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 0.3mLmin-1 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). Limit of quantification was 0.045ng injected, corresponding to 0.25μgg-1 in cartilage. Linearity was obtained up to 20μgg-1 (R2>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 98mgkg-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 1040ngg-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. © 2011 Elsevier B.V.