**2.2.2 PCA-loadings**

26 Principal Component Analysis

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

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,

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

**-24 -20 -16 -12 -8 -4 0 4 8 b**

**PC2 (6%)**

**Non-matured Mezcals**

**-30 -15 0 15 30 45 60**

**PC1 (91%)**

**Matured Mezcals**

(◄) distilled (white/young coloured), (▼) rested and (♦) aged.

**Matured Mezcals**

**-20 -15 -10 -5 0 5 10 15 20**

**PC1 (95%)**

remained constant for all measurements.

**2.2.1 PCA-scores** 

**-4.5 -3.0 -1.5 0.0 1.5 3.0 4.5 a**

**PC2 (4%)**

**Non-matured Mezcals**

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 rested and aged mezcals.

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 standardized spectra.

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

Application of Principal Component Analysis

not to remove them.

benefit for the spectral data analysis.

**white w/worm**

our results.

**-20**

**-10**

**0**

**10**

**PC2 (7%)**

**20**

model to describe the data structure in a more efficient way.

to Elucidate Experimental and Theoretical Information 29

the case of the aged sample, the higher concentration of wood extracts in comparison to the rested samples originates a noticeably different spectrum, thus explaining the observation of this aged sample as an isolated object. In order to improve the distribution of objects, samples detected as outliers were removed. Fig. 3 (b) shows the scores plot after outliers removal. Similarly to Fig. 3(a), two main groups can be observed. However, the percentage of explained variance for PC1 increases to 97%. This fact indicates that, even when the object distribution does not depict significant variations, the removal of these outliers, allows the

There are other samples that can be considered as outliers [indicated with numbers 1-3 in Fig. 3(a)]. Among them, number 1 corresponds to white mezcal with worm (some mezcal producers add a worm to their product as a distinctive), the worm probably providing certain organic molecules. These organic molecules would have similar emission properties than those of the rested ones. Hence, number 1 can be taken as outlier or not. We decide to take it, as correctly classified. On the contrary, objects 2 and 3, were considered as outliers, because they do not have any particular characteristics like object 1. Furthermore, when they were removed from the PCA (data not shown), distribution of objects and explained variance percentages remained similar. For this reason, we decided

In conclusion, only the joint analysis of the scores plot, the raw spectra, the loading plots and all other available information of the samples can give a correct interpretation of

Figure 4 (a) and (b) shows the PCA-scores plots obtained from standardized spectra before and after the removal of outliers. As it was described before, the similar object distribution observed in Fig. 3 and 4 indicates that standardization does not provide any additional

Fig. 4. PCA-scores plots obtained for the mean centred and standardized fluorescence spectra. Samples correspond to different stages of maturation. (a) Scores plot before the removal of outliers, (b) scores plot after the removal of outliers. (**□**) White or young, (△)

**-8**

**-4**

**0**

**4**

**PC2 (3%)**

**8**

**white w/worm**

**-30 0 30 60 90 120**

**PC1 (90%)**

**Aged mezcal**

**<sup>12</sup> b**

**Aged mezcal**

abocado, (**○**) rested, (▽) aged, (◁) distilled and (◇) ethanol.

**Outliers**

**-40 -20 0 20 40 60 80 100 120**

**PC1 (85%)**

**<sup>30</sup> a**

the spectral features belonging to authentic matured mezcals. On the other hand, onedimensional loadings plot obtained from standardized spectra [Fig. 2 (c)], lacks in the spectral information provided, thus limiting its uses for interpretation purposes. In spite of that, standardization may be useful for certain applications (*i.e.* calibration of prediction/classification by PLS or PLS-DA) (Esbensen, 2005).
