Attenuated total reflectanceFourier transform infrared spectrometry and chemometrics model was used for determination of physicochemical properties (pH, redox potential, free acidity, electrical conductivity, moisture, total soluble solids (TSS), ash, and HMF) in honey samples. The reference values of 189 honey samples of different botanical origin were determined using
Honey is a natural, sweet, and syrupy fluid collected by bees from the nectar of flowers. The taste and aroma of this liquid vary according to its floral origin and geographical and seasonal conditions. A large number of melliferous sources give therefore the opportunity to produce much characteristically monofloral and a high number of polyfloral nectar honeys [
The composition and sensory attributes of honey vary considerably depending on its botanical and geographical origin. Mexico has a great geographical and botanical diversity; for this reason there are many types of blooms that produce different types of honey with a multitude of tastes, colors, and aromas, which are in demand in the European and American markets. Mexico is the fifth largest producer of honey with about 57,000 tons per year and the third largest exporter in the world [
The physicochemical quality criteria for honey are well specified by
In contrast to the timeconsuming analysis techniques near infrared spectroscopy (NIRS) and midinfrared spectroscopy (MIR), both vibrational spectroscopy techniques, when combined with multivariate calibration, can be simple, fast execution and low cost [
The aim of this study was to determine eight physicochemical properties for Campeche honey, with the aim to develop PLS regression models based on the FTIRATR spectroscopy transform for to be used as a rapid and nondestructive analytical tool for quality control of honey.
For this work a total of 189 honey samples were collected in 43 communities from eight different municipalities of the state of Campeche, 172 honey samples were of the
Geographical origin of the honey samples collected from different regions of the state of Campeche, Mexico.
The reference methods used for the quantitative determination of free acidity, pH, hydroxymethylfurfural (HMF), electrical conductivity, ash content, moisture content, total soluble solids (TSS), and redox potential of the samples honey were in agreement with the standardized methods proposed by the official methods
The pH in honey samples was measured in a solution of 10 g of honey in 75 mL ultrapure water free of carbon dioxide, at 20°C using a pHmeter Thermo Scientific, model Orion Star A211. The pHmeter was calibrated using buffer standard solution between 4–7 and 7–10 pH values [
The concentration of free was determined by a titrimetric method [
The electrical conductivity in honey samples was measured at 20°C, by dissolution 20 g honey sample in a 100 mL in ultrapure water with Thermo Scientific conductimeter; the results were expressed as mS/cm [
Ash percent was measured by calcination in muffle furnace Lindberg/Blue, for one night in a furnace at 550°C, until reaching a constant mass [
The moisture and the total soluble solids in honey samples were determinate based on the refractometry method. The refractometry indexes in honey samples were measured at 25°C using an Atago refractometer model PAL2SS and the reading was further corrected to a standard temperature of 20°C by adding the correction factor of 0.00023/°C [
Hydroxymethylfurfural was determined by spectrometry UVvisible, about 5 g honey sample after clarifying with Carrez reagents I and II and the addition of sodium bisulfate [
Redox potential was measured at 20°C using a pHmeter Thermo Scientific, model Orion Star A211. Honey samples were diluted with deionized water, ranging from 10% to 100% (w/v) [
The FTIR spectra of honey were acquired with an Agilent Model 660 spectrometer, equipped with a diamondtip single reflection attenuated total reflectance (ATR), Pike Technologies model Gladi. The software Resolution 4.0 pro Variant served as an interface between the computer and the spectrophotometer. For the analysis of the honey about 0.3 mL of honey was placed on the diamondtip of ATR; all spectra were recorded at a controlled temperature (
The Pirouette software was employed for modeling and data treatment. In total 567 spectral fingerprint data obtained from 189 honey samples were used for the construction of each calibration model, in the FTIR region from 700 to 3700 cm^{−1} (Figure
Characteristic of FTIRATR spectrum from all honeys samples, acquired from 3700 to 700 cm^{−1}.
The preprocessing of the spectra set improves the performance of the calibration models and capacity of predictions. Different treatments were applied to matrix MIR spectra: meancenter, autoscale, baseline correct, normalize, smooth, first derivate, align, logarithm, and standard normal variate (SNV). The reference values determined by chemical methods and the spectral data of both calibration and validation sets were analyzed to detect any outliers using a general Mahalanobis distance (
To determine the robustness of the calibration models, these models were evaluated using crossvalidation with “leave fifteen out” data. The predictive abilities of the models were evaluated through external validation, using a set of 38 samples, and not involved in the construction of the calibration model. To determine the predictive capacity of the calibration models the following statistical parameters were calculated: (PCs) optimum number of principal components, (SECV) standard error of crossvalidation, (
For a better analysis of the standard deviation between the data obtained by the reference methods and predicted by the models calibration of each physicochemical property was necessary to determine the coefficient of variation (CV). The CV is a measure of reproducibility of the model and as a general rule a model can be considered reasonably reproducible if its CV is not greater than 10%.
The 189 samples of pure honey were analyzed by triplicate using spectroscopy FTIRATR. Figure
To construct, the PLS calibration model was used, the FTIR spectral information contained between 3700 and 700 cm^{−1}, interference free region. Several pretreatments were realized to spectral fingerprints to reduce error; the Mahalanobis distance was established for each calibration model in order to determine the outliers points Table
PLS calibration models were developed applying different mathematics treatments to reduced errors in predicting of the properties of honeys.
Honey properties  Different spectral treatments sequence  Mahalanobis distance, criterion  Outliers points 

pH  Autoscale, baseline correct (quadratic), normalize, smooth (order polynomial 25) 

23 
Free acidity  Autoscale, 1st derivate (order polynomial 15), log_{10}, baseline correct (quadratic), normalize 

43 
Electrical conductivity  Autoscale, baseline correct (quadratic), normalize, 1st derivate (order polynomial 15), smooth (order polynomial 25) 

27 
Ash  Autoscale, 1st derivate (order polynomial 15), log_{10}, normalize, baseline correct (quadratic) 

23 
TSS (Brix°)  Meancenter, log_{10}, baseline correct (quadratic), normalize, smooth (order polynomial 25) 

21 
Moisture  Autoscale, baseline correct (quadratic), log_{10}, normalize, align (15) 

19 
HMF  Autoscale, baseline correct (quadratic), log_{10}, 1st derivate (order polynomial 15), align (15) 

22 
Redox potential  Autoscale, 1st derivate (order polynomial 15), log_{10}, baseline correct (quadratic), normalize 

19 
Statistical parameters obtained for each of the calibration models.
Honey properties  PCs  SECV 

PCs  SEP 

Coefficient of variation 

pH 







Free acidity 







Electrical 







Ash 







TSS (Brix°) 







Moisture 







HMF 







Redox potential 







The results of analysis physicochemical of honey samples for each municipality of the state of Campeche, medium, maximum, and minimum values for each property, are presented in Table
Physicochemical for honey samples of Campeche state.
Municipalities  pH  Free acidity 
Electrical conductivity 
Ash 
TSS 
Moisture 
HMF 
Redox potential 

Calakmul 








Calkiní 








Campeche 








Champotón 








Escarcega 








Hopelchén 








Hecelchakán 




82.44 ± 0.46 



Sabancuy 








The pH is a parameter that is correlated with honey storage and with microorganism growth that could change the texture and the honey stability; the pH value can be used for the discrimination of floral and honeydew honey [
Figure
PLS validation plots of pH, contribution of PCA, and regression vector (a)–(c); PLS validation plots of free acidity, contribution of PCA, and regression vector (d)–(f).
Honey acidity is due to the presence of organic acid, mainly gluconic acid, and to inorganic ions such as sulfate, phosphate, and chloride. Acid measurement is useful for evaluation of honey fermentation, authentication of unifloral honey, and differentiating nectar from honeydew [
The electrical conductivity depends on the ash, organic acid, mineral compounds such as sulfates and phosphates, protein, and some complex sugar and polyols contents and varies with botanical origin. Electrical conductivity is widely used for discrimination between honeydew and blossom and also for the characterization of unifloral honey [
PLS validation plots of electrical conductivity, contribution of PCA, and regression vector (a)–(c); PLS validation plots of Ash, contribution of PCA, and regression vector (d)–(f).
Figure
Figures
The Brix° scale is used in the food industry for measuring the approximate amount of sugars. The honey is mainly composed of sugars; about 25 different oligosaccharides have been detected in the composition of honey. The fructose and glucose are present in a higher concentration and provides the honey with its extreme sweetness. The total soluble solids (TSS) which are directly related to sugar content may be a reliable index of adulteration. Honey samples municipalities like Calkini and Hecelchakan had the lowest concentration of sugar,
PLS validation plots of TSS (Brix°), contribution of PCA, and regression vector (a)–(c); PLS validation plots of moisture contribution of PCA and regression vector (d)–(f).
The honey moisture content depends on the environmental conditions, and also the honey degree of maturity and the manipulation from beekeepers at the harvest period, and it can vary from season to season and from year to year. The moisture is an important parameter of control quality in honey; higher moisture content could lead to undesirable honey fermentation during storage. The honey fermentation produces the formation of ethyl alcohol and carbon dioxide; the alcohol can be oxidized to form acetic acid and water, which is undesirable because it alters the quality of the honey [
The HMF content is widely recognized as an indicator of freshness and related to storage, processing, and possible adulteration with invert syrup, unrelated to the botanical or geography origin. Several factors influence the levels of HMF, such as temperature, time of heating, pH, storage conditions, and floral source [
PLS validation plots of HMF, contribution of PCA, and regression vector (a)–(c); PLS validation plots of Redox potential, contribution of PCA, and regression vector (d)–(f).
The redox potential gives information about the real oxidation/reduction ability of a molecule and its prevalent form oxidized or reduced in the system. Therefore, the redox potential may be also an interesting indicator of the antioxidant efficiency of food [
The ATRmidinfrared spectroscopy combined with chemometrics techniques based on PLS makes the development of calibrations models that have a satisfactory power prediction for the quality parameters of honey studies possible: pH, electrical conductivity, ash, moisture, TSS (Brix°), and redox potential. The honeys of the state of Campeche presented low concentrations of organic acids and HMF, so that the absorbance bands of their functional groups in the infrared spectrum were not useful, to be able to construct calibration models that allowed predicting insatisfactorily the free acidity and HMF in the honeys of the state of Campeche.
The determination of measurands such as pH and redox potential is valuable for the evaluation of storage and heat damage, while the determination of measurands for electrical conductivity and ash could allow determining the geographical origin of honey. Redox potential could be used to determine the concentration of antioxidants in Campeche honeys.
The main advantage of ATRMIR combined with chemometrics methods is that it can provide valuable information of several measurands used for quality control of honey from the state of Campeche, with the advantage that is fast, low cost, and environment friendly.
The authors declare that there are no competing interests.
The authors are grateful to PROMEP for the financial support through the Project DSA/103.5/14/10634 (UNACAREXB076) and to the Mayan beekeepers from state of Campeche for the donation of honey samples.