Talanta 65 (2005) 686691

Mineral content and botanical origin of Spanish honeys

Rut Fern andez-Torres , Juan Luis P erez-Bernal, Miguel Angel Bello-L opez, Manuel Callej on-Moch on, Juan Carlos Jim enez-S anchez, A. Guira um-P erez
Department of Analytical Chemistry, Faculty of Chemistry, University of Seville, Seville 41012, Spain Received 10 March 2004; received in revised form 28 May 2004; accepted 23 July 2004 Available online 26 August 2004

Abstract Eleven elements (Zn, P, B, Mn, Mg, Cu, Ca, Ba, Sr, Na and K) were determined by inductively plasma coupled spectrometry in 40 honey samples from different places of Spain and four different botanical origins: Eucalyptus (Eucalyptus sp.), Heather (Erica sp.), Orange-blossom (Citrus sinensis) and Rosemary (Rosmarinus ofcinalis). K, Ca and P show the higher levels with average concentrations ranged between 434.11935 mg kg1 for K; 42.59341.0 mg kg1 for Ca and 51.17154.3 mg kg1 for P. Levels of Cu (0.5312.117 mg kg1 ), Ba (0.1061.264 mg kg1 ) and Sr (0.2571.462 mg kg1 ) are the lowest in all honey samples. Zn (1.3327.825 mg kg1 ), Mn (0.1339.471 mg kg1 ), Mg (13.2674.38 mg kg1 ) and Na (11.69218.5 mg kg1 ) concentrations were found strongly dependent on the kind of botanical origin. Results were submitted to pattern recognition procedures, unsupervised methods such as cluster and principal components analysis and supervised learning methods like linear discriminant analysis in order to evaluate the existence of data patterns and the possibility of differentiation of Spanish honeys from different botanical origins according to their mineral content. Cluster analysis shows four clusters corresponding to the four botanical origins of honey and PCA explained 71% of the variance with the rst two PC variables. The best-grouped honeys were those from heather; eucalyptus honeys formed a more dispersed group and nally orange-blossom and rosemary honeys formed a less distinguishable group. 2004 Elsevier B.V. All rights reserved.
Keywords: Honey; Metals; Chemometrics; Pattern recognition; Eucalyptus (Eucalyptus sp.); Orange-blossom (Citrus sinensis); Rosemary (Rosmarinus ofcinalis); Heather (Erica sp.)

1. Introduction Honey is produced by bees from nectar of plants, as well as from honeydew. Bees and plants are the sources of some honey components as: carbohydrates, water, traces of organic acids, enzymes, aminoacids, pigments; and others like pollen and wax arise during honey maturation. Honey composition depends on great extent on the nectar sources. The monosacharides, glucose and fructose are
Abbreviations: ICP-AES, inductively coupled plasma-atomic emission spectroscopy; NW, North-western; PR, Pattern recognition; CA, Cluster analysis; PCA, Principal components analysis; LDA, Linear discriminant analysis; DF, discriminant function; PC, principal component Corresponding author. Tel.: +34 954556442; fax: +34 954557168. E-mail address: rutft@us.es (R. Fern andez-Torres). 0039-9140/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2004.07.030

the main components of honey, mineral content ranges from about 0.04% in pale honeys to 0.2% in some dark honeys and protein content of honey is usually lower than 0.5% [1]. The Commission of European Union has adopted a new Council Directive, 2001/110/ECC, which repeals the Directive 74/409/ECC [2]. The new Directive establishes the types of honeys that can be marketed in the European Union and gives general denitions related to honey, including general and specic honey compositional characteristics such as, hydroximethyl furfural content, humidity, enzymatic activities and pesticide levels in honey, but those parameters have not been found to have a real relationship to geographical or botanical origin of honey. Pollen recognition has been the traditional method to determine the botanical origin of honey, but this technique is tedious and has limitations:

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counting procedure, identication and interpretation are difcult and requires trained analysts. Other studies [1] have sought analytical markers, such as aroma compounds, sugar prole, avonoid pattern, non-avonoid phenolics, isotopic relations, protein or amino acid content to honey classication. Pattern recognition techniques have been widely applied to food chemistry in recent years [36]. The subject of food authenticity covers a wide range of products, and it is of a great economic and social importance, for sectors involved in food production and consumers. Authenticity helps to guarantee the characteristics and quality of food products and helps to prevent overpayment due to adulteration or consumers mislead as a result of ambiguous, improper product labelling. Honey dilution with water and addition of sugars and syrups are the main honey adulteration methods. Besides, in recent years, there has been a rising presence of monooral honeys in markets, more expensive than multi-oral ones, so possible adulteration by honey mixtures must be controlled. Different procedures for the determination of honey mineral content have been proposed in some previous papers [727]; some of them with classication purposes [2426], but always according to geographical origin and none of them established a classication of honey according to its botanical origin. Stein [9] determined Cd, Pb and Mn in honey samples, in different seasons of the year, using atomic absorption furnace and three different methods, they obtained identical results by the three methods; Serra [11] determined the levels of Ca, Mg, K, Na, Fe, Cu, Cr and Pb in Spanish eucalyptus honey; Sevimli et al. [12] determined As, Cr, Sb, K, Br, Zn, Fe and Co in Turkish honey by neutron activation analysis; Fodor and Molnar [13] analysed seven honey samples produced in industrial areas to investigate environmental contamination, Al, B, Ca, Cd, Cu, Fe, K, Mg, Mn, Na, P, Pb and Zn were determined; Rodriguez Otero et al. [14] determined Na, K, Ca, Mg, Cu, Fe, Mn and P in Galician honeys; Vi nas et al. [16] determined Pb, Zn, Cu and Cd in honey samples from different type and origin by electrothermal atomic absorption spectrometry; Latorre et al. [25,26] determined 11 metals in honeys from Galicia with classication purposes, they differentiate between honeys from Galicia and non-Galician ones, and processed and industrially commercialised Galician honeys using pattern recognition methods. Despite the fact that some authors correlate the mineral content of honey only with its geographical origin [1], the aim of this paper is, rst to contribute to the scarce data about mineral content of Spanish honeys and secondly establish whether mineral content can explain botanical origin.

Table 1 Operating parameters for ICP-OES RF frequency Operating power Coolant argon ow rate Plasma/nebuliser argon ow rate Burner type Nebuliser type Viewing height Sample ow rate Metals Zn P B Mn Mg Cu Ca Sr Ba Na K 27.12 MHz 650 W 7.5 l min1 0.8 l min1 Minitorch Meinhard 8 mm 2.3 ml min1 Emision wavelength (nm) 213.868 214.914 249.796 257.610 279.553 324.772 393.369 407.771 455.424 589.003 766.473

(Fisons Instruments, Valencia, CA). Table 1 shows the analytical lines for each element, as well as the instrumental conditions. 2.2. Reagents and solutions All chemicals were of analytical-reagent grade or better. Standard solutions were prepared by adequate dilution of a multi-element standard (1000 mg l1 ) obtained from Merck (Darmstadt, Germany). Nitric acid 65% and perchloric acid 85% were from Merck. All solutions and dilutions were prepared with ultrapure water (Milli-Q, Milipore, Bedford, MA). 2.3. Honey samples Forty samples of honey were analysed. The samples were obtained from stores and cooperatives, representing the most common types of honey readily available to consumers in Spain, i.e. eucalyptus (Eucalyptus sp.), orangeblossom (Citrus sinensis), rosemary (Rosmarinus ofcinalis) and heather (Erica sp.). A palinological study of the honey samples was performed in order to guarantee the labelled botanical origin [28]. An identication code was assigned to each group of samples, E for eucalyptus, OB for orangeblossom, R for rosemary and H for heather ones. All the samples from the same botanical origin are from different geographic origin to avoid formation of natural grouping between samples due to the same geographic origin. In addition, samples from the same geographic origin but different botanical class have been analysed to guarantee classication is due to botanical origin. Table 2 shows the origin of the analysed samples.

2. Materials and methods 2.1. Apparatus Elemental analysis was carried out on a Fisons-ARL 3410 inductively coupled plasma-atomic emission spectrometer

688 Table 2 Studied honey samples Sample E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 OB1 OB2 OB3 OB4 OB5 OB6 OB7 OB8 OB9 OB10 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 Geographical origin Huelva Madrid Badajoz Sevilla Salamanca Cadiz Caceres Ciudad Real Vizcaya La Coru na Huelva Caceres Avila Galicia Asturias Cadiz Guadalajara Palencia Badajoz Castellon Huelva Murcia Castellon Valencia Alicante. Tarragona Sevilla Malaga Cordoba Almeria Huelva Avila Caceres Cuenca Guadalajara Badajoz Cadiz Granada Salamanca Segovia

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Botanical origin Eucalyptus Eucalyptus Eucalyptus Eucalyptus Eucalyptus Eucalyptus Eucalyptus Eucalyptus Eucalyptus Eucalyptus Heather Heather Heather Heather Heather Heather Heather Heather Heather Heather Orange-blossom Orange-blossom Orange-blossom Orange-blossom Orange-blossom Orange-blossom Orange-blossom Orange-blossom Orange-blossom Orange-blossom Rosemary Rosemary Rosemary Rosemary Rosemary Rosemary Rosemary Rosemary Rosemary Rosemary

analytical data with adequate precision and accuracy; good results were obtained. The statistical package STATISTICA version 6.0 (2001) for windows, from Statsoft was used for all the chemometric calculations.

3. Results and discussion 3.1. Mineral content Metal content of the studied honeys is shown in Table 3. Heather and eucalyptus, darker honeys, have a higher mineral content than the rest of the analysed honeys. This result is consistent with Ankalm [1] that concludes that dark honeys have a higher mineral content than pale ones. In general, K, Ca and P show the highest levels with average concentrations ranged between 6391845 mg kg1 ; 111257 mg kg1 and 63.8143 mg kg1 , respectively. Latorre et al. [25] obtained similar values for K in Spanish honeys (botanical origin not specied). Eucalyptus honeys from Spain were analysed by Serra [11] showing similar levels for Ca, but slightly lower levels of K and Cu than in the present study. K and Ca levels also agree with those reported by Packer and Gin e [21] in eucalyptus honey. Cu, Ba and Sr are the elements with the lowest concentrations in all samples. Cu has been widely analysed in previous papers [8,11,13,1422,24,25], with levels ranging from 0.057 to 2,200 mg kg1 in honeys from different botanical and geographical origin; the values obtained in the present study are comprise in that range. Zn, Mn, Mg and Na concentrations are strongly dependent on the honey botanical origin. Results obtained for Zn (1.3327.825 mg kg1 ), Mn (0.1339.471 mg kg1 ), Mg (13.2674.38 mg kg1 ) and Na (11.69218.5 mg kg1 ) agree with values found previously [19]. 3.2. Data analysis Using the mineral content found in the analysed honey samples as chemical descriptors, pattern recognition (PR) methods [30,31] (cluster analysis, principal components analysis and linear discriminant analysis) were applied to establish whether there are dened groups between eucalyptus, heather, orange-blossom and rosemary honey samples. A data matrix was constructed using the analysed elements as columns and the honey samples as rows. Cluster analysis was applied to the auto-scaled data, the Euclidean distance was used to calculate the sample similarities and a hierarchical agglomerative procedure was employed to establish clusters. The results obtained are shown as a dendogram in Fig. 1. Four clusters corresponding to each botanical origin were found at a linkage distance level of 4.6. From the left, the rst cluster (A) is composed of rosemary honey samples; cluster (B) is composed of orange-blossom samples; cluster (C) is formed by eucalyptus samples and cluster (D) by heather honey samples.

2.4. Analytical determinations Eleven metals (Zn, P, B, Mn, Mg, Cu, Sr, Ca, Ba, Na and K) were determined in each honey sample. 1 g of honey was treated with 10 ml of concentrated nitric acid, in open PTFE vessels, and heating until nearly dry. This procedure was repeated with 15 ml of a 2:1 (HNO3 /HClO4 ) mixture until complete mineralization [29]. The residue was dissolved, at room temperature, in 1 ml of concentrated nitric acid, transferred to a 25 ml volumetric ask and diluted with milli-Q water. Samples were measured by ICP-AES according to the parameters shown in Table 1. Blank solutions were prepared under identical conditions and the average signal was subtracted from analytical signals of honey samples. Recovery assays and standard addition procedures with known spiked samples were carried out in order to assure

Table 3 Honey samples mineral content (mg kg1 )a Zn H1 H3 H10 H7 H6 H4 H8 H9 H5 H2 E1 E4 E2 E5 E3 E8 E6 E9 E7 E10 O1 O6 O3 O7 O2 O8 O4 O5 O9 O10 R1 R9 R2 R3 R7 R8 R4 R6 R5 R10 LD LC
a

P 154.31.191 144.21.081 142.65.821 121.65.611 130.83.185 145.83.335 148.55.884 143.55.834 136.75.884 146.95.984 113.00.048 101.90.062 140.30.942 119.30.732 96.612.098 111.92.248 116.01.662 111.01.612 116.50.777 127.00.877 76.720.477 75.610.367 53.270.458 51.170.248 57.642.191 59.152.341 63.194.466 58.094.416 68.324.362 69.354.462 77.042.027 75.951.918 78.391.768 76.421.571 87.912.016 89.352.160 105.81.376 105.21.316 87.2911.49 88.281.267 0.957 3.192

B 6.9880.169 6.8780.059 5.1500.665 4.9400.455 2.9620.471 3.1120.621 6.0690.919 6.0190.869 4.0561.094 4.1561.194 7.0650.935 4.9670.825 3.1631.272 2.9531.062 2.3140.862 2.4671.012 4.8420.612 4.7920.562 4.3461.814 4.4511.914 8.1201.042 8.010.93 6.9260.725 6.7160.515 5.4191.133 5.1541.283 6.5061.345 6.4541.295 5.7230.412 5.8260.512 6.6570.374 6.5480.265 5.3402.132 5.1431.935 2.6460.896 5.7901.040 8.4561.984 8.3961.924 5.7742.118 5.8732.217 0.160 0.532

Mn 4.5390.721 4.4290.611 3.4850.936 3.2750.726 2.4380.295 2.5880.445 4.0120.527 3.9620.477 2.9610.524 3.0610.624 7.4270.300 7.3160.190 6.6000.408 6.3900.198 9.3180.195 9.4710.345 7.9500.547 7.9000.497 6.3240.201 6.4290.145 0.4560.127 0.350.02 0.1330.006 0.2000.059 4.2680.056 0.2760.071 0.2860.058 0.2340.008 0.4910.076 0.3880.060 0.5860.042 0.6950.031 0.5250.119 0.7220.099 1.3740.164 1.2300.179 0.8190.143 0.7590.083 0.8260.335 0.9250.434 0.032 0.106

Mg 61.346.815 55.846.705 66.262.152 61.161.942 71.184.673 74.384.823 63.802.459 61.802.409 68.722.460 73.222.560 36.615.447 35.515.337 47.642.970 45.542.760 36.881.505 38.381.655 41.501.627 41.001.577 40.654.477 41.704.577 19.222.779 18.112.67 15.351.947 13.261.737 42.341.482 23.851.632 20.861.059 20.361.009 19.411.268 20.441.368 19.703.013 20.792.904 20.970.929 22.940.732 33.462.281 34.902.425 37.821.221 37.221.161 26.991.578 27.981.964 0.003 0.011

Cu 0.8090.048 0.6990.062 2.1170.126 1.9070.084 0.5540.097 0.7040.247 1.4630.654 1.4130.200 1.3360.781 1.4360.090 0.5780.057 0.5420.046 0.6450.091 0.5330.070 0.5170.003 0.6700.018 0.6360.088 0.5860.083 0.5940.052 0.6990.062 <0.531 <0.531 <0.531 <0.531 <0.531 <0.531 <0.531 <0.531 0.548 <0.531 0.6360.057 <0.531 <0.531 0.6930.025 0.5740.043 <0.531 <0.531 <0.531 0.5500.060 0.6490.069 0.160 0.532

Ca 263.020.95 252.020.84 201.627.97 195.627.76 326.031.72 341.031.87 232.330.71 227.330.66 263.826.95 273.830.95 184.422.28 173.312.08 177.710.75 156.74.897 269.311.93 254.012.08 203.011.22 198.011.17 208.66.35 219.15.575 42.704.512 42.594.40 51.524.842 49.434.632 80.702.021 82.212.171 59.24.02 54.03.09 89.63.102 90.93.202 152.917.91 142.016.90 206.728.93 187.028.73 128.320.12 142.720.98 140.428.59 134.428.53 121.020.03 130.918.90 0.006 0.021

Sr 0.2710.068 0.2600.042 0.3540.036 0.3330.174 0.4610.016 0.4760.166 0.3130.042 0.3080.008 0.4080.053 0.4180.153 0.4080.013 0.5970.008 0.8230.058 0.6130.052 0.6350.048 0.7880.025 1.1750.011 1.1250.039 0.7600.034 0.8650.040 0.6910.132 0.580.02 0.6650.038 0.4560.012 0.5730.075 0.7240.225 1.4620.028 1.4100.022 0.9160.046 1.0190.146 0.2570.023 0.3660.012 0.4800.076 0.2830.056 0.3860.049 0.5300.193 0.5310.032 0.4710.026 0.4130.104 0.5120.203 0.003 0.011

Ba 0.2250.051 0.2140.042 0.2150.065 0.1940.004 0.2170.032 0.2320.010 0.2200.005 0.2150.045 0.2160.001 0.2260.011 0.4440.001 0.5550.010 0.4930.174 0.3930.036 0.5220.099 0.6750.114 0.3800.018 0.3300.032 0.3600.072 0.4650.082 <0.106 <0.106 <0.106 <0.106 1.1130.124 1.2640.089 <0.106 <0.106 <0.106 <0.106 0.1220.023 0.1110.012 0.1750.076 0.1550.056 0.1550.047 0.1700.061 0.0440.049 0.1380.043 0.1240.050 0.1340.060 0.032 0.106

Na 97.668.35 86.667.65 47.097.860 48.905.761 63.605.957 78.606.107 72.389.87 67.386.21 55.348.255 65.347.900 168.61.689 157.51.579 116.21.820 137.21.610 218.59.639 203.24.095 150.01.724 145.01.674 197.03.995 186.58.987 43.447.124 42.342.09 11.690.960 23.591.501 69.378.379 70.888.529 39.398.762 38.878.710 27.415.485 28.445.588 33.1910.84 32.105.897 22.054.931 20.084.734 16.170.157 16.310.301 36.800.962 36.200.902 27.058.308 28.047.045 1.596 5.320

K 177121.0 176038.21 184542.20 182441.99 192021.93 193522.08 180836.86 180336.81 188237.38 189227.89 109419.44 108318.34 142520.72 140418.62 121713.78 123217.89 158130.71 157625.90 132921.73 134022.88 618.98.31 608.88.98 455.07.671 434.17.461 839.57.374 854.67.524 634.96.190 629.75.670 647.321.30 657.65.987 494.019.55 483.115.76 630.918.29 611.216.09 872.113.01 886.510.09 754.28.466 748.28.971 687.813.20 697.78.453 15.960 53.19 689

3.2111.053 3.1010.943 2.6320.568 2.4220.358 2.2750.430 2.4250.580 2.9210.290 2.8710.240 2.4540.178 2.5540.278 5.4381.037 5.3270.927 5.8210.979 5.6110.769 7.6721.017 7.8251.167 5.4260.816 5.3750.766 6.0890.927 6.1941.027 5.2970.617 3.9430.200 4.0540.286 3.8450.076 2.9500.021 3.1010.171 3.2680.739 3.2170.689 4.2720.345 4.3750.445 1.4410.007 1.3320.006 3.2540.624 3.0570.427 3.8640.151 4.0080.295 4.4960.584 4.4360.524 3.2630.140 3.3620.239 0.032 0.106

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Average of three determinations S.D.

690

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Fig. 1. Dendogram of cluster analysis. (Category codes: E, eucalyptus; H, heather; OB, orange-blossom; R, rosemary).

Principal components analysis was performed on the autoscaled data. From the loadings of original variables in the two rst considered principal components (Table 4), P, Mg, Ca and K are the dominant variables in the rst principal component (PC1) that represents 43.48% of the total variance, while Ba, Sr, Cu and Zn show the lower values. The dominant variables appear at negative values of PC1, all being very close. From this fact, it could be inferred that they provide the same kind of information. Principal component two (PC2) explains up to 28.11% of the total variance. Examining the loadings, Zn results the most dominant variable in this PC. The rst two principal components account for 71.59% of the total variance and were considered to be sufcient for such data. Examining the score plot of the objects in the space dened by the two principal components considered, Fig. 2, a natural separation of the four honey groups of different botanical origin was found. On the bottom left side of the biplot a compact group composed of heather honey samples can be observed. Most of the eucalyptus honeys appear at the top left side as a more dispersed group. rosemary honeys forms a
Table 4 Loadings of the variables for the two rst PCs (principal components) Variable Zn P B Mn Mg Cu Ca Sr Ba Na K PC1 0.220 0.902 0.617 0.768 0.873 0.202 0.896 0.223 0.022 0.657 0.929 PC2 0.843 0.289 0.307 0.551 0.401 0.651 0.180 0.625 0.601 0.688 0.236

compact group on the bottom right side, and a more dispersed group of orange-blossom honeys appear on the top right side of the biplot. Despite rosemary and orange-blossom samples appear very close in the biplot, it could be considered they formed two different groups, rosmary honeys are on the negative side of the biplot and orange-blossom on the positive side. Principal components analysis shows that metal content could provide enough information to develop a botanical classication for the studied honey. In addition, the agreement between the two unsupervised techniques used, PCA and cluster analysis, is consistent with this idea. Unsupervised techniques only have visualising capabilities, so a supervised pattern recognition method such as linear discriminant analysis (LDA) was performed with the purpose of obtaining classication rules for assigning categories to samples. After applying LDA, three discriminant functions

Fig. 2. Principal components analysis (PC1 vs. PC2 ).

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tained classication rules and it was validated by means of cross-validation, obtaining good recognition and prediction abilities, 100 and 97%, respectively. The discriminant model obtained could save time and money in the determination of the botanical origin of honey.

References
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Fig. 3. Linear discriminant analysis (DF1 vs. DF2 ).

were calculated. Fig. 3 shows the scatter plot of honey samples in the plane dened by the discriminant functions DF1 and DF2 . The four types of honeys appear completely separated in the plot. The samples corresponding to heather honey are situated as a compact group at the top left side of the plot. At the right side appears the rosemary and orange-blossom honeys forming two clearly dened groups and at the lower side of the plot forming a more dispersed group, it appears the eucalyptus honey samples. Validation was performed using a cross-validation method with 25% of samples as the evaluation set. The same process was repeated several times with different compositions of both training and evaluation sets, ensuring that all samples were included in the evaluation set at least once. Recognition ability, calculated as the percentage of members of the training set that were correctly classied, and prediction ability, calculated as the percentage of members of the evaluation set correctly classied were 100 and 97%, respectively. Using LDA analysis a highly successful classication between the four honey groups studied was achieve, so, the determination of the mineral content of monooral honeys and the later chemometric study can be an useful alternative to the usual pollen studies to obtain botanical classication.

4. Conclusions In this work, an attempt to establish a classication of some Spanish honeys from different botanical and geographical origin was made using pattern recognition techniques. Until now, few attemps have been made on honey from different botanical origin classication attending its mineral content. PCA and cluster analysis show that metal contents could provide enough information to develop a classication between the four botanical origins studied. LDA was applied to ob-