Development and validation of a HPLC-ES-MS/MS method for the determination of glucosamine in human synovial fluid.

ABSTRACT

A new HPLC method for the determination of glucosamine (2-amino-2-deoxy-D-glucose) in human synovial fluid was developed and validated. Synovial fluid samples were analyzed after a simple protein precipitation step with trichloroacetic acid using a polymer-based amino column with a mobile phase composed of 10 mM ammonium acetate (pH 7.5)-acetonitrile (2080, v/v) at 0.3 mL/min flow rate. D-[1-13C]glucosamine was used as internal standard. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in positive ionization mode and in multiple reaction monitoring acquisition (m/z 180-->72 and 181-->73 for glucosamine and internal standard, respectively). The limit of quantification (injected volume=3 microL) was 0.02 ng, corresponding to 10 ng/mL in synovial fluid. Calibration curves obtained using matrix-matched calibration standards and internal standard at 600 ng/mL were linear up to 2000 ng/mL. Precision values (%R.S.D.) were < or = 14% in the entire analytical range. Accuracy (%bias) ranged from -11% to 10%. The recoveries measured at three concentration levels (50, 800, and 1500 ng/mL) were higher than 89%. The method was successfully applied to measure endogenous glucosamine levels in synovial fluid samples collected from patients with knee osteoarthritis and glucosamine levels after oral administration of glucosamine sulfate (DONA) at the dose of 1500 mg/day for 14 consecutive days (steady-state).