title: Identification and quantification of glucosamine in rabbit cartilage and correlation with plasma levels by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry.
abstract: A new HPLC-ESI-MS/MS method for the determination of glucosamine (2-amino-2-deoxy-d-glucose) in rabbit cartilage was developed and optimized. Glucosamine was extracted from cartilage by cryogenic grinding followed by protein precipitation with trichloroacetic acid. The HPLC separation was achieved with a polymer-based amino column using a mobile phase composed of 10mM ammonium acetate (pH 7.5)-acetonitrile (20:80%, v/v) at 0.3 mL min flow rate. d-[1-(13)C]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). Limit of quantification was 0.045 ng injected, corresponding to 0.25 μg g⁻¹ in cartilage. Linearity was obtained up to 20 μg g⁻¹ (R(2)>0.991). Precision values (%R.S.D.) were <10%. Accuracy (% bias) ranged from -6.0% to 12%. Mean recoveries obtained at 3 concentration levels were higher than 81% (%R.S.D.≤8%). The method was applied to measure glucosamine levels in rabbit cartilage and plasma after single oral administration of glucosamine sulfate at a dose of 98 mg kg⁻¹(n=6). Glucosamine was present in cartilage in physiological condition before the treatment. After dosing, mean concentration of cartilage glucosamine significantly increased from 461 to 1040 ng g⁻¹. Cartilage glucosamine levels resulted to be well correlated with plasma concentrations, which therefore are useful to predict the target cartilage concentration and its pharmacological activity.
authors: Department of Pharmaceutical Sciences, University of Bologna, Italy.
authors: Elisabetta
authors: E
authors: Pastorini
authors: Stefania
authors: S
authors: Vecchiotti
authors: Carolina
authors: C
authors: Colliva
authors: Stefano
authors: S
authors: Persiani
authors: Roberto
authors: R
authors: Rotini
authors: Giulia
authors: G
authors: Roatti
authors: Lorenzo
authors: L
authors: Zaccarelli
authors: Lucio Claudio
authors: LC
authors: Rovati
authors: Aldo
authors: A
authors: Roda