Identification and quantification of glucosamine in rabbit cartilage and correlation with plasma levels by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry

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 10 mM ammonium acetate (pH 7.5)–acetonitrile (20:80%, v/v) at 0.3 mL min−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 0.045 ng injected, corresponding to 0.25 μg g−1 in cartilage. Linearity was obtained up to 20 μg g−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 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.

Figure optionsDownload as PowerPoint slideHighlights
► Optimization of an HPLC-ESI-MS/MS method for glucosamine in rabbit cartilage.
► Application of the method to an in-vivo study.
► Glucosamine presence in cartilage in physiological condition.
► Significant increase of cartilage glucosamine concentration after dosing.
► Good correlation between cartilage glucosamine levels and plasma concentrations.