## 3 Results and Discussion
### Fingerprinting for Determining the Agronomic Practice Adopted for Saffron Cultivation  
The non-targeted NMR approach applied to the spectroscopic data derived from the analysis of the aqueous extracts of pure safron allowed extrapolation of insightful information related to the agronomic practice adopted for this spice cultivation. In the PC1/PC2 scores plot (see Supplementary Materials, Figure S11), a clear clustering of the samples was observed when the regular-sized (0.04 ppm) spectral buckets were subjected to the unsupervised PCA. Through a more in-depth examination of the characteristics of the sample, including the geographical origin, the producer, and the agronomic practice adopted for cultivation (see Table S5 in Supplementary Materials for the complete list of available information relating to the investigated samples), the agronomic practices were found to be the most influencing factor on samples distribution. The supervised PLS-DA (see Figures S15 and S16 in Supplementary Materials for further details on the model overview and model validation) was conducted to obtain more information regarding changes in metabolic composition associated with the agronomic practice employed. The analysis of  
Figure 4: Comparison of typical 1D \({}^{1}\)H NOESY NMR spectra (Bruker Avance 400 MHz, D\({}_{2}\)O) of aqueous extracts of authentic saffron (blue spectrum), adulterated samples with 20 wt% of safflower (red spectrum), and adulterated samples with 20 wt% of turmeric (green spectrum).  
Figure 5: Results of OPLS-DA performed on the spectral data obtained from the extracts of authentic (PS, represented as circles) and adulterated (AS, represented as triangles) saffron in DMSO-\(d_{6}\): (**a**) Scores plot between the selected PCs. The explained variances are shown in brackets. (**b**) Analysis of the Variable Importance in Projection (VIP) identified by OPLS-DA. The colored boxes on the right indicate the relative concentrations of the corresponding metabolite in each group under study.  
the Variable Importance in Projection, VIP (see Table S5 in Supplementary Materials for the full list of 78 variables with VIP > 1), along with the visual inspection of the 1D \({}^{1}\)H NOESY NMR spectra allowed the selection of a pool of 15 spectral regions acting as diagnostic variables towards the discrimination between the saffron samples obtained through conventional cultivation compared to those derived from organic cultivation (Figure 7b).  
The hierarchical clustering heatmap performed on the normalized data (distance measure, Euclidean; Clustering method, Ward) clearly shows that the aqueous extracts of saffron cultivated according to an organic agronomic practice were characterized by a relatively higher content of sugars, like sucrose and gentiobiose, along with organic acids, as succinic and malic acids. On the other hand, it was found that the two most typical metabolites of saffron, namely picrocrocin and crocin, were contained majorly in those samples derived from a conventional agronomic practice (Figure 8).  
Figure 6: Results of the hierarchical clustering heat map performed on the normalized data (distance measure using _Euclidean_, and clustering algorithm using _Complete_). Each colored cell on the map corresponds to a concentration value in the data table, with samples in columns and metabolites in rows. The belonging class of samples is indicated as red and green squares for adulterated saffron (AS) and authentic one (PS), respectively.  
Figure 7: Results of PLS-DA performed on the spectral data obtained from the aqueous extracts of authentic saffron cultivated through conventional cultivation (C, represented as triangles) and organic one (B, represented as circles): (**a**) Scores plot between the selected PCs. The explained variances are shown in brackets. (**b**) Analysis of the Variable Importance in Projection (VIP) identified by PLS-DA. The colored boxes on the right indicate the relative concentrations of the corresponding metabolite in each group under study.