Improved Sensitive High Performance Liquid Chromatography Assay for Glucosamine in Human and Rat Biological Samples with Fluorescence Detection
Purpose. An improved HPLC method with fluorescence detection was developed and validated for determination of glucosamine in human and rat biological samples. Method. Aliquot of 0.1 mL plasma was spiked with mannosamine HCl as the internal standard (IS); proteins were precipitated with acetonitrile; the clear layer was derivatized with 9-fluorenylmethyl chloroformate (8 mM/acetonitrile) in presence of borate 0.2 M buffer at 30o C for 30 min. The excess derivatizing agent was removed with 1-aminoadamantane HCl (300 mM in acetonitrile-water 1:1). Chromatographic separation was achieved on a C18 (100mm X 4.6 mm, id 3μm) reversed phase column using 0.1% acetic acid/acetoniltrile gradient mobile phase at 1 mL/min flow rate. Glucosamine was determined in the plasma of a human and rats and also in rat urine. Results. The analytes were detected at excitation and emission wavelengths of 263 and 315 nm, respectively. The assay was linear over the range of 0.05-20 µg/mL with a typical correlation coefficient of 0.999 and intra-day and inter-day coefficient of variation of <15%. The lowest limit of quantification was set at 50 ng/mL. The recovery for glucosamine and mannosamine was 98 and 96%, respectively. Conclusion. We were able to improve glucosamine assay suitable to quantify glucosamine in both human and rat plasma and rat urine.