A comparative study of several HPLC methods for determining free amino acid profiles in honey

Abstract

A study of the viability of three derivatizing reagents for obtaining amino acid profiles in honey through high performance liquid chromatography (HPLC) is presented. A method using diode array detection based on a reaction with diethyl ethoxymethylene malonate (DEMM) and two other methods using fluorescence detection based on derivatization with fluorenylmethyl chloroformate (FMOC-Cl) and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) have been developed. The three methods yield detection limits close to the ppb level, but vary in relation to other analytical characteristics. The use of methyl chloroformate derivatives allows the profile to be obtained with the greatest sensitivity within a short time frame. On applying such methods to honey samples of diverse botanical origin, we observe that the proline values obtained are always lower than those found using the official spectrophotometric method, thereby underlining the advisability of using HPLC methods to reduce uncertainty in these results.

A sensitive method for free amino acids analysis by liquid chromatography with ultraviolet and mass spectrometric detection using precolumn derivatization with diethyl ethoxymethylenemalonate: Application to the honey analysis

“…Various articles have used different reaction conditions to address this problem. Bernal et al [19] used the following procedure. After 20 s of reaction, the reaction mixture was placed in an oven at 90 • C for 50 min.…”

“…Simultaneous chromatographic separation of that many rather similar compounds can already be challenging. Quite often complete separation is not achieved [17,[19][20][21]. One must also consider peaks caused by derivatization residue and matrix.…”

Analysis of free amino acids during fermentation by Bacillus subtilis using capillary electrophoresis

“…Conventional techniques for analysis of amino acids include ion exchange chromatography (IEC), using amino acid analyzer [9] and reverse-phase (RP)-HPLC [10,11], usually using a post-or pre-column derivatization with various kinds of chromophores [12] or fluorophores [13,14]. Amino acid analysis using an amino acid analyzer is expensive and time-consuming.…”

New derivatization strategies for the ultrasensitive analysis of non-aromatic analytes with APLI-TOF-MS

“…The concept of adding an ionizable section to a molecule is comparable to labeling analytes with fluorescence tags [24][25][26][27]. This approach is also used for other ionization techniques in mass spectrometry to enhance or enable ionization [9,28,29].…”