Amino Acids In Honey Research Articles

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.

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.

Analysis of 22 free amino acids in honey from Eastern Europe and Central Asia using LC-MS/MS technique without derivatization step

This study aimed to investigate the presence of 22 free amino acids (FAAs) in six honey types from Belarus (BY), Kazakhstan (KZ) and Poland (PL), for the first time. A short sample preparation step based on extraction by formic acid/water/methanol system and a relatively simple and easy clean-up stage with graphitized carbon black sorbent were performed. Using a HILIC (hydrophilic interaction liquid chromatography) column, the separation of 22 FAAs was successfully carried out. Liquid chromatography coupled with mass spectrometry detectors (LC–MS/MS) were used in 22 FAAs analysis without a derivatization step. Eighty one samples of honey from 6 types and 3 origins with 13 combinations: honeydew (16), multifloral (18), rape (16), linden (8), buckwheat (9) and dandelion (5) contained FAAs from 127.7 mg/kg (dandelion, PL) to 1523.2 mg/kg (multifloral herbs, KZ). Proline ranged from 70.8 to 461.4 mg/kg, tyrosine was characteristic for KZ honey (429 mg/kg), and cystine was present only in PL linden honey (0.1 mg/kg). Summarized results confirm presence and the diversity of FAAs in honey of different types and origin. Honey samples were classified using chemometric methods based on the amino acid concentration. Amino acid profiling of honey is useful, additional tool, expanding standard quality and melissopalynological analysis.

A novel fluorescent labeling reagent, 2-(9-acridone)-ethyl chloroformate, and its application to the analysis of free amino acids in honey samples by HPLC with fluorescence detection and identification with online ESI-MS.

In this study, a novel fluorescent labeling reagent 2-(9-acridone)-ethyl chloroformate (AEC-Cl) was designed, synthesized and applied for the determination of free amino acids by high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). The free amino acids were rapidly and efficiently labeled by AEC-Cl in the presence of basic catalyst (pH9.0) within 5min at room temperature (25°C). The derivatives exhibited excellent stability and fluorescence properties, with maximum excitation and emission wavelengths at 268nm and 438nm, respectively. Derivatives of 22 kinds of natural amino acids were completely separated by gradient elution on a Hypersil ODS C18 column. Under the optimal conditions, the calibration curves exhibited excellent linear responses, with correlation coefficients of R2 > 0.9994. The detection and quantification limits were in the range of 0.61-2.67μgkg-1 and 2.07-8.35μgkg-1, respectively. Therefore, AEC-Cl was successfully applied for the detection of trace levels of free amino acids in honey samples. Graphical abstract A novel fluorescent labeling reagent was applied for the determination of free amino acids in honey by high-performance liquid chromatography with a fluorescence detector.

The effects of different thermal treatments on amino acid contents and chemometric-based identification of overheated honey

This study was dedicated to distinguish overheated honey under simulated industrial thermal treatment by analyzing the amino acid contents. In this research, jujube honey and chaste honey samples were characterized by amino acids contents, color values and 5-hydroxymethylfurfural (5-HMF) contents after different thermal treatments. According to the results and multivariate statistical analysis, the contents of most amino acids in honey decreased after heat treatment, and there were significant differences between moderate and over processed honey samples. It also turns out that the sensitive markers of thermal treating are 5-HMF, b*, L*, a* and proline in jujube honey, 5-HMF, L*, b*, a* and phenylalanine in chaste honey. The above researches indicate that all honey samples in present study should be subject to category division of whether overheating.

Free amino acids profile of Polish and Slovak honeys based on LC-MS/MS method without the prior derivatisation.

LC-MS/MS method was applied for determination of free amino acids in honey without derivatisation steps. Twenty free amino acids including aspartic acid, asparagine, glutamic acid, glutamine, alanine, arginine, glycine, leucine, histidine, hydroxyproline, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine and ornithine were analyzed in thirty honey samples from Poland and Slovakia. The analysis covered: acacia, lime, rape, multifloral and forest types of honey. Applied method was characterized to had good sensitivity with limit of detection ranged from 3.0ng/cm3 for valine to 13.0ng/cm3 for hydroxyproline. Average content of proline (main amino acid component in honey) ranged from 151.46μg/g (rape honey from Slovakia) to 389.66μg/g for forest honey (honeydew honey) from Poland. In analyzed honeys large quantities of glutamine, glutamic acid, lysine, phenylalanine, asparagine, alanine, and valine were also found.

Biochemical and nutritional components of selected honey samples.

The purpose of this study was to investigate the relationship of biochemical (enzymes) and nutritional components in the selected honey samples from Malaysia. The relationship is important to estimate the quality of honey based on the concentration of these nutritious components. Such a study is limited for honey samples from tropical countries with heavy rainfall throughout the year. A number of six honey samples that commonly consumed by local people were collected for the study. Both the biochemical and nutritional components were analysed by using standard methods from Association of Official Analytical Chemists (AOAC). Individual monosaccharides, disaccharides and 17 amino acids in honey were determined by using liquid chromatographic method. The results showed that the peroxide activity was positively correlated with moisture content (r = 0.8264), but negatively correlated with carbohydrate content (r = 0.7755) in honey. The chromatographic sugar and free amino acid profiles showed that the honey samples could be clustered based on the type and maturity of honey. Proline explained for 64.9% of the total variance in principle component analysis (PCA). The correlation between honey components and honey quality has been established for the selected honey samples based on their biochemical and nutritional concentrations. PCA results revealed that the ratio of sucrose to maltose could be used to measure honey maturity, whereas proline was the marker compound used to distinguish honey either as floral or honeydew.

Determination of amino acids in honey by capillary electrophoresis with indirect ultraviolet detection

A method of capillary electrophoresis with indirect ultraviolet (UV) detection was developed for the separation and determination of nine amino acids such as lysine, tryptophan, glutamic acid, etc. The effects of sodium dihydrogen phosphates concentration, pH of buffer and sample injection type and time on the reproducibility and efficiency were investigated. The optimum injection time was 5 s at 5 kPa. The optimum electrophoretic conditions were as follow: 10 mmol/L sodium dihydrogen phosphates (pH 10. 2) containing 0. 5 mmol/L cetrimonium bromide, 20 mmol/L nicotinic acid and 10% (v/v) methanol as running buffer, applied voltage of - 15 kV, detection wavelength of 220 nm. The base line separation of the nine amino acids was achieved successfully within 11 min. The lowest detection limit was 0. 3 mg/L. All of the nine analytes showed good linearities within 1. 0 - 1000 mg/L. The relative standard deviations of migration time and peak area were 0. 64% - 5. 83%. The recoveries of the eight amino acids spiked in a real sample were between 60. 00% and 118.37%. The method was applied in the determination of the amino acids in honey samples from different nectar plants and origins. Prolin, serine and aspartic acid were found in five honey samples, and tryptophan was only found in a litchi honey sample. This method can provide good reference to the evaluation of the quality and nectar origin of honey.

Botanical origin of monovarietal dark honeys (from heather, holm oak, pyrenean oak and sweet chestnut) based on their chromatic characters and amino acid profiles

An early optimisation of the o-phthaldialdehyde (OPA) method for the HPLC-fluorimetric analysis of amino acids in honey and bee-pollen made feasible the quantification of 23, which included five infrequently searched for. Forty samples of monovarietal dark honeys, from four botanical origins, were analysed for their amino acids’ profile (which included proline) and their colour. In order to test for their botanical classification stepwise linear discriminant analysis was applied to a number of variables made from a selection of analytical results and simple mathematical functions of them. This allowed, firstly, distinguishing honeys from these four botanical sources with 85.0% cross-validated grouped cases correctly classified. A second application was made for the group of honeydew honeys—a matter of particular interest since no chemical or microscopic method exists for the honeys from holm oak and oak. Ninety-five percent of the cross-validated samples were correctly classified, only one sample was misclassified.

Use of Selective TOCSY NMR Experiments for Quantifying Minor Components in Complex Mixtures: Application to the Metabonomics of Amino Acids in Honey

The application of the traditional methods of multivariate statistics, such as the calculation of principle components, to the analysis of NMR spectra taken on sets of biofluid samples is one of the central approaches in the field of metabonomics. While this approach has proven to be a powerful and widely applicable technique, it has an inherent weakness, in that it tends to be dominated by those chemical species present at relatively higher concentrations. Using a set of commercial honey samples, a comparison of this classical metabonomics approach to one based on the use of the selective TOCSY experiment is presented. While the NMR spectrum of honey and its classical metabonomic analysis is completely dominated by a very few chemical species, specifically alpha-glucose and fructose, the statistical signal carried by minor honey components, such as amino acids, may be accessed using a selective TOCSY-based approach. This approach has the intrinsic virtue that it focuses the statistical analysis on a set of predefined chemical species, which might be chosen for their metabolic significance, and could be composed of either major or minor mixture constituents. Furthermore, the selective TOCSY method allows for more certain chemical identification, acquisition times of approximately 1 min, and accurate quantification of the species contributing to the statistical discriminatory signal.

Rapid and sensitive method for determining free amino acids in honey by gas chromatography with flame ionization or mass spectrometric detection

This paper describes a rapid, sensitive and specific method for determination of free amino acids in honey involving a new reaction of derivatization and gas chromatography (GC) with flame ionization (FID) and mass spectrometric (MS) detection. The method allows the determination of 22 free amino acids in honey samples in a short time: 8 and 5 min for GC–FID and GC–MS, respectively. Quantitation was performed using Norvaline as internal standard, with detection limits ranging between 0.112 and 1.795 mg/L by GC–FID and between 0.001 and 0.291 mg/L by GC–MS in the selected-ion monitoring mode. The method was validated and applied to a set of 74 honey samples belonging to four different botanical origins: eucaliptus, rosemary, orange and heather. The statistical treatment of data shows a correct classification of different origins over 90%.

Evaluation of enantiomeric purity of selected amino acids in honey

Highly sensitive and accurate HPLC methods were used for the determination of total amounts of proline, leucine and phenylalanine and their enantiomeric ratios in a variety of different honey samples. Significant amounts of D-leucine and D-phenylalanine and relatively low concentrations of D-proline have been found in honeys of different botanical and geographical origins. It is suggested that the enantiomeric ratios of amino acids could be used to test for storage effects, age, and the quality of the processing of the honey.

Capillary gas chromatographic determination of free amino acids in honey as a means of discrimination between different botanical sources

AbstractThe chemical analysis of honey tends to concentrate on factors related to its state of preservation, e.g. HMF, diastase activity, and water content. Although there is no characteristic of honey officially regarded as suitable for certification of its botanical origin, literature is available in which several of the “minor” components of honey, such as flavors, di‐ and trisaccharides, and free amino acids, have been used to certify the botanical origin of the product.In this paper, six kinds of honey from different botanical sources (acacia, citrus fruit, chestnut‐tree, rhododendron, rosemary, and lime‐tree) were analyzed by capillary gas chromatography, and the data obtained evaluated statistically to determine whether the amino acid profile could be used to verify the botanical source of the material.The results have shown that the presence of amino acids such as arginine, tryptophan, and cystine is characteristic of a particular kind of honey, and that others, such as proline, asparagine, lysine, and methionine, can be used for discrimination if quantitative data is available about the levels of the compounds present.Evaluation of optimum split ratio for amino acid determination, and problems concerning the derivatization process, are also discussed.

Quantitative Determination of Amino Acids in Some Australian Honeys

SummaryThe amino acids in seven Australian honeys were investigated qualitatively and quantitatively using an automatic amino acid analyser; 17 were separated, identified and measured quantitatively. In general the same amino acids were found in all the honeys (7 Eucalyptus, 1 Trifolium), but the amounts of the different acids varied greatly, and there were important differences between species. The probable origins of the amino acids in honey are discussed.