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1 available online at Pelagia Research Library Der Chemica Sinica, 2014, 5(5):52-60 ISSN: 0976-8505 CODEN (USA) CSHIA5 52 Pelagia Research Library Validated RP-HPLC method for the determination of Benzoic acid in bulk and pharmaceutical formulation M. Madhu1, V. Sreeram*2, A. V. D. Nagendrakumar3 and T. V. Reddy4 1 Department of Chemistry, P. B. Siddhartha College of Arts & Science, Vijayawada, Krishna (Dt.), A. P., India 2 Department of Chemistry, A. G. & S. G. Siddhartha College of Arts & Science, Vuyyuru, Krishna (Dt.), A. P., India 3 Department of Chemistry, GITAM University, Visakhapatnam, Andhra Pradesh, India 4 Department of H.

2 & S. Malla Reddy College of Engineering, Secunderabad, India _____ ABSTRACT A simple, selective, linear, precise and accurate RP-HPLC method was developed and validated for rapid assay of Benzoic acid in Bulk and Pharmaceutical Formulation. Isocratic elution at a flow rate of ml/min was employed on symmetry C18 5 m ( x 150 mm) column at ambient temperature. The mobile phase consisted of Acetonitrile: Buffer in the ratio of 45:55 v/v. The UV detection wavelength was 254nm and 20 l sample was injected. The retention time for Benzoic acid was 18 min. The percentage RSD for precision and accuracy of the method was calculated.

3 The method was validated as per the ICH guidelines. The method was successfully applied for routine analysis of Benzoic acid in the rapid and reliable determination of Benzoic acid in Pharmaceutical formulation. Key words: Benzoic acid, HPLC, UV detection, recovery, precision. _____ INTRODUCTION The molecular formula for benzoic acid was found to be C7H6O2. Benzoic acid is a white, crystalline powder or colourless crystals, odorless or with a very slight characteristic odour. It is slightly soluble in water, soluble in boiling water, freely soluble in alcohol and in fatty oils.

4 Benzoic acid occurs naturally in many plants [1] and it serves as an intermediate in the biosynthesis of many secondary metabolites. Salts of benzoic acid are used as food preservatives and benzoic acid is an important precursor for the industrial synthesis of many other organic substances. Benzoic acid is a constituent of Whitfield's ointment which is used for the treatment of fungal skin diseases such as tinea, ringworm, and athlete's foot.[2][3] Benzoic acid is relatively nontoxic. It is excreted as hip uric acid.[4] Benzoic acid is metabolized by butyrate-CoA ligase into an intermediate product, benzoyl-CoA,[5] which is then metabolized by glycine N-acyltransferase into hip uric acid[6].

5 Cornelia Petronela ENE et. al., [7]described the determination of the antimicrobial preservative benzoic acid in different food matrix. The determination of the preservative was performed employing a HPLC system equipped with UV diode array detection. The HPLC working parameters were optimized and the method was validated by establishing the analytical criteria of performance. The results obtained for the determination of benzoic acid on real soft drink samples from different Romanian companies showed values of preservative content between 0 and 110 mg/L, meeting the national regulated limit.

6 Jankana Burana-osot ,[8]proposed a simple, sensitive and specific HPLC method was validated for the simultaneous determination of benzoic acid and sorbic acid in noodles. Sample preparation involved the extraction with methanol and water (60:40, v/v). The separation was achieved on a V. Sreeram et al Der Chemica Sinica, 2014, 5(5):52-60 _____ 53 Pelagia Research Library Germini- C18 modified silica column (50 mm x mm , 5 m particle diameter). The mobile phase consisted of M ammonium acetate (pH ) and methanol in the ratio of 60:40 (% v/v) at a flow rate of 1 mL/min and detection was performed at 234 nm using a diode-array detector (DAD).

7 The method was validated with respect to specificity, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ) and robustness. The calibration curve for benzoic acid and sorbic acid was found to be linear. The values of LOD and LOQ were and g/mL and and g/mL respectively. The accuracy determined by spike recovery measurement was for benzoic acid and for sorbic acid. Sonal Shah ,[9] descried the identification and quantification of p-hydroxy benzoic acid and agnuside in the extracts of Vitex negundo and Vitex trifolia.

8 The separation was achieved using acetonitrile and O-phosphoric acid water ( , v/v) as the mobile phase in an isocratic elution mode. The developed method was validated as per the ICH guidelines for limit of detection, limit of quantification, linearity, accuracy and precision. Good linearity (r2 ) was observed for both the compounds in wide concentration range. Relative standard deviation values for intra-day and inter-day precision studies were less than 2%. The analytical recoveries of p-hydroxy benzoic acid and agnuside by the developed HPLC method were and respectively.

9 Chiara Guarino [10]Demonstrated a single method, based on RP-HPLC with UV detection, was developed with the aim of simultaneously quantifying four preservatives in cheeses: benzoic acid, sorbic acid, natamycin and lysozyme. The preservatives were extracted from different cheeses by using the same procedure, and separated by a single RP-HPLC gradient elution showing good resolution, in a short time. Recoveries were always higher than 91%; MDLs ranged from to g g 1, and MQLs were included between and g g 1; RDS ranged from 1% to 7%.

10 Quantitation was performed in reference to a matrix matched calibration curve. The method was also applied to real samples for the determination of the four preservatives, with satisfying results. Fig: 1 Structure of Benzoic Acid MATERIALS AND METHODS Materials: Instrumentation: Peak HPLC containing LC 20AT pump and variable wavelength programmable UV-Visible detector and Rheodyne injector was employed for investigation. The chromatographic analysis was performed on a C18 5 m ( x 150 mm). Degassing of the mobile phase was done using a Loba ultrasonic bath sonicator.