Validated RP-HPLC Method for Determination of Bromhexine Hcl, Chlorpheniramine Maleate, Dextromethorphan Hbr and Guaiphenesin in Pharmaceutical Dosage Forms

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 standard solution containing BROM, CHL, DEX and GUA were prepared by 160 mg, 40 mg, 200 mg and 2000 mg of each reference standard respectively and transferring it to 1000 ml volumetric flask and 200 ml of diluents (HPLC grade methanol) was added to the volume and sonicated in bath sonicator for 10 minutes to ensure complete solubilization. After sonication, the volume was made up to the mark 1000 ml. with same diluent, to result in a final concentrations of 160 µg/ml of BROM, 40 µg/ml of CHL, 2 µg/ml of DEX and 2000 µg/ml of GUA of each reference standard respectively. 2.5 Estimation fromAccepted pharmaceutical dosage form Manuscript Twenty tablets of MERICOF were taken and calculated average weight of one tablet. They were homogenized to a fine powder and it was transferred to a 1000.0 ml volumetric flask and dissolved in 200.0 ml of diluents (HPLC grade methanol) and sonicated in bath sonicator for 20.0 minutes to ensure complete solubilization. After sonication, the supernatant liquid was transferred to 1000.0 ml of volumetric flask through whatmann paper no. #41 filters paper. The residue was washed three times with 10.0 ml portion of methanol and the combined filterate was made up to

Page 5 of 17 the mark 1000.0 ml with the same diluents to result in a final concentration 160.0 µg/ml of BROM, 40.0 µg/ml of CHL, 2.0 µg/ml of DEX and 2000.0 µg/ml of GUA respectively.

A constant volume of sample solution was injected six times under the conditions described above. From the chromatogram it was found that BROM, CHL, DEX and GUA retention time 16.254, 12.219, 6.156 and 9.432 respectively with a resolution 3.15 between DEX and GUA, 2.72 between GUA and CHL, 3.98 between CHL and BROM. The result of capacity factor, tailing factor, theoretical plate number are reported in Table 1.The total runtime was 20 min. The peak areas were measured at 265 nm for BROM, CHL, DEX and GUA, respectively, and their concentrations in the samples were determined using multi-level calibration curve developed on the same HPLC system under the same conditions using linear regression equation.

2.6 Preparation of linearity solution

From the above standard stock solution 1, 100.0 µg/ml of each (BROM, CHL, DEX and GUA), different working standards were prepared for each drugs concentration 4.0, 8.0, 12.0, 16.0, 20.0 and 24.0 µg/ml for BROM, 5.0, 10.0, 15.0, 20.0, 25.0 and 30.0 µg/ml for CHL, 10.0, 20.0, 30.0, 40.0, 50.0 and 60.0 µg/ml for DEX and GUA respectively for linearity determination. Six replicates of each different working standard were prepared for each drug. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs.

2.7 Analytical method validation

The method was validated for analytical procedures according to ICH guidelines in order to determine the linearity, sensitivity, precision and accuracy for the analyte. System suitability test of the chromatography system was performed before each validation run. Five replicate injections of a system suitability standard and one injection of a check standard were made. Regression characteristics, validation and system suitability parameters analysis of BROM, CHL, DEX and GUAAccepted in pharmaceutical dosage form Manuscript are shown in Table 3. 2.7.1 Linearity:

The method was linear from 4.0 µg/ml – 24.0 µg/ml for BROM, 5.0 – 30.0 µg/ml for CHL, 10.0 – 60.0 µg/ml for both DEX and GUA respectively. The calibration curve was plotted using area vs. concentration each compound had R2 value of 0.9980 or greater.

2.7.2 Accuracy:

Page 6 of 17 Recovery study of MERICOF was carried out as per ICH guidelines [37], where to a preanalyzed solution of tablet formulation, a known concentration of all four standards solutions was added that are equivalent to 80, 100, 120 % of total drug content and the % age recovery was calculated.

2.7.3 Precision:

The intra and inter day precision study of MERICOF were calculated by assay of sample solution (marketed formulation) on the same day and different days at different time in interval respectively. It was performed with at least six replicates of assay of sample solution. An amount of the sample powder equivalent to 100% of the label claim of BROM, CHL, DEX and GUA was accurately weighed and assayed. Method repeatability was achieved by repeating the same procedure six times on the same day for intra-day precision. The intermediate (inter-day) precision of the method was checked by performing the same procedure on different days under the same experimental conditions.

2.7.4 Limit of Detection and Limit of Quantitation (LOD and LOQ):

The LOD and LOQ were calculated using the following equation as per ICH guidelines:

LOD=3.3 X σ /S, LOQ = 10 X σ /S

Where is the standard deviation of -intercepts of regression lines and S is the slope of the

calibration curve.

For LOD and LOQ,Accepted 10.0 µg/ml. of all four standard solutionManuscript were prepared from the 100.0 µg/ml of each standard stock 1 solution (BROM, CHL, DEX and GUA) 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 µg /ml working dilution for BROM and CHL, 0.3, 0.5, 0.7, 0.9, 1.1 and 1.3 µg/ml for working dilution DEX, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 µg/ml working dilution for GUA. LOD and LOQ values were calculated to check the detection limit of the method by using following equation:

2.7.5 Selectivity and Specificity:

Page 7 of 17 A combination of methanol: acetonitrile: 0.025M phosphate buffer pH 5.5 ratio (50: 25: 25 v/v) was used as a specific mobile phase and 265.0 nm selected as a specific analytical wavelength for simultaneous determination of BROM, CHL, DEX and GUA in a marketed formulation (MERICOF) analysis in HPLC method. Specificity was assessed by a qualitative comparison between chromatograms obtained from sample, standard, blank and placebo solutions. Diluent was injected as a blank. Placebo (Fig:2) interference study was conducted by injecting placebo solution prepared from the excipients most commonly used in pharmaceutical formulations including starch, lactose monohydrate, and magnesium stearate.

3. Results and Discussion

A new, rapid, sensitive and accurate RP- HPLC method for simultaneous estimation of Bromhexine hydrochloride, Chlorpheniramine maleate, Dextromethorphan hydrobromide and Guaphenesine in Pharmaceutical formulation has been developed. After trying different column, the final choice of the stationary phase giving satisfactory resolution and run time was the

reversed phase column C18 cromatopack Peerless (250.0 mm. X 4.6 mm. X 5µm. i.d). There were many mobile phases that tried to resolve all four chromatographic peaks, in that first with simple methanol: water (80: 20, v/v), methanol: water (50:50, v/v), but found broadness of peaks and could not get satisfactory results in these chromatograms. To improve these, with phosphate buffer and for sharpness of chromatographic peaks we worked with acetonitrile with slightly acidic condition. Finally, the mobile phase methanol: acetonitrile: 0.025 M phosphate buffer (50: 25:25, v/v/v) (pH: 5.5) adjusted with O-phosphoric acid was found to be satisfactory which gave four symmetric peaks for BROM - CHL - DEX and GUA. Total run time was 20 minutes at a 1.0 ml min-1 and ambient temperature. 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 (Fig:3) respectively. The best fit for the calibration curve could be achieved by a linear regression equation, which was found to be y = 9040x + Accepted9993 (BROM), y = 9579x – 308 (CHL),Manuscript y = 25935x + 25500 (DEX) and y = 34255x + 14194 (GUA) separately by plotting a calibration curve of peak area against their respective concentration. The proposed method was evaluated in the assay of formulation containing BROM, CHL, DEX and GUA. Four replicate determinations were carried out on capsules. % assay found was 100.45 % for BROM, for 100.25 % CHL, for DEX 99.88 % and that for GUA was, 100.56 %. Under the proposed chromatographic condition all four drugs were completely separated from each other with a resolution 3.15 between DEX and GUA, 2.72

Page 8 of 17 between GUA and CHL and 3.98 between CHL and BROM. It indicates that the method is selective for simultaneous estimation. Specificity was assessed by comparing the chromatogram of tablet solution with the placebo solution and also with the chromatograms obtained from standard drugs. As the retention time of all four drugs were same in quaternary mixed standard solution’s well as marketed formulation (MERICOF) solution and also there was no interference found of excipients. It indicates the specificity of the method for quantitative estimation of BROM, CHL, DEX and GUA in marketed formulation (Table 2). The recoveries of the all component were found between 99.0-102%. The LOD and LOQ values were found to be 21.49 and 65.13 ng ml-1, 29.17 and 88.39 ng ml-1, 11.65 and 35.51 ng ml-1 , 8.1 and 24.57 ng ml-1 for BROM, CHL, DEX and GUA respectively Table 2. No interference of the excipients with the peaks of interest appeared; hence the proposed method is applicable for the routine simultaneous estimation of BROM, CHL, DEX and GUA in pharmaceutical dosage forms.

4. Conclusion: In the present work a successful attempt for simultaneous analysis of BROM, CHL, DEX and GUA in a single marketed formulation (MERICOF) by RP-HPLC method were performed by laboratory research experiment based on literature survey. All of the critical steps in method development have been summarized and prioritized. In order to develop a RP-HPLC method effectively, most of the effort should be spent in method development and optimization as this will improve the final method performance. The method validation, however, should be treated as an exercise to summarize or document the overall method performance for its intended purpose. Thus the present method is rapid, easy and accurate for the simultaneous estimation of BROM, CHL, DEX and GUA in a commercially available pharmaceutical formulation. 5. Acknowledgement:

The authors are thankful to TABLIKE and SCHON Pharmaceutical Jumbu Di Hapsi Hatod Road, Indore, M.P, for providing gift samples. One of author VJ thanks the All India Council for Technical EducationAccepted (AICTE), New Delhi, India, forManuscript the financial support for this research. We are grateful to referee to given valuable suggestion.

References

1. Parvez L, Vaidya M, Sakhardande A, Subburaj S, Rajagopalan TG. Evaluation of antitussive agents in man. Pulm.Pharm. 1996; 9: 299-308.

Page 9 of 17 2. Cobbin DM, Elliott FM, Rebuck AS. The mucolytic agent bromhexine (bisolvon) in chronic lung disease. A double-blind crossover trial. Aust.New Zea J Med. 1971; 1: 137-40.

3. Rauha JP, Salomies H, Aalto M. Simultaneous determination of bromhexine hydrochloride and methyl and propyl p-hydroxybenzoate and determination of dextromethorphan hydrochloride in cough-cold syrup by high-performance liquid chromatography. J. Pharm. Biomed. Anal. 1996; 15: 287-293.

4. Rao EV, Rao GR, Raghuveer S, Khadgapathi P. Gas-liquid chromatographic and ion-pair high-performance liquid chromatographic determination of pseudoephedrine hydrochloride and bromhexine hydrochloride in pharmaceuticals. Analyst. 1987; 112:871–874.

5. Lau OW, Cheung YM. Simultaneous determination of some active ingredients in cough—cold syrups by gas-liquid chromatography. Analyst. 1990; 115: 1349–1353.

6. Boh CE, Rudy JA, Soma LR, Fennell M, May L, Sams R, Railing FA, Shellenberger J, Kahler M. Characterization of bromhexine and ambroxol in equine urine: effect of furosemide on identification and confirmation. J. Pharm. Biomed. Anal. 199; 19: 33-9.

7. Rauha JP, Salomies H, Aalto M. Simultaneous determination of bromhexine hydrochloride and methyl and propyl p-hydroxybenzoate and determination of dextromethorphan hydrobromide in cough-cold syrup by high-performance liquid chromatography. J. Pharm. Biomed. Anal. 1996; 15: 287-93.

8. Vasudevan M, Ravisankar S, George M, Ravi J. Simultaneous estimation of terbutaline, bromhexine and guaiphenesin in soft gelatin capsules by HPLC method. Ind. Drug. 2000; 37: 489-492.

9. Dave HN, Mashru RC, Patel AK. Thin layer chromatodraphy method for the determination of ternary mixture Accepted containing salbutamol sulphate, bromhexineManuscript hydrochloride and Etofylline. J .Pharma .Sci .Res. 2010; 2:143-148.

10. Gupta V, Verma M, Misra U, Nema RK. Simultaneous spectrophotoetric estimation of bromhexine hydrochloride and pseudoephedrine hydrochloride in tablet dosages. Asian. J. Chem. 2009; 21:1633-1635.

11. Gupta AK, Kaskhedikar S.G. Derivative spectrophotometric estimation of amoxycillin and bromhexine hydrochloride in tablets. Asian .J. Chem. 2003; 15:977-980.

Page 10 of 17 12. Panda SK, Sharma AK, Sahu L K. Simultaneous analysis of phenylpropanolamine, chlorpheniramine and bromhexine in syrups by derivative spectrophotometry.Ind. J. Pharma. Sci. 2002; 64:540-544.

13. Gangwal S, Trivedi P. Simultaneous determination of terbutaline sulphate, bromhexine hydrochloride and guaiphenesin in three-component tablet formulation by UV spectrophotometry. Ind. J. Pharma .Sci. 1999;61:128-130.

14. Paton DM, Webster DR. Clinical pharmacokinetics of H1-receptor antagonists (the antihistamines). Clin. Pharmaco. 1985; 10:477-97.

15. Maithani M, Raturi R, Vertika G, Kumar D. Development and validation of RP-HPLC method for the determination of chlorpheniramine maleate and phenylephrine HCl in pharmaceutical dosage form. Int. Res. J .Pharm . 2010;5:1-4.

16. Wanjari DB, Parashar VV, Lulay S N, Tajne MR, Gaikwad NJ. Simultaneous HPLC estimation of acetaminophen, chlopheniramine maleate, dextromethorphan hydrobromide and pseudoephedrine hydrochloride in tablets. Ind. J. Pharma. Sci. 2004; 66:345-34.

17. Senyuva H, Özden T. Simultaneous high-performance liquid chromatographic determination of paracetamol, phenylephrine HCl, and chlorpheniramine maleate in pharmaceutical dosage forms. J. Chromatogr. Sci. 2002; 40:97–100.

18. Hunan N, Multal S.Densitometric analysis of chlorpheniramine maleate, phenylephrine and acetaminophen by HPTLC method. Analy. Lett. 1986; 19: 7-8.

19. Murtha J, Julian T, Radebaugh G. Simultaneous determination of pseudoephedrine hydrochloride, chlorpheniramine maleate and dextromethorphan hydrobromide by Second- Derivative photodiode array spectroscopy. J. Pharma. Sci. 1988; 77:715-717.

20. Suntornsuk AcceptedL, Pipitharome O, Wilairat P. Simultaneous Manuscript determination of paracetamol and chlorpheniramine maleate by micellar electrokinetic chromatography. J. Pharm. Biomed. 2003;33:441-449.

21. United States Pharmacopoeia, 25th Review, The National Formulary, 19th Review, US Pharmacopoeia Convention,Rockville, MD, 2002.

Page 11 of 17 22. KuKanich B, Papich MG. Plasma profile and pharmacokinetics of dextromethorphan after intravenous and oral administration in healthy dogs. J. Vete. Pharma. Therap. 2004;27: 337-341.

23. Shervington L A. A quantitative simultaneous HPLC determination of Pseudoephedrine HCl. Guaifenesin and dextromethorphan hydrobromide. Anal. Lett . 1990;30: 927.

24. Mistry B, Leslie J, Eddington NE. A sensitive assay of metoprolol and its major metabolite alpha-hydroxy metoprolol in human plasma and determination of dextromethorphan and its metabolite dextrorphan in urine with high performance liquid chromatography and fluorometric detection. J. Pharm. Biomed. Anal. 1998; 16:1041-9.

25. Bendriss EK, Markoglou N, Wainer IW. High-performance liquid chromatography assay for simultaneous determination of dextromethorphan and its main metabolites in urine and in microsomal preparations. J. Chromatogr. B. Biomed. Sci. Appl . 2001; 754:209-15.

26. Tantishaiyakul V, Poeaknapo C, Sribun P, Sirisup K. Derivative spectrophotometric determination of Dextromethorphan HBr and bromohexine HCl in tablet. J. Pharma. Biomed. Anal.1998; 17:237-243.

27. Davidson G, Mkoji LMM. Simultaneous assay of Triprolidine, pseudoephedrine and Dextromethorphan in combined preparation by derivative difference spectrophotometry. J. Pharm. Biomed. Anal. 1988; 6:449-460.

28. Suntornsuk L. Separation of cold medicine ingredients by capillary electrophoresis. Electrophoresis. 2001; 22:139-43.

29.Statheropoulos M, Tzamtzis N, Mikedi K. Short column gas chromatography-mass spectrometry and principal component analysis for the identification of co eluted substances in doping control analysis. J .Chromatogr. B. Biomed. Sci. Appl. 1998; 706:245-51.

30. The British Pharmacopoeia,Accepted 2007. Her Majesty’s, Manuscript The Stationary Office, London.

31. Martindale-Extra Pharmacopoeia, 2005. The Complete Drug References, 34th Ed. The pharmaceutical Press, London, UK.

32. Sharaf Maged HM, Stiff Dwight D. Determination of guiafenesin in human serum by capillary gas chromatography and electron capture detection. J. Pharm. Biomed. Anal . 2004; 35:801-806.

Page 12 of 17 33. El-Gindy A, Emara S, Shaaban H. Development and validation of chemometrics assisted spectrophotometric and liquid chromatographic methods for the simultaneous determination of two multi-component mixtures containing bronchodilator drugs. J. Pharm. Biomed. Anal. 2007; 43:973-982.

34. Wen J, Zhang H, Xia C, Hu X, Xu W, Cheng X, Gao J, Xiong Y. A sensitive liquid chromatography-electrospray ionization-mass spectrometry method for the Simultaneous determination of pentoxyverine citrate and guifenesin in human plasma- application to pharmacokinetic and bioequivalence studies. Biomed. Chromatogr. 2010; 24:351-357.

35. Foster J. Determination of Guifenesin in human plasma using automated liquid- liquid extraction and tandem mass spectrometric detection, The AAPS. Abstracts AM-2004; 002586.

36. Eichhold TH, McCauley-Myers DL, Khambe DA, Thompson G A, Hoke SH. Simultaneous determination of dextromethorphan, dextrorphan and Guaifenesin in human plasma using semi- automated liquid/liquid extraction and gradient liquid chromatography tandem mass spectrometry. J. Pharm. Biomed. Anal . 2007; 43: 586-600.

37. International Conference on Harmonization (ICH), Q2B: Text on validation of analytical procedures: Methodology; vol.62: US FDA Federal register, (1997).

Accepted Manuscript

Page 13 of 17 Table 1: Data for the evaluation of the system suitability

Property Bx HCl CPM Dx HBr GPH

R t 16.254 12.219 6.156 9.432

T f 1.09 1.04 1.13 1.29 K' 1.562 1.217 1.764 4.571 N 6359 1276 4925 18743

Rs 3.98 2.72 0.00 3.15

t R , retention time; Tf, tailing factor; k′, capacity factor; N, number of theoretical plates Rs, resolution

Table 2: Results from assay of the marketed formulation

Drug Label claim Amount found Label claim S.D. S.E. % COV (mg/tab) (mg/tab) (%) n=6

Bx HCl 8 8.0365 100.45 0.5902 0.2410 0.5875

CPM 2 2.0052 100.25 1.2891 0.5263 1.2858 Dx HBr Accepted10 9.9887 99.88Manuscript0.7494 0.3059 0.7502 GPH 100 100.5601 100.56 0.7991 0.3262 0.7946

S.D- standard deviation; COV- coefficient of variance; S.E- standard error; n- Number of replicates

Page 14 of 17 Table.3: Summary of validation parameters Parameter Bx HCl CPM Dx HBr GPH Linearity range 4 - 24 5 – 30 10 - 60 10 – 60 Slope 9040 9579 25935 34255 Interept (y) 9993 -308 25500 14194 R2 0.9987 0.9988 0.9981 0.9988 Accuracy (Percentage of recovery) 80% 99.67 100.0521 101.2917 101.7354 100% 101.07 100.9583 101.225 101.185 120% 100.15 101.0417 100.9931 100.1035 Intraday Precision %COV 0.1755 - 0.6333 0.3050 0.6715 0.1806 - 0.6929 0.2813 - 0.8976 Interdays Precision %COV 0.1018 - 0.2804 0.0675 - 0.1639 0.1529 - 0.4252 0.1227 - 0.2925 L.O.D(ng/ml) 21.49 29.17 11.65 8.1 L.O.Q.(ng/ml) 65.13 88.39 35.51 24.57 Specificity/selectivity No interference Robustness Reliable results Ruggedness Reproducible results

Accepted Manuscript

Page 15 of 17 (a) (b)

Br Cl

CH3 HC COOH

N Br HCl N CH HC COOH CH3 NH2 CH2CH2N CH3

H3CO

OCH3 OH

O CH OH HBr 2 H H

N CH3

Fig-1. Chemical structure of interacting compounds of (a) Bromhexine Hydrochloride (b) Chlorpheniramine Maleate (c) Dextromethorphan Hydrobromide (d) Guaphenesine

Accepted Manuscript

Page 16 of 17 Fig.2: RP-HPLC Chromatogram of Placebo

Accepted Manuscript

Fig.3: HPLC-Chromatogram of marketed formulation

Page 17 of 17