Method optimization and validation for the determination of glucosamine active ingredient in food supplement tablets by high performance liquid chromatography (HPLC) method

This study involves the method optimization and validation of the glucosamine agent contained in dietary supplement tablets with a high-performance liquid chromatography (HPLC) device. Detection of glucosamine from dietary supplement tablets was performed using an Agilent brand 1100 model HPLC device with diode array detector (DAD). Prodigy™ C18 (150 mm length, with a particle size of 5 μm and an inner diameter of 3.2 mm) the reversed phase liquid chromatography column was used as a stationary phase. Glucosamine sulphate was used as a reference standard substance for detecting glucosamine in the dietary supplement tablets and derivatization process was realized with Fmoc-OSu. In order to determine the mobile phase in the method optimization studies of the glucosamine agent, different mobile phase compositions were tested and the acetonitrile (ACN):H2O mixture (50:50) (pH adjusted to 4 with trifluoroacetic acid (TFA)) was selected as the appropriate mobile phase. Studies were carried out with the DAD dedector at different wavelengths, and the wavelength at which glucosamine performed a maximum absorbance was obtained at 206 nm. The optimum value for band width (bw) was selected as 4. Van Deemter equation was used for determining the flow rate of the mobile phase and the optimum flow rate was found to be 0,6 mL/min. The optimum column temperature value was assigned as 35 ºC and the optimum injection volume was selected as 5 μL. The values of the limit of detection (LOD) and the limit of quantification (LOQ) were calculated as 0,014 mg/L and 0,046 mg/L, respectively. For determination of precision, intra-day and inter-day studies were carried out and percent relative standard deviation (%RSD) values were calculated. Glucosamine agent was added to the dietary supplement tablets at certain concentrations for the trueness parameter and %recovery values were obtained between 85,15% and 116,88%. For selectivity parameter, studies were carried out with a dietary supplement tablets with a different formulations than the dietary supplement tablet samples used in the experiments. For stability tests, control samples were prepared and their short-term stability values were compared at room temperature on different days for 5 days. When the results of optimization and validation studies for the glucosamine agent were evaluated, it was seen that the system was steady and applicable.