## 3 Results and Discussion  
### Fingerprinting for Saffron Authenticity  
#### 3.1.1 Metabolic Analysis of Authentic Saffron Aqueous Extracts  
The NMR analysis (1D \({}^{1}\)H NOESY, COSY, and TOCSY) performed on the aqueous extracts of the authentic pure saffron enabled the identification of a pool of metabolites (see Table S1 in Supplementary Materials for the full list of metabolites). The main representative classes of metabolites detectable in the aqueous extracts of authentic saffron (see Figure S2 in Supplementary Materials for further details) include organic acids (lactic, acetic, malic, fumaric, and formic acids), amino acids (alanine, asparagine, glycine), and carbohydrates (D-glucose, fructose, D-gentiobiose, and D-sucrose). Additionally, the colorless monoterpene glucosideprocrocin (4-[\(\beta\)-d-glucopyrananoyl]-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (hereafter referred to as picrocroccin) has been reported as abundantly present in the aqueous extracts of saffron. It represents one of the metabolites majorly responsible for the typical bitter taste of saffron [45]. However, it has been previously reported as occurring from degradation of picroccin in aqueous saffron spice extracts upon thermal treatment [46]. Therefore, since the analyzed samples were subjected to a pasteurization process at 80 \({}^{\circ}\)C to keep the samples stable for 24 h (see Figure S1 in Supplementary Materials for stability studies), alycone 4-hydroxy-2,6,6-trimethylcyclohex-1-enecarbaldehyde (HTCC) was expected to be detected as a degradation product. Indeed, characteristic signals as singlets ascribable to this compound were detected at 1.20, 1.23, and 9.97 ppm. Correspondingly, the saccharidic moiety, namely \(\beta\)-D-glucose, derived from the degradation of picroccin was identified by the well-resolved doublets at 5.24 and 4.65 ppm, and the doublet of doublets at 3.26 ppm. Moreover, it has been reported that the analyzed aqueous extracts contain significant amounts of a group of water-soluble carotenoids derived from crocetin (8,8\({}^{\prime}\)-diapo-\(\Psi\),\(\Psi^{\prime}\)-carotenedioic acid) which are responsible for the coloring capacity of saffron and the use of this spice as food colorant [47]. While a pool of carotenoids derived from crocetin (8,8\({}^{\prime}\)-diapo-\(\Psi\),\(\Psi^{\prime}\)-carotenedioic acid) has been identified through LC-ESI-MS using water (or acidified with 0.25% formic acid) and acetonitrile [48], the 1D \({}^{1}\)H NOESY spectrum in D\({}_{2}\)O, in agreement with the literature [26], contains predominantly the signals related to one of the identified derivatives, namely _trans_ crocetin (\(\beta\)-D-gentibiosyl) ester, commonly appointed as crocin with the _trans_ isomer significantly more abundant than the _cis_ one, which has not been detected. Indeed, in our case, characteristic signals ascribable to the polyconjugated olefinic protons of _trans_ crocetin (\(\beta\)D-gentibiosyl) ester are present at 7.26, 6.42, and 6.12 ppm, while the broad singlets related to the methyl moieties can be found at 1.87 and 1.77 ppm. In addition, kaempfer, which is a flavonol under the class of flavonoids, is contained in the saffron aqueous extracts. While stigma samples have been reported to contain high levels of crocin, picrocrocion, and their derivatives, the stamens and sepals of saffron have been reported as rich in flavonoids which are present under three forms: kaempfer-3-sophoroside, kaempfer-3-sophoroside-7-glucoside, and kaempfer-3,7,4-triglucoside [49]. Kaempfer can be detected in the 1D \({}^{1}\)H NOESY NMR spectrum of aqueous extracts of stigmas as a mixture of two glycosylated forms, which have been characterized as 7-O-glucopyranoside-3-O-sophoroside, and the 7-O-sophoroside glycosides of kaempfer [49]. In agreement with the literature [26], two doublets at 8.03 and 7.94 ppm, and, two further doublets at 6.97 and 6.89 ppm can be ascribed to these two forms of kaempfer.