**2.2 Collection of UV absorption spectra**

Spectra were collected in the 285-450 nm spectral range, using an UV/Vis spectrometer model USB4000 from the Ocean Optics company, coupled to the Deuterium tungsten

Application of Principal Component Analysis

**2.2.2 PCA-loadings** 

rested and aged mezcals.

standardized spectra.

**0.036 0.040 0.044 0.048 0.00 0.02 0.04 0.06 0.08 0.0 0.6 1.2 1.8**

**PC1-loadings**

**PC1-loadings**

**Absorbance**

to Elucidate Experimental and Theoretical Information 27

sample non-related with the other objects. This unexpected observation can be explained considering the similarity between the spectra of the rested and aged mezcals [see Fig. 2 (a)]. For this reason, the PCA-scores plot corresponding to standardized spectra, must be

Once the distribution of objects in the PC-space has been interpreted, the analysis of the onedimensional loadings plots has been carried out in order to find the relationship between the original variables (wavelength) and the scores plots (Esbensen, 2005; Geladi & Kowalski, 1986; Martens & Naes, 1989). In this case, PC1 is the component discriminating mezcal samples according to their maturation stage and time. Consequently, the PC1-loadings provide information about the spectral variables contributing to that discrimination. Fig.2 (a) shows four representative absorption spectra in the 290-450 nm ultraviolet range for white, abocado, rested and aged mezcals. According to the Figure, the absorption spectra of white and abocado samples look similar, and different from those corresponding to the

Fig. 2. (a) Representative raw UV absorption spectra for each of the four types of mezcals, (b) PC1-loadings plot for the centred spectra, and (c) PC1-loadings plot for the centred and

**300 320 340 360 380 400 420 440**

**Abocado Rested Aged**

**c**

**b**

**a**

**Wavelength (nm)**

The loading plots indicate that the 320-400 nm region [blue dashed rectangle, Fig. 2 (a)], is the best region to evaluate the authenticity of matured mezcals because the wood compounds extracted, produced and added to mezcals during the aging process absorb in this region. The 290-320 nm range [red dashed rectangle, Fig. 2 (a)], provides the signature for non-maturated mezcals. Fig. 2 (b) and (c) depict one-dimensional PC1 loadings plots corresponding to mean centred and standardized spectra, respectively. From Fig. 2 (b), it is feasible to observe the great similarity between the one-dimensional PC1 loadings plot and the representative spectrum of rested and aged mezcals, suggesting that PC1 mainly models

considered cautiously since they can lead to incorrect interpretations.

**White/Young**

halogen light source and cuvette holder by means of optical fibers, and with a spectral resolution of ~1.5 nm. The mezcal samples were deposited in disposable 3.0 mL cuvettes, specially designed for UV/Vis spectroscopy under a transmission configuration, which remained constant for all measurements.

#### **2.2.1 PCA-scores**

Fig. 1 (a) and (b), depict the distribution of objects (samples/spectra) corresponding to the two pre-treatment options (MC and 1/Std) in the PC-space. In both graphs, a similar distribution of objects along PC1-axis was observed. The groupings along PC1 indicate a good discrimination between matured and non-matured mezcals. Additionally, samples corresponding to mezcals *a priori* known as artificially matured (*i.e.* abocado and distilled samples) and other few, labeled as rested but presumably artificially matured, cluster together with the non-maturated ones. This indicates that the UV absorbance properties of compounds and molecules naturally generated in the wood cask, are significantly different from those from other compounds used with counterfeit purposes (Boscolo et al, 2002).

Fig. 1. PCA-Scores plots obtained from raw UV absorption spectra (a) mean centred, (b) standardized. (■) White/young, (●) White w/worm, (▲) abocado or artificially matured, (◄) distilled (white/young coloured), (▼) rested and (♦) aged.

A central region, delimited with dashed lines and mainly including samples corresponding to rested mezcals and a few artificially mature samples (abocado and white mezcal w/worm) can be considered as an "indecisive zone". However, taking into account that some samples analysed in this study were directly purchased from liquor stores, it may be possible that few of them, claimed as authentic rested, have been artificially matured. In addition, the sample corresponding to aged mezcal is separated from all the other samples in both graphs, but always clustering together with the rested samples. This indicates that the cluster of objects/samples is related not only with their maturation stage, but also with their maturation time. This behaviour points out that the standardization pre-treatment does not affect significantly the distribution of objects in the scores plots. However, there are some issues that must be considered: in Fig. 1 (a), the aged sample is located close to the rested group, but non as part of it. This can be explained in terms of their different times of maturation. On the other hand, in Fig. 1 (b), the aged sample seems to be an outlier or a sample non-related with the other objects. This unexpected observation can be explained considering the similarity between the spectra of the rested and aged mezcals [see Fig. 2 (a)]. For this reason, the PCA-scores plot corresponding to standardized spectra, must be considered cautiously since they can lead to incorrect interpretations.
