Development and validation of a sensitive solid phase extraction/hydrophilic interaction liquid chromatography/mass spectrometry method for the accurate determination of glucosamine in dog plasma.

A sensitive and accurate LC/MS method was developed for the monitoring of glucosamine (GLcN) dog plasmatic concentration. In this scope, relatively low plasmatic concentrations of GLcN were expected, ranging from 50 to 1000 ng/mL. Liquid chromatography coupled to simple quadrupole mass spectrometry detection (LC/MS) was selected bringing the selectivity and the sensitivity needed for this application. Additionally, a solid phase extraction (SPE) step was performed to reduce matrix and ion suppression effects. Due to the ionisable character of the compound of interest, a mixed-mode strong cation exchange (Plexa PCX) disposable extraction cartridge (DEC) was selected. The separation was carried out on a Zorbax SB-CN column (5 microm, 4.6mm i.d. x 250 mm), considering hydrophilic interaction liquid chromatography (HILIC). Indeed, the mobile phase was made of methanol and 5mM ammonium hydrogen carbonate buffer at pH 7.5 (95/5, v/v). The detection was led at m/z ratios of 180.0 and 417.0, for GLcN and IS, respectively. Reliability of the results was demonstrated through the validation of the method using an approach based on the accuracy profile allowing managing the risk associated to the use of these methods in routine analysis: it is thus guaranteed that each future result will fall in the +/-30% acceptance limits with a probability of at least 90%. Successful application of the method to a preliminary pharmacokinetic study illustrated the usefulness of the method for pre-clinical studies.