Accepted Manuscript Title: Validated RP-HPLC Method for determination of Bromhexine HCl, Chlorpheniramine Maleate, Dextromethorphan HBr and Guaiphenesin in Pharmaceutical Dosage Forms Author: Vishal Jain Mukesh C. Sharma PII: S1658-3655(15)00063-1 DOI: http://dx.doi.org/doi:10.1016/j.jtusci.2015.02.019 Reference: JTUSCI 172 To appear in: Received date: 15-11-2014 Revised date: 7-2-2015 Accepted date: 15-2-2015 Please cite this article as: Vishal, Validated RP-HPLC Method for determination of Bromhexine HCl, Chlorpheniramine Maleate, Dextromethorphan HBr and Guaiphenesin in Pharmaceutical Dosage Forms, Journal of Taibah University for Science (2015), http://dx.doi.org/10.1016/j.jtusci.2015.02.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Validated RP-HPLC Method for determination of Bromhexine HCl, Chlorpheniramine Maleate, Dextromethorphan HBr and Guaiphenesin in Pharmaceutical Dosage Forms Vishal Jain and Mukesh C. Sharma* * School of Pharmacy, Devi Ahilya Vishwavidyalaya, Takshashila Campus, Indore (M.P.) 452001, India * Corresponding Author E-mail: [email protected], [email protected] Accepted Manuscript Page 1 of 17 Abstract A simple, precise and accurate reverse phase high performance liquid chromatographic method has been developed for simultaneous estimation of Bromhexine hydrochloride, Chlorpheniramine maleate, Dextromethorphan hydrobromide and Guaiphenesin in their tablet dosage form. The chromatographic conditions were standardized using Chromatopak C18 (25cm×4.6mm i.d.×5µm) with UV detection at 265 nm and mobile phase consisting of methanol: acetonitrile: 0.025 M phosphate buffer (50: 25: 25 v/v/v). The retention times of Bromhexine hydrochloride, Chlorpheniramine maleate, Dextromethorphan hydrobromide and Guaiphenesin have been found to be 16.254 min, 12.219 min, 6.156 min and 9.432 min respectively. Calibration curves were linear with correlation coefficient 0.9987, 0.9988, 0.9981 and 0.9981 over a concentration range of 4.0 µg/ml-24.0 µg/ml for Bromhexine hydrochloride, 5.0-30.0 µg/ml for Chlorpheniramine maleate, 10.0-60.0 µg/ml for both Dextromethorphan hydrobromide and Guaiphenesin respectively. The proposed method has been validated as per guideline of ICH and successfully applied to the estimation of four drugs in combined formulation to the determination of the examined drugs with good accuracy and precision. Keywords: RP-HPLC, Bromhexine hydrochloride, Chlorpheniramine maleate, Dextromethorphan hydrobromide, Guaiphenesin, ICH guideline Accepted Manuscript Page 2 of 17 1. Introduction Combinations of decongestant, and antihistaminic, preparations are widely used for cough and cold treatment. Bromhexine HCl (Fig:1), chemically named 2-amino-3, 5-dibromo-N- cyclohexyl-N-methyl benzenemethanamine hydrochloride, is a mucolytic agent used in the treatment of respiratory disorders associated with viscid or excessive mucus [1, 2]. Its mechanism is by increasing the production of serous mucus in the respiratory tract and makes the phlegm thinner and less viscous. Bromhexine HCl (BROM) is a mucous modifying drug helps to improve the flow properties of bronchial mucous and eases expectoration. A literature survey reveals some HPLC methods reported for the simultaneous determination of along with some other active ingredients which exist as various combinations in cough-cold mixture [3], liquid chromatography [4], liquid gas chromatography [5], Gas Chromatography with mass detection [6], combined formulations using HPLC [7-9] and UV spectrophotometric [10-13]. Chlorpheniramine maleate (CHL), 3-(p-chlorophenyl)-3-(2-pyridyl)-N,N-dimethyl propylamine (Fig:1), is a powerful first-generation alkyl amine antihistamine, H1-receptor antagonist, widely used for symptomatic relief of common cold and allergic rhinitis, with weak sedative properties [14]. These symptoms include rash, watery eyes, itchy eyes, throat, cough, and sneezing. It is also effective against nausea and motion sickness, with its primary mechanism of action being its ability to reduce acetylcholine levels in the brain. Literature survey shows that several HPLC methods have been reported for chlorpheniramine maleate single and in combinations in pharmaceuticals liquid chromatographic [15-17], HPTLC [18], spectrophotometry [19] and micellar electrokinetic chromatography [20]. Dextromethorphan hydrobromide (DEX), [(+)-3- Methoxy-17-methyl-9α, 13α, 14α morphinan hydrobromide monohydrate] is a cough suppressant, used for the relief of non-productive cough; it has a central action on the cough centre in the medulla [21]. Dextromethorphan hydrobromide (DEX) is an antitussive drug and used for pain relief and psychological applications [22]. The chemical structures of DEX are shown in Fig: 1.Accepted The combination of these drugs Manuscript is used as antitussive and mucolytic in bronchitis and chronic pulmonary conditions. Several analytical techniques have been reported in the literature, most commonly liquid chromatography [23-24], first and second-derivative technique UV spectrophotometric [25-27] capillary electrophoresis [28] Gas Chromatography [29]. Guaiphenesin (GUA; Fig: 1) is (2RS)-3-(2-methoxyphenoxy) propane-1, 2-diol [30], it is reported to increase the volume and reduce the viscosity of tenacious sputum and is used as an expectorant for productive cough. It is the glyceryl ether of guaiacol (a constituent of guaiac resin from the wood of Guajacum officinale Linne), it act as expectorant by increasing the Page 3 of 17 volume and reducing the viscosity of secretions in the trachea and bronchi. It is the component of numerous cough cold preparations available worldwide. Also it has been given to patients with altered nasal mucociliary clearance associated with HIV infection [31]. The literature survey revealed that some techniques have been published for the determination of Guaifenesin either in their combined form or in combinations with other drugs by capillary gas chromatography [32], HPLC [33], LC-MS [34], LC-MS/MS [35-36]. There is no method reported for the simultaneous estimation of Bromhexine hydrochloride (BROM), Chlorpheniramine maleate (CHL), Dextromethorphan hydrobromide (DEX) and Guaiphenesin (GUA) in combined dosage form. Therefore, communicate here rapid and cost- effective quality-control tool and reliable method for simultaneous assay of these four drugs in mixture seemed to be necessary. The method should have sufficient accuracy and precision and permit a simple and time-saving assay of BROM - CHL - DEX and GUA in mixtures. 2. MATERIALS AND METHODS 2.1 Apparatus To develop a suitable LC method for the analysis of BROM, CHL, DEX and GUA in their combined dosage form, different mobile phases were tried. The Chromatographic system consists of pump (Shimadzu LC 10AT VP ) with universal loop injector (Rheodyne 7725i) of injection capacity 20 µL. Detector consists of photodiode array detector (PDA) SPD-10 AVP UV-Visible detector and column used was Chromatopak C18 (25cm×4.6mm i.d.×5µm). The equipment was controlled by a PC work station equipped with software CLASS M 10-VP software (Shimadzu, Kyoto, Japan). UV/Visible double beam spectrophotometer (Shimadzu Model 1700) was employed with spectral bandwidth of 1nm and wavelength accuracy of 0.3 nm (with automatic wavelength correction with a pair of 1cm matched quartz cells). 2.2. Reagents and Materials Pure drugs sampleAccepted BROM, CHL, DEX and GUA were Manuscript generously obtained as a gift sample from TABLIKE and SCHON Pharmaceutical, Indore, India. The tablet doses form, MARICOF, (Label claim: 8.0 mg BROM, 2.0 mg CHL, 10.0 mg DEX and 100.0 mg GUA) was procured from the local market (Manufactured by G.S. Pharmaceuticals Pvt. Ltd., Roorkee, India). HPLC grade methanol and acetonitrile were obtained from Merck (Mumbai, India). 2.3 Chromatography conditions Page 4 of 17 The solubility of the four drugs indicated that reversed phase chromatographic method would be best option for simultaneous estimation of BROM, CHL, DEX, and GUA. The mobile phase consists of organic phase methanol, acetonitrile and 0.025M phosphate buffer in the ratio of 50:25:25 (v/v/v adjusted to pH 5.5 using orthophosphoric acid). Mobile phase and working solutions were filtered through 0.2 µm nylon and degassed using sonicator before use. To determine the appropriate wavelength for simultaneous determination of BROM, CHL, DEX, and GUA solutions of these compounds were scanned by UV-visible spectrophotometer in a range 200- 400nm. From the overlain UV spectra suitable wavelength choices considered for monitoring these drugs was 265 nm. 2.4 Preparation of standard stock solutions Standard stock solution of BROM, CHL, DEX and GUA were prepared separately by dissolving accurately weighed 10.0 mg of each BROM, CHL, DEX and GUA (reference standard) transfer it to 20.0 ml of HPLC grade methanol in 100 ml volumetric flask. Sonicated in bath sonicator for 10 minutes to ensure complete solubilization. After sonication, the volume was made up to the mark 100 ml. with same solvent HPLC grade methanol, to result in a final concentration of 0.1 mg/ml (100µg/ml) of each reference standard. A combined