As Salbutamol Sulphate) by Use of a Validated High- Performance Liquid Chromatographic Method
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Acta Chromatographica 23(2011)1, 109–119 DOI: 10.1556/AChrom.23.2011.1.7 Simultaneous Analysis of Phenylephrine Hydrochloride, Guaiphenesin, Ambroxol Hydrochloride, and Salbutamol (as Salbutamol Sulphate) by Use of a Validated High- Performance Liquid Chromatographic Method S. JOSHI1,*, C. BHATIA1, C.S. BAL2, AND M.S.M. RAWAT3 1Department of Chemistry, K.L.D.A.V. (P.G.) College, Roorkee, Uttarakhand, India-247667 2Talwar Pharma, Jhabrera Road, Manglore, Roorkee, Uttarakhand, India-247656 3Department of Chemistry, H.N.B. Garhwal University, Srinagar, Uttarakhand (India) E-mail: [email protected] Summary. A simple, reversed-phase HPLC method has been developed for rapid, si- multaneous quantification of phenylephrine hydrochloride, guaiphenesin, ambroxol hy- drochloride, and salbutamol (as salbutamol sulphate) in a commercial cough–cold liquid formulation. The compounds were separated on a 250 mm × 4.6 mm C8 column with a gradient prepared from pH 3.0 phosphate buffer and 1:1 methanol–acetonitrile as mobile phase at a flow rate of 1.0 mL min−1. Elution of the analytes was achieved in less than 15 min. Detection was by UV absorbance at 273 nm for phenylephrine hydrochloride and guaiphenesin and 225 nm for ambroxol hydrochloride and salbutamol. Percentage recovery and RSD were, respectively, 100.09% and 0.22% for phenylephrine hydrochlo- ride, 100.43% and 0.50% for guaiphenesin, 100.91% and 0.70% for ambroxol hydrochlo- ride, and 100.54% and 0.55% for salbutamol. The components of the syrup formulation were quantified on the basis of the peak areas obtained from freshly prepared standard solutions. The method was validated in accordance with ICH guidelines. Key Words: simultaneous quantification, validation, cough–cold syrup, RP-HPLC Intro-duction Complete prevention of infections induced by the cold virus is not yet pos- sible, which has led to increasing demand for formulations for relief of cough and allergy symptoms. Cough and cold formulations are available commercially as over-the-counter (OTC) products. These multicomponent formulations include dextromethorphan hydrobromide, ambroxol hydro- chloride, guaiphenesin, phenylephrine hydrochloride, levocetirizine hydro- chloride, salbutamol, and pseudoephedrine hydrochloride, among others. The presence of different classes of active chemicals and the disparity in 0231–2522 © 2011 Akadémiai Kiadó, Budapest Unauthenticated | Downloaded 09/29/21 08:49 PM UTC 110 S. Joshi et al. concentration and formulation matrix poses an analytical challenge which is attracting the attention of analytical chemists. Our earlier work on formulations of non-steroidal anti-inflammatory drugs (NSAIDs) [1] and anti-hypertensive drugs [2] has directed us toward developing rapid and reliable HPLC methods for analysis of a multi- component cough formulation containing phenylephrine hydrochloride, guaiphenesin, ambroxol hydrochloride, and salbutamol (as salbutamol sul- phate) as active ingredients. Guaiphenesin and ambroxol hydrochloride are expectorants, phenylephrine hydrochloride is an alpha-adrenergic receptor agonist, and salbutamol sulphate is a beta-adrenoceptor agonist [3]. The chemical structures of these compounds are shown in Fig. 1. H CH 2 HC CH2OH O H OH OH OCH HO N .HCl 3 CH3 Phenylephrine Hydrochloride Guaiphenesin OH H CH Br 3 N HO CCH3NC CH3 .H2SO4 H . HCl OH CH HOH C 3 NH2 2 Br 2 Ambroxol hydrochloride Salbutamol Sulphate Fig. 1. The chemical structures of phenylephrine hydrochloride, guaiphenesin, ambroxol hydrochloride, and salbutamol sulphate Analysis of multi-component anti-cough formulations with two or three active ingredients by HPLC [4–9] is more common than analysis of those with four or more ingredients [10, 11]. Other techniques, including micellar electrokinetic chromatography [12], non-aqueous capillary electro- phoresis [13], gas chromatography [14], and derivative spectrophotome- try [15], have been used to determine a few of these compounds. To the best of our knowledge, however, no analytical method has been reported for si- multaneous determination of these four compounds in the presence of pre- servative and excipients which might interfere with analysis of the active ingredients. Unauthenticated | Downloaded 09/29/21 08:49 PM UTC HPLC Analysis of a Cough–Cold Formulation 111 In this investigation a simple HPLC method with gradient elution was used for analysis of a cough–cold formulation. The method was validated in accordance with ICH guidelines [16]. Experimental Chemicals, Reagents, and Solutions Reference standards were gifts from Roorkee Research and Analytical Laboratories, Roorkee, India. Solvents were of HPLC-grade and chemicals were of analytical grade (Qualigens India, Mumbai). The commercial for- mulation Viscodyne-S syrup containing phenylephrine hydrochloride, guaiphenesin, ambroxol hydrochloride and salbutamol (as salbutamol sul- phate), manufactured in India by Wockhardt, was purchased locally. Approximately 100 mg phenylephrine hydrochloride, 500 mg guai- phenesin, 150 mg ambroxol hydrochloride, and 10 mg salbutamol sulphate (reference standards) were accurately weighed, transferred to a 100-mL volumetric flask, dissolved in diluent prepared by mixing methanol and water in the ratio 20:80, sonicated for 10 min, and made up to volume with diluent to produce a stock solution. Aliquots of the stock solution were suitably diluted with diluent to produce sample solutions of suitable con- centration. Sample solutions were filtered through a 0.45-μm pore size membrane filter, injected (20 μL) in triplicate on to the column, and the peak areas and retention times were recorded. Average peak areas were used for calculations after ensuring that the RSD was <2%. Sample Preparation Syrup equivalent to approximately 10 mg phenylephrine, 50 mg guaiphene- sin, 15 mg ambroxol hydrochloride, and 1 mg of salbutamol was accurately weighed and transferred to a 100-mL volumetric flask. The weighed sample was dissolved in 70 mL diluent, sonicated for 10 min, and diluted to volume with diluent. The solution was filtered through a 0.45 μm pore size Nylon filter discarding the first 5–10 mL of filtrate. The dilution step prevents problems because of the presence of hard syrup matrix in the sample. Unauthenticated | Downloaded 09/29/21 08:49 PM UTC 112 S. Joshi et al. Chromatography Analysis was performed with a Shimadzu gradient high-performance liquid chromatograph (Prominence model), binary pump (model LC-20AT), man- ual injector (Rheodyne model 7725i) of capacity 20 μL, and a UV–visible de- tector (model SPD-20A). Compounds were separated on a 250 mm × 4.6 mm i.d., 5-μm particle, Luna C8 column (Phenomenex, USA). The mobile phase was a gradient prepared from aqueous buffer solution (component A) and a 1:1 (v/v) mixture of methanol and acetonitrile (component B). To prepare solution A (phosphate buffer, 10 mM, pH 3.0), potassium dihydro- gen phosphate (2 g) and hexane sulphonic acid sodium salt (750 mg) were accurately weighed and dissolved in approximately 800 mL water. Tri- ethylamine (2 mL) was added and the pH was adjusted to 3.0 with ortho- phosphoric acid. The resulting solution was diluted to 1000 mL with water. Before use the mobile phase components were filtered through a 0.45-μm membrane filter, degassed by sparging, and pumped from the respective solvent reservoirs to the column at a flow rate of 1 mL min−1. The run time was set at 15 min, the column temperature was ambient, and the injection volume was always 20 μL. Before analysis, the column was equilibrated for at least 15–20 min with the mobile phase. Data were acquired, stored, and analyzed with LC Solutions software (Shimadzu). Results and Discussion To develop and validate a simple HPLC method for simultaneous analysis of the active ingredients phenylephrine hydrochloride, guaiphenesin, am- broxol hydrochloride, and salbutamol in a pharmaceutical dosage form, de- tection wavelength and mobile phase composition were investigated. Be- cause buffered low pH mobile phases keep basic compounds in their ion- ized form, it reduces their retention and hence makes the method selec- tive [17, 18]. Thus phosphate buffer (pH 3.0) was used with acetonitrile which also supports easy sample elution, thereby maintaining column life. It was further modified by addition of ion-pairing reagent hexane sulphonic acid sodium salt to affect retention of the ion-paired solutes expected at this pH. To further improve resolution between peaks, methanol was added to the organic component of mobile phase (methanol–acetonitrile 1:1). System- atic variation of their ratio and the detection wavelength resulted in good peak separation and short analysis time. The detection wavelengths were 273 nm for guaiphenesin and phenylephrine and 225 nm for salbutamol and ambroxol. The mobile phase gradient is given in Table I. A gradient was pre- Unauthenticated | Downloaded 09/29/21 08:49 PM UTC HPLC Analysis of a Cough–Cold Formulation 113 ferred for good resolution of all the compounds because salbutamol and ambroxol required a low buffer to organic solvent ratio whereas guaiphene- sin and phenylephrine required a high ratio. System suitability tests were performed (Table II). All four components of the formulation were clearly resolved with retention times (RT) between 0 and 11 min. Before implemen- tation for quantitative analysis of the active ingredients in the pharmaceuti- cal preparation, this method was thoroughly validated in accordance with ICH guidelines [16]. Table I. The optimum multi-linear gradient Mobile phase Time (min)