Recent achievements in element analysis of bee honeys by atomic and mass spectrometry methods

Accepted Manuscript Recent achievements in element analysis of bee honeys by atomic and mass spectrometry methods Pawel Pohl, Aleksandra Bielawska-Pohl, Anna Dzimitrowicz, Piotr Jamroz, Maja Welna, Anna Lesniewicz, Anna Szymczycha-Madeja PII:

S0165-9936(17)30044-4

DOI:

10.1016/j.trac.2017.05.009

Reference:

TRAC 14928

To appear in:

Trends in Analytical Chemistry

Received Date: 3 February 2017 Accepted Date: 22 May 2017

Please cite this article as: P. Pohl, A. Bielawska-Pohl, A. Dzimitrowicz, P. Jamroz, M. Welna, A. Lesniewicz, A. Szymczycha-Madeja, Recent achievements in element analysis of bee honeys by atomic and mass spectrometry methods, Trends in Analytical Chemistry (2017), doi: 10.1016/ j.trac.2017.05.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Recent achievements in element analysis of bee honeys by atomic and mass spectrometry methods Pawel Pohl*a, Aleksandra Bielawska-Pohlb, Anna Dzimitrowicza, Piotr Jamroza, Maja Welnaa, Anna Lesniewicza, Anna Szymczycha-Madejaa a

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Wroclaw University of Technology, Faculty of Chemistry, Division of Analytic Chemistry and Chemical Metallurgy, Wyspianskiego 27, Wroclaw, 50-370 Poland b Laboratory of Glycobiology and Cellular Interactions, Polish Academy of Sciences, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Weigla 12, 53-114 Wroclaw, Poland * Corresponding author. Tel.: +48 (0) 71 320 2494. E-mail address: [email protected] (P. Pohl).

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Abstract Consumption of honey cannot raise any concern about its wholesomeness, safety and quality in reference to the content of different contaminants, particularly including trace and hazardous elements. Element analysis of honey by atomic and mass spectrometry methods is important part of its quality and safety. The present paper comprehensively reviews recent achievements in element analysis of honey that have been reported since 2012. The survey is focused on different research aspects of such analysis, including assessment of biological and geographical origin of honey by using chemometric methods, quality and safety of honey, and sample preparation of honey prior to element analysis by atomic and mass spectrometry methods. Calibration strategies and ways of quality assurance and control of the results are surveyed as well.

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Keywords: Elements, Honey, Botanical origin, Geographical origin, Chemometrics, Sample preparation, Atomic spectrometry, Mass spectrometry

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1. Introduction Honey is a natural food product made by Apis mellifera bees from nectar of melliferous and flowering plants and/or secretions of other plants [1]. Beekeeping in European and Asiatic, i.e. China [1], as well South American, i.e. Argentina [2], countries is significant agricultural, semi-industrial or even industrial activity. In the European Union countries, honey trade is important so as its quality is regulated by a special directive, which covers specific characteristics of honey [3]. To protect honey authenticity, safety and quality, honeys coming from the EU countries or sold on their territories have to possess specific compositional characteristics, related to the content of carbohydrates, humidity, acidity, electrical conductivity, diastase activity and the content of hydroxymethylfurfural (HMF) [2]. Because the composition and properties of honeys strongly depend on type of forage sources of bees, i.e. nectar and pollen of blooming plants that are available for them within their flight range [2,4], environmental, pedological, and anthropological factors in addition to specific melliferous plants give a distinctive and unique character to honey from a certain region. In reference to this, concentrations of different elements in honey can be distinguishing both to its botanical and geographical origin and hence, used for authentication and safety assurance and control of this natural food product [4-6]. Unfortunately, honey can be subjected to adulteration during its production, e.g. by addition of glucose solutions, sucrose syrups or sugarcane juice [7,8], or improper beekeeping practices [9]. With no doubt, consumption of honey cannot raise any concern about its wholesomeness, safety and quality in reference to the content of different contaminants, particularly including trace and hazardous elements [5]. Therefore, element analysis of honey by atomic and mass spectrometry methods is important part of its quality and safety assurance and control. Results of such analyses, regarding the content of various environmentally, toxicologically and nutritionally relevant elements, can have a notable informative value for consumers, giving them reliable information about quality, safety, authenticity and nutritional status of honey ingested by them. It can also give an instructive feedback to beekeepers whose may improve their practices referred to harvest and extraction of honey and enhance final quality of honey. Indirectly, farmers, knowing about the effect of their plants cultivation and agriculture practices in a given territory on quality of honey reflected by the content of hazardous trace elements, may change the means of irrigation, fertilization and spraying of flower plants planted by them. It is not surprising that honey and its quality is more and more the subject of many research papers and several review papers related to general chemical composition of honey, including minerals and trace elements [10-12], or entirely devoted to element analysis [1315]. It appears that interest in element analysis of honey has increased over the past five years. The present review surveys original scientific works devoted to element analysis of honey that were published since 2012. It is focused on different research aspects treated by these works, i.e. general necessity and significance of element analysis of honey in terms of assessing its biological and geographical origin by using chemometric methods as well as quality and safety, in addition to sample preparation of honey prior to its element analysis by atomic and mass spectrometry methods. In the latter case, means of calibration and quality assurance of the results are surveyed as well.

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2. Sources of elements in honey and their role 2.1. Biological and geographical origin of elements in honey It appears that presence of elements in honey is relevant to its quality, safety and dietary value [2,16-19]. Considering major elements, honeys is an solid source of K, which is the most abundant element among others, in addition to Ca, Mg, Na, P and S, which are also 2

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present in high to moderate amounts [2,5,6,16,18,19-47]. Minor and trace elements of honey are Al, B, Ba, Cd, Co, Cr, Cu, Fe, Mn Ni, Pb, Sn, Sr and Zn [28,29,37,40]. Major and minor elements of honey, can provide a certain nutritional value, therefore, element analysis of honey helps in assessing and checking this dietary content. In reality, it appears that ingestion of honey does not contribute to cover recommended dietary allowances (RDAs) for minerals and minor elements in high regard. Safety of honey is related to the content of toxic and hazardous trace elements, but importantly, there is low chance of poisoning with Cd, Cr or Pb through honey consumption because these elements do not rather exceed their acceptable limits and/or tolerable daily intakes (TDIs) [48,49]. Accordingly, daily consumption of certain amounts of honey, e.g. 20 g [20,21,49,50], 25 [33], 43 g (two spoons) [6], 50 g [48,49] or even 100 g [51], can supply, with some exceptions, only up to few percents of RDAs or TDIs, e.g. 0.2-52.7% for Zn, 0.2-1.1% for Fe, 0.1-334% for Cu, 1.8-47.0% for Cr, 0.1-12% for Mn, 0.1-5.4% for Ca, 0.2-5.3% for K, 0.2-2.1% for Mg, 0-0.4% for Na, up to 1.3% for P, up to 2.6% for Mo, up to 5.3% for As, up to 1.3% for Se, 0.5-4.0% for Al, 0.2-3.6% for Pb, 0-0.7% for Ni and up to 1.8% for Cd. Nevertheless, presence of elements in honey have a physiological effect in daily diet [33,42]. The content of elements in honey is heterogeneous and vary considerably [6,16,17,22,25,26,29,33,38,43,48,49,51-57]. Researchers are agreed that presence of major and minor elements is mostly related to biological origin of honey, i.e. variety of flowers that bloom in a given territory and composition of floral sources that are foraged by bees, including nectar and pollen [4,5,16,17,20,22,25,26,28,30-33,40,43,44-46,49,51-54,57-60]. Nevertheless, even within the same botanical origin of blossom plants, concentrations of various elements can greatly vary from country to country or region to region, showing the effect of exposition of mentioned bee resources to different environmental and pedological changes, strong dependence of the content of elements on climate, vegetation season, geochemistry and composition of soils on a given territory [6,17,20,22,23,2529,32,33,35,43,45,46,48,49,51,53,54,57-63]. Correspondence between specific profiles of elements and ratios of isotopes in honey, on one hand, and in soil and water, on the other hand, is recognized as high, particularly considering Co, Ni, Sr, Mn, Rb and ratio of K/Rb (correlation observed between honey and soil) or Ca, B, Rb, Mg and ratio of Ca/Sr (correlation observed between honey and water) [6]. Absorption of elements from environment by blossom plants differs due to plant variety and availability of elements in soil [35]. Pronounced differences in accumulation of elements by different honeys due to botanical provenience of plant sources from which they are produced are primarily established between honeydew, forest and other dark color honeys, on one side, and blossom amber to light color honeys, on the other side. The first type of honeys typically contain higher concentrations of many elements as compared to pale honeys [38,47,62], e.g. Al [64], As [9], Ca [38,43,52], Cd [9,64], Cu [35], Fe [43,45,64], K [38,43,45,52,64], Mg [38,43,45,52,64], Mn [35], Na [38,43,45], Ni [65], P [52,60], Pb [9,64], Zn [35,43,45]. This is reflected by positive correlation found between color of honeys and their mineral content, i.e. Ca, K, Mg and Na [4,38,40,47]. In addition, strong correlation between the content of elements and antioxidant activity [4,5,40,41,52] or the content of proteins [52], the content of total phenolic compounds [4,5,34,41] and flavonoids [4,41] is also described. This points out that electrons donating metals ions are complexed by polyphenolic structures [4,52]. Due to higher contents of phenolics, proteins and minerals, honeydew [52] and dark color honeys [5] express in general higher antioxidant activity than respective light color honeys.

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2.2. Contamination of honeys with minor and trace elements In reference to anthropogenic activity and contamination of water, air and soil with hazardous elements on a given territory, the element composition of honey is changeable and 3

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seems to give important information about environmental pollution and contamination with those trace elements [1,6,16,21,25,26,30,37,39,47-51,55,57,59,61-64,66-70]. Collecting contaminated resources from element-accumulating plants or being chronically exposed to pollution on large industrial or urbanized areas, bee heaves and bees are subjected to contamination with many toxic elements, e.g. Al, Cd, Co, Cu, Cr, Fe, Pb, Se and Sn [37,49,69,71]. There is evidence that honeys originating from such industrialized (due to mines and steelworks, smelters and refiners of non-ferrous metals), urbanized (due to power plants, municipal wastes, transportation), agricultural or highly populated regions present higher levels of selected trace and minor elements, e.g. Al [37], As [57], Ba [37], Cd [37,48,49,55,57,66,68], Co [37,69], Cr [37,48,49,63,69], Cu [37,49,68,69], Fe [37,49,62,68], Hg [57], Mn [68], Ni [37,63,68], Pb [19,37,48,49,55,66-69], Sb [57], Se [37,68], Sn [37], Sr [37], Zn [16,68,70], as compared to those coming from non-industrialized and rural regions. Therefore, some researchers conclude that honey can be regarded as a reliable biological marker for environmental pollution assessment, primarily of notoriously polluted zones, where air, water, vegetation and soil are loaded with trace elements of anthropic origin [37,48,55,57,68,69]. In this case, the influence of botanical origin of honey on its characteristic profile of elements is weaker than the effect of environmental pollution and/or the mineral composition of soil on certain territory. On the other hand, there are few researchers of honey who assert that specific capacity of individual plants to assimilate the elements from soil and accumulation yields of certain elements in pollen and nectar of these plants are stronger and more important than the effects related to environmental pollution originated from proximity to large cities or industrial districts [16,64,65,70]. These researchers report no difference in the content of certain trace hazardous elements, i.e. As, Cd, Pb and Tl, in honeys collected from apiaries located in ecologically devastated and of intensive human activity areas and honeys originating from other regions [16,64,70]. Honeys originated from industrial regions, i.e. power plant areas, can even represent lower concentrations of Cd, Cr, Ni and Pb than those determined in honeys from clear areas [65]. Therefore, the use of honey for environmental monitoring of pollution with trace elements is not so obvious and sometimes can not provide quantitative information on environmental pollution with these elements [16,65,70,72]. All researchers consider that high levels of trace hazardous elements, particularly As, Al, Ba, Be, Cd, Ce, Co, Cr, Dy, Er, Ho, La, Ni, Pb, Se, Sm, Th, Tl, U and V, in honeys raise concern about eventual adverse effects on consumers health [1,2,21,37,51,52,61,67,69,72,73]. For that reason they admit that constant monitoring of trace elements, particularly heavy metals, in honey and other apiculture products is critical for the whole community of honey recipients. In addition, presence of elements listed above may ultimately indicate increased industrial activity (e.g. through mining), increased automobile exhaust emission in the area of bee forage or intensive agriculture activity (e.g. through extensive application of pesticides, natural or industrial fertilizers or atmospheric fallout) [1,6,21,23,25,26,37,48,49,51,53,62,66,69,73]. Apart from sources of trace elements in honey mentioned above, presence of elevated amounts of some of them can be either associated with improper beekeeping practices used for harvesting and processing of honey, e.g. extraction by centrifuging in stainless steel centrifuges or pressing in stainless steel press machines [46]. Contamination occurred during harvesting, processing, preparation or storage of honey, connected with the use of inappropriate materials and tools (e.g. stainless steel or galvanized containers for honey storage, stainless steel presses for honey extraction), or practices (e.g. pharmacological treatment of bees, conservation of honey, feeding of bees with sugar syrups) is commonly reflected by higher concentrations of certain major, minor and trace elements [2,8,9,23,25,46,49,51,54,56,62,74]. Release of elements, particularly of Cd, Cr, Pb and Zn, is facilitated by natural acidity of honey [49,56].

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Considering possibility of contamination of honey with different trace elements from described external sources, many researchers highlight that safety of honey for consumption has to be customarily verified by assessing what is the amount of elements that are hazard to human health and what is the eventual degree of intake of various elements [19,23,48,49,57,66,75,76]. Hence, very low levels or absence of As, Al, Cd, Co, Cr, Hg, Ni, Pb, Se or Te is normally a good indicator of high quality of honey, showing that there is no health hazard associated with its consumption [1,2,6-8,16,17,19-21,23,25,32,33,37,39,4547,50,54-57,59,61,63,65,69,72,74,76-79]. In a similar way, other elements, e.g. Ba, Be, Bi, Cu, Li or even Zn, present at very low levels or not detected at all points out that honey is safe and of high quality [2,6,7,17,19,39,42,47,54,59,61,62,72,76-78]. From this point of view, consumption of honey is typically considered as safe for human health, however, an eventual intoxication level has to be controlled since some of the elements tend to accumulate in the human body. Low levels of hazardous elements in honey can either reflect that it comes from clean environment and/or regions free from contamination [75,76,78].

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3. Chemometric evaluation of honey due to elements profiles Elements are very good variables for chemometric classification of honeys because they are stable over a long period of time, while their content is determined by plant species sources of pollen and nectar (biological or floral origin) as well as geochemical composition of soil on which these plants are grown [35,45]. Existence of similarities and differences between honeys of different botanical origin and coming from different regions has its justification in physiology of plants and their collection of nutrients from soil. Major and minor elements found in honeys are those being the most and medium abundant in plants, while trace elements can result from environmental contamination [66]. It appears that different unsupervised pattern-recognition chemometric techniques, i.e. clustering by hierarchic cluster analysis (HCA) [7,2,28,31,33,37,43,45,57,66,69] and dimensionality reduction by principle component analysis (PCA) [2,5,7,19,22,25,28,29,31,34,37-41,45,47,48,57,62,64,66,69,80] and factor analysis (FA) [52,33], as well as supervised pattern-recognition and learning techniques, i.e. canonical discriminant analysis (CDA) [20], linear discriminant analysis (LDA) [2,6,21,38,41,54,60,62,80], partial least-squares discriminant analysis (PLS-DA) [22] and back-propagation artificial neural network (BP-ANN [22], are used for data exploration, and identification and categorization of honeys of different botanical and geographical provenience. In addition, classification trees analysis (CTA) [6] and generalized procrustes analysis (GPA) [6] are used.

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3.1. Cluster analysis HCA is useful to separate honeys due to honeybee species (entomological origin) [7,45]. This is possible just with concentrations of a limited number of elements, i.e. Ca, Mg, Na and Sr [7], or two major minerals (K, Na) along with other important parameters, i.e. the ash content, the total content of proteins, the total content of carbohydrates, the concentrations of fructose, glucose and sucrose [45]. HCA is also applied to distinguish production sites related to countries of origin [2,66] or certain geographical regions [37,57,69]. In this case, information about concentrations of a number of elements, i.e. As, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Se, Tl, U, V and Zn [2], Ca, Cd, Cu, Fe, K, Mg, Mn, Pb and Zn [66], Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P Pb, Se, Sn, Sr and Zn [37], Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sr and Zn [69] or Al, As, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, Ge, Hg, Mn, Mo, Pb, Sb, Se, Sn, Sr, Tl, U, V and Zn [57] is used to establish similarities between the samples. In 5

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the same way, HCA helps in finding interrelationships between concentrations of selected elements, e.g. Ca, Cu, Fe, K, Mg, Na, P and Zn [33] or Ca, K, Mg, Na and Zn [43], and belonging to a particular group of honeys, i.e. dark and light color honeys. The data matrix taken for HCA and separation of honeys due to geographical origin can include information about elements as well as additional physicochemical properties of honey, e.g. the concentrations of major elements (Ca, K, Mg, Na) along pH, free acidity, moisture, electrical conductivity, the ash content, the concentrations of fructose, glucose, saccharose, turanose and maltose [31].

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3.2. Principle component analysis PCA is often used for finding relationships between the concentrations of elements and samples having the same botanical origin [20,22]. Accordingly, Ca, K, Ni and Zn are those variables which are responsible for discriminating the samples of different botanical provenience [20]. Other sets of elements, e.g. Ba, Ca, Cu, Fe, K, Mg, Mn, Na, Rb and Sr [22], can be applied for that purpose as well. Interestingly, PCA can point out differences between the samples and distinguish the groups of honeys originating from different honeybee species (entomological origin) [7,45]. Such discrimination is possible with the concentrations of just four elements (Ca, Mg, Na and Sr) [7] or the concentrations of K and Na in addition to the ash content, the total contents of proteins and carbohydrates and the concentrations of fructose, glucose and sucrose [45]. On the other hand, PCA is applied for distinguishing the samples of honeys coming from certain regions or countries (geographical origin) [19,20,25,29,37,39,47,48,57,62,66,69]. Accordingly, Ca, Cr, Cu, Mg, Ni, Pb and Zn [20], Al, Ca, Cd, Cu, Fe, K, Mg, Mn, Na, Ni, Pb and Zn [29], Ca, Cd, Cu, Fe, K, Mg, Mn, P, Pb and Zn [66], Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Se, Sn, Sr and Zn [37], Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sr and Zn [69], As, Ca, Cd, Co, Cu, Fe, K, Mg, Na, Pb and Zn [19], Al, As, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, Ge, Hg, Mn, Mo, Pb, Sb, Se, Sn, Sr, Tl, U, V and Zn [57] or Ag, Al, As, Ba, Be, Ca, Cd, Co, Cr, Cu, Cs, Fe, Ga, In, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr Ti, U, V and Zn [47] are those elements, which concentrations in honey are established to vary greatly due to location of apiaries and the effect of lithological and anthropic origin of elements in soil and other parts of environment. Even concentrations of Cd and Pb are acknowledged to be indicative for geographical origin of samples due to characteristic geochemistry of soil and environmental contamination of soil, water and air on a given territory [48]. In a similar way, concentrations of As, Ca, Cd, Co, Cr, Cu, K, Fe, Mg, Mn, Na, Ni, P, Pb and Zn can be used to distinguish the samples of honeys originated from regions of certain types of soil [25]. In this case, the highest differences are particularly evident for Ni, Mg, Mn and P [25]. To evaluate relationship between geographical origin of honeys and the element content, the concentrations of Ca, Cu, Fe, K, Mg, Na and Zn [62] or Ca, Cu, Fe, K, Pb and Zn [39] are considered. Except for the concentrations of elements, the data correlation matrices for PCA usually include other physicochemical parameters (see details in Table 1), e.g. pH, free acidity, the moisture content, electrical conductivity, the contents of fructose, glucose, sucrose, turanose and maltose and the ash content [31], the isotope ratios of C, H and O [38], the total contents of phenolic compounds and the total content of flavonoid compounds, color, electrical conductivity, and antioxidant activity [41]. Such complementary data are very useful for distinguishing different floral types of honeys [38,41] or determining differences between honeys of different geographical provenience [31]. It can also be the concentration of Cu along with to the content of vitamin B2 and antioxidant activity [34] or the total mineral content (as sum of the concentrations of Ca, Fe, K, Mg and Na) along with the total content of phenolic compounds and antioxidant activity [5].

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The number of additional physicochemical properties of honeys taken for distinguishing and separating the groups of similar samples of honey by PCA can be really quite high, e.g. Cd, Cu, Fe, Mg, Ni and Pb along with moisture, pH, total acidity, ash, fructose, glucose and sucrose, the ratio of fructose to glucose, invertase and diastase activities, proline, HMF, total phenolic compounds, total flavonoid compounds, antioxidant activity and color [80]; Al, Ca, Cd, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, Pb and Zn along with total phenolic compounds, total flavonoid compounds, total proteins and total non-protein tiols [64]; Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, S, Sb, Se, Sr, V and Zn in addition to total phenolic compounds, antioxidant activity, twenty five individual phenolics and fourteen individual carbohydrates [28]; Ca, K, Mg and P along with fructose, glucose and malezistose, antioxidant activity, diastase activity, color, electrical conductivity, pH, total phenolic compounds and total flavonoid compounds [40]. FA shows that nutritional components of honey, including K, Mg and Na, fructose, glucose maltose and melezitose, total proteins, total phenolics and total flavonoids, can indicate botanical origin of the compared samples [52]. FA, performed on the data matrix composed of the concentrations of Ca, Cu, Fe, K, Mg, Mn, Na, P and Zn, can denote differences between honeys of different botanical origin [33].

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3.3. Discriminant analysis Classification and categorization of honeys due to elements profiling can be successfully achieved with the aid of supervised techniques, particularly LDA. Accordingly, LDA enables to discriminate the samples of honey coming from different regions due to the concentrations of Ca, Cr, Cu, Mg, Ni, Pb and Zn [20], B, Ca, Cu, Li, Mo, Ni, P, Sb, Se, Si, Sn, Sr and Ti [54], or Ca, Cu, Fe, K, Mg, Na, Zn along with other physicochemical properties, i.e. antioxidant activity, the total content of phenolic compounds, color and the ash content [62]. In a similar way, geographical discrimination made by LDA can be obtained for the concentrations of B, Na, La, Rb, U and Zn in addition to the concentrations ratios of K/Rb and 87Sr/86Sr that are related to geological and geochemical features of soil on which the blossom plants are grown [6]. LDA is also capable of categorization of honey due to its type and/or floral origin. Such categorization can be done considering the concentrations of Ca, Cu, Fe, K, Mg, Mn, P, S and Zn [21]. Except for elements, additional physicochemical properties of honey are also considered (see details in Table 2), e.g. pH, total acidity, moisture, ash, fructose, glucose and sucrose, ratio of fructose to glucose, invertase and diastase activities, proline, HMF, total phenolic compounds, total flavonoid compounds, antioxidant activity and color [80]; isotope ratios of C, H and O [38], color, electrical conductivity, total phenolic compounds, total flavonoid compounds, and antioxidant activity [41], or color, antioxidant activity an the C isotope ratio [60]. Discriminant analysis (DA) on PCA factors, derived from the data matrix constituted by such loadings as the concentrations of Ca, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Zn in addition to the moisture content, the total content of sugars and pH, is also indicative to the production site [2]. PLS-DA and BP-ANN are capable of predicting botanical origin of honeys due to the concentrations of Ba, Ca, Cu, Fe, K, Mg, Mn, Na, Rb and Sr [22]. In addition, machine learning algorithms, i.e. support vector machine (SVM), multi-player perception network (MLP) and random forest (RF), are used for identification of geographical origin due to content of five elements, i.e. Er, Ho, Pb, Pt and Tl [53].

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4. Samples and their botanical origin 7

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Honeys are collected directly from beekeepers [1,2,7-9,16-19,20-23,25,26,28-32,3445,49,51-53,55,57,59-67,70,73,74,76,78,80-83]. If comb honey samples are available, they are mechanically filtrated or extracted at first [46,61,62,84]. Samples can also be purchased from local companies and beekeeping associations engaged in purchase and sale of honey [36,45,48,50,51,53,54,56,60,73,85,86] or shops and stores [8,9,30,33,42,45,50,51,54,58,75,76,79,81-83,87-90]. Except for honeys produced by Apis mellifera, honeys produced by other bee species, i.e. Apis dorsata [8,17,19,45,47], Apis cerana [19,45], Heterotrigona itama [19,45], Melipona fasciculate [7], Melipona flavoneata [7], Tertragonisca angustula [84], or other subspecies, i.e. black bees [27], are collected and analyzed. The list of unifloral and monofloral honeys and their botanical origin is given in Table 3. Poly- or multi-floral honeys derived from nectar of numerous species of flowers and blossoms [2,4,6,8,9,18-21,25,26,30,31,33,36,39,42,43,49-52,56-59,66,67,69,7476,82,83,85,87,89,91], honeydew honeys [19,21,33,34,42,47,49,64-66,69,82,83,89] or not specified types of honeys [6,7,16,23,46,48,51,53,55,61,63,71,73,75-79,81,84,86,88,90] are also analyzed. Samples are kept in dark until analysis, usually at 4-8 oC [1,2,6,7,9,17,19,20,22,23,26,28,30,31,36,41,43,50,54,58,60,61,63,66,74,76,80,84], at 10 oC [37,69] or at room temperature [5,18,21,38,39,47,48,55,57,67,73,78]. Freezing of honeys to 20 oC is also practiced [32,46,62,65,71]. To homogenize the samples and make their handling simpler, they are initially heated o to 30 C [16,70], 40-50 oC [20,22,26,27,35,38,52,57,84,90] or 65 oC [74] and liquefied in this way. In addition, the resulting samples aliquots are sonicated [22,27,35,52]. This step ensures uniform dispersion of the analytes in fluid bulk material. The samples of honey can also be initially dissolved with water at 1:1 (g/ml) [53], 1:2 (g/ml) [63] or 1:3 (g/ml) [74] and such mixtures are subsequently handled, e.g. taken for mineralization.

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5. Sample preparation Honey is regarded as a highly concentrated aqueous solution of different carbohydrates, primarily fructose, glucose, maltose and sucrose, and to smaller degree other oligo- and polysaccharides [52,79]. All carbohydrates are as high as 95% of dry weight of honey [52]. This matrix comprises the elements, including minerals (major elements), minor and trace elements. Considering importance of reliable element analysis of honey by atomic and mass spectrometry methods, effectiveness of prior sample preparation is quite significant. Sample preparation procedures have to provide quantitative release of elements from the complex organic matrix, reduce eventual losses of elements and avoid any contamination of the resulting samples solutions [79]. Therefore, it is necessary to completely remove the organic matrix of the samples of honey by their decomposition and quantitatively extract the elements ions from the samples into the respective solutions prior to their analysis. Indeed, it seems that the strategy based on wet decomposition of samples and elimination of the organic matrix constituents is preferred by the overwhelming majority of researchers. Apparently form literature, small, i.e. 0.2 g [66,77], 0.25 [6,24,79], 0.3 g [74], 0.4 g [20], 0.5 g [19,26,34,35,37,40,45,52,53,56,69,88,90], 0.6-0.7 g [28], 0.8 g [2], and 1 g [1,5,7,22,23,27,30-33,38,44,46,47,49-51,57,58,65,68,76,80,84], or moderate, i.e. 2 g [64], 2.5 g [89,91], 3 g [16,21,70], 5 g [9,43,72], and 10 g [25,54,60,81-83], masses of samples are taken for wet digestion. Wet digestion procedures are normally carried out in closed-vessel systems assisted by microwaves action [1,2,6,19,20,22-24,26-30,33-35,37,39,40,44,45,50,53,56-59,6466,68,69,71,74,76,77,79,80,84,87,88,90] or in open-vessel systems using digestion blocks [5,7,32,38,46,74], hot plates [9,16,21,25,31,43,47,50,51,70,72,81-83,89,91], water baths

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[54,60] or a heating mantle [63]. Comparison of different wet digestion procedures points out that better recoveries of trace elements are obtained when closed-vessel microwave-assisted digestion procedures are used [50]. In addition, time required for digestion of samples in microwave ovens is shorter, while loss of the elements from the honey matrix due to volatility of their species is completely eliminated [79]. Therefore, closed-vessel microwave-assisted wet digestion is preferred in element analysis of honey [79]. As shown by the cited works, it is now the most reliable and often used sample preparation procedure of honey prior to its element analysis by atomic and mass spectrometry methods. Reagents used for wet oxidative digestion of honey are commonly mixtures of concentrated HNO3 and 30% H2O2, differing due to their volume ratios, i.e. 3:4 [56], 1:1 [1,74,89,91], 3:2 [45], 5:3 [22,27], 2:1 [7,16,19,26,31,53,66,70,79], 7:3 [2], 5:2 [34], 3:1 [43,50,57,65,68,80], 10:3 [21,44,63], 4:1 [49,77], 9:2 [35,40,52], 5:1 [5,81], 7:1 [20,28,84], 15:2 [25,82,83], 9:1 [37,58,69], or not specified [72]. Other mixtures are also applied, i.e. concentrated HNO3, 30% H2O2 and concentrated HF at 5:2:1 [24], HNO3 and concentrated HClO4 at 6:1 [32,46] or another ratio [76]. Remnants of HF have to be removed by boiling the samples solutions on a hot plate [24]. Wet digestion is also carried out only in concentrated HNO3 [6,9,23,33,38,47,51,54,60,64,74,88,90]. Initially, the samples are treated with the reagents at room temperature and left for pre-digestion [1,7,21,25,34,38,77,82-84,90]. Finally, the resulting digests of samples are diluted exclusively with water or a 1% HNO3 solution [24] to the required volume so as the original samples are finally diluted 2.5-fold [25,81-83], 5-fold [9,72], 10-fold [31,33,37,50,54,60,65,68,69,80], 14-fold [44], 16.7-fold [21], 18.8-fold [2], 20-fold [7,53,63,64,89,91], 25-fold [1,32,46], 30-fold [77], 40-fold [74], 50-fold [5,22,27,30,34,35,38,43,47,52,57,66,84,90], 62.5-fold [20], 83.3-fold [28], or even 100-fold [19,26,45,56,76,88] and 200-fold [24]. Dry ashing of honey samples is also used [4,8,17,18,36,41,55,61,62,67,75,78,86], however, it has to be noted that the procedure takes long time to prepare suitable samples solutions, while risk of losses of trace elements due to volatilization is quite high and frequent. In this case, 1-g [17], 5-g [4,8,36,41,55,61,62,67,75] or 10-g [18] samples of honey are placed in porcelain or quartz crucibles and their combustible organic matrices are destroyed at high temperature. Typical ashing temperatures, to which the samples are heated up and kept at this temperature for incineration of the organic matter, are 450 oC [17,55,67], 550 oC [4,8,18,36,41,61,62,86] or 600 oC [75,78]. The resulting noncombustible ash residues are dissolved in water [75], diluted HNO3 [4,55,61] or HCl [8,36,41] solutions, or their mixtures [18], concentrated HNO3 [62,78] or HCl [67] solutions, or their mixtures [17]. This is done by warming the residues with the solutions on hot plates. Finally, the samples solutions are diluted with water to 100 ml [41], 50 ml [18,67], 25 ml [4,61,62] or 10 ml [36,55,75]. It is reasonable to suppose that sample preparation procedures alternative to wet or dry oxidative high-temperature treatments are favorable to simplify and shorten element analysis of honey. Such simplified sample preparation procedures with minimal handling and preparation can certainly reduce the risk of contamination of samples and losses of analytes due to prolonged sample treatment. Unfortunately, as mentioned before, the researchers primarily choose well recognized, tested and reliable microwave assisted wet digestions. Simplified sample preparation procedures based on nondestructive dissolution of samples are rarely used. Accordingly, in case of graphite furnace atomic absorption spectrometry (GFAAS), 7% (m/v) honey solutions are prepared in 0.1 mol L-1 HNO3 and contain 3% H2O2 (Cd, Pb) or 9% H2O2 (Cr) [48,73]. Both reagents are used to decompose the organic matter of the samples during the heating program in a graphite tube. Before injection, the sample solutions are sonicated to assure their homogenization [48,73]. More concentrated solutions are also prepared, i.e. 50% honey solutions in 2.5% HNO3 and 12.5% H2O2 [85] (Cd, Pb).

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In case of flame atomic absorption spectrometry (FAAS), 3% [42] or 5% [89] water solutions of honey are prepared and directly subjected to element analysis versus simple standard solutions for calibration. 5% solutions of honey can be subjected to dispersive liquidliquid micro-extraction (DLLME) in the system with sodium diethyldithiocarbamate (NaDDTC) for complexing the Cd(II) and Pb(II) ions, acetonitrile (dispersive solvent) and carbon tetrachloride (CC49, extraction solvent) [90]. The resulting organic phase is then measured by FAAS [90]. 10% honey solutions [82,83,91], acidified with HNO3 to ~0.2 mol L-1 [82,83], or not [82,91], can be prepared and treated by solid phase extraction (SPE) using Dowex 50W×8-400 SPE tubes prior to quantitative retention of traces of Cu [82,91] and Zn [82]. Latter on, the analytes are completely eluted using 2 [91] or 3 [82] mol L-1 HCl solutions and the resulting eluates are subjected to analysis by FAAS. For inductively coupled plasma mass spectrometry (ICP MS), 50% honey solutions are prepared and then small portions of such solutions are incubated for 20 min with concentrated HNO3 and then diluted with water so as to reach its final concentration of 0.3 mol L-1 and the final honey content of 2% [53].

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6. Spectrometric methods of element analysis of honey Inductively coupled plasma optical emission spectrometry (ICP OES) is quite often selected by the researchers for multi-element analysis of digested samples of honey, including major, minor and trace elements [1,7,20,21,25,28,30,31,34,37,47,50,54,60,63,69,71,75,78,79,82,83,88]. ICP MS is applied for the same reason, however, due to higher sensitivity and better detectability, it is possible to highly dilute the samples solutions [2,6,9,16,2024,27,29,38,44,45,47,53,55,57,59,70,74,77,84,87,90]. In the latter case, element analysis of digested samples solutions is virtually free from any chemical interference. Nevertheless, polyatomic ions, which are formed due to presence of mineral sample matrix constituents, reagents used for sample decomposition and plasma gas, can give rise to undesirable spectral interferences. Therefore, the ICP MS instruments with octopole/collision reaction cells [2,6,20,23,27,38,44,55,57,74,77,84,90] or sector field mass spectrometers [16,70] are used to remove them. FAAS is quite frequently used to quantify major and minor elements i.e. Ca, Cu, Fe, K, Mg, Mn, Na and Zn in solutions of digested samples of honey [46,8,18,19,26,32,33,35,36,39,40,42,43,45,46,49,51,52,56,58,62,64,66,68,80,82,86,89,91]. In addition, the method is accepted to be used for quantification of some trace elements, i.e. Al, As, B, Ba, Cd, Co, Cr, Hg, Ni, Pb [32,33,46,51,58,61,67,81,83,90]. Owing to relatively low temperature of acetylene-air flames, served as atomization cells in FAAS, possible chemical interferences are controlled by using appropriate buffers. For example, when K is quantified, a NaCl solution is added to standards and samples to eliminate ionization effects on the analyte [5]. A KCl solution is applied in a similar way in case of determination of Ca, Mg and Na [5]. A CsCl solution is used when both K and Na are determined [18,33,43]. Finally, a LiNO3 solution is taken to decrease ionization interferences for Ca [66]. On the other hand, La2O3 [33] or LiCl3 [43] solutions are applied as releasing buffers in case of determination of Ca and Mg. In this case, chemical interferences related to formation of some refractory compounds of the analytes, e.g. phosphates, are readily overcome. A mixture of La2O3 and KCl solutions can also be used for measuring Ca and Mg and preventing chemical interferences caused by ionization of the analytes and formation of their refractory compounds [18]. Solid phase extraction (SPE) with newly synthesized chelating [81] or commercially available cation-exchange [82,83,91] resins can be used for pre-concentration of Cd [81,83], Co [81,83], Cr [81], Cu [81,82,91], Fe [81], Mn [81], Ni [83], Pb [81,83] and

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Zn [81,82] prior to their determination by FAAS. In a similar way, DLLME can be used to pre-concentrate Cd and Pb [90]. GFAAS is the less frequently used spectrometric method for element analysis of honey. However, it seems that it is irreplaceable in case of quantification of few trace and ultratrace elements, e.g. As, Cd, Co, Cr, Hg, Ni, Pb and Se [17,19,26,36,39,48,49,56,66,68,72,76,80,85]. It can also be used for determination of some minor elements, i.e. Cu, Fe, Mn and Zn [17,26,49,65,72,76,80]. To thermally stabilize the analytes in the digested honey matrix, different matrix modifiers are used, including Pd(NO3)2 for As [19], Cd [19], Co [19], Cu [19] and Pb [19], a Pd(NO3)2-Mg(NO3)2 mixture for As [26], Cd [26,48,73], Cu [26] and Se [26], NH4H2PO4 for Cd [49,56,85] and Pb [49,56], Mg(NO3)2 for Cr [49], Fe [49] and Mn [49] or a NH4H2PO4-Mg(NO3)2 mixture for Pb [26,85]. The detail list of elements measured by ICP OES, ICP MS, FAAS and GFAAS is given in Table 4.

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7. Calibration and quality assurance/quality control Simple standards solutions are commonly used for calibration of the analytical methods and quantification of elements in solutions of digested honeys [1,4-9,16-41,43-47,49-51,54-56,5860,62-68,73-80,82-84,87-89]. In case of simplified sample preparation, when the respective samples are not digested, matrix matching standards solutions, i.e. containing fructose [55,73] or honey [53], are used to overcome matrix effects occurring during measurements. Rarely, external calibration is applied in such case [89]. Matrix matching standard solutions are used as well in case of determination of the analytes in eluates resulted from SPE treatment of undigested honey solutions; in this case the concentration of HCl in the solutions used for elution is considered [82,83,91]. To overcome eventual chemical interferences, calibration by additions of standards solutions to samples solutions is either used when simplified sample preparation is proposed [2,85] or when the analytes are pre-concentrated by DLLME from undigested samples solutions [90]. The mentioned calibration method by standards solutions additions can also be applied to correct matrix interferences in the ICP MS measurements of digested samples solutions [57]. Calibration using both the method of standards solutions additions and the method of simple standards solutions (external calibration) can be used to find out if matrix effects are present or absent in case of the simplified analytical methods with non-digestive sample preparation [73,90] or the method with microwave-assisted wet digestion [84]. In case of ICP MS, calibration by means of simple standard solutions with the added internal standard (IS) is quite frequently applied to verify efficiency of sample digestion (the IS, e.g. Re [20], is added to the samples before their preparation) and correct any instrumental drift and variations due to the matrix composition (the ISs, e.g. Bi, Ge, In and Sc [20], are added to the sample solutions and standards). Other ISs used in the ICP MS measurements are Bi [16,47,77], Ge [27], In [2,16,21,27], Pt [2], Rh [21,27,53,55,59], Sc [2,23,27,47,77], Tb [23,27,47,84] and Y [21,23,47,77]. Taking into account quality of the results of element analysis of honey by atomic and mass spectrometry methods, assessment of their reliability is hampered because there is no matrix matching certified reference material (CRM) of honey. Although a proficiency test material, i.e. spiked honey [87], is reported to be developed, it unfortunately concerns a limited number of elements, i.e. Cd and Pb. As a result, traceability and trueness of the results achieved in the course of element analysis of honey is verified by using CRMs of other matrices [2,21,22,59,64,81], e.g. waste water (CWW-TM-D) [81], tea leaves (GBW 10016 [22,27], INCT-TL-1 [81,50], NCS DC 73351 [33]), plant material (INCT MPH-2) [66],

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tomato leaves (SRM 1573a) [77], cabbage (IAEA-359) [33], peach leaves (SRM 1547) [77], apple leaves (SRM 1515) [2,37,50,65,68,84], yellow clover (WEPAL IPE 110) [16,70], lichen (BCR 482) [49], wheat flour (SRM 1567a) [6], rice flour (SRM 1568a) [53], Antarctic krill (MURST-ISS-A2) [2], oyster tissue (SRM 1566b) [71], chicken powder (GBW 10018) [56], and lobster hepatopancreas (TORT-2 [74]). This is, however, exceptional because quite often accuracy of the results of element analysis of honey is not verified at all. Otherwise, spike-and-recovery experiments are carried out, however it does not have a high metrological priority when another analytical method is available and can be used for comparison of the results [1,7,9,16,17,19,20,22,23,26,37,38,46,47,51,55-57,64,69,71,73,74,77,79,82-84, 90,91]. Reference methods (RMs) are more adequate in this case but they are used occasionally, particularly when new sample preparation procedures are developed. Accordingly, the results are verified using, e.g. microwave assisted wet digestion followed by GFAAS or ICP MS [85] (RM for simple dissolution and acidification followed by GFAAS detection), microwave assisted wet digestion followed by ICP MS detection (RM for simple dissolution, incubation with concentrated HNO3 and dilution followed by ICP MS detection [74,53] and simple dissolution followed by DLLME treatment and FAAS detection [90]) or hot plate wet digestion followed by FAAS [91,89] or ICP OES [82,83] detection (RMs to simple dissolution followed by FAAS detection [89], simple dissolution followed by SPE treatment and FAAS [91] or ICP OES [82,83] detection).

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8. Conclusions Quest for a healthy life style and demand for healthy and safe food have made increased consumption of honey. It appears that this natural nutritious food product play an important role in a habitual diet of different age groups in many countries. Apart from good taste, pleasant aroma and the exceptional nutritional value, growing interest in honey is related to scientific evidence about its health benefits to humans, e.g. antioxidant and antiinflammatory activities. Honey is also an invaluable source of many essential elements that has to be considered in provision and assessment of nutrition support, primarily of infants and young children. Conversely, quality of honey depends on presence of non-essential elements, e.g. heavy metals, that being in elevated amounts are harmful and even toxic. Different environmental and non-environmental factors are responsible for a large variety in the content of trace elements contaminating honey, i.e. Al, As, Cd, Cr, Cu, Hg, Pb, Se. The latter elements constitute major public health concern because some of them, e.g. Cd, Pb, can be accumulated in the body, leading to health deterioration and imposing some epidemiological problems. In view of food safety and nutritional considerations, it appears that the need for determining and controlling the content of major, minor and trace elements honey is important and highly justified. This is of particular significance due to seasonal variations in quality and chemical composition of melliferous plants. On the other hand, increased consumption of honey and higher consumers’ awareness about safe food products entail more extensive production efficiency and higher nutritional quality of this food product. Popularity and high price of commercially available honey have also led to concerns about its authenticity and origin. A helpful approach to assessing authenticity of honey is to reveal its chemical composition and indicate differences from the established reference guidelines. The elements patterns with chemometric exploratory data analysis provide at present the most robust and reliable tool for determining origin and provenience of honey. However, to build dependable databases of different types of mono- and multifloral honeys, which can be applied to establish authenticity of unknown samples by comparing their

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element composition with the genuine control samples and detect any adulterations or frauds, information on the content of different elements has to be of the highest quality, i.e. reliable and verified in reference to accuracy and precision. Such data can be used to identify the useful key markers, univocally differentiating the regions and countries plants from which given honeys come from and specific blossoms and plants from which they are made. Finally, it is beyond doubt that increased production of honey should strictly be supported by reliable analytical methods enabling to determine various (major, minor and trace) elements in a fast and uncomplicated way. Such dependable methods are of particular importance in terms of routine element analysis of honeys undertaken for controlling and assuring their quality and safety.

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Acknowledgments This work was financed by a statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wroclaw University of Science and Technology.

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[79] B. Akbari, F. Gharanfoli, M. H. Khayyat, Z. Khashyarmanesh, R. Razaee, G. Karimi, Determination of heavy metals in different honey brands from Iranian markets, Food Additives and Contaminants B 5 (2012) 105-111. [80] R. Kamboj, M. B. Bera, V. Nanda, Evaluation of physico-chemical properties, trace metal content and antioxidant activity of Indian honeys, International Journal of Food Science and Technology 48 (2013) 578-587. [81] T. Dasbasi, S. Sacmaci, N. Cankaya, C. Soykan, A new synthesis, characterization and application of chelating resin for determination of some trace metals in honey samples by FAAS, Food Chemistry 203 (2016) 283-291. [82] H. Stecka, D. Jedryczko, M. Welna, P. Pohl, Determination of traces of copper and zinc in honeys by the solid phase extraction pre-concentration followed by the flame atomic absorption spectrometry detection, Environmental Monitoring and Assessment 186 (2014) 6145-6155. [83] H. Stecka, D. Jedryczko, P. Pohl, M. Welna, Pre-concentration of traces of cadmium, cobalt, nickel and lead in natural honeys by solid phase extraction followed by their determination using flame atomic absorption spectrometry, Journal of the Brazilian Chemical Society 25 (2014) 331-339. [84] F. A. de Oliveira, A. T. de Abreu, N. de Oliveira Nascimento, R. E. S. Froes-Silva, Y. Antonini, H. A. Nalini Jr., J. C. de Lena, Evaluation of matrix effects on the determination of rare earth elements and As, Bi, Cd, Pb, Se and In in honey and pollen of native Brazilian bees (Tetragonisca angustula – Jatai) by Q-ICP-MS, Talanta 162 (2017) 488-494. [85] A. Colabucci, A. C. Turco, M. Ciprotti, M. Di Gregorio, A. Sorbo, L. Ciaralli, Analysis of cadmium and lead in honey: direct determination by graphite furnace atomic absorption spectrometry, Acta Imeko 5 (2016) 10-14. [86] F. O. Omoya, O. A. Ijabadeniyi, O. B. Ogonnoh, Physicochemical properties of honey samples from Ondo state, Nigeria, and their bioactivity against spoilage and pathogenic organisms, Journal of Food, Agriculture and Environment 12 (2014) 104-107. [87] L. Ciaralli, A. C. Turco, M. Ciprotti, A. Colabucci, M. Di Gregorio, A. Sorbo, Honey as a material for proficiency testing, Accreditation and Quality Assurance 20 (2015) 359365. [88] M. M. Ozcan, F. Y. Al Juhaimi, Determination of heavy metals in bee honey with connected and not connected metal wires using inductively coupled plasma atomic emission spectrometry (ICP-AES), Environmental Monitoring Assessment 184 (2012) 2373-2375. [89] P. Pohl, H. Stecka, K. Greda, P. Jamroz, Bioacessibility of Ca, Cu, Fe, Mg, Mn and Zn from commercial bee honeys, Food Chemistry 134 (2012) 392-396. [90] F. C. Rosa, F. A. Duarte, J. N. G. Paniz, G. M. Heidrich, M. A. G. Nunes, E. M. M. Flores, V. L. Dressler, Dispersive liquid-liquid microextraction: an efficient approach for the extraction of Cd and Pb from honey and determination by flame atomic absorption spectrometry, Microchemical Journal 123 (2015) 211-217. [91] P. Pohl, H. Stecka, P. Jamroz, Interference-free determination of trace copper in freshly ripened honeys by flame atomic absorption spectrometry following a preconcentration by solid-phase extraction and a two-step elution process, Archives of Environmental Contamination and Toxicology 66 (2014) 287-294.

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Table 1. Application of principal component analysis (PCA) for chemometric characterization of honey

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PURPOSE -identification of relations and correlations between different variables within the data matrix composed of different samples -identification and characterization of groups/types of honey

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APPLICATION Distinguishing geographical origin/production area or region of honey #Concentrations of As, Ca, Cd, Co, Cu, Fe, K, Mg, Na, Pb and Zn [19] #Concentrations of Al, As, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Se and Zn [20] #Concentrations of As, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb and Zn [25] #Concentrations of Al, Ca, Cd, Cu, Fe, K, Mg, Mn, Na, Ni, Pb and Zn [29] #Concentrations of Ca, K, Mg and Na along with pH, free acidity, the moisture content, the ash content, the concentrations of fructose, glucose, saccharose, turanose and maltose, sum of the fructose and glucose concentrations and concentrations ratio of fructose to glucose [31] # Concentrations of Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P Pb, Se, Sn, Sr and Zn [37] #Concentrations of Ca, Cu, Fe, K, Pb and Zn [39] #Concentrations of Ag, Al, As, Ba, Be, Ca, Cd, Co, Cr, Cu, Cs, Fe, Ga, In, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr Ti, U, V and Zn [47] #Concentrations of Cd, Cr and Pb [48] #Concentrations of Al, As, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, Ge, Hg, Mn, Mo, Pb, Sb, Se, Sn, Sr, Tl, U, V and Zn [57] #Concentrations of Ca, Cu, Fe, K, Mg, Na, Zn [62] #Concentrations of Ca, Cd, Cu, Fe, K, Mg, Mn, Pb and Zn [66] #Concentrations of Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sr and Zn [69] Distinguishing botanical origin and types of honeys #Sum of concentrations of Ca, Fe, K, Mg and Na (total mineral content) along with total phenolics content and antioxidant activity (as DPPH and FRAP assays) [5] #Concentrations of Ca, Mg, Na and Sr along with the total soluble solids content, the moisture content and free acidity [7] #Concentrations of Ba, Ca, Cu, Fe, K, Mg, Mn, Na, Rb and Sr [22] #Concentrations of Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, S, Sb, Se, Sr, V and Zn along with the total content of phenolic compounds, antioxidant activity (as DPPH), the concentrations of 25 individual phenolics, the concentrations of 14 individual carbohydrates (sugars and sugar alcohols) [28]

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DPPH 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. FRAP Ferric reduction antioxidative power. CUPRAC Cupric reducing antioxidant capacity. ABTS 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity. AAEC Ascorbic acid equivalent content.

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#Concentration of Cu along with vitamin B2 content, antioxidant activity (as CUPRAC and ABTS assays) [34] #Concentrations of Ag, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Se, Sr, Th, Tl and U along with the C, H and O isotope ratios (δ13C, δ2H, δ18O) [38] #Concentrations of Ca, Cd, Cu, Fe, K, Mn, Na, P, Pb, Zn with color, electrical conductivity, the total concentration of phenolic compounds, the total concentration of flavonoid compounds, antioxidant activity (as DPPH and AAEC) [41] #Concentrations of Al, Ca, Cd, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, Pb and Zn along with the sum of the concentrations of flavonoids, sum of the concentrations of benzoic acids, sum of the concentrations of cinnamic acids, the total concentration of soluble proteins, the total concentration of non-protein tiols, and the total content of phenolic compounds [64] #Concentrations of Cd, Cu, Fe, Ni, Mn and Pb along with pH, total acidity, the moisture content, the ash content, the concentrations of fructose, glucose and sucrose, the concentrations ratio of fructose to glucose, invertase activity, diastase activity, the concentration of proline and hydroxymethylfurfural (HMF), the total concentrations of phenolic compounds, the total concentrations of flavonoids, antioxidant activity (as DPPH assay), color [80]

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Table 2. Application of linear discriminant analysis (LDA) for chemometric characterization of honey

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APPLICATION Classification of honeys due to geographical origin/production area #Principle component analysis factors for concentrations of As, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Se, Tl, U, V and Zn along with pH, total sugars content and the moisture content [2] #Concentrations of B, Na, Rb, La and Zn and concentrations ratios of K/Rb, 87Sr/86Sr [6] #Concentrations of Al, As, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Se and Zn [20]a #Concentrations of B, Ca, Cu, Li, Mo, Ni, P, Sb, Se, Si, Sn, Sr and Ti [54] #Concentrations of Ca, Cu, Fe, K, Mg, Na, Zn along with antioxidant activity, the total phenolics content, color and the ash content [62]

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Classification of honeys due to botanical origin and type #Concentrations of Ca, Cu, Fe, K, Mg, Mn, P, S and Zn [21] #Concentrations of Ag, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Se, Sr, Th, Tl and U along with the C, H and O isotope ratios (δ13C, δ2H, δ18O) [38] #Concentrations of Ca, Cd, Cu, Fe, K, Mn, Na, P, Pb, Zn with the color intensity, electrical conductivity, the total concentration of phenolic compounds, the total concentration of flavonoid compounds, antioxidant activity (as DPPH and AAEC) [41] #Concentrations of P and S along with the color intensity, antioxidant activity (as DPPH assay) and the C isotope ratio (δ13C) [60] #Concentrations of Cd, Cu, Fe, Ni, Mn and Pb along with pH, total acidity, the moisture content, the ash content, the concentrations of fructose, glucose and sucrose, the concentrations ratio of fructose to glucose, invertase activity, diastase activity, the concentration of proline and hydroxymethylfurfural (HMF), the total concentrations of phenolic compounds, the total concentrations of flavonoids, antioxidant activity (as DPPH assay), color [80] Canonical discriminant analysis (CDA). DPPH 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. FRAP Ferric reduction antioxidative power. CUPRAC Cupric reducing antioxidant capacity. ABTS 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity. AAEC Ascorbic acid equivalent content. 22

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-(False) Acacia (Robinia pseudoacacia or Acacia [8,19,20,22,24,26,29,30,33,34,38,39,42,47,49,56-58,64,66,72,82,83,89,91] -Alfalfa or lucerne (Medicago sativa) [8,31,66] -Algarrobo (Prosopis caldenia) [2] -Anise (Pimpinella sp.) [72] -Apple (Malus pumila) [33,41] -Astragalus (Astragalus microcephalus) [34,72] -Avocado (Persea americana) [43] -Bitter gourd (Momordica charantia) [19] -Black-berry (Rubus fruticosus) [21,52] -Borneo (Acacia mangium) [19,45] -Buckwheat (Fagopyrum esculentum) [33,51,66,82,83,89] -Cardoon (Carlina racemosa) [4] -Carambola tree (Averrhoa carambola) [19] -Carob tree (Ceratonia siliqua) [4,5,72] -Cashew tree (Anacardium occidentale) [36] -Catanduva (Piptadenia moniliformis) [36] -Cedar (Cedrus libani) [72] -Chasteberry (Vitex agnus-castus) [72] -Cherry (Prunus avium) [41] -Chestnut (Castanea sativa) [20,26,33,34,40,43,44,52,57,66,70,72,85] -Chilca (Baccharis articulata) [2] -Cilantro (Coriandrum sativum) [57] -Citrus (Citrus sp.) [2,4,37,54,56,69,72] -Clover (Trifolium sp.) [8,62,66] -Coffee (Coffea arabica) [32] -Coriander (Coriander sativum) [80] -Cotton (Gossypium hirsutum) [8,69,72,80] -Dandelion (Taraxacum officinale) [33] -Eucalyptus (Eucalyptus sp.) [2,4,5,8,20,35,52,56,62,66,67,69,72,85] -Euphorbia (Euphorbia sp.) [72] -Facelia (Phacelia tanacetifolia) [21] -False buttonweed (Spermacoce verticillata) [36] -Ferula (Ferula sp.) [72] -Fir (Abies cephalonica) [60] -Gelam tree (Melaleuca cajuputi) [19,45,47] -Ghaf (Prosopis juliflora) [30] -Goldenrod (Solidago virgaurea) [82,83,91] -Hawthorn (Crataegus sp.) [21] -Heather (Calluna vulgaris, Errica umbellate) [4,5,33,34,52,66,69,82,83,89,91] -Indian rosewood (Dalbergia sissoo) [80] -Jua (Ziziphus joazeiro) [36] -Jujube (Ziziphus jujuba) [1,56,58] -Jurema-branca (Mimosa verrucosa) [36] -Kadi Patta (Murraya koenigii) [80] -Khalsi (Aegiceras corniculatum) [17] -Lavender (Lavandula stoechas) [4,5,34,43]

AC C

1 2

23

ACCEPTED MANUSCRIPT

Brassica

napus)

AC C

EP

TE D

M AN U

SC

RI PT

-Lemon (Citrus limon) [20] -Lime (Citrus sp.) [33] -Litchi (Litchi chinensis) [17,56] -Linden (Tilia amurensis) [21,22,26,39,42,56,66,72,89,91] -Lippia (Lippia sidoides) [36] -Longan tree (Dimocarpus longan) [19] -Loquat tree (Eriobotrya japonica) [56] -Mango (Magnifera indica) [67] -Manuka (Leptospermum scoparium) [8,17,19,30,45] -Melon (Cucurbitaceae sp.) [85] -Mint (Mentha sp.) [28,72] -Mistol (Ziziphus mistol) [2] -Mustard (Brassica juncea) [17] -Oak (Quercus robur) [34] -Oiticica (Licania rigida) [36] -Orange (Citrus sinensis) [5,20,33,43,60,66] -Parsley (Petroselinum sp.) [69,72] -Pignut (Hyptis suaveolens) [36] -Pine (Pinus sp.) [34,60,65,66,69,72] -Pineapple (Ananas comosus) [19,45] -Pennyroyal (Mentha pulegium) [4] -Pongamia (Pongamia pinnata) [67] -Rape or colza (Brassica campestris or [21,22,33,38,39,62,64,66,82,83,91] -Rhododendron (Rhododendron sp.) [72] -Rosemary (Rosmarinus officinalis) [43] -Rubber tree (Hevea brasiliensis) [19,67] -Saffron (Crocus sativus) [41] -Sage (Salvia officinalis) [26,28] -Sainfoin (Onobrychis sp.) [72] -Sapindus (Sapindus saponaria) [67] -Serjania (Serjania sp.) [36] -Shefallah (Capparis sp.) [8] -Silk grass (Asclepias sp.) [21] -Sourwood tree (Oxydendrum arboreum) [19] -Strawberry tree (Arbutus unedo) [4,5,72] -Sulla or sweetvetch (Hedysarum coronarium) [20,31,57,66] -Sunflower (Helianthus annuus) [4,21,39,42,50,57,62,66,68,72] -Sweet clover (Melilotus albus) [62] -Thistle (Cardus nutans) [42] -Thyme (Thymus vulgaris or Thymus capitatus) [4,5,9,28,43,60,72] -Tualang tree (Koompassia excelsa) [8,17,19,45,47] -Urundeuva (Myracrodruon urundeuva) [36] -Vetch (Vicia sp.) [21] -Vitex (Vitex negundo) [22,38] -White mangrove (Laguncularia racemosa) [18] -Wild bush (Plectranthus rugosus) [41] -Wild jujube (Ziziphus spina-christi) [8,24,30,38] -Wild mountain (Acacia tortilis) [30]

24

ACCEPTED MANUSCRIPT -Winter savory (Satureja montana) [28] -Yellow star-thistle (Centaurea solstitialis) [72]

AC C

EP

TE D

M AN U

SC

RI PT

1

25

ACCEPTED MANUSCRIPT Table 4. Spectrometric methods used for element analysis of honey samples ICP OES

ICP MS

Ag

FAAS

GFAAS

[65]

[38,47] [1,7,20,21,28,30,37 ,50,54,69,75,79]

[6,24,29,47,53,57,59,74,7 7]

[32,46,61,64]

As

[1,7,25,28,30,54,79 ]

[2,9,16,20,21,23,38,45,47 ,53,55,57,59,70,74,77,84]

[67]

B

[28,37,54,69,88]

[6,59]

[61]

Ba

[7,28,37,54,69]

[6,22,24,27,38,47,53,57,7 4,77]

[61]

Be

[7,54]

[2,47,53,57]

Bi

[7]

[53,84]

Ca

[1,7,20,21,25,28,30 ,31,34,37,54,69]

[2,6,16,22,24,27,29,38,44 ,47,57,59,70,74,77]

Cd

[1,7,25,28,30,37,54 ,63,69,71,75,78,79, 88]

Co

Cr

TE D

M AN U

SC

[19,26]

[4,5,8,18,19,26, 32,35,36,4043,45,46,52,58,6 1,62,64,66,86,89 ]

[8,58,41,61,64,6 7,81a,83a,90c]

[17,19,26,48 ,49,56,65,66 ,68,72,73,80 ,85]

[6,53,84]

[1,7,25,28,30,37,54 ,69,75,88]

[2,6,24,38,47,53,57,59,74 ]

[8,58,81a,83a]

[19]

[1,7,25,28,30,37,54 ,63,69,75,78,88]

[2,20,21,23,24,38,47,53,5 5,57,59,74,77]

[58,61,64,67,81a ]

[17,36,48,49 ,65,68,72,73 ]

[32,35,36,3943,46,52,56,58,6 1,62,6466,68,80,81a,82a ,89,91a]

[17,19,26,49 ,72]

Cs Cu

[2,6,9,16,20,21,23,29,38, 45,47,53,55,57,59,70,74, 77,84,85,87,90]

EP

Ce

RI PT

Al

AC C

1 2

[6,47,74] [1,7,21,25,28,30,34 ,37,50,54,69,71,75, 78,79,88]

[2,6,20,2224,29,38,44,45,47,53,57, 59,74,77]

26

ACCEPTED MANUSCRIPT Dy

[53,84]

Er

[53,84]

Eu

[6,53,84]

[6,47,74]

Gd

[53,84]

Ge

[57]

Hg

[54,79]

[45,57,59] [53]

Ho

[53]

I

[59]

In

[47,84]

Ir [1,7,20,21,25,28,30 ,31,34,37,47,69]

Li Lu

AC C

La

[67]

[74]

[2,6,22,24,27,29,38,44,59 ]

EP

K

TE D

Hf

[17,49,72]

SC

Ga

[5,18,19,26,32,3 5,36,3943,45,46,52,58,6 1,62,6466,68,80,81a,89]

RI PT

[2,29,38,44,47,53,57,59,7 4,77]

[1,7,20,21,25,28,30 ,34,37,50,54,69,75, 79,88]

M AN U

Fe

[1,7,28,54]

[4b,5,8,18,19b 32,35,36,40-42, 43b,45,46,52,58, 61,62b,64,66b,86 ]

[6,53,84] [6,47,59,74]

[8,32,42,46]

[6,53,84]

Mg

[1,7,20,21,25,28,30 ,31,37,54,69]

[2,6,16,22,24,27,29,38,44 ,47,53,59,70,74,77]

[4,5,8,18,19,32, 35,36,40,42,43,4 5,46,52,58,61,62 ,64,66,89]

Mn

[1,7,21,25,28,30,34 ,37,50,54,69,75,79,

[2,6,16,20,22,24,27,29,38 ,44,47,53,57,59,70,74,77]

[8,36,41,42,58,6 1,64-

[17,49,65,72 ,80] 27

ACCEPTED MANUSCRIPT 66,68,81a,89]

Mo

[28,30,54,88]

[6,21,38,53,57]

Na

[1,7,20,25,28,30,31 ,37,47,69]

[2,22,24,27,29,38,44,59]

Nd

[4b,5,6,8,18,19b, 32,35,36,40,41,4 2,43b,45,46,52,5 8,61,62b,64,86]

RI PT

88]

[6,53,84] [1,7,25,28,30,37,50 ,54,63,69,75,78,88]

[2,6,20,23,24,29,38,53,59 ,74]

[8,32,46,58,61,6 4,83a]

P

[21,25,28,30,37,54, 60]

[16,22,27,44,45,53,59,70, 77]

[18,35,40,41,52, 86]

Pb

[1,25,28,30,37,50,5 4,63,69,71,75,78,7 9]

[2,6,9,16,20,21,23,24,29, 45,47,53,55,57,59,70,74, 77,84,85,87,90]

Pr

[53,84]

Pt

[53]

Rb

Sb

[28,54]

Si

AC C

[21,28,30]

EP

[74]

S

Se

[17,19,26,39 ,48,49,56,66 ,68,72,73,80 ,85]

[6,22,24,27,47,53,74]

Ru

Sc

M AN U

[53,74]

[8,41,58,61,64,8 1a,83a,90c]

[65,68,72,80 ]

TE D

Pd

SC

Ni

[16,53,57,74]

[84]

[28,37,54,71,79]

[2,6,20,21,38,47,53,57,74 ,77,84]

[54]

[59]

Sm

[18]

[26,36,68,72 ] [18]

[6,53,84]

Sn

[37,54,60]

[9,53,57,59]

Sr

[7,28,30,37,54,69,7 5]

[6,22,24,27,38,47,57,59,7 4] 28

ACCEPTED MANUSCRIPT Tb [54]

[53,74]

Ti

[54]

[47]

Tl

[54]

[2,6,16,38,53,57,70,74]

Tm

[84]

U

[2,6,38,47,53,57] [28,54]

[2,6,47,53,57,59,74,77]

Y

[84]

Yb

[6,53,84]

a

[1,7,20,21,25,28,30 ,37,50,54,69,75,78, 79,88]

[16,22-24,27,29,44,47, 53,57,59,70,74,77]

[8,18,19,32,35,3 6,39,40,41,43,45 ,49,52,56,58,61, 62,6466,68,81a,82a,89 ]

[17,72]

TE D

Zn

M AN U

V

EP

With solid phase extraction (SPE) pre-concentration. Flame photometry. c With dispersive liquid-liquid micro-extraction (DLLME) pre-concentration. b

AC C

1 2 3

[38,53]

RI PT

Th

SC

Te

[53]

29

ACCEPTED MANUSCRIPT #Element analysis of honey by atomic and mass spectrometry reviewed #Elements profiling suitable for honey classification #Elements profiling suitable for honey quality and safety assessment

AC C

EP

TE D

M AN U

SC

RI PT

#Adequate sample preparation important for reliable analysis