Development and validation of a liquid chromatographic method for the simultaneous determination of Bromhexine, Guaifenesin, Ambroxol, Salbutamol/Terbutaline, Pseudoephedrine, Triprolidine and Chlorpheniramine Maleate in Cough-cold syrups
Cough is one of the most common symptoms for which patients seek treatment from primary health care providers. Cough syrups are usually prescribed for relieve of coughs and most of them are formulated as combination of several ingredients. Currently there is no single high performance liquid chromatography (HPLC) method for the analysis of the active ingredients simultaneously as the official methods' available specify analysis of the active ingredients individually. This makes the analysis of the components time consuming and expensive. In the present study, a simple, reliable,' accurate, precise, robust and isocratic HPLC method was developed for simultaneous determination of salbutarnol (SBT)/terbutaline (TBT), pseudoephedrine (PED), guaifenesin (GFN) , ambroxol (AMB), chlorpheniramine (CPM), triprolidine (TPN) and bromhexine (BXN) in cough-cold mixtures in the presence of excipients. The optimized chromatographic conditions were a mobile phase consisting of acetonitrile-0.25 I M sodium hexanesulphonate-0.2 M ammonium acetate pH 3.0-water (35:4:10:51, % v/v/v/v) delivered through the HPLC system at a flow rate of 1.0 mLimin. The stationary phase was a reversed phase octadecylsilane (CIS) measuring 250 mm in length, 4.6 mm internal diameter, 5 um particle size and 1 io A pore size. The ultra-violet (UV) detection wavelength was set at 254 run while the injection volume was 20 ul., The diluent consisted of acetonitrile-water (40:60, % v/v). The retention time of salbutamollterbutaline, pseudoephedrine, guaifenesin, ambroxol, chlorpheniramine, triprolidine and bromhexine was 3.0, 3.5, 4.3, 5.9, 7.8, 9.4 and 18.3 minutes respectively, giving a total run time of21 minutes. The developed method was validated for accuracy, precision, linearity, limit of detection, limit of quantitation and robustness. The validation was carried out according to the International Conference on Harmonization (ICH) guidelines. The accuracy of the method was demonstrated by the recovery values obtained, being 99.9 % (SBT), 100.5 % (TBT), 99.2 % (PED), 99.8 % (GFN), 98.5 % (AMB), 99.7 % (CPM), 99.9 % (TPN) and 99.2 % (BXN). The precision of the method was shown through adequate repeatability or intra-day precision (RSD, 0.15-0.56 %) and intermediate precision (RSD, 0.45-1.53 %). The linearity range for the eight compounds was 25- 200 % with coefficient of determination (R2) being> 0.999 for all the compounds. The limit of detection values for salbutamol, pseudoephedrine, guaifenesin, ambroxol, chlorpheniramine, triprolidine, bromhexine and terbutaline were 1.28, 12.45, 7.68, 1.29,3.27,31.03, 1.67 and 10.08 ng, while the limit of quantitation values were 4.00,18.68,10.24,3.22,17.45,51.72,3.34 and 25.21 ng respectively. The robustness ranges for the three critical factors, mobile phase pH, column temperature and acetonitrile concentration, were 2.5-3.5 pH units, 35-45 °C and 33-37 % (v/v) respectively. The variations did not significantly affect separation of the components. The method was applied in the analysis of nine commercial products obtained from pharmacy outlets within the city of Nairobi. Six products had three batches analyzed while two batches of the remaining three products were analyzed. All the samples complied with the general United States Pharmacopoeia (USP) specifications for assay (90.0-110.0 %' label claim). The results obtained also demonstrated that there were minimal batch-to-batch variations. Extraction procedures were not applied during the assay of the samples thus significantly shortening the analysis time. It was concluded that the method is useful in quality control laboratories for the routine analysis of these compounds in cough-cold medications.