Development and validation of a liquid chromatograpidc method for the simultaneous determination of diphenhydramine, promethazine, chlorpheniramine and ephedrine in cold-cough syrups

Abstract

The quality of essential drugs used to treat diseases such as HIV / AIDS, tuberculosis and malaria is often monitored locally through market surveillance initiatives driven by different Ministry of Health departments in Kenya. The same scrutiny is not applied to drugs used in the treatment of less serious ailments. This is the case especially with drug mixtures used to relieve the symptoms associated with conditions such as the common cold. One of the reasons for the absence of quality monitoring is the lack of reliable, accurate or cost-effective analytical methods capable of simultaneously analyzing the multiple active ingredients in such products. In this study, a simple, rapid, precise and sensitive isocratic elution reversed-phase liquid chromatographic method was developed for the simultaneous determination of the antihistamine drugs diphenhydramine, promethazine and chlorpheniramine as well as the decongestant ephedrine contained in commercially available cold-cough syrups in the Kenyan market. The effect of chromatographic parameters including use of both inorganic and organic mobile phase buffers, pH, column temperature, organic modifier concentration and ion pairing agents were studied during method development. Combinations of these drugs were separated by the liquid chromatography method comprising a mobile phase consisting of methanol-water-triethylamine-0.2 M ammonium acetate pH 5.0 (50:9.85:0.15:40, % v/v/v/v) delivered at a flow rate of 1.0 mL per minute with ultraviolet detection at 254 nm. The stationary phase used was a silica based octyldecyl silane Gemini-NX column of dimensions 250 mm length and 4.6 mm internal diameter with particle size 5 urn maintained at a temperature of 40°C. Validation of the method showed that it exhibited good linearity over the 25% to 150% range of the analytical concentration with linear regression coefficient R2 values of 0.9999 for ephedrine, 0.9997 for both chlorpheniramine and diphenhydramine and 0.9979 for promethazine. Limits of detection were 234 ng, 0.2 ng, 13 ng and 0.04 ng while the limits of quantitation were 1125 ng, 1.1 ng, 32.6 ng and 0.7 ng for ephedrine, chlorpheniramine, diphenhydramine and promethazine respectively. The method was found to exhibit good precision with the intra-day analysis coefficients of variation ranging from 0.9% to 1.7% and inter-day coefficients of variation being 1.7% to 2.1 % for the four drug compounds. The developed method was used to analyze twelve batches of commercially available samples of cold-cough syrups from the Kenyan market. Results obtained indicated low levels of ephedrine in all the samples tested with assay values ranging from 71.8% to 89.7% of the labeled amount. Promethazine was also noted to exhibit inter-batch variation with assay values ranging from 69.1 % to 103%. Chlorpheniramine and diphenhydramine were found to exhibit the least degree of inter-product and inter-batch variation with assay values ranging from 93.4% to 100% and 86.1% to 96.0% respectively. From the findings obtained in the evaluation of commercial samples, it can be concluded that the developed method can be adopted for the routine quality analysis of these cold¬cough medicine ingredients by the pharmaceutical industry and drug regulatory authority quality control laboratories.