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.

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

PASTORINI, ELISABETTA;COLLIVA, CAROLINA;ROTINI, ROBERTO;RODA, ALDO
2011

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.
ANALYTICA CHIMICA ACTA
Pastorini E; Vecchiotti S; Colliva C; Persiani S; Rotini R; Roatti G; Zaccarelli L; Rovati LC; Roda A
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/125515
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