Determination of the nutraceutical, glucosamine hydrochloride, in raw materials, dosage forms and plasma using pre-column derivatization with ultraviolet HPLC

A selective and specific high performance liquid chromatography method was developed to quantitate glucosamine hydrochloride in raw materials, dosage forms and plasma. Reverse phase chromatography using pre-column derivatization with phenylisothiocyanate, and ultraviolet detection (lambda = 254 nm) was used to quantify the eluate. The mobile phase consisted of MeOH/H2O/CH3COOH (10:89.6:0.04) and was pumped at a flow rate of 1.2 ml/min. The standard curves for glucosamine hydrochloride showed linearity (r > or = 0.99) over the selected concentration range from 6.65 to 16.63 microg/ml for raw materials and dosage forms. The precision of the dosage form assay, expressed as the % relative standard deviation (R.S.D.), was < 5% at all concentrations. The intra-day and inter-day accuracy, as indicated by the relative error (R.E.), ranged from - 2.54 to 2.70% for glucosamine hydrochloride. For the plasma assay, beagle dog plasma was used to prepare standard curves in the concentration range of 1.25-20 microg/ml. Precipitation of plasma proteins was accomplished with acetonitrile to separate interfering endogenous products from the compound of interest. The supernatant was derivatized using phenylisocyanate in phosphate buffer (pH = 8.3) and subsequently evaporated to dryness under a nitrogen stream at 42 degrees C. The residue was dissolved in 250 microl mobile phase and injected onto the chromatographic system. The assay was linear in concentration ranges of 1.25-20 microg/ml (r > or = 0.999). Intra- and inter-day precision was < or = 5.23 and 5.65%, respectively and the intra- and inter-day accuracy, indicated by R.E., ranged from - 8.6 to 10.35%. The method was found to be specific and with excellent linearity, accuracy and precision and is well suited for the quantitation of glucosamine hydrochloride in raw materials, dosage forms, and pharmacokinetic studies.