|dc.description.abstract||Honey, a natural product produced by honeybees, has a complex matrix of sugars proteins, minerals, vitamins, enzymes and free amino acids. Amino acids, obtained from the nectar of plants, account for 50-30 mg/kg of honey, the most abundant of which is proline, a secondary amino acid originating mainly from the haemolymph of bees.
Three methods were investigated for the analysis of amino acids in honey. The first used HPLC-UV with pre-column derivatisation, the second HPLC-MS with hydrophobic interaction chromatography, and finally HPLC-MS with aTRAQ™ derivatisation.
The HPLC-UV method involves derivatisation of amino acids by OPA-MPA and FMOC. A fully automated injection program analysed seventeen primary amino acids in 19 minutes. Ultimately, the detection by UV had inadequate sensitivity, and the secondary amino acid proline could not be detected. The method was rejected for these main reasons.
ZIC-pHILIC chromatography paired with LC-MS-MS gave high-quality separation of twenty one amino acids, detected using scheduled MRM, in 10 minutes. Amino acid recovery out of vial was low for the majority, this variation originating from sample preparation. In vial loss of amino acids could not be recovered and so investigation into the last method was initiated.
The final method used an aTRAQ™ kit which labels amino acids with a Δ8 reagent for analysis and also provides Δ0 labelled internal standards for comparison. Forty eight amino acids and internal standards can be accurately detected by MRM’s in 18 minutes. Sample preparation was optimised for honey and the method was validated.
The amino acid content of ten honeys were compared to values obtained from Massey University. Small differences in the majority of amino acids were observed. Mānuka and clover honeys from this data set were also compared, it was found that phenylalanine and tyrosine were at much higher concentrations in clover honey.
Seven mānuka honeys stored in different conditions, warm and cold, were analysed. Applied statistical analysis with the hypothesis that the warm honeys would have lower amino acid concentrations than the cold, found this to be true for seventeen amino acids. Glutamine and then lysine were at decreased concentrations after warm storage in the most honey samples.
The amino acid content of honey, analysed by this method can be used to investigate: botanical origin of honey, speed of honey production/harvest, effect on DHA conversion, inaccurate labelling, and sugar syrup addition.||