig. 6: Standard graph of felodipine
Fig. 7: Drug release kinetics of felodipine
Table 1: Accuracy of felodipine
| S. No. | Recovery level | Amount added (µg/ml) |
Amount spiked (µg/ml) |
% recovery | Statistical analysis of % recovery |
| 1. | 80% | 10 10 10 |
8 8 8 |
99.75 99.3 100.4 |
Mean Std. dev %RSD |
| 2. | 100 % | 10 10 10 |
10 10 10 |
100.35 99.9 99.5 |
Mean Std. dev %RSD |
| 3. | 120% | 10 10 10 |
12 12 12 |
99.81 100.06 100.15 |
Mean Std. dev %RSD |
Table 2: Robustness of felodipine
| S. No. | Concentration(µg/ml) | Peak area at different wavelengths |
| 228 nm | ||
| 1. | 10 | 3321.038 |
| 2. | 3283.931 | |
| 3. | 3318.351 | |
Mean = 3341.106 SD = 50.437 %RSD=1.509 |
Mean = 3605.878 SD = 50.437 %RSD=1.509 |
Table 3: Ruggedness of felodipine
| S. No. | concentration(µg/ml) | Peak area at different analysts | Peak area of different columns |
| Analyst-1 | Analyst-2 | ||
| 1. | 10 | 1270.200 | 1266.450 |
| 2. | 1260.458 | 1284.904 | |
| 3. | 1266.449 | 1281.802 | |
Mean = 1275.702 SD = 6.949 %RSD=0.5 |
Mean = 1285.012 SD = 9.83 %RSD=0.7 |
Mean = 1439.10 SD = 21.075 %RSD=1.4 |
Table 4: Validation and system suitability parameters
| Parameters (units) | Results |
| Linearity range(µg/ml) | 5-25 |
| Correlation coefficient | 0.994 |
| Slope | 149.9 |
| Intercept | 153.2 |
| Recovery (%) | 100 |
| Assay | 100% |
| System precision | 1.3 |
| Method precision | 1.4 |
| Intraday precision | 0.7 |
| Interday precision | 0.9 |
| Robust | Robust |
| Retention time | 3.140 |
| Tailing factor | 1.5 |
Table 5: Drug in vitro protein binding study data
| S. No. | Time (min) | % drug release |
| 1. | 10 | 21.038 |
| 2. | 20 | 32.076 |
| 3. | 30 | 49.114. |
| 4. | 40 | 66.123 |
| 5. | 50 | 82.345 |
| 6. | 60 | 96.806 |
CONCLUSION
There is no specific and short retention time analytical method reported in literature for felodipine. The developed method gaveadequate resolution with a short analysis time. The method was validated as per ICH guidelines. All the validation parameters were found to be well within the acceptance criteria. We conclude that the method was accurate, reproducible, repeatable, linear, precise, and reliable. This method is used to perform protein binding study of felodipine with egg membrane and the total drug release after 60 min was found to be 96.8% and it was good agreement with reported pharmacokinetic data and drug release kinetics.
Hence the developed methodis used for routine analysis of felodipine in bulk and pharmaceutical formulations in quality control and pharmacokinetic studies.
ACKNOWLEDGEMENT
Authors are thankful to Dr. Mohan Babu, chairmen, Sree Vidyanikethan College of Pharmacy Sree Sainath Nagar, Chandragiri (Mandal), Tirupati, Chittoor (Dist), A. P. for providing the necessary facilities to carry out this research work.
CONFLICT OF INTERESTS
The authors declare no conflict of interest
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How to cite this article
Swarna Vijitha, P Rajavel, K Udayasree. Protein binding study of felodipine using validated liquid chromatographic method. Int J Curr Pharm Res 2017;9(5):134-139.