**1. Introduction**

Due to its sweet taste and antibacterial properties, honey is in high demand in today's market. In 2018, approximately 2000 tons of honey from 103,000 beehives were produced in Latvia. In Europe more generally, the demand for honey is higher than local producers can produce, and therefore a large part is imported [1]. Honey is an expensive product when compared to other sweeteners. Counterfeit honey is considered to be honey that contains added amounts of other cheaper sweeteners. Directive 2014/63/EU of the European Parliament and the Council clearly defines what constitutes natural honey. To protect the interests of consumers and regulate the fair price of honey in today's market, methods of honey authenticity and quality indicators are constantly evolving. One or several modern instrumental methods are increasingly used with which quality characteristics are determined, as well as the authenticity of honey is assessed by applying chemometric methods [2].

Although IRMS has been used mainly to determine the presence of C4 sugar additives, measurements of light stable isotopes have increasingly been used to determine the

**Citation:** Labsvards, K.D.; Rudovica, V.; Kluga, R.; Rusko, J.; Busa, L.; Bertins, M.; Eglite, I.; Naumenko, J.; Salajeva, M.; Viksna, A. Determination of Floral Origin Markers of Latvian Honey by Using IRMS, UHPLC-HRMS, and 1H-NMR. *Foods* **2022**, *11*, 42. https://doi.org/10.3390/ foods11010042

Academic Editors: Olga Escuredo and M. Carmen Seijo

Received: 22 November 2021 Accepted: 22 December 2021 Published: 24 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

botanical or geographical origin of honey. δ13C and δ15N values for honey and proteins provide useful information in distinguishing samples of different floral types of honey (acacia, chestnut, citrus, eucalyptus, rhododendron, and polyfloral honey) [3].

Polyphenol compound concentrations are considered as potential useful variables for floral origins [4]. The polyphenol profile is a useful tool for geographical and floral origin assessment. A robust UHPLC-HRMS method for polyphenol quantification is often used [5–7]. Sugaring-out assisted liquid–liquid extraction (SULLE) sample preparation has been proven to be an optimal choice of honey studies using HRMS equipment [5].

Nuclear magnetic resonance (NMR) is increasingly used to evaluate the authenticity of honey. The information provided by the proton NMR spectrum, in combination with various chemometric methods, is used to distinguish between honey of different botanical origins. Depending on the task to be performed, chemometrics can be performed for the whole spectrum or only for a certain interval. In most cases, the region characteristic of aromatic compounds (9–6 ppm) or the region characteristic of aliphatic compounds (3–0.5 ppm) are used [8,9]. A study in Brazil successfully distinguished between citrus, eucalyptus, and wildflower honey, and some honey was found to be counterfeit [10]. A similar approach was used by Spiteri et al. for the assessment of geographical origin [11]. Samples of acacia honey from Eastern Europe and Italy were compared. Due to the different flora, characteristic floral markers were found in the samples [12].

Principal component analysis (PCA) in chemistry allows for the study of the properties of different datasets of chemical compounds. Determining which compounds have similar properties and which study objects form groups, one can also try to predict the properties of the study object or belonging to a group. Various instrumental analyses are practically effective for the analysis of principal components, wherein the spectral image is obtained, for example, the total ion chromatogram after the retention time, under different conditions [13]. Quantitative values of various honey compounds, isotope ratio values, etc., quality indicators can be used to analyze the principal components. Depending on the purpose of the study (counterfeits, origin of flowers, geographical regions, etc.), honey types are selected, chemical instrumental analyses are performed, and the results are used for the analysis of principal components to determine the formation of groups [10,14–16].

The main aim of this study was the use of different methodologies to classify the botanical origin of various types of monofloral Latvian honey to target the mislabeling of protected destination of origin (PDO) products. One of the goals was to gather the data on fresh samples collected directly from the beekeepers of Latvia instead of processed and commercially available honey. Further, we validated the true floral origin using melissopalynology analysis. Finally, we evaluated multiple criteria to classify individual monofloral variety honeys by using a combination of analytical methods (IRMS, UHPLC-HRMS and NMR) and statistical treatment of experimental data and PCA analysis.

### **2. Materials and Methods**
