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This study aimed at an experimental design of response surface methodology (RSM) in the optimization of the dominant volatile fraction of Greek thyme honey using solid-phase microextraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS). For this purpose, a multiple response optimization was employed using desirability functions, which demand a search for optimal conditions for a set of responses simultaneously. A test set of eighty thyme honey samples were analyzed under the optimum conditions for validation of the proposed model. The optimized combination of isolation conditions was the temperature (60 °C), equilibration time (15 min), extraction time (30 min), magnetic stirrer speed (700 rpm), sample volume (6 mL), water: honey ratio (1:3 v / w ) with total desirability over 0.50. It was found that the magnetic stirrer speed, which has not been evaluated before, had a positive effect, especially in combination with other factors. The above-developed methodology proved to be effective in the optimization of isolation of specific volatile compounds from a difficult matrix, like honey. This study could be a good basis for the development of novel RSM for other monofloral honey samples.

Background: The authenticity of honey is of high importance since it affects its commercial value. The discrimination of the origin of honey is of prime importance to reinforce consumer trust. In this study, four chemometric models were developed based on the physicochemical parameters according to European and Greek legislation and one using Raman spectroscopy to discriminate Greek honey samples from three commercial monofloral botanical sources., Results: The results of physicochemical (glucose, fructose, electrical activity) parameters chemometric models showed that the percentage of correct recognition fluctuated from 92.2% to 93.8% with cross-validation 90.6-92.2%, and the placement of test set was 79.0-84.3% successful. The addition of maltose content in the previous discrimination models did not significantly improve the discrimination. The corresponding percentages of the Raman chemometric model were 95.3%, 90.6%, and 84.3%., Conclusion: The five chemometric models developed presented similar and very satisfactory results. Given that the recording of Raman spectra is simple, fast, a minimal amount of sample is needed for the analysis, no solvent (environmentally friendly) is used, and no specialized personnel are required, we conclude that the chemometric model based on Raman spectroscopy is an efficient tool to discriminate the botanical origin of fir, pine, and thyme honey varieties. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Thyme honey is the most appreciated unifloral Greek honey in Greece as well as around the world. In an effort to investigate the headspace composition of this type of honey, 28 samples were analyzed by means of solid-phase microextraction coupled to a gas chromatography-mass spectrometry system. The botanical origin of the samples was ascertained by pollen analysis, and samples displayed relative frequencies of thyme pollen between 18 and 41%. A total of 62 compounds were isolated, and phenylacetaldehyde was the most abundant (32.9% of the total peak area). Possible botanical markers are 1-phenyl-2,3-butanedione (13.4%), 3-hydroxy-4-phenyl-2-butanone, 3-hydroxy-1-phenyl-2-butanone (14.7%), phenylacetonitrile (4.8%), and carvacrol (0.9%), since these compounds are found only in thyme honey. Additionally, high proportions of phenylacetaldehyde are also characteristic ( F = 12.282, p < 0.001). The average concentrations of seven compounds were significantly different ( p < 0.05), namely phenylacetaldehyde, acetophenone, octanoic acid, carvacrol, phenylethyl alcohol, nonanal, and hexadecane. Applying principal component analysis to the data, six components were extracted, explaining 85.4% of the total variance. The first component explained 46.2% of the total variance and was positively correlated to phenylacetaldehyde, nonanoic acid, acetophenone, decanoic acid, benzaldehyde, phenylacetonitrile, isophorone, and nonanal. The extracted components were used as variables to the discriminant analysis, which showed good discrimination, especially for samples from Crete. A leave-one-out classification showed 85.7% of cross-validated grouped cases correctly classified. These results are promising to establish a discrimination model for these geographical regions. This is crucial for local beekeeper corporations on their effort to produce honey with geographical origin label.

A new method for mass percentage determination of fructose and glucose based on FT-Raman spectroscopy is evaluated with a standard HPLC-based method. FT-Raman spectra manipulation is done via the spectrometer software, and a PLS (partial least squares) method is developed with the TQ Analyst software (Ver 1. 1a). The simultaneous quantitative determination uses an input range from 1700 to 700 cm(-1) without correction or baseline factors. The standards used in the PLS method are honey samples previously analyzed by HPLC to obtain their mass percentage concentrations in fructose and glucose. The returned results are statistically tested with those of the HPLC method. Both methods appear to score equally in terms of reproducibility. The honey content of the two sugars in total was found up to 40-74%. The honey samples content in fructose and glucose was determined by HPLC (24.1-42.9% and 16.2-33.1%, respectively) and FT-Raman (24.0-40.8% and 21.1-32.2%, respectively).

Definition: DefinitionHoney is a functional, honeybee product with a useful role in human nutrition and several health benefits. Greece is a Mediterranean region with several types of monofloral honey. Today, Greek honey has acquired an important position in national and international markets. Due to this increased industrialization and globalization, quality control is a necessity. Mislabeling constitutes one of the most notable types of fraudulence, while most consumers are looking for authentic honey. Moreover, producers and suppliers are searching for rapid and analytical methodologies to secure Greek honey in a competitive environment. In this context, we aimed to describe the classical (melissopalynological, physicochemical) and analytical (chromatographic, spectrometric, and spectroscopic) methods for the standardization of the botanical origin of Greek honey. [ABSTRACT FROM AUTHOR]

The standardization of the botanical origin of honey reflects the commercial value and quality of honey. Nowadays, most consumers are looking for a unifloral honey. The aim of the present study was to develop a novel method for honey classification using chemometric models based on phenolic compounds analyzed with right angle fluorescence spectroscopy, coupled with stepwise linear discriminant analysis (LDA). The deconstructed spectrum from three-dimensional-emission excitation matrix (3D-EEM) spectra provided a correct classification score of 94.9% calibration and cross-validation at an excitation wavelength (λex) of 330 nm. Subsequently, a score of 81.4% and 79.7%, respectively, at an excitation wavelength (λex) of 360 nm was achieved. Each chemometric model confirmed its power through the external validation with a score of 82.1% for both. Differentiation could be correlated with hydroxycinnamic and hydroxybenzoic acids, which absorb in this region of the spectrum. Fluorescence spectroscopy constitutes a rapid and sensitive technique, which, when combined with the stepwise algorithm and LDA method, can be used as a reliable and predictive authentication tool for honey. This study indicates that the developed methodology is a promising technique for determination of the botanical origin of common Greek honey varieties. Our long-term ambition is to support producers and suppliers to remain in a competitive national and international market. [ABSTRACT FROM AUTHOR]

Among the variants of Greek honey, the most commonly available are pine, fir, thyme, and citrus honey. Samples of the above kinds of honey, identified according to European and Greek legislation, were studied using gas chromatography coupled with mass spectrometry (GC-MS) and the attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic techniques. Two chemometric models were developed based on statistically significant volatile compounds (octane; 2-phenylacetaldehyde; 1-nonanol; methyl 2-hydroxybenzoate; 2-(4-methylcyclohex-3-en-1-yl); nonanoic acid) and the 1390–945 and 847–803 cm−1 spectral regions, mainly vibrations of fructose and glucose, combined with the stepwise linear discriminant analysis (stepwise LDA) statistical technique. In total, 85.5% of standard samples, and 82.3% through internal validation and 88.5% through external validation, were identified correctly using the GC-MS-stepwise-LDA chemometric model. The corresponding results for the ATR-FTIR-stepwise-LDA chemometric model were 93.5%, 82.5%, and 84.6%. The double validation (internal, external) enhances the robustness of the proposed chemometric models. The developed models are considered statistically equivalent, but FTIR spectroscopy is simple, rapid, and more economical. [ABSTRACT FROM AUTHOR]

Abstract: A validated method for the discrimination and classification of honey samples performing GC/MS fingerprinting of headspace volatile compounds was developed. Combined mass spectra of honey samples originated from different plants and geographical regions of Greece were subjected to orthogonal partial least squares-discriminant analysis™ (OPLS™-DA), soft independent modelling of class analogy (SIMCA), and OPLS™-hierarchical cluster analysis (OPLS™-HCA). Analyses revealed an excellent separation between honey samples according to their botanical origin with the percentage of misclassification to be as low as 1.3% applying OPLS™-HCA. Fragments (m/z) responsible for the observed separation were assigned to phenolic, terpenoid, and aliphatic compounds present in the headspace of unifloral honeys. On the other hand, a variable classification for citrus and thyme honeys according to their geographical origin could be achieved. Results suggested that the developed methodology is robust and reliable for the botanical classification of honey samples, and the study of differences in their chemical composition. [Copyright &y& Elsevier]

The volatile fraction of unifloral Greek thyme honey was investigated by means of ultrasound-assisted extraction followed by GC–MS analysis. Phenolic compounds were the most abundant, followed by short chain fatty acids. In total, 14 compounds are proposed as potent botanical markers for thyme honey, 12 of which are phenolics. Among them, 3-hydroxy-4-phenyl-2-butanone and 3-hydroxy-1-phenyl-2-butanone were the most important, as their concentration averages more than 35 mg/kg of honey. Significant average proportions were found for 1-phenyl-2,3-butanedione, 3-hydroxy-4-phenyl-3-buten-2-one, 3,4,5-trimethoxybenzaldehyde and 3,4,5-trimethoxybenzoic acid. Minor phenolics with statistical significance ( p < 0.0001) are phenylacetonitrile, vanillin, 4-hydroxyphenylethanol, 4-hydroxyphenylacetonitrile, and 2-hydroxyacetophenone. Phenylacetaldehyde, a common honey constituent is present at higher concentrations in thyme honey ( p < 0.0001). Important non-phenolic components are 2-methylpropionic acid which was found at higher proportions in thyme honey ( p = 0.002) and 4-(4-hydroxy-2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-3-buten-2-one, a norisoprenoid found solely in this type of honey. [ABSTRACT FROM AUTHOR]

For long heather honey has been a special variety due to its unique organoleptic characteristics. This study aimed to characterize and optimize the isolation of the dominant volatile fraction of Greek autumn heather honey using solid-phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS). The described approach pointed out 13 main volatile components more closely related to honey botanical origin, in terms of occurrence and relative abundance. These volatiles include phenolic compounds and norisoprenoids, with benzaldehyde, safranal and p-anisaldehyde present in higher amounts, while ethyl 4-methoxybenzoate is reported for the first time in honey. Then, an experimental design was developed based on five numeric factors and one categorical factor and evaluated the optimum conditions (temperature: 60 °C, equilibration time: 30 min extraction time: 15 min magnetic stirrer velocity: 100 rpm sample volume: 6 mL water: honey ratio: 1:3 (v/w)). Additionally, a validation test set reinforces the above methodology investigation. Honey is very complex and variable with respect to its volatile components given the high diversity of the floral source. As a result, customizing the isolation parameters for each honey is a good approach for streamlining the isolation volatile compounds. This study could provide a good basis for future recognition of monofloral autumn heather honey. [ABSTRACT FROM AUTHOR]

The aim of this review is to describe the chromatographic, spectrometric, and spectroscopic techniques applied to honey for the determination of botanical and geographical origin and detection of adulteration. Based on the volatile profile of honey and using Solid Phase microextraction-Gas chromatography-Mass spectrometry (SPME-GC-MS) analytical technique, botanical and geographical characterization of honey can be successfully determined. In addition, the use of vibrational spectroscopic techniques, in particular, infrared (IR) and Raman spectroscopy, are discussed as a tool for the detection of honey adulteration and verification of its botanical and geographical origin. Manipulation of the obtained data regarding all the above-mentioned techniques was performed using chemometric analysis. This article reviews the literature between 2007 and 2020. [ABSTRACT FROM AUTHOR]