Development and validation of an HPLC method for the determination of glucosamine hydrochloride in a medicinal product in the form of a cream

Abstract

The development of medicinal products requires the use of selective and reproducible analytical methods that ensure reliable determination of active ingredients. The analysis of glucosamine hydrochloride as a medicinal product in the form of a cream has specific challenges due to its high polarity and lack of a chromophore; therefore, its determination within the dosage form requires a specially adapted analytical approach. The aim. To develop and validate a liquid chromatographic method for the determination of glucosamine hydrochloride in a medicinal product in the form of a cream in accordance with the requirements of ICH Q2(R2), the State Pharmacopoeia of Ukraine (SPhU), and the European Pharmacopoeia (EP). Materials and methods. The object of the study was an experimental batch of a cream containing glucosamine hydrochloride (1%). The analysis was performed using a KNAUER Smartline chromatograph equipped with a UV detector and a Zorbax SB-C8 column (150 × 4.6 mm, 5 μm). Phenyl isothiocyanate was used for derivatization, resulting in the formation of a UV-active glucosamine derivative. Validation was carried out with respect to specificity, linearity, accuracy, precision, system suitability, and robustness. Results. Optimal chromatographic conditions were established (mobile phase: acetonitrile–water, 40:60, acidified with phosphoric acid to pH 3.0; detection wavelength 230 nm; flow rate 1.0 mL/min; column thermostat temperature 25°C), ensuring complete separation of the glucosamine derivative (retention time 2.21 min) from sodium benzoate (retention time 3.55 min) and other excipients in the cream formulation. The minimum required concentration of the derivatization reagent was determined to be 0.008–0.01 g/mL. Evaluation of validation parameters confirmed the specificity of the method, its linearity within the 80–120% range (r = 0.9991), precision (ΔZ = 0.61%), accuracy (δ = 0.31%), repeatability (≤ 2%), and robustness (analytical solutions stable for 1 hour). Metrological assessment (n = 6) demonstrated that the systematic error was statistically insignificant, while the relative uncertainty of a single determination was 7.88% at a confidence level of P = 0.95. The detection limit of glucosamine hydrochloride is 0.23 μg/ml. Conclusions. An HPLC method for the determination of glucosamine hydrochloride in a cream formulation after derivatization with phenyl isothiocyanate has been proposed and experimentally substantiated. The method complies with the requirements of international guidelines and can be applied in quality control for the identification and quantitative determination of the active pharmaceutical ingredient in the investigated medicinal product