. Development of analytical method for determination of free amino acids in honey using by solid-phase extraction clean-up coupled to ultra-high-pressure liquid chromatography-tandem mass spectrometry.

The quality of honey is usually evaluated by its sensorial, chemical, physical and microbiological properties. The Commission of the European Union (EU) has adopted a proposal to amend the Council Directive 74/409/EEC concerning honey [1]. This Directive laying down common rules and quality parameters, such as humidity, diastatic index, HMF content and pesticides levels, but some of these parameters have no relationship to geographical or botanical origin of honey. The amino acid profile could be useful tools to identify the botanical or geographical origin of honey samples. For this reason, the aim of this work was the development of analytical method for determination of free amino acids in honeys. The analytical procedure is based on solid-phase extraction (SPE) cleanup of diluted honey (1:10 w/w) using a cation exchange cartridge (MCX). The purified sample was directly analysed by Hydrophilic interaction chromatography-tandem mass spectrometry (UHPLC–MS/MS) for sensitive and selective determination of 20 amino acids. The parameters of mass spectrometry source and MRM transition were carefully optimized in order to get high sensitivity and selectivity for each analyte. Moreover, in order to achieve the best chromatography separation and a good peak resolution, different columns and mobile phases were tested. BEH Amide stationary phase using a gradient with acetonitrile and water containing formic acid, ammonium acetate and ammonium formate, has been used to obtain, in just 15 minutes, the complete separation of 20 α-amino acids investigated. The proposed method was applied to Calabrian honeys of different botanical origins (Citrus, Chestnut, Robinia, Eucaliptus and Sulla). The obtained results suggested that the developed methodology is robust and reliable for the analysis of free amino acids in honey, showing an efficient reduction of matrix effect.