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1 ISSN: X CODEN: IJPTFI Available Online through Research Article METHOD DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC METHODS FOR THE SIMULTANEOUS ESTIMATION OF CAPTOPRIL AND HYDROCHLOROTHIAZIDE IN BULK AND TABLET FORMULATION Seeta Vitthal Mali* Ashpak Mubarak Tamboli, Mr.kale S.S. Department of Pharmaceutical Chemistry, Sahyadri College of Pharmacy, Methwade,Sangola , Solapur, Maharashtra, India. Received on: Accepted on: Abstract: The method for the simultaneous estimation of Captopril and hydrochlorothiazide in bulk and tabletdosage form have been developed.this method was simple, accurate and results of analysis have been validated statistically and by recovery studies. This work was carried out on Shimadzu UV-Spectrophotometer model-1800 with 1cm quartz cell spectrophotometer. The absorption spectra of reference and test solutions were carried out in 1 cm matched quartz cell over the range of nm. Methanol and distilled water are used as solvent. Where the Captopril shows absorption spectra at 220nm and Hydrochlorothiazide shows at 271 nm. The isobestic point was found to 207 nm. Where the linearity ranges for both the drugs were 2-10 μg/ml. The Simultaneous equation method was validated in terms of accuracy, precision, linearity, and recovery, limit of detection and limit of quantitation and can be used for the estimation of these drugs in combined pharmaceutical dosage forms. All these parameters were within the limit Key words: Captopril, Hydrochlorothiazide, Methanol, Water, Sonicator, Weighing Balance, UV- Visible Spectrophotometer. Introduction: Captopril is chemically 1-(2 methyl-3-sulphonylpropanoyl) pyrolidine-2-carboxylic acid. With molecular formula C 9 H 15 NO 3 S and molecular wt g/mol. Its beneficial effects in hypertension and heart failure appear to result primarily from suppression of the renin-angiotensin-aldosterone system. Captopril prevents the conversion of angiotensin I to angiotensin II by inhibition of ACE, a peptidyl dipeptide carboxy hydrolase. This inhibition has been demonstrated in both healthy human subjects and in animals by showing that the elevation of blood pressure caused by exogenously administered angiotensin I was attenuated or abolished by captopril. Inhibition of ACE results in decreased plasma angiotensin II and increased plasma renin activity (PRA), the latter resulting from loss of negative IJPT June-2017 Vol. 9 Issue No Page 30087

2 feedback on renin release caused by reduction in angiotensin II. The reduction of angiotensin II leads to decreased aldosterone secretion, and, as a result, small increases in serum potassium may occur along with sodium and fluid loss. Hydrochlorothiazide is chemically 6-chloro-1, 1-dioxo-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine-7- sulfonamide.with molecular formula C 7 H 8 ClN 3 O 4 S 2 and molecular wt g/mol.It belongs to Thiazide class of diuretics. It reduces blood volume by acting on the kidneys to reduce sodium (Na) reabsorption in the distal convoluted tubule. The major site of action in the nephron appears on an electro neutral Na + -Cl co-transporter by competing for the chloride site on the transporter. By impairing Na transport in the distal convoluted tubule, hydrochlorothiazide induces a natriuresis and concomitant water loss. Thiazides increase the reabsorption of calcium in this segment in a manner unrelated to sodium transport. Additionally, by other mechanisms, HCTZ is believed to lower peripheral vascular resistance. Fig.No1.Captopril. Fig.No.2 Hydrochlorothiazide. Materials and methods: Instrumentation: Absorbance measurements were made on UV-Visible spectrophotometer Lab India, model is Shimadzu UV Spectrophotometermodel-1800 with 1cm quartz cell. The absorption spectra of reference and test solutions were carried out in 1 cm matched quartz cell over the range of nm. Isobestic point was found to be 205nm. Captopril shows spectra at 220nm and Hydrochlorothiazide at 271nm.In this spectrophotometer single and multicomponent drugs spectra determined UV and visible region. Chemicals and reagents: Captopril and hydrochlorothiazide were obtained as gift samples from Wockhardt Limited Aurangabad (India).Methanol (AR) grade was purchased and distilled water were purchased and used. The combination of CAP of HTZ are available as tablet dosage forms with trade names CAPOTRIL-H (25 mg captopril and 15 mg hydrochlorothiazide) (Lupin ltd. India) IJPT June-2017 Vol. 9 Issue No Page 30088

3 Preparation of stock solution: Standard Stock Solution of Captopril: 25 mg of Captopril was weighed accurately and transferred to a 10 ml volumetric flask, dissolved in sufficient quantity of methanol and water (80:20) then sonicated for 15 min and diluted to 10 ml with the same solvent so as to get the concentration of 1000 µg/ml. Standard stock solution of Hydrochlorothiazide: 15 mg of Hydrochlorothiazide was weighed accurately and transferred to a 10 ml volumetric flask, dissolved in sufficient quantity of methanol and water (80:20) then sonicated for 15 min and diluted to 10 ml with the same solvent so as to get the concentration of 1000 µg/ml. Procedure: Determination of λmax of Captopril: Standard solution of Captopril was prepared. The standard drug solution at different concentration was scanned on UV spectrophotometer in the range of nm and the λ max was found to be 220 nm against methanol: water. The UV spectrum is shown in Fig. No. 3 Fig. No. 3 Individual spectra of Captopril. Determination of λ max of Hydrochlorothiazide: Standard solution of Hydrochlorothiazide was prepared. The standard drug solution at different concentration was scanned on UV spectrophotometer in the range of nm &the λ max was found to be nm against methanol: water. The UV spectrum is shown in Fig. No.4. Fig. No. 4 Individual spectra of Hydrochlorothiazide. IJPT June-2017 Vol. 9 Issue No Page 30089

4 Determination of absorption maxima: Appropriate dilution of two drugs were prepared separately using standard stock solutions containingcaptopril andhydrochlorothiazide were scanned in the range of 400 nm to 200 nm to determine the wavelength of maximum absorption for both the drugs.captopril and Hydrochlorothiazide showed absorbance maxima at 220 nm and 271 nm respectively. The overlain spectra showed λ max of both drugs Fig. No. 5. Fig. No. 5 Overlain spectra of Captopril& Hydrochlorothiazide Determination of absorptivity values:- The standard drug solutions were prepared to get a concentration of 20 µg/ml of Captopril and 20 µg/ml of and Hydrochlorothiazide volume was made up to the mark with methanol: water. The absorbance of each solution was measured at 220 nm and 271. The absorbances were then divided concentration to get absorptivity. The details shown in Table no.1. Table No.1 Absorptivity value of Captopril and Hydrochlorothiazide. Components Absorptivity at 220nm Absorptivity of 271nm Captopril(x) ax1= ax2= Hydrochlorothiazide(y) ay1= ay2= Analysis of standard mixture by proposed method: In order to observe the feasibility of proposed method for simultaneous estimation of Captopril and Hydrochlorothiazide in pharmaceutical formulations, the method was tried on standard mixture. Accurately weighed quantities of Captopril (25 mg) and Hydrochlorothiazide (15 mg) were taken in 10 ml volumetric flask separately and dissolved in methanol: water (80:20) by vigorous shaking. The volume was made up to the mark using IJPT June-2017 Vol. 9 Issue No Page 30090

5 methanol: water. The aliquot portions of above solution were further diluted with solvent to get Captopril (10 mg/ml) and Hydrochlorothiazide (10 mg/ml). The absorbances of the resulting solutions were measured at nm and nm. Concentration of each drug was determined using simultaneous equation: A 2 ay 1 - A 1 ay 2 C x = ax 2 ay 1 - ax 1 ay 2 A 1 ax 2 - A2ax 1 C y = ax 2 ay 1 - ax 1 ay 2 Where, C x = concentration of Captopril. C y = concentration of Hydrochlorothiazide. ax 1 = absorptivity value of Captopril at nm. ax 2 = absorptivity value of Captopril at nm. ay 1 = absorptivity value of Hydrochlorothiazide at nm. ay 2 = absorptivity value of Hydrochlorothiazide at nm. A 1 = absorbance of standard mixture at nm. A 2 = absorbance of standard mixture at271.0 nm. Table No. 2 Results of analysis of Captopril and Hydrochlorothiazide in standard. Sr. No. Concentration present (µg/ml) Concentration found (µg/ml) % Estimated CAP HTZ CAP HTZ CAP HTZ Mean SD %RSD IJPT June-2017 Vol. 9 Issue No Page 30091

6 Analysis of tablet formulation: Brand Name: - Capotril-H Each tablet content: Captopril: 25mg Hydrochlorothiazide: 15mg Manufacturer:Lupin Ltd, India. Analysis of marketed formulation by proposed method: Ten tablets of brand name Capotril-H were accurately weighed and were reduced to fine powder and mixed thoroughly. A quantity of tablet powder equivalent to Captopril (25 mg) and Hydrochlorothiazide (15 mg) was transferred to 10 ml volumetric flask and dissolved in methanol: water (80: 20) with vigorous shaking. The volume was made up to the mark using methanol: water. The solution was filtered through Whatmann filter. The aliquot portion of filtrate was further diluted to Captopril (10 mg) and Hydrochlorothiazide (10 mg) respectively. The absorbance of sample solution was measured at 220 nm and 271 nm and the results are shown in (Table No. 3) Table No.3 Result of analysis of Captopril and Hydrochlorothiazide in tablet Formulation. Concentration present Concentration found Sr. No. (µg/ml) (µg/ml) % Estimated CAP HTZ CAP HTZ CAP HTZ Mean SD %RSD Validation Parameters: 1. Linearity: Linearity of analytical procedure is its ability (within a given range) to obtain test results, which are directly proportional to concentration of an analyte in the sample. The calibration plot was constructed after analysis of five IJPT June-2017 Vol. 9 Issue No Page 30092

7 Abs different (from 2 to 10µg/ml for Captopril and 2-10 µg/ml for Hydrochlorothiazide) concentrations from standard stock solution respectively. Absorbances for each concentration were measured at 220 nm (λ max of Captopril) and 271 nm (λ max of Hydrochlorothiazide) respectively. (Table No. 4, 6). The calibration curves are shown in (Fig. No. 6, 7). The result of optical parameters for Captopril Hydrochlorothiazide are shown in (Table No. 5, 7) respectively. Table No. 4 Linearity of Captopril. Sr. No. Concentration µg/ml Absorbance Concentration y = 0.048x R² = Absor Линейная (Absor) Fig. No. 6 Standard calibration curve of Captopril. Optical parameter: Table No. 5 Result of optical parameter of Captopril. Sr. No. Parameters Observation 1 λ max nm 2 Beer s law limit (µg/ml) 2-10 µg/ml 3 Regression equation X Correlation Coefficient ( r 2 ) IJPT June-2017 Vol. 9 Issue No Page 30093

8 Abs. Table No. 6 Linearity of Hydrochlorothiazide. Sr. No. Concentration µg/ml Absorbance y = 0.072x R² = Concentration Fig. No. 7 Standard calibration curve of Hydrochlorothiazide. Optical parameter: Table No. 7 Result of optical parameter of Hydrochlorothiazide. Sr. No. Parameters Observation 1 λ max nm 2 Beer s law limit (µg/ml) 2-10 µg/ml 3 Regression equation X Correlation Coefficient ( r 2 ) Recovery studies: Recovery studies were performed to validate the accuracy of developed method. To preanalysed tablet solution, a definite concentration of standard drug (80%, 100%, and 120%) was added, the absorbances are recorded at 220 nm, 271 nm.( λ max of Captopril & Hydrochlorothiazide respectively) and then its recovery was analyzed and result was IJPT June-2017 Vol. 9 Issue No Page 30094

9 shown in (Table No.8) and statistical validation of recovery studies shown in (Table No. 9). Table No.8: Recovery studies of Captopril & Hydrochlorothiazide. Level of Recovery (%) Amount present (mg) CAP HTZ CAP HTZ CAP HTZ Amount of Std. added (mg) Amount Recovered (mg) % Recovery Table No.9 Statistical Validation of Recovery Studies. Level of Mean Standard Drug Recovery (%) % Recovery Deviation* %RSD CAP HTZ CAP HTZ CAP HTZ *Denotes average of three determinations. 3. Precision: The method was established by analysing various replicates samples of Captopril and Hydrochlorothiazide. Precision was carried out by performing inter-days variation and intra-day variation. In inter-days variation the sample was analyzed on three consecutive days. In intraday variation the absorbances were measured three times in a day. The results for intra-day precision are shown in (Table No.10&11) and for inter-day precision are shown in (Table No. 12&13). IJPT June-2017 Vol. 9 Issue No Page 30095

10 Table No. 10Intra-day precision study of Captopril. Conc. µg/ml Absorbance Trial 1 Trial 2 Trial 3 Mean Area SD %RSD Table No. 11 Intra-day precision study of Hydrochlorothiazide. Conc. µg/ml Absorbance Trial 1 Trial 2 Trial 3 Mean Area SD %RSD Table No.12 Inter-day precision study of Captopril. Conc. µg/ml Absorbance Day 1 Day 2 Day 3 Mean Area SD %RSD Table No. 13 Inter-day precision study of Hydrochlorothiazide. Conc. µg/ml Absorbance Day 1 Day 2 Day 3 Mean Area SD %RSD LOD (Limit of detection) and LOQ (Limit of quantitation):- The Limit of Detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD IJPT June-2017 Vol. 9 Issue No Page 30096

11 was calculated using the following formula and shown in (Table No.14) LOD = 3.3 (σ / S) Where, S = slope of calibration curve, σ = standard deviation of the response. The Limit of Quantitation (LOQ) is the smallest concentration of the analyte, which gives a response that can be accurately quantified. LOQ was calculated using the following formula and shown in (Table No.14) LOQ = 10 (σ / S) Where, S = slope of calibration curve, σ = standard deviation of the response. Table No. 14 Result of LOD and LOQ. Sample LOD (µg/ml) LOQ(µg/ml) CAP HTZ Range: The range shown by CAP and HTZ is given as follows. CAP: 2-10 µg / ml HTZ: 2-10 µg / ml Discussion: The analysis of tablet formulation was done and results obtained within the limits. The results obtained for validation study were also within the limit specified by the ICH guidelines and hence the method was found to be accurate, linear, and precise. The method developed can be used for the routine analysis of Captopril and Hydrochlorothiazide in bulk and tablet dosage form. Conclusion: For the Spectrophotometric methods Methanol: Water (80:20) used as a solvent. The wavelength used for Captopril and Hydrochlorothiazide was nm and nm respectively.the simultaneous equation method was developed and validated according to the ICH guidelines. The linearity, precision, LOD, LOQ, recovery by the simultaneous equation method was within the limits as specified by the ICH guidelines. Hence the method was found to be simple, accurate, precise, and economical for routine analysis of Captopril and Hydrochlorothiazide in bulk as well as tablet formulations. Acknowledgement: The authors sincerely thanks to Sahyadri College of Pharmacy, Methwade, Sangola, Solapur, Maharashtra, India for providing experimental facilities to carry out this work successfully. IJPT June-2017 Vol. 9 Issue No Page 30097

12 References: 1. Chatwal G.R., Anand S.K. A Text Book of Instrumental Method of Chemical Analysis. 5 th ed n 2002; pp Dr. Kasture A.V., Dr. Mahadik K.R., Dr.Wadodkar S.G., Dr. More H.N. Text book of Pharmaceuticals Analysis Instrumental Methods. 2002, Vol. 2: Audumbar Digambar Mali, Seeta vitthal Mali, Ashpak Tamboli, Ritesh Bhate. Simultaneous UV Spectrophotometric Methods for Estimation of Metformin HCL and Glimepiride in Bulk and Tablet Dosage Form.2015, vol.4 (6): Kareti Srinivasa Rao, Minakshi Panda, Nargesh Kumar Keshar, Sushil Kumar Yadav. Simultaneous estimation of captopril and hydrochlorothiazide in combined dosage forms. Chronicles of Young Scientists. 2012; 3(1): Audumbar Mali, Ankush Hake, Manoj Patil, Ritesh Bathe, Manojkumar Patil, Ashpak Tamboli. Estimation of metronidazole benzoate in bulk and formulation by first order derivative area under curve UVspectrophotometric methods. ; March 2016, Volume 1; Issue 1; Page No Audumbar Digambar Mali Estimation of Domperidone in Bulk and Formulation by First Order Derivative Area Under Curve UV-Spectrophotometry Methods,July-2015, Vol. 7, Issue No Mali Audumbar Digambar, Hake Gorakhnath and Patil Manojkumar. Zero order and area under curve spectrophotometric methods for determination of Paracetamol in pharmaceutical formulation.2015, 01 (03); Audumbar Digambar Mali, Ritesh Bathe, Manojkumar Patil and Ashpak Tamboli. Zero order and area under curve spectrophotometric methods for determination of Levocetirizine in pharmaceutical formulation. 2015; 1(06): Siladitya Behera, Subhajit Ghanty, Fahad Ahmad, Saayak Santra, and Sritoma Banerjee. UV-Visible Spectrophotometric Method Development and Validation of Assay of Paracetamol Tablet Formulation. J Anal. Bioanal.Techniques. 2012; 3 (6): Chatwal G.R., Anand S.K. Instrumental Methods of Chemical Analysis, 5 th ed n.himalaya Publishing House, Mumbai; 2002; , Bekett A.H., Stenlake J.B., Practical Pharmaceutical Chemistry, CBS Publishers and Distributors, New Delhi, IJPT June-2017 Vol. 9 Issue No Page 30098

13 Part-2, 2002: Dr. Kasture A.V., Dr. Mahadik K.R., Dr. Wadodkar S.G., Dr. More H.N. Text book of Pharmaceutical Analysis Instrumental Methods, 12 th ed n. Nirali Publication; 2005; Sharma B.K. Instrumental Methods of Chemical Analysis, 26 th edn. Goal Publishing House, Meerut; 2007; S Parimo P.A. Text book of Pharmaceutical Analysis, 1 st ed n. CBS Publishers &Distributors; 1998: Jasmine Chaudhary, Akash Jain, Vipinsaini. Simultaneous Estimation of Multicomponent Formulations by UV- Visible Spectroscopy: An Overview. Int. Res. J. Pharm. 2011; 2(12): Manohar A. Potdar.A Textbook of Pharmaceutical Quality Assurance, 4 th ed n. Nirali Prakashan.2015; Ich Harmonized Tripartite Guideline. Validation of Analytical Procedures: Text and Methodology. Q2 (R1): ICH Q2A Text on validation of analytical procedures, International conference on harmonization, tripartite guideline, 1994; 1-5. Corresponding Author: Seeta Vitthal Mali * IJPT June-2017 Vol. 9 Issue No Page 30099

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