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CC8�"8�"�"�k4�6�����5u�z�W�!^�4�7480d85x=B"vx=(6x=C6��"	8	8�"d8��������������������������������������������������������������������x=		:	@A Validated Rp-Hplc Method for Tablets Containing Amlodipine Besylate and Telmisartan Hcl as Active Pharmaceutical Ingredient
Shaila Sumaiya*, Anjali Bharadwaj
Department of Chemistry, Arya College of Pharmacy, India
*Correspondence to: Shaila Sumaiya, Department of Chemistry, Arya College of Pharmacy, India, TeL: 9154504625, E-mail: shailasumaiya1997@gmail.com
Rec date: July 05, 2020; Acc date: Aug 06, 2020; Pub date: Aug 12, 2020
Citation: Sumaiya S (2020) A Validated Rp-Hplc Method for Tablets Containing Amlodipine Besylate and Telmisartan Hcl as Active Pharmaceutical Ingredient. Mod Chem Appl Vol: 8 Iss: 3:276. doi:10.35248/2329-6798.20.8.276.
Copyright: � 2020 Sumaiya S. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACT 
The LC method for the determination of test procedures of Assay for Amlodipine Besylate and Telmisartan HCl in bulk drug, and tablets were simple, reliable, sensitive and less time consuming. The positivity of the present working procedures was a simple isocratic method. The present method can be used for rapid and precise quantification of Amlodipine Besylate and Telmisartan HCl. The present work showing a validation with highly sensitive and selective method for the estimation of Amlodipine Besylate and Telmisartan HCl in pharmaceutical dosage forms.
Keywords: HPLC; Amlodipine Besylate; Telmisartan HCl; Chromatogram; Validation

INTRODUCTION 
Importance of Chromatography
The importance of Chromatography is increasing rapidly in pharmaceutical analysis. The precise differentiation, selective identification and quantitative determination of structurally closely related compounds [1,2].
High performance Liquid Chromatography
The cutting edge kind of chromatography has been called superior, high, high goal and fast fluid chromatography. 
Guideline of division in HPLC
The standard of partition in typical stage HPLC is adsorption. At the point when blends of segments are acquainted in with a HPLC section, they travel predictable with their relative affinities towards the fixed stage. (Figure 1) The segment which has greater partiality towards the adsorbent ventures more slowly [3].
Solvent Reservoir
The dissolvable or portable stage store has a few attributes. The arrangement of repository should render it latent to an assortment of watery and non-fluid is to be pressurized; glass is to be kept away from. 
Pumps
HPLC is every now and again conveyed as elite fluid chromatography; the siphon must be equipped for creating weights of up to 3ml/min for diagnostic arrangements. The dissolvable stream from the siphon ought to be beat less or ought to be hosed so as to expel beats, since the weight of heartbeats in the stream may cause deceptive outcomes with certain indicators.
Injection Device
The infusion of the example on to the segment presents some one of a kind issues in light of the high-pressure associated with HPLC. While septum injectors can be utilized in HPLC, the circle injectors find greatest use in HPLC. Initially, the example ought to infuse in tight fitting [4].
Column, Detector, and Recorder are according to the experimentally needed.

MATERIAL AND METHODS
Instrument used: Younglin HPLC - ACME-9000
Detector: UV/Visible detector 
Integration: done by using AutoChro-3000 software package
Solvents used: HPLC Grade (Merck)
Reagents and Chemicals
Water (purified using Millipore Milli Q system), Methanol, Acetonitrile, Amlodipine Besylate and Telmisartan Hcl, Preparation of 0.02M Ammonium acetate buffer,Buffer was prepared by dissolving 2.72g of Potassium dihydrogenorthophosphate (KH2PO4) in 1L of water and followed by the degassing of the solution. (Figure 2)
Diluent Preparation
1L of diluent was prepared by mixing 450 ml of 0.02 M Phosphate Buffer, 300 ml of Acetonitrile and 250 ml of Methanol.
Preparation of stock solution
Accurately weighed 10 mg of the both Amlodipine Besylate and Telmisartan Hcl was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the Methanol and mixed well. This yielded a stock solution with concentration 1000 ppm of Amlodipine Besylate and Telmisartan Hcl mixture. (Figure 3)
Preparation of standard solution
Accurately amount of 0.1 and 0.8 ml of the Amlodipine Besylate and Telmisartan Hcl stock was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the diluent and mixed well. (Figure 4) This yielded a standard stock solution with concentrations of 10 ppm and 80 ppm of Amlodipine Besylate and Telmisartan Hcl respectively [5].

DISSCUSSION
Standard solution were prepared as per the test method and injected six times to test the performance of the chromatographic instrument.
Method precision
Six preparations were prepared individually using single batch of Amlodipine Besylate and Telmisartan Hcl working standard as per test method and injected each solutions in duplicate [6].
Limit Detection and Limit of Quantitation
Procedure: RSD criteria: Series of working standard solutions of different concentrations below two specification level were prepared (generally about 10%, 20%, 30%, 40% and 50% of the specification concentration) and injected six replicate injections into HPLC. The solution was prepared at predicted concentration (for LOQ/LOD) and injected six replicates as per methodology.
Acceptance criteria for LOD and LOQ
RSD Criteria
Concentration at which RSD< 33.0%(LOD)	
Concentration at which RSD<10.0 (LOQ)
 
Linearity
Procedure: Prepared a series of standard solutions not less than five in the specified concentration range and analyze them as per method.
Acceptance Criteria: The Correlation Coefficient Should Be Not Less Than 0.9990.
Accuracy
Accuracy normally refers to the difference between the mean of the set of results and the true or correct value for the quantity measured. According to IUPAC accuracy relates to the difference between results (or mean) and the true value. For analytical methods, there are two possible ways of determining the accuracy, absolute method and comparative method [7].
Accuracy is best reported as percentage bias, which is calculated from the expression.
Since for real samples the true value is not known, an approximation is obtained based on spiking drug-free matrix to a nominal concentration. The accuracy of analytical method is then determined at each concentration by assessing the agreement between the measured and nominal concentrations of the analytes in the spiked drug�free matrix sampler. Acceptance criteria: Assay recovery should be between 98%-102%.
Specificity
Procedure: Acid hydrolysis: Sample was treated with 5ml of acid (1N Hcl) and kept for 1 hr.After 1 hr the solution was neutralized with 1N NaOH and analyzed using HPLC.
Oxidation: Amlodipine Besylate and Telmisartan Hcl solution 100 ppm was mixed with 5 mL of 30% aqueous hydrogen peroxide solution.
Alkali hydrolysis: Sample was treated with 5ml of alkali (1N NaOH) and kept for 1 hr.After 1 hr the solution was neutralized with 1N Hcl and analyzed using HPLC.
Photolysis: Samples were kept under UV light for different time intervals (15-60 mins) and observed by HPLC. [8].
Heat: Samples were heated at 90 0 C for 1-2 hrs and analyzed.

Range
The range of an analytical procedure is the interval between the upper and lower concentrations (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.
Acceptance criteria
Linearity, Precision and Recovery should be shown.
The logic behind this parameter was�typical concentration range was essential between which the actual concentration should fall when performing real sample analysis.
Robustness
Measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides indication of its reliability during its normal usage.
Procedure: samples were analyzed under the following conditions.
Ruggedness
Method/Procedure: samples were prepared as per the test method the test method and injected in duplicate using different system, analyst on different day.
Calculated Mean area, SD, %RSD.
Stability Studies
In the rational design and evaluation of dosage forms for the drugs, the stability of the activity components must be a major criterion in determining their stability. The medicine has to reach the patient in an active and acceptable form maintaining the criteria for acceptable equality.

CONCLUSION AND RESULTS
Method Validation: System Suitability 
Acceptance Criteria: 1. RSD should not be more than 2.0% for five replicate injections of standard. 2. USP Tailing for Amlodipine Besylate and Telmisartan Hcl peak in not more than 2.0. 3. The column efficiency as determined for Amlodipine Besylate and Telmisartan Hcl, USP Plate Count should not be more than 2000.
Preparation of standard solution: Accurately amount of 0.1 and 0.8 ml of the Amlodipine Besylate and Telmisartan Hcl stock was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the diluent and mixed well. This yielded a standard stock solution with concentrations of 10 ppm and 80 ppm of Amlodipine Besylate and Telmisartan Hcl respectively (Table 1).
Hplc Method Development
The above trials indicating that RT for the drug was not constant and elution time was faster which not prefered for the analysis. The above trials indicating that RT for the drug was not constant and elution time was faster which not prefered for the analysis. (Table 2)
Discussion: Remaining experiments were done with above method and the results are satisfactory. (Table 3)
System Suitability for Amlodipine Besylate and Telmisartan Hcl
Precision
Acceptance Criteria: RSD should not be more than 2.0% for five replicate injections of standard.
Preparation of standard solution: Accurately amount of 0.1 and 0.8 ml of the Amlodipine Besylate and Telmisartan Hcl (Figure 5) stock was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the diluent and mixed well. (Table 4) This yielded a standard stock solution with concentrations of 10 ppm and 80 ppm of Amlodipine Besylate and Telmisartan Hcl respectively (Table 5).
Method Precision: Six individual preparations were prepared using single batch of Amlodipine Besylate and Telmisartan Hcl working standard as per test method and injected each solutions.
Summary of results of Method Precision parameter for Amlodipine Besylate and Telmisartan Hcl
Injection Precision
Standard solution were prepared as per test method and injected six times.
Summary of results of Injection Precision parameter for Amlodipine Besylate and Telmisartan Hcl

Limit of detection
LOD: Lowest amount of analyte in a sample that can be detected but not necessarily quanities, under the stated experimental conditions. 
Preparation of standard solution: Accurately amount of 0.1 and 0.8 ml of the Amlodipine Besylate and Telmisartan Hcl stock was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the diluent and mixed well. This yielded a standard stock solution with concentrations of 10 ppm and 80 ppm of Amlodipine Besylate and Telmisartan Hcl respectively. (Table 6)
Method Procedure: The mobile phase was allowed to run equilibrate with stationary phase upto good baseline was obtained. The different concentration ranging from 0.001 to 0.1ppm of Amlodipine Besylate and 0.008 to 0.8ppm Telmisartan Hcl was injected and peaks were recorded. 0.003 and 0.024ppm for Amlodipine Besylate and Telmisartan Hcl concentrations were detected respectively.
Chromatogram obtained for Chromatogram of Amlodipine Besylate and Telmisartan Hcl showing LOD

Limit of quantification
LOQ: Lowest amount of analyte in a sample, which can be quantitatively, determined with suitable precision and accuracy.
Preparation of standard solution: Accurately amount of 0.1 and 0.8 ml of the Amlodipine Besylate and Telmisartan Hcl stock was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the diluent and mixed well. (Figure 6) This yielded a standard stock solution with concentrations of 10 ppm and 80 ppm of Amlodipine Besylate and Telmisartan Hcl respectively. (Table 7)
Method Procedure: The mobile phase was allowed to run equilibrate with stationary phase upto good baseline was obtained. The different concentration ranging from 0.001 to 0.1ppm of Amlodipine Besylate and 0.008 to 0.8ppm of Telmisartan Hcl was injected and peaks were recorded. 0.01 and 0.08ppm for Amlodipine Besylate and Telmisartan Hcl concentrations were detected respectively. (Table 8)
Summary of results of LOQ for Amlodipine Besylate and Telmisartan Hcl
Linearity & range
Acceptance Criteria: R2 value should be not more than 1.
Preparation of standard stock solution:
Accurately weighed 10 mg of the both Amlodipine Besylate and Telmisartan Hcl was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the Methanol and mixed well (Table 9). This yielded a stock solution with concentration 1000 ppm of Amlodipine Besylate and Telmisartan Hcl mixture.
Preparation of standard solution: Accurately amount of 0.1 and 0.8 ml of the Amlodipine Besylate and Telmisartan Hcl stock was transferred to 10 ml clean and dry volumetric flask. 

Summary of results of Linearity parameter for Amlodipine Besylate and Telmisartan Hcl
Then the volume was made up to the mark with the diluent and mixed well. This yielded a standard stock solution with concentrations of 10 ppm and 80 ppm of Amlodipine Besylate and Telmisartan Hcl respectively.
Method Procedure: The mobile phase was allowed to run equilibrate with stationary phase upto good baseline was obtained. (Figure 7) The various concentrations ranging from 0.1 to 20ppm for Amlodipine Besylate and 0.8 to 160ppm for Telmisartan Hcl was injected then peaks were detected. (Figure 8)The linearity graph was plotted as concentration Vs peak area is depicted. (Figure 9)
Accuracy
Acceptance Criteria: Recovery should be in between 95 to 105 %.
Preparation of standard solution: Accurately amount of 0.1 and 0.8 ml of the Amlodipine Besylate and Telmisartan Hcl stock was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the diluent and mixed well. This yielded a standard stock solution with concentrations of 10 ppm and 80 ppm of Amlodipine Besylate and Telmisartan Hcl respectively. (Table 10)
Method precision: Prepared solutions in triplicate at levels 80%, 100% and 120% of test concentrations using for Amlodipine Besylate and Telmisartan Hcl working Standards as per the test method and injected each solution in triplicate. (Figure 10)
Summary of results of Accuracy parameter for Amlodipine Besylate and Telmisartan Hcl
Robustness
Acceptance Criteria: Overall RSD should not be more than 2.0 %.
Preparation of standard solution: Accurately amount of 0.1 and 0.8 ml of the Amlodipine Besylate and Telmisartan Hcl stock was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the diluent and mixed well. This yielded a standard stock solution with concentrations of 10 ppm and 80 ppm of Amlodipine Besylate and Telmisartan Hcl respectively.
Method procedure: 1. Flow: The standard solution was prepared and injected for the two times with (+1) flow rate. 2. Mobile Phase: The standard solution was prepared and injected for the two times with (+5) Mobile Phase composition
Ruggedness
Acceptance Criteria: Overall RSD should not be more than 2.0 %.
Preparation of standard solution: Accurately amount of 0.1 and 0.8 ml of the Amlodipine Besylate and Telmisartan Hcl stock was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the diluent and mixed well. (Table 11) This yielded a standard stock solution with concentrations of 10 ppm and 80 ppm of Amlodipine Besylate and Telmisartan Hcl respectively (Table 12).
Method Procedure: The standard solution was individually prepared as per the test method and injected each solution in six times using different system, analyst, date.

DISCLOSURE
There is no conflict of interests.

ACKNOWLEDGMENTS
No disclosures or acknowledgments.

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