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

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 (20:80, 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 μL) 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 ≤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).