Development of a Simple, Underivatized Method for Rapid Determination of Free Amino Acids in Honey Using Dilute-and-Shoot Strategy and Liquid Chromatography-Tandem Mass Spectrometry

Abstract

A simple, fast and reliable analytical method was developed for 20 free amino acids (FAAs) determination in honey samples through a dilute-and-shoot strategy and hydrophilic interaction liquid chromatography tandem mass spectrometry. Compared with previous reports, direct dilution by water has significantly reduced the matrix effect and facilitated full extraction of FAAs. Further, a 5 min determination method was established with an acetonitrile–water mobile phase system with 0.1% formic acid addition. The established method was validated and demonstrated several advantages including short detection time, wide linear range over 3–4 orders of magnitude, high sensitivity down to 0.1 ng/mL and negligible matrix effect. Twenty FAAs were determined in 10 honey samples from different botanical origins by this method, and 19 FAAs were found. This general applicable method was also promising for fast determination of FAAs in other practical samples.

Underivatized Amino Acid Chromatographic Separation: Optimized Conditions for HPLC-UV Simultaneous Quantification of Isoleucine, Leucine, Lysine, Threonine, Histidine, Valine, Methionine, Phenylalanine, Tryptophan, and Tyrosine in Dietary Supplements

“…Several publications have reported AA analysis in foods and biological fluids using GC–MS, capillary electrophoresis, HILIC-MS/MS, ion-paired LC–MS/MS, electrochemical detection (CE), ion exchange chromatography, and HPLC. , Most of these methods depend on pre- or postcolumn derivatization to improve the detection sensitivity. However, elimination of derivatization steps attracts many researchers, and many studies have described the analysis of AAs without derivatization steps such as underivatized determination of free AAs in honey using LC–MS/MS, branched-chain AAs in maple syrup urine disease using LC–MS/MS, evaluation embryo viability using CE-MS, urine analysis by LC–MS/MS, ion exchange, CE-MS coupled to a porous layer-gold nanoparticle-modified chiral column, AAs and peptides in nutritive mixtures in the total parenteral nutrition solution, and online coupling of UV, fluorescence, and electrochemical detection . The elimination of the derivatization step is advantageous for many reasons as the selection of an appropriate derivatization reagent is challenging as the reagents differ in their selectivity toward each amino acid; also, additional peaks usually appear in chromatograms, side reactions usually occur, complete derivatization cannot be ensured, long sample preparation time is required, and unstable derivatizations occur.…”

Overview of chromatographic and electrophoretic methods for the determination of branched‐chain amino acids

“…AMF may potentially result in decreased efficiency of ESI, thereby significantly suppressing the MS signal response. Moreover, certain AAs such as Asp and His exhibit tailing peaks, which negatively impacts the sensitivity of the analysis [160, 161]. The removal of AMF would lead to an increase in peak area (it is 4–74 times higher compared to using 5 mM AMF) and a decrease in peak width, resulting in a significant increase in sensitivity, which would be more favorable for the quantitation of Leu and Ile.…”

“…Ma et al. [161] employed three chromatographic columns to separate 20 AAs in honey. AAs were poorly retained by the Polar C18 and HSS T3 columns.…”

Analysis of 18 Free Amino Acids in Honeybee and Bumblebee Honey from Eastern and Northern Europe and Central Asia Using HPLC-ESI-TQ-MS/MS Approach Bypassing Derivatization Step

“…With the advancement in analytical chemistry and instrumentation, the analysis of FAAs becomes more easily done by bypassing derivatization steps and without taking a risk of environmental pollution and human health matter. The advantages of liquid chromatography-mass spectrometry (LC-MS) in the analysis of FAAs in various foodstuff, including barley extracts [ 13 ], orange juice [ 14 ], mammalian milk [ 15 ], and honey [ 16 , 17 ] has repeatedly been confirmed, highlighting also robustness, sensitivity, and selectivity of this approach compared with HPLC-DAD [ 18 ] or GC-MS [ 19 ] techniques. Selective analysis of FAAs utilizing the LC-MS system makes it attainable to distinguish both the botanical origin of the honey and verify its authenticity [ 17 ] in a relatively shorter time.…”