Reversed Phase Chromatographic Analysis of 13 Amino Acids in Honey Samples

A simple method using reversed phase high-performance liquid chromatography (RP-HPLC) was developed for the simultaneous analysis of 13 amino acids. Amino acids were pre-column derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl) before analysis by RP-HPLC. Experimental parameters affecting the derivatization and chromatographic separation were investigated. Amino acids were derivatized with FMOC-Cl under alkaline condition in 0.1 mol/L borate buffer pH 10.0 at room temperature. The FMOC-amino acid derivatives were separated on an Atlantis C18 column under the gradient elution of 0.05 % trifluoroacetic acid and acetonitrile and UV detection at 265 nm. Linear ranges were 0.2–100.0 μg/mL with the correlation coefficients greater than 0.992. Limits of detection and limits of quantitation were in the range of 0.05–2.0 and 0.2–5.0 µg/L, respectively. The intra-day precision (n = 3) of retention time was less than 1 %, while for the peak area was less than 4 %. The inter-day precision (n = 3 × 3) of retention time was less than 2 % and the peak area was less than 8 %. This method was applied in honey samples and the results showed that proline is the major amino acids in honey samples.