Development and Validation of Stability Indicating RP-HPLC Method for Amlodipine Besylate and Perindopril Arginine in Synthetic Formulation

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evelopment and Validation of Stability Indicating RP-HPLC Method for and in Synthetic Formulation Priya R. Rajput, Atul Bendale, Shailesh V. Luhar 3, r. Sachin B. Na

1 evelopment and Validation of Stability Indicating RP-HPLC Method for and in Synthetic Formulation Priya R. Rajput, Atul Bendale, Shailesh V. Luhar 3, r. Sachin B. Narkhede. M. pharm Student, Smt. B.N.B Swaminarayan Pharmacy College, Salvav, Vapi-3, Gujarat, India. Assistant Proffessor of epartment of Quality Assurance, Smt. B.N.B Swaminarayan Pharmacy College, Salvav, Vapi-3, Gujarat, India 3. Head of the department of quality assurance department, Smt. B.N.B Swaminarayan Pharmacy College, Salvav, Vapi-3, Gujarat, India. Principal of Smt. B.N.B Swaminarayan Pharmacy College, Salvav, Vapi-3, Gujarat, India ABSTRACT: A simple, rapid, precise and accurate stability-indicating RP-HPLC method was developed and validated for the simultaneous determination of (AM) and (PR)pharmaceutical dosage form. Method was carried out by using Sheisedo C (5 *. mm, 5µm) column and Acetonitrile : Methanol : water (3::3 % v/v/v) as mobile phase at. ml/min flow rate. etection was carried out at nm. Rt was found to be 3. min for AM and. min for PR. For stability study drugs were subjected to acid hydrolysis, alkaline hydrolysis, oxidative degradation and thermal degradation. AM was highly susceptible to acidic and thermal condition. Pharmaceutical dosage form was more stable than active pharmaceutical ingredient. The linearity of the proposed method was investigated in the range of 5-5 μg/ml (r=.) for AM and 5-5 μg/ml(r=.) for PR. The limit of detection were.μg/ml and.μg/ml and the limit of quantification were.3μg/ml and.μg/ml for AM and PR respectively. KEY WORS:,, Stability indicating RP-HPLC Method, Validation Article history: Received April Revised April Accepted April Available online April Citation: Rajput P. R., Bendale A., Luhar S. V., evelopment and Validation of Stability Indicating RP-HPLC Method for and Perindopril in Synthetic Formulation J Pharm SciBioscientific Res., (3): *For Correspondence: Priya R. Rajput M. pharm Student, Smt. B.N.B Swaminarayan Pharmacy College, Salvav, Vapi-3, Gujarat, India. ( INTROUCTION [-] 3-ethyl 5-methyl -[(-aminoethoxy) methyl]--(- chlorphenyl) -methyl-,-dihydropyridin dicarboxylate. It isdihydropyridine calcium channel blocker and used for treatment of antihypertensive. It is official in Indian pharmacopeia. It is freely soluble in methanol. Molecular weight of is 5.5 gm/mol and formula isc H 3 ClN O S. is chemically L-arginine (S,3aS,aS)--[(S)--[[(S)- (ethoxycarbonyl) butyl] amino]propanoyl] octahydro-h-indole-- carboxylate. It is the Competitive inhibitor of ACE.It is not official in any Pharmacopeia. It is slightly soluble in Readily soluble in purified water. Molecular weight of is 53. gm/mol and formula is C H 3 N O 5 C H N O. Literature Review revels that there was no reported Stability Indicating RP- HPLC method for Simultaneous Estimation of and in combined dosage form. So the present work is aimed for To develop an accurate, specific, repeatable Stability Indicating RP-HPLC 3

2 method for Simultaneous estimation of Amlodipine Besylate and in synthetic formulation.. MATERIALS AN METHOS.. Method development:.. Materials: was obtained as gift sample fromalkem laboratories,ltd.,vapi..perindopril was obtained as gift sample from Arti Pharmaceuticals, Mumbai. Methanol, Acetonitrile, Water and Ortho phosphoric acid HPLC grade were purchased from Ran Kem Lab... Instrumentation and Chromatographic method: The analysis of the drug was carried out on a Peak HPLC system equipped with a reverse phase Shisedo C column, peak pump with auto samplerand a detector running on Peak LC Solution Software. The mobile phase consists of Acetonitrile:Methanol: Water (3:: 3% v/v/v)(ph 5.) and the flow rate were maintained at. ml/min. The mobile phase was freshly prepared and passed through nylon membrane filter of pore size of.5µm and it was degassed by sonicating for min. before it was used. The elution was monitored at wavelength of nm with UV detector, and the injection volume was µl...3 etermination of maximum absorbance: The standard solutions of and were scanned in the range of - nm against mobile phase as blank. Isobestic point of and atnm. Thus the wavelength selected for the determination of and was nm... Preparation of stock and standard solutions: Accurately weighed mg of and 5mg of were dissolved in ml volumetric flask containing ml of Methanol which is considered as stock solution. Working standard solution of and esloratdine were prepared by making various dilutions of the drug solution from the stock solution. Fivesets of the drug solution were prepared in the mobile phase containing 5-5µg/ml of and 5-5 µg/ml of Perindopril. Each of this drug solution was injected into the column and the peak area and retention time was recorded...5 Assay of marketed formulation (Brand name of tablet Ventidox - L): Twenty tablet were weighed and average weight of a single tablet was calculated. Tablets were crushed and mixed using a mortar and pestle. Then drug sample equivalent to mg of and mg of were accurately weighed and transferred into a ml volumetric flask and mixed with known amount of methanol and the active pharmaceutical ingredients were extracted into the methanol followed by ultra-sonication and then filtered through a nylon membrane of pore size.5µm. The drug sample was diluted by adding methanol to obtain a stock solution of µg/ml of and µg/ml of.. Method validation [] The Proposed method was validated according to ICH guidelines. The parameters assessed were linearity, precision, accuracy, LO and LOQ... System Suitability System suitability tests are an integral part of liquid chromatography. They are used to verify that resolution and reproducibility of chromatography system are adequate for the analysis to be done. System Suitability was performed on standard solution and system suitability parameters were calculated at the start of study for each parameter... Linearity and Range. The linearity was determined at Three levels over the range of 5-5 μg/ml and 5-5 μg/ml. Peak area of above linearity solution preparations were taken at each concentration three times...3 Accuracy Recovery studies were carried out by addition of standard drug to the sample at 3 different concentration levels (%, % and %) taking into consideration percentage purity of added bulk drug samples. These 3

3 solutions were subjected to re-analysis by the proposed method and Results are Calculated... Precision A. Repeatability Study: Standard solutions of,, μg/ml Amlodipine Besylate and,, μg/ml Perindopril were prepared and chromatograms were recorded. Area was measured of the same concentration solution three times and was calculated. B. Intra-day precision Mixed solutions containing,,5 μg/ml and,, μg/ml Perindopril were analyzed three times on the same day,% R.S. was calculated. C. Inter-day precision Mixed solutions containing,,5 µg/ml and,,µg/ml Perindopril were analyzed on three different days and % R.S. was calculated...5 Limit of etection and Limits of Quantitation Limit of etection (LO) From the linearity curve equation, the standard deviation (S) of the intercepts (response) was calculated. The limit of detection (LO) of the drug was calculated by using the following equation designated by International Conference on Harmonization (ICH) guideline: LO = 3.3 Intercept / Slope Limit of Quantitation (LOQ) The limit of quantitation (LOQ) of the drug was calculated by using the following equation designated by International Conference on Harmonization (ICH) guideline: LOQ = Intercept / Slope.. Robustness The robustness of the method was established by making deliberate minor variations in the following method parameters a) ph of mobile phase: ±. b) Flow rate : ±. ml/min c) Change in the ratio of component in the mobile phase: ± %..3 Stability studies [] Stability Studies was carried out on the drug in order to check the stability of the drug by providing various stress conditions like acid, base, oxidation and thermal degradation compared with normal conditions. The purpose of force degradation method is to provide evidence that the analytical method is efficient in determination of drug substances in commercial drug product in the presence of its degradation products..3. Acidic hydrolysis Take ml solution of AM µg/ml and.5 ML ofpr µg/ml, ml of.m HCl was added. The solution was heated for hr at :C and transferred to a ml volumetric flask, cooled, neutralized by.m NaOH and diluted up to mark with methanol to get final concentration µg/ml of AM and µg/ml of PR..3. Alkaline hydrolysis Take ml solution of AM µg/ml and.5ml ofpr µg/ml, ml of.m NaOH was added. The solution was heated for hr at :C and transferred to a ml volumetric flask, cooled, neutralized by.m HCl and diluted up to mark with methanol to get final concentration µg/ml of AM and µg/ml of PR..3.3 Oxidative degradation Take ml solution of AM µg/ml and.5 ml ofpr µg/ml, ml % H O was added at room temperature for hours at :C and transferred to a ml volumetric flask, cooled diluted up to mark with methanol to get final concentration µg/ml of AM and µg/ml of PR..3. Thermal degradation Take ml solution of AM µg/ml and.5ml ofpr µg/ml, heat the solution for hr at :C and transferred to a ml volumetric flask, cooled diluted up to mark with methanol to get final concentration µg/ml of AM and µg/ml of PR. 3

4 3. RESULTS AN ISCUSSION 3. Linearity:The calibration curve showed (Fig.) good linearity in the range of 5--5μg/ml for Amlodipine Besylate and 5-5μg/ml for with correlation coefficient (r ) of. and. for and Perindopril respectively. Results are given in Table. 3. Recovery:Recovery studies were carried out by addition of standard drug to the sample at 3 different concentration levels (%, % and %) taking into consideration percentage purity of added bulk drug samples.and % recovery values found to be in the range of.% -.% for and..3% for. The result are given in table. 3.3 Precision:Intraday precision was carried out using test samples analyzed on the same day. Interday precision was assessed by analysis of the same solutions on consecutive days. The % RS value were below indicate that the method is precise. The results are given in table, and. 3. Robustness:Small deliberate changes in chromatographic conditions such as change in mobile phase ratio (+ %), change in ph (± units) and flow rate (± units) were studied to determine the robustness of the method. The results were in favor of (% RS< %) the developed RP-HPLC method for the analysis of and. The results are given in table and Limit of etection (LO) and Limit of Quantification (LOQ): The LO was found to be.µg/ml and the LOQ.3 µg/ml for and the LO was found to be.µg/ml and the LOQ.µg/ml for Perindopril estimated by using the standard formulas. 3. Stability studies:stability indicating RP HPLC method was performed in different stress condition using the Acetonitrile : Methanol : water (3::3 % v/v/v) as mobile phase suggested the following degradation behavior. The chromatograms obtained on stress degradation, like photolytic degradation and similarly other conditions were shown in figure,,,,, 3,,,,, and. 3. ISCUSSION RP-HPLC method was found to be linear over the range of 5-5 µg/ml for and 5-5 µg/ml for. The method has been validated by studying accuracy and precision, LO, LOQ and system suitability according to ICH guideline.the Stability study of and Perindopril indicates that the drug significantly degrade under acidic and thermal conditions.. CONCLUSION: A simple, economic, accurate and robust RP-HPLC method has been developed and validated for the estimation of and Perindopril bulk and pharmaceutical dosage form. The reverse phase liquid chromatography was performed using Sheisedo-C (5mm x.mm, 5 μm) column. The mobile phase used was Acetonitrile: Methanol: Water (ph-5.) (3::3 %v/v/v) with flow rate ml/min. The detection was carried out at nm. The retention time were found be 3. ±. min and. ±. min for and Perindopril respectively. There was no interference from any excipients in the determination of drugs in dosage form which indicates the method is specific. All method validation parameters lie within its acceptance criteria as per ICH Q (R) guideline so we can conclude that method is simple, linear, accurate and precise. Hence, it can be successfully used for the routine analysis of and pharmaceutical dosage forms. Stability indicating RP HPLC method has been developed for and Perindopril pharmaceutical dosage form. Amlodipine Besylate is easily degrade in acidic and thermal condition while was slightly degrade. is more stable than Amlodipine Besylate in various stress condition. From the result, We can conclude that Pharmaceutical dosage form was more stable than Active pharmaceutical ingredient. 5. REFERENCES:. Sharma BK, Instrumental Method of Anal. ; th Edn, Goel Publishing House, Merrut,, pp -3. rug Information 35

3. rug Information http://www.drugbank.ca/drugs/b3. Indian Pharmacopoeia, Govt. of India Ministry of Health and Family Welfare, The Indian Pharmacopoeia Commission, Ghaziabad, Vol & 3, pp- 5-

5 3. rug Information Indian Pharmacopoeia, Govt. of India Ministry of Health and Family Welfare, The Indian Pharmacopoeia Commission, Ghaziabad, Vol & 3, pp- 5-, British Pharmacopoeia (BP) The Stationery Office. London, U.K, Vol I, pp -3-3,-. ICH guidelines, Validation of analytical procedure: Methodology QB, I.C.H. Harmonized Tripartite Guidelines,, pp.-3.. ICH Harmonized Tripartile Guideline [ Nov. ], Validation of Analytical Procedures; Text and Methodology Q[R], International Conference on Harmonization, Geneva, Switzerland. Vora N and KadavAA. evelopment and Validation of a Simultaneous HPLC Method for Estimation of Bisoprolol Fumarate and from Tablets,Indian Journal of Pharmaceutical Science,; ():5 5.. Sarat MP andmurali KP. evelopment and validation of stability indicating RP-HPLC method for simultaneous estimation of and Benazepril HCl in tablet dosage form,international Journal of Current Pharmaceutical Research,, (3):-.. Chebrolu HP and Vadlamudi GS. evelopment and validation of RP-HPLC method for the simultaneous determination of Hydrochlorothiazide, Amlodipine Besylate and Telmisartan in bulk and pharmaceutical formulation,international Journal of pure and applied chemistry, ;3():3-3. Sahoo S and Panda PK Stability indicating HPLC method development for simultaneous estimation of Hydrochlorothiazide and Perindropril in tablet dosage form, Asian International Journal of and clinical Research, ;5():3-3. Nekkala KPand Bhatt SK Method development and validation of stability indicating RP-HPLC method for simultaneous estimation of Perindopril Erbumine and in bulk and its pharmaceutical formulations, American Journal of Advanced rug elivery, ;(5): -5 A R3 E A 5 Figure Calibration curve of Perindopril y =.x +.5 R² =. 3 Concentration Figure 5 Calibration curve of Perindopril Figure Acid hydrolysis peak of TABLES AN FIGURES 35

Figure Acid hydrolysis peak of Figure Acid hydrolysis peak of Pharmaceutical dosage form Figure Oxidative degradation peak of Perindopril Figure Alkali hydrolysis peak of Figure Oxidative degradation

6 Figure Acid hydrolysis peak of Figure Acid hydrolysis peak of Pharmaceutical dosage form Figure Oxidative degradation peak of Perindopril Figure Alkali hydrolysis peak of Figure Oxidative degradation peak of Pharmaceutical dosage form Figure Alkali hydrolysis peak of Figure Thermal degradation peak of Amlodipine Besylate Figure 3 Alkali hydrolysis peak of Pharmaceutical dosage form Figure Thermal degradation peak of Perindopril Figure Oxidative degradation peak of Amlodipine Besylate Figure Thermal degradation peak of Pharmaceutical dosage form 35

7 Factor AM PR Parameters Specifications Column Sheisedo C (5mm*.mm, 5µm) Mobile phase Acetonitrile : Methanol :Water (35 : : 5 % V/V/V) ph 3. Flow rate ml/min Run time min etection nm wavelength Retention time 3. min for AM and. min for PR Table Finalization of Chromatographic condition Mean. Mean.Area±S. (μg/ml) Area±S. (μg/ml) 5 3.±. 5.3 ±.5.5±. ±..±..3 ±.3.5± ±.3 5.± ±.3 Table Calibration curve for and Parameters Result Amlodipine Besylate Perindopril Linearity Range (μg/ml) Slope 35.. Intercept.5.5 Retention Time 3.. (min) Correlation Coefficient.. Table 3 and ru g AM Actu al of rug Obtaine d %ru g Foun d PR Mean S Table Assay Result of Synthetic Formulation.5.5 Table suitablility parameters for and (µg/ml ) Area Mean ± S ±..5±..±..5±.3 5.± (µg/ml ) Area Mean ± S ±.5 ±..3 ± ± ± Table Linearity for and % Recov ery Targ et, (µg/ ml) % RT 3.. Tailing Factor.5. Theoritical Plates.5. Resolution 5.. Spike d conc., (µg/ ml) + Final, (µg/ ml) +, Obtained MT KT. ES. MTK T. % Assay ES

8 3 5 % % ± ± ±...3± ± ± Table Intra-day precision for Amlodipine Besylate and Table Accuracy for and.. ±. rug (μg /ml) Mean Abs. ± S.. Amlodipine 3.± ±.5 ±.. Perindopril (µg/ml ) Area Mean ± S.. (n=3)..5 5.±.5.3 ±. RT 3.5 ±..53 Table Inter-day precision for Amlodipine.5 ±..3 Besylate and (µg/ml ) Area Mean ± S.. (n=3).3.5±.35..5± ± ±.3 Sr..5 ( µg/ml) ±. no.. ph Flow rate Mobile phase RT. ± units units units units % % Table Repeatability for and (µg/m l) Area Mean ± S.. (n=3) (µg/m l) Area Mean ± S.. (n=3) Me

an 5 3 LOQ.3. S...3 3.3.3.. %Recovery ( Accuracy, n = 3).-..-.3 % RS.3..53..3. Repeatability (RS, n = 3) %.-.3.3-. Table Robustness for Precision ata Sr. no.

9 an 5 3 LOQ.3. S %Recovery ( Accuracy, n = 3) % RS Repeatability (RS, n = 3) % Table Robustness for Precision ata Sr. no. ( µg/ml) Ph Flow rate Mobile phase Interday Intraday units Me an S.. -. units units units % % Robustness () ph(+. units).3. ph(-. units)..3 Flow Rate(+. units) Flow Rates(-. units) Mobile Phase(+%) Mobile Phase(-%) Table Summary of RP HPLC Method % R.S SR. NO. STRESS TYPE % EGRAATION of SAMPLE % EGRAATION of SYNTHETIC MIXTURE Table 3 Robustness for Parameters AM PR AM PR AM PR Acidic...5. etection wavelength (nm) Concentration range (mcg/ml) nm Alkaline Oxidation.... Thermal Slope Intercept.5.5 Table Summary of Stability indicating RP HPLC Method Correlation coefficient.. Regression Coefficient y =35.x+.5 y=.x+.5 LO.. 355

Development and Validation of Stability Indicating RP-HPLC Method for Montelukast Sodium and Desloratadine in Pharmaceutical Dosage Form

Development and Validation of Stability Indicating RP-HPLC Method for Montelukast and in Pharmaceutical Dosage Form Krupali K. Patel*, Bhumika Suthar, Shailesh V. Luhar, Sachin B. Narkhede Department of