HPTLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF AMLODIPINE BESYLATE AND CELECOXIB IN PURE AND COMBINED DOSAGE FORM
HTML Full TextHPTLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF AMLODIPINE BESYLATE AND CELECOXIB IN PURE AND COMBINED DOSAGE FORM
Tejaswini Rajaram Mandale * and Manish S. Kondawar
Department of Quality Assurance, ABCP, Sangli - 416416, Maharashtra, India.
ABSTRACT: HPTLC is simple, reliable, precise and accurate for separation and identification of drug in a combined dosage form which gives satisfactory accuracy in results when applied on amlodipine besylate (Amlo) and celecoxib (Celo). Toluene, ethyl acetate, methanol and conc. ammonia (6:5:1.5:0.3 v/v/v/v) was required as a mobile phase for separation and identification of drugs. These drugs were scanned by densitometry at 310 nm. The developed method was validated for linearity, precision, accuracy, LOD, and LOQ. The linearity range and correlation coefficient for Amlo and Celo were obtained to be 100 to 600 ng/ml and 2 to 12 μg/ml, 0.9971, and 0.9989, respectively. The results of the recovery study for Amlo and Celo have obtained the range of 99.52% to 99.83% and 99.83% to 99.98%, respectively. The method precision was established by repeatability study. LOD and LOQ are 17.88µg/ml and 54.20 µg/ml for Amlo and 0.39µg/ml and 1.20µg/ml for Celo, respectively. The statistical parameters and recovery data indicated that the method may be engaged for efficient, rapid drug analysis from tablet formulation. The percentage label claim present in tablet formulation was obtained to be 100.25% and 99.93% for Amlo and Celo, respectively. The outcomes of analysis clearly specified that with no interference from excipients in the formulation.
| Keywords: |
HPTLC, Amlodipine besylate, Celecoxib, Method development, Validation
INTRODUCTION: Amlodipine besylate (Amlo) chemically designed as 3-ethyl 5-methyl (4RS)-2-[(2-aminoethoxy) methyl]- 4- (2-chlorophenyl)-6-methyl- 1, 4-dihydropyridine- 3, 5- dicarboxylate benzene sulfonic acid Fig. 1A is an antihyper-tensive agent used for the treatment of hypertension 1, 2. Celecoxib (Celo) chemically known as 4-[5-(4- methyl phenyl) –3-(trifluoromethyl) pyrazol-1–yl] benzene sulphonamide Fig. 1B. Celecoxib is a NSAID (Non-Steroidal Anti-inflammatory Disorder) that exhibits anti-inflammatory, analgesic, and antipyretic activities 3.
FIG. 1A: CHEMICAL STRUCTURE OF AMLODIPINE BESYLATE
FIG. 1B: CHEMICAL STRUCTURE OF CELECOXIB
It is a purpose for preventing rheumatoid arthritis & osteoarthritis. In some cases, patients have problems, hypertension, and osteoarthritis. In that condition, they used to take both tablets and sometimes they may face the non- compliance for taking two tablets. Such conditions can be overcome in a single tablet, i.e., combination, by administering both drugs. Therefore, Amlo and Celo in a combination used for both conditions. These combinations were prepared to overcome the possibility of osteoarthritis in hypertension patients and vice versa.
As per the literature review, for estimating Amlo using HPLC, HPTLC, and UV spectrophotometry methods have been indicated for the analysis of alone or in combination with other drugs 4-11. Various methods have been stated that the analysis of Celo in a pharmaceutical formulation such as HPLC and UV spectrophotometry 12-14. Therefore, on HPTLC method developed for the simultaneous estimation of Amlo and Celo. This method shows better mobile phase composition for the determination of drugs. HPTLC method is a modification of TLC for the identification and separation of drug analysis. This technique is widely used in very many fields both for the qualitative & quantitative (identification and estimation) analysis of single and mixture of substance 15.
Due to its benefits of low operating costs, elevated sample throughput, and essential for minimum sample preparation. TLC today is quickly becoming a routine analytical technique. TLC's main benefit is that multiple samples may be run simultaneously by a small amount of mobile phase, not like HPLC, thus reducing the time and cost of analysis. It is an improved form of TLC. There are some developments to the basic thin-layer chroma-tography method to computerize the different steps, which increase the resolution and allows for more precise quantitative measurements 16.
The main objective is to develop a rapid, sensitive, and accurate HPTLC method for the simultaneous estimation of amlodipine besylate and celecoxib in pure and combined dosage form.
MATERIALS AND METHODS:
Reagents and Chemical: Standard drug sample of Amlodipine besylate and Celecoxib was provided as a gift sample from Smruthi Organics Limited, Solapur, and Aarti Drugs Limited, Mumbai respectively. Toluene, ethyl acetate, methanol, conc. Ammonia (analytical grade) was procured from Research Lab (Mumbai, India).
Instrumentation: In this HPTLC method, samples were spotted in the form of bands of width 8 mm with 100 μl sample syringe on 10 cm × 10 cm aluminum plates precoated with silica gel 60 F 254 using a Spraylin Aetron sample applicator. Densitometric scanning was performed on a Camag TLC Scanner 3. The source of radiation was a deuterium lamp emitting a continuous UV spectrum between 200 and 400 nm. An evaluation was performed using peak areas with linear regression.
Experimental Work for HPTLC Method:
Selection of Common Solvent: The solubility of drugs was estimated in different solvents by using Indian pharmacopeia standards. Solubility was performed in polar to non-polar solvents. The common solvent was established to be methanol, and it was chosen as a solvent for the HPTLC method, and it was selected on account of its readily available, cost factor, and solubility for the analysis of Amlo and Celo.
Selection of Mobile Phase: Selection of the mobile phase for the further experiment of HPTLC was prepared by using precoated thin layer plates made by silica gel 60 F254. As per the literature survey, a variety of mobile phases were tried for proper identification of Rf values of Amlo and Celo. From trial and error basis combination of mobile phase were selected.
Selection of Chromatographic Condition: After the selection of the mobile phase, the next important part was the selection of chromatographic conditions and selection of proper parameters for the sample application, plate development, documentation, and determination of plate parameters. The following are the parameters selected for the study of Amlo and Celo.
Preparation of Standard Solution: Standard drug solution (stock solution) of both drug was prepared by dissolving 10mg Amlo with sufficient to produce 10ml methanol and 200mg of Celo with sufficient 10ml methanol to produce 10ml solution separately to obtained stock solution having 1000 µg/ml of Amlo and 20,000µg/ml of Celo. All solutions were protected from the light to avoid degradation.
Selection of Analytical Wavelength: The solution of both standard drugs was suitably dilute with methanol, to acquire the concentration of 10µg/ml of Amlo and Celo separately and they can also be scanned separately in a wavelength range of 200-400 nm against blank as methanol to estimate the wavelength of maximum absorption for the drugs. From the overlain spectrum, 310 nm of wavelength was selected for Amlo & Celo detection by densitometry for measured peak area.
Preparation of Calibration Curve: For the calibration curve, dilution of 1, 2, 3, 4, 5, and 6μl of a standard solution of Amlo and Celo were applied to the TLC plate. The range of concentration Amlo was between100 to 600 ng/spot, whereas Celo has a range between 2 to 12 µg/spot. TLC plates were developed, dried and examined photometrically. The calibration curve was plotted between concentration vs. respective area for Amlo and Celo separately.
Analysis of Sample Preparation: A total of 20 tablets were correctly weighed and grinding by using a glass mortar pestle. A quantity equivalent to one tablet (containing 10 mg Amlo and 200 mg of Celo) was transferred to 10 ml flask of volumetric. The powder was firstly dissolved in few ml of methanol, and the flask were sonicated for 5 min, the volume was up to 10 ml and then filtered through Whatman filter paper, the resultant clear solution was diluted with methanol to get the concentration of 1000 µg/ml of amlodipine besylate and 20,000 µg/ml of celecoxib. The concentration of Amlo and Celo per tablet was estimated by plotting the value of the area from the calibration curve.
Method Validation: The method was validated as per the guidelines of ICH Q2 (R1), in terms of parameters like linearity, accuracy, precision, and specificity of the sample application. All the results were discussed in the result and discussion.
Linearity: The linearity of the method was investigated by serial dilution of standard solution. Six different concentrations of the standard drugs of Amlo and Celo were prepared for linearity studies and injected into the system. The response was measured as peak areas. The linearity was calculated for two drugs, amlodipine besylate, and celecoxib, separately by plotting a calibration curve for peak area against their respective concentration. From the calibration curve, it was clear that Amlo has better linearity between 100 to 600 ng/spot, whereas Celo has a range between 2 to 12 µg/spot.
Precision: The precision of the analytical method is the degree of arrangement among the individual test results when the method is useful for repeated the multiple aliquots of the same samples. The method precision was tested by determining the percentage relative standard deviation (%RSD) for three determination of peak areas of Amlo (500 ng per band) and Celo (10 µg per band).
Accuracy: In order to ensure the accuracy of the proposed method, recovery studies were performed. The recovery studies were carried out by standard addition method at the level of 80%, 100% & 120%. Known amounts of standard solutions of Amlo (400, 500 & 600 ng/band) and Celo (8, 10 & 12 μg/band) were added to pre-analyzed sample solutions of Amlo (500 ng/band) and Celo (10 μg/band). The concentration of Amlo and Celo was determined by applying obtained values to the regression equation of the calibration curve.
Limit of Detection: It is the lowest concentration of an analyte in a sample that may be detected but not essentially quantitated under the indicated experimental conditions. It can be calculated by using the following equation,
LOD = 3.3 × (σ/S)
Where, σ = response of Standard deviation, S = calibration curve slope
Limit of Quantitation: It is the lowest concentration of an analyte in a sample that may be quantitated with acceptable precision and accuracy under indicated experimental conditions. It can be determined by using the following equation,
LOQ = 10 × (σ /S)
Where, σ = response of Standard deviation, S = calibration curve slope.
RESULTS AND DISCUSSION:
Selection of Detection Wavelength: After chromatogram development, bands was scanned at a range of 200-400 nm. It was observed that both the drugs show considerable absorbance at 310 nm. So, 310 nm was selected as the wavelength for detection Fig. 2.
FIG. 2: OVERLAY SPECTRUM OF AMLO AND CELO
TABLE 1: MACHINE PARAMETERS USED FOR HPTLC METHOD
| S. no. | Parameters | Chromatographic condition | |
| Amlodipine Besylate | Celecoxib | ||
| 1 | Syringe size (µ/l) | 100 | 100 |
| 2 | Syringe volume (µ/l) | 80 | 80 |
| 3 | Pre dosage volume (µ/l) | 2 | 2 |
| 4 | Dead volume (µ/l) | 5 | 5 |
| 5 | Dosage speed (sec/µl) | 5 | 5 |
As per trial and error basis, mobile phases were selected in different ratios but only Toluene: Ethyl acetate: Methanol: Glacial acetic acid (6:5:1.5:0.3 v/v) gives good resolution, minimum tailing and values of Rf are 0.28 and 0.73 for Amlo and Celo respectively.
Amlo and Celo has good solubility in methanol as compared to ethanol, acetone, and water. Toluene: Ethyl acetate: Methanol: Conc. Ammonia was finalized as a mobile phase by using various ratios, in which 6:5:1.5:0.3 (v/v) ratio shows comparatively good resolution of a drug. For detection of a drug at 310 nm wavelength was selected Fig. 3 and 4.
TABLE 2: PLATE PARAMETERS USED FOR HPTLC METHOD
| S. no. | Parameters | Chromatographic condition | |
| Amlodipine Besylate | Celecoxib | ||
| 1 | Plate length (mm) | 100 | 100 |
| 2 | Plate width (mm) | 100 | 100 |
| 3 | Start –X (mm) | 10 | 10 |
| 4 | Start –Y (mm) | 10 | 10 |
| 5 | Band space (mm) | 5 | 5 |
| 6 | Band length (mm) | 8 | 8 |
| 7 | No. of tracks | 6 | 6 |
| 8 | Actual no. of tracks | 6 | 6 |
FIG. 3: PHOTOGRAPH OF DEVELOPED HPTLC PLATE
FIG. 4: DENSITOGRAM OF AMLO ANDCELO AT 310 nm
Analysis of Sample Solution: A sample solution containing Amlo 10 mg and Celo 200 mg was used for analysis. The sample solution was analyzed three times, and the concentration of drugs determined by the HPTLC method was is good with the label claim. The results for the sample solution was observed to be 100.02% and 99.93% for Amlo and Celo, respectively. The result for analysis of formulation shows that % RSD values are < 2%, which indicates within limit Table 4.
TABLE 3: CHROMATOGRAPHIC CONDITIONS USED HPTC METHOD
| S. no. | Parameters | Chromatographic condition | |
| Amlodipine Besylate | Celecoxib | ||
| 1 | Stationary phase | Silica gel 60 F254 | Silica gel 60 F254 |
| 2 | Mobile phase | Toluene: Ethyl acetate: Methanol: Conc. Ammonia (6:5:1.5:0.3 v/v) | Toluene: Ethyl acetate Methanol: Conc. Ammonia (6:5:1.5:0.3 v/v) |
| 3 | Chamber saturation | 10 min | 10 min |
| 4 | Plate development time | 25 min | 25 min |
| 5 | Migration distance | 7 cm | 7 cm |
| 6 | Radiation source | D2& W | D2&W |
| 7 | Scanning wavelength | 310nm | 310nm |
TABLE 4: RESULT OF ANALYSIS OF SAMPLE PREPARATION
| Drug | Label claim
(mg/tab) |
Amount taken (mg) | AUC (Mean area)* | Amount found (mg) | % Drug content | ±SD | %
RSD |
| Amlo | 10 | 10 | 2366.44 | 10.02 | 100.25 | 27.55686 | 1.16 |
| Celo | 10 | 10 | 2549.97 | 199.86 | 99.93 | 23.50914 | 0.92 |
*n=3 (Three measurement)
Method Validation: As per ICH Q2 (R1) guidelines, the method validation parameters like linearity, precision, accuracy, the limit of detection and limit of quantitation were performed.
Linearity: Linearity was determined by using different concentrations of amlodipine besylate and celecoxib. The stock solution of Amlo (1000 µg/ml) and Celo (20,000 µg/ml) was prepared in flask of volumetric and applied on the pre-coated TLC plate in a range of concentration 100-600 ng/spot for Amlo and 2-12 µg/spot for Celo. After plate development, the peak area was determined for each concentration, and a calibration graph was plotted between peak area vs. drug concentrations.
From the graph, the value of R2 was observed to be 0.9971 for Amlo, and the value of R2 was observed to be 0.9989 Table 5-8, Fig. 5-9.
TABLE 5: CALIBRATION CURVE OF AMLO
| S. no. | Sample volume in µl | Conc. of Amlo (ng) | AUC (Mean area)* | ±SD | % RSD |
| 1 | 0.1 | 100 | 569.8867 | 9.544927 | 0.208 |
| 2 | 0.2 | 200 | 1110.547 | 18.27891 | 0.509 |
| 3 | 0.3 | 300 | 1499.953 | 25.69189 | 0.462 |
| 4 | 0.4 | 400 | 1950.627 | 30.14439 | 0.458 |
| 5 | 0.5 | 500 | 2360.507 | 40.40886 | 0.449 |
| 6 | 0.6 | 600 | 2927.61 | 24.51116 | 0.439 |
*n=3 (Three measurements)
TABLE 6: CALIBRATION CURVE OF CELO
| S. no. | Sample volume in µl | Conc. of Celo (ng) | AUC (Mean area)* | ±SD | % RSD |
| 1 | 0.1 | 2 | 841.36 | 9.905004 | 1.177 |
| 2 | 0.2 | 4 | 1225.817 | 13.38723 | 1.092 |
| 3 | 0.3 | 6 | 1721.647 | 15.89962 | 0.923 |
| 4 | 0.4 | 8 | 2144.78 | 33.82947 | 1.577 |
| 5 | 0.5 | 10 | 2551.73 | 39.91856 | 1.564 |
| 6 | 0.6 | 12 | 3053.633 | 46.89966 | 1.535 |
*n=3 (Three measurements)
TABLE 7: PRECISION STUDIES FOR AMLO AND CELO
| Drug | Concentration | *Mean AUC | ±SD | %RSD |
| Amlo | 500ng/band | 2360.50 | 40.40886 | 1.71 |
| Celo | 10µg/band | 2551.73 | 39.918557 | 1.56 |
*n=3 (Three measurements)
TABLE 8: RECOVERY STUDY OF AMLO AND CELO
| Drug name | Initial Amount (ng/band) | Added Amount (ng/band) | *Mean AUC | ±SD | %RSD | %Recovery |
| 500 | 400 | 4254.483 | 8.596705 | 0.202 | 99.52 | |
| Amlo | 500 | 500 | 4728.063 | 10.05794 | 0.212 | 99.61 |
| 500 | 600 | 5206.613 | 20.00885 | 0.384 | 99.83 | |
| 10 | 8 | 4647.383 | 33.31653 | 0.716 | 99.98 | |
| Celo | 10 | 10 | 5162.413 | 34.17049 | 0.661 | 99.9 |
| 10 | 12 | 5676.037 | 28.48506 | 0.501 | 99.83 |
*n=3 (Three measurements)
FIG. 5: 3D CHROMATOGRAM OF AMLO AND CELO SPOT FOR EACH CONCENTRATION DETECTED AT 310 nm
FIG. 6: 3D CHROMATOGRAM OF AMLODIPINE BESYLATE
FIG. 7: CALIBERATION CURVE AT 310 nm OF AMLO
FIG. 8: 3D CHROMATOGRAM OF CELO AT 310 nm
FIG. 9: CALIBERATION CURVE OF CELECOXIB
Precision: The precision study of Amlo and Celo shows that the developed method is precise with %RSD within limit i.e., ˂2%.
Accuracy: The recovery of Amlo and Celo was described in terms of ±SD and %RSD. Percentage recovery values were observed to be 99.52% to 99.83% for Amlo and 99.83% to 99.98% for Celo in a range with low values of %RSD of drug content.
Limit of Detection and Limit of Quantitation (LOD and LOQ): It is determined by the slope (s) of calibration curve and means of SD (σ) of response. These were observed to be 17.88 µg/ml and 54.20 µg/ml for Amlo and 0.39 µg/ml and 1.20 µg/ml for Celo, respectively.
CONCLUSION: It is concluded that the developed HPTLC method is simple, new, fast reproducible and sensitive methods for the simultaneous estimation of Amlo and Celo in the pure and combined dosage form. This method is successfully validated using ICH Q2 (R1) guidelines for analytical method validation. This method help for routine drug analysis in pharmaceutical dosage form with no interference of excipient with good sensitivity.
ACKNOWLEDGEMENT: We thankful to the principle of the Appasaheb Birnale College of Pharmacy, Sangli. Institute for providing excellent laboratory facilities. We are grateful thanks to Smruthi Organics Limited, Solapur, and Aarti Drugs Limited, Mumbai, for a gift sample of Amlo and Celo, respectively. I would be thankful to Dr. Manish S. Kondawar, HOD of Department Quality Assurance, for guidance.
CONFLICTS OF INTEREST: Nil
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How to cite this article:
Mandale TR and Kondawar MS: HPTLC method development and validation for simultaneous estimation of amlodipine besylate and celecoxib in pure and combined dosage form. Int J Pharm Sci & Res 2020; 11(10): 5198-04. doi: 10.13040/IJPSR.0975-8232.11(10). 5198-04.
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