Summary:
The present diploma thesis had as its aim the development of a RP-HPLC method for the simultaneous determination of the active pharmaceutical ingredient (API) “Butamirate Citrate” and of the preservative “Benzoic Acid” in syrup preparation. The analytical procedure was designed and subsequently validated so that its performance characteristics be rationally assessed, throughout an experimental documentation, and the method itself prove to be suitable for its intended purpose (fitness for purpose). The experimental part was conducted at the Greek Military Pharmaceutical Laboratories in Tauros, Athens.
Butamirate is a widely used suppressant for dry, non-productive cough which is most commonly prescribed in the form of its citrate salt (Butamirate Citrate, BC). Benzoic acid and its respective salt compounds constitute essential preservatives of the pharmaceutical industry, ensuring the structural stability of a formulation as a whole.
Even though Pharmacopoeial assays often rely quite heavily on direct UV spectroscopy, yet in industry this kind of detection usually succeeds a “preliminary separation” by HPLC. Approximately, 3⁄4 of all HPLC applications are carried out employing reversed phase packings (RP-HPLC). Regarding this study, the leading guideline for the method development stage was the achievement of an optimum “compromise” between the conflicting goals of adequate resolution vs as short as possible run time. Various preliminary tests eventually led to the selection of a cyanopropyl silica gel stationary phase (-CN column). Columns of this kind are considered to be as the most polar amongst reversed phased packings and as the least polar amongst normal phased packings. The mobile phase consisted of equal volumes (1:1) of MeOH and NaH2PO4*H2O 50 mM, 1% Et3N, pH=3±0,1. The column’s temperature was set at 36 °C, while the detection of target analytes was achieved with a diode array detector (DAD) at 210 nm.
In spite of demonstrating a congenitally satisfactory separation, yet the analytical method produced quite asymmetrical peaks and that is why corrections were immediately made. All the same, the observed peak asymmetry was far from a magnitude which could be detrimental in terms of peak overlap or integration integrity. We aimed at avoiding a long delay elution of butamirate and a quite rapid elution of benzoic acid (close to the solvent front). The method was found to fulfill the required specifications with respect to specificity, linearity, accuracy (trueness), precision and stability of both standard solutions and of samples. What is more, an experimental design was set in order to provide documented assessment and evidence in relation to method ruggedness.
Keywords:
Butamirate Citrate, Benzoic Acid, development and validation, PDA detector, syrup
