Simultaneous determination of chito-oligosaccharides in rat plasma by the LC-MS/MS method: application to a pharmacokinetic study

Abstract

A simple and sensitive method for the simultaneous determination of chito-oligosaccharides (COSs) with degrees of polymerization (DPs) from 2 to 7 was developed and used for COS quantification in rat plasma. Samples were separated on a Waters XBridge Amide column (3.5 μm, 2.1 × 150 mm) by isometric elution with 10 mM aqueous ammonium acetate (pH = 9) in acetonitrile and 10 mM aqueous ammonium acetate (pH = 9) (v/v, 50 : 50) employing multiple reaction monitoring (MRM) detection. Analytes and internal standards (IS) were extracted from rat plasma by protein precipitation with acetonitrile. The assay was linear over a concentration range of 20–10 000 ng mL⁻¹ for COS2–7. The intra-day and inter-day precision of the investigated components exhibited an RSD within 15%, and the accuracy (RE%) ranged from −7.3% to 7.6%. The extraction recoveries of the six constituents were determined to be between 82.5% and 94.3%. No significant matrix effects for COS2–7 were observed in rat plasma. COS in plasma remained stable for 24 h at room temperature (short-term), after freeze–thaw cycles, and 30 days in a −40 °C freezer. In comparison to reported COS quantitation methods, this method is simple, sensitive and cost-effective and could be used for the simultaneous quantitation of COS2–7. This method meets the Food and Drug Administration guidelines and had been successfully applied to the analysis of pharmacokinetic samples collected from rats.