## 3 Results
### Phenolic Composition of Saffron Leaf Extracts
Additionally, the ion at \(m/z\) 803 was dismissed as an artifact. Peak **(3)**, which shows an [M\(-\)H]\({}^{-}\) ion at \(m/z\) 787, has been variously assigned to a quercetin trihexoside [15] or to myricetin 3-rutinoside-7-O-glucose [18], though both occur only in trace amounts in saffron petals. Its UV-Vis absorption, however, aligns more closely with kaempfer derivatives. Since multiple studies identify a kaempfer trihexoside as the major flavonoid in saffron extracts [4,16,18,19], we tentatively attribute peak 3 to kaempfer-3-O-glucose-7-O-sophoroside. Peak **(4)** showed UV peaks at 253 and 352 nm, characteristic of the flavonoid isorhamnetin (C\({}_{16}\)H\({}_{12}\)O\({}_{7}\)). The ions at \(m/z\) 963 [C\({}_{16}\)H\({}_{12}\)O\({}_{7}\)+162+162+162]\({}^{-}\) suggest the presence of an isorhamnetin tetrahexoside, as also supported by the ions at \(m/z\) 801 [M\(-\)H\(-\)162]\({}^{-}\) and 681 [M\(-\)H\(-\)162-120]\({}^{-}\), previously interpreted by [PERSON] et al. (2004) [15] as major fragments of isorhamnetin-3-O-sophorotrioside-7-O-glucose. Moreover, the ion at \(m/z\) 771 may correspond to the kaempfer-3-O-glucose-7-O-sophoroside [C\({}_{15}\)H\({}_{10}\)O\({}_{6}\)+162+162+162]\({}^{-}\) also suggested for peak (3) and present in trace amounts. Peak **(5)** presented a UV spectrum that matches with a kaempfer glycoside (C15H10O6). The 609 \(m/z\) [C\({}_{15}\)H\({}_{10}\)O\({}_{6}\)+162+162]\({}^{-}\) ion confirmed the presence of a kaempfer diglucose, which is consistent with the ions 489 [M-H-120]\({}^{-}\) and 429 [M-H-180]\({}^{-}\) of kaempfer-3-O-sophoroside reported by [PERSON] et al. (2004) [15]. Peak **(6)** was identified as luteolin 6-C-glucose by comparing it to a known commercial standard, as showed the characteristic fragmentation of this C-glucosyl flavone with ions at 447 [M\(-\)H]\({}^{-}\), 357 [M\(-\)H\(-\)90]\({}^{-}\) and 327 \(m/z\) [M\(-\)H\(-\)120]\({}^{-}\), but also to the retention time. Interestingly, another luteolin isomer, luteolin 8-C-glucose, has also been previously spotted in saffron leaf extracts [19]. Peak **(7)** presented an unexpected UV spectrum similar to that of a flavanone and an MS pattern with peaks at \(m/z\) 771, 609, 429 and 181. Fragment ions at \(m/z\) 771 [C\({}_{15}\)H\({}_{10}\)O\({}_{6}\)+162+162+162]\({}^{-}\), 609 [M\(-\)H\(-\)162]\({}^{-}\) and 429 \(m/z\) [M\(-\)H\(-\)162-180]\({}^{-}\) were in agreement with the fragmentation described by different authors for kaempfer-3-O-sophoroside-7-O-glucose ([M\(-\)H]\(-\) at \(m/z\) 771) [1,15,16,17]. Although the mass spectrometry (MS) pattern matches this compound, the UV spectrum suggests verifying the presence of a contaminating flavanone or co-elution. Peak (**8**) showed a complex mixture of components, as deduced by the different UV spectrum observed. The major and more interesting compound was identified as vitexin (431 [M\(-\)H]\(-\), 341 [M\(-\)H\(-\)90]\({}^{-}\) and 311 [M\(-\)H\(-\)120]\({}^{-}\)) on the basis of the retention time and fragmentation pattern of the commercial standard. Peak (**9**) presented a flavanone UV pattern, but was not definitively attributed to any possible compound. In terms of quantification (Table 1), the semi-purified fraction (SLPF) exhibited a significantly higher concentration of phenolic compounds compared to the crude hydroalcoholic extract (SLE). The novel purification method resulted in a more than fourfold increase in total phenolic content, from 75.3 mg/g to 330.0 mg/g of dry extract, highlighting the efficiency of the purification process in concentrating these bioactive compounds. The substantial increase in phenolic compounds through this developed methodology underlines the potential of the semi-purified fraction as a rich source of phenolics. With respect to individual phenolic components, their concentrations showed considerable variation, with increases ranging from 2- to 5-fold depending on the compound, demonstrating the selective enrichment of specific compounds during the purification.