Saffron contains more than 150 compounds (volatile and non-volatile) including carotenoids (crocetin, crocin, β-carotene, lycopene, and zeaxanthin), monoterpene aldehydes (picrocrocin and safranal), monoterpenoids, and isopherones [8,28]. However, it also contains other compounds such as flavonoids, vitamins, proteins, and amino acids [51]. Saffron owes its sensory and functional properties mainly to the presence of its carotenoid derivatives, synthesized throughout flowering but also during the whole production process [43]. These compounds include crocin, crocetin, picrocrocin, and safranal, which are the secondary or bioactive metabolites [8,43,44,52]. Saffron’s quality depends on its chemical profile and is directly related to the geographic area, climate variability, environmental practices, genetic traits, soil composition, cultivation conditions, and processing and storage methods [53,54]. Nevertheless, according to the ISO standards (3632-1:2011 and ISO 3632-2:2010), the value and quality of the stigma are measured based on the content of the color components (crocin and crocetin), the bitter taste component (picrocrocin), and the volatile compounds responsible for the odor and aroma (safranal). These specific parameters are influenced by the environmental conditions, extraction method, purification, etc. [14,28,55,56,57]. Some studies have been conducted on the extraction of bioactive compounds from saffron using the concept of green chemistry [58]. Some research on saffron stability demonstrates that temperature and humidity exert a strong influence on the degradation of the principal active ingredients [8].

Crocin: The main bioactive compound of saffron was isolated by Aschoff in 1818, reporting a family of yellowish-red water-soluble carotenoids (mono-glycosyl or di-glycosyl-polyene esters) of 20 carbons [8,34,58,59,60]. In other words, this was a group of compounds formed by crocetin esterification (dicarboxylic carotenoid), which were classified according to their sugar fractions [59]. The abbreviations used in this review are as follows. The cis/trans-X-R1R2 crocin abbreviation system is used based on three main characteristics: (a) cis/trans isomers, (b) X: number of glucose components (1–5), and (c) type of structure in R1 and R2 (acid form: H; glucose: g; gentiobiose: G; Neapolitan: n; or triglucose: t.) (Suchareau et al. (2021)). The most represented crocins are trans-4-GG, trans-3-Gg, trans-2-G, trans-2-gg, trans-5-tG, and trans-1-g, among others [19,59,61,62,63,64,65,66,67,68,69,70].

Crocins are unusual apocarotenoids since their terminal glycoside rings confer high solubility. These pigments are detected in the red lobes of the stigmas of the Crocus sativus flower [14,19] and their content is proportional to the color and quality index. However, it should be noted that zeaxanthin (fat-soluble carotenoid) can also influence the color [35]. Crocins as such have low stability and lose their functionality during exposure to heat, oxygen, light absorption, acidic environments, and/or due to the presence of additives [43]. Therefore, the drying and storage temperatures are important for proper color development [68]; poor storage conditions lead to color pigment degradation [71]. Several factors are related to the concentration of these pigments in saffron stigmas, which are mainly the geographical growing region, crop conditions, type of soil, plant genetic traits, climate, planting time (rate), seed/crown rate, planting depth, corm size/weight, crop density, nutrient management, weed management, growth regulators, harvest and postharvest management, and drying conditions [49,72]. Finally, crocin (digentiobiose ester of crocetin) is recognized as a natural food-grade dye that displays biological activity such as antigenotoxic, cytotoxic, antioxidant, anti-inflammatory, anti-atherosclerotic, anti-diabetic, hypotensive, hypolipidemic, hypoglycemic, and antidepressant properties [14,28,55].

Crocetins are lipophilic carotenoids derived from the hydrolysis of crocin glycosides, which is a crocin aglycone [67]. It contains a carboxyl group at each end of the polyene chain [19]; these groups of compounds (α-crocetin or crocetin I, crocetin II, β-crocetin, γ-crocetin) are produced from the degradation of zeaxanthin [73].

Picrocrocin’s structure was established by Khun and Winterstein in 1934 [60]. It is a colorless and odorless glycoside monoterpene (4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde or hydroxy-β-cyclocitral: HTCC and glucose), a product of the degradation of zeaxanthin, and is responsible for saffron’s bitter taste [8,28,34,52,58,74]. Picrocrocin is the second most abundant component in dry matter content [66,73,75]. During the drying process (35–50 °C for 4–7 h), picrocrocin’s temperature and/or hydrolysis form an aglycone [73,76]. Therefore, picrocrocin decreases during dehydration, whereas safranal is absent before drying [17].

Safranal is an aldehyde monoterpene and the volatile component responsible for saffron essential oil. HTCC (hydroxy-β-cyclocitral or 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde) is regarded by many authors as a safranal precursor. This compound is obtained by chemical or enzymatic hydrolysis (dissociation) or when the vegetal material is dehydrated and transformed into safranal, but this also happens due to the handling and storage processes [8,53,58,63,75,77,78]. The safranal content changes according to the duration and intensity of drying, causing quality fluctuations [34], whereas its concentration increases with the storage and timely harvesting of flowers. However, heat and sunlight decrease the final quality and price [28].

There are various hypotheses on the method of obtaining these important apocarotenoids from saffron. The first theory focuses on synthesizing these compounds in the plant from protocrocin (glycosyl derivative of zeaxanthin), the substrate of an oxidative enzyme that produces a molecule of crocin and two molecules of picrocrocin. Regarding safranal, it has been described that only a minimal concentration is detected in the fresh spice [79]. Fallahi et al. [80] described another pathway wherein apocarotenoids, which are commercially important, are obtained by the cleavage of carotenoids (zeaxanthin and β-carotene) by the carotenoid dioxygenase enzyme, giving rise to crocetin and hydroxy-β-cyclocitral as products. Later, they propose a glycosylation (glycosyltransferases) step, which produces crocins and picrocrocin, respectively. Finally, they describe that picrocrocin is hydrolyzed to form safranal. This hypothesis is consistent with that described by Sereshti et al. [81], who also describe other, more specific enzymes and substrates, as seen in Figure 1.

The enzyme dioxygenase performs a 7–8C and 7′–8′C symmetric cleavage on the carotenoid zexanthin, converting it to 3-hydroxy-𝛽-cyclocitral and dialdehyde crocetin. Crocetin dialdehyde undergoes oxidation by aldehyde dehydrogenase to crocetin. Crocetin further undergoes glycosylation at the carboxyl group by the enzyme UDP-glucuronosyl transferase, forming crocin. Picrocrocetin is obtained from 3-hydroxy-𝛽-cyclocitral by glycosylation at the hydroxyl group by the enzyme UDP-glucuronosyl transferases. Picrocrocin is converted to safranal by the action of the enzyme 𝛽-glucosidase along with heat during drying [14].

Saffron’s quality depends on its chemical profile, which provides the bitter taste, desirable aroma, and attractive yellowish-red color of this spice [29,82]. Several studies on saffron stability are related to temperature, humidity, pH, light, oxygen [76], geographical growth location, and drying and storage conditions [83]. Since 1980, a standard quality procedure has been employed for saffron classification according to the International Standard Organization (ISO/TS 3632), which was updated in subsequent years (2003, 2010, 2011). This regulation allows saffron to be classified into distinct categories based on physical and chemical criteria: Category I—high quality; Category II—±medium quality; and Category III—low quality [61,84,85]. The grouping parameters used are moisture content, flower residues, foreign material, ash, and coloring power. However, external parameters, such as the absence of other plants, biological micro-flora, and pesticide residues, are also used. The methodology to determine saffron’s quality using these regulations is the spectrophotometric quantification of the stigmas’ aqueous extracts (1%) at three maximum wavelengths, namely 257 nm to indicate flavor strength (picrocrocin), 330 nm related to aroma (safranal), and 440 nm for coloring force (crocins), using a 1 cm pathway quartz cell [85,86,87,88,89]. The results are reported according to Equation (1): where λmax is the wavelength (257, 330, or 420 nm), A is the absorbance, m is the saffron sample weight (g), and H is the moisture content (%) [20,79,88,90,91,92]. The color intensity is the most important characteristic related to quality and is used to establish the market price of saffron [93]. The crocin content (degraded carotene) [32] determines the market color specifications. Category I includes a minimum value of 200 units of coloring strength (ucs) and for Category III, the minimum value is 120 ucs [61]. Saffron merchants usually consider a 3-4-year shelf life for saffron when stored under suitable conditions (at room temperature without light exposure). The color intensity decreases by nearly 30 to 40 units per year and is a significant determinant of the final quality of saffron [94]. Diverse drying methods affect crocins, which may be related to the time, temperature, and resistance used [35]. Other factors that affect color are geographic location, harvest, storage, and mixing with additional non-colored parts of the plant (stems and other adulterating materials) [91]. Saffron’s bitter taste is attributed to picrocrocin, a compound present in the plant’s stigmas. The ISO standard determines the flavor strength with values of 70 (Category I), 55 (Category II), and 40 (Category III) [61]. The final picrocrocin content varies according to the dehydration process used [94]. The spice’s flavor can suffer significant losses during processing [1]. Safranal is the active odor in this spice [18,94,95]. The ISO 3632 method determines three categories of aroma strength in safranal, with values within a range of 20–50 [61,96]. It is important to emphasize that during dehydration and storage, there are modifications in saffron’s sensory characteristics [94,97].

Therefore, the chemical components of saffron quality are crocin, picrocrocin, and safranal. Lage and Cantrell [21] established that crocins are found in a more significant range (18–37%), followed by picrocrocin (4.2–28%) and, in a lower proportion, safranal (0.04–0.48%). This is consistent with the results described by various authors [21,64,72,90,96], who determined crocins as the major components, specifically trans-4-GG and trans-3-Gg crocins [61,64,98].

Concerning crocins, Chaouqi et al. [87] demonstrated that these coloring components are extracted in a more considerable proportion at 40 °C than at room temperature; the authors suggested the use of short dehydration times since an increase in temperature allows for the maximum crocin content, which also depends on the production [94]. However, Rocchi et al. [68] found that the use of elevated temperatures (125–200 °C) in the drying treatment can influence the pigments’ degradation (glucose hydrolysis), and fresh samples (<1 year) retain a significant amount of glycosylated crocin, which is hydrolyzed after storage. Sereshti et al. [81] described that freshly dried samples have an intense color due to crocins since during storage, these pigments decrease (enzymes, temperature, light, hydrolysis), with a negative correlation with odor (the color is reduced, whereas the aroma increases). Saffron storage causes apocarotenoids’ glycosidic bonds to break down (band at 1028 cm), which was confirmed using FT-IR spectroscopy, and is associated with the presence of glucose, together with intensities in the region of 1175–1157 cm linked with glucosidic bonds [99]. The second quality component in the percentage is picrocrocin, which increases with the dehydration temperature (40 °C) [21] but decreases with storage time [87]. Ordoudi et al. [78] determined that saffron produced under optimal processing and storage conditions retains its organoleptic characteristics for 1 to 4 years. Meanwhile, samples stored for more than four years produce low amounts of crocetin and picrocrocin esters. This is related to the findings described by Sereshti et al. [81], who determined that during storage, picrocrocin loses its sugar residues and becomes HTCC and safranal (fresh samples are more bitter). In other words, fresh samples contained a higher concentration of crocins and picrocrocins, whereas the level of safranal (the most abundant volatile component, but with a minimum total concentration in the aromatic spice) was higher in the stored samples; therefore, the relationship between time and safranal content was demonstrated by the higher concentration in the samples with extended storage. García-Rodríguez et al. [96] determined that the aged spice produces safranal from HTCC. The safranal concentration depends on the drying and storage conditions [97].

The ISO standard proposes a fast, economical, and easy-to-implement spectrophotometric UV-vis method for aqueous saffron extracts. However, this technique does not allow for the actual determination of the quality compounds [87]. ISO 3632 proposes the quantifications of picrocrocin, safranal, and crocins at a maximum of 257 nm, 330 nm, and 440nm, respectively. However, Cossignani et al. [88] and Aiello et al. [86] determined that crocins show an absorption spectrum between 250 and 470 nm that overlaps at various wavelengths between the compounds. Trans-crocin isomers showed two bands: the first at 260 nm (glycosidic ester bond) and the second band between 400 and 470 nm (typical of carotenoids). Meanwhile, the cis-crocin isomers showed three bands: two bands as previously described and a third band of medium intensity at 328 nm. This indicates that the amount of picrocrocin is affected by the concentration of cis and trans-crocins. Meanwhile, the safranal concentration obtained by UV-vis is not precise since cis-crocins interfere. In summary, overlapping causes quantification errors and limitations in this technique [57,90,96,100,101,102]. Another group of compounds that could interfere with saffron’s quality is the kaempferol derivatives, which absorb UV-vis light at 264 and 344 nm [88,103]. Moreover, safranal is slightly soluble in water and therefore the use of hexane and chloroform has been determined as the best strategy for the extraction and detection of adulterants [101,104].

Color, flavor, and odor are the quality parameters for saffron aqueous extract according to ISO 3632. They are determined by a non-specific spectrophotometric technique, albeit with limitations in assessing the authenticity of saffron. In the search for a more effective technique, liquid chromatography (LC) or HPLC have been proposed to separate and identify the components contained in a sample [89]. Various studies have described the identification and detection of saffron metabolites by HPLC including safranal, crocins, picrocrocin, and kaempferol and its derivatives [86]. For its part, a mass spectrometry (MS) detector coupled to HPLC and/or DAD could improve quantification [105,106], and MS/MS could facilitate the identification of compounds through structural elucidation [107]. The key quality parameter of saffron is color and the compound to which it is attributed is crocin, which must be quantified in order to determine the market price. For the qualitative and quantitative determinations of crocins, it is necessary to implement standards (quantification by internal and external standards) such as trans-4-GG-crocin (high price and questionable purity ~80%) [53,67,102].

The MS detector has been of considerable help since the lack of suppliers and the high prices of the standards make the structural elucidation (fragmentation patterns) of each crocin important (the different crocins can be identified by the number of hexoses and the molecular weight provided by the mass spectra) to compare them with the patterns in the scientific literature [102,107]. Crocin determination was carried out by Aghhavani et al. [28]; they determined no correlation between the color indexes obtained with spectrophotometry and HPLC data. They concluded that one could use the most accurate, easiest, and low-cost method depending on the experimental conditions to evaluate the quality of saffron. Rocchi et al. [68], demonstrated a poor correlation between the total crocin content (quantification) obtained by the ISO method and by UHPLC-MS/MS.

García-Rodríguez et al. [96] and Kabiri et al. [90] found that the quantification of safranal obtained by UV-vis does not correlate with HPLC data due to the interferences (overestimation by interference) generated by cis-crocetin esters and other compounds with λmax 330 nm. They also demonstrated that crocins interfere with picrocrocin and safranal, resulting in overestimates of the latter compounds in samples with large amounts of crocin. They concluded that semipreparative HPLC could represent an efficient method for the quantification of apocarotenoids. Similar results were presented by Moras et al. [106]; they reported that safranal content is more accurately calculated using UHPLC-DAD-MS because it is not influenced by the overestimation of safranal (with cis-crocetin esters at λmax 310–330 nm), which is shown when using the ISO methodology. They recommend determining, separating, identifying, and quantifying the metabolite content using the UHPLC-DAD-MS method as a unique and rapid analysis technique. Maggi et al. [104] and Bononi et al. [100] reported a null correlation between safranal content obtained by ISO 3632 and the GC method, as many other saffron substances display absorbance at a maximum of 330 nm.

For this reason, several instruments and analytical methods have been developed for saffron quality control, including chromatography, spectroscopy, molecular biology, and biomimetic techniques, with varying degrees of success and benefits [89]. HPLC is used to isolate, identify, quantify, purify, and determine the quality or adulteration; reverse-phase chromatography is widely used as it is capable of detecting compounds of different polarities and molecular masses [108]. Some authors have pointed out that HPLC-DAD is a selective, precise, sensitive, and specific technique that could evaluate the commercial quality of saffron [27,109].

In Table 1, the major commercial-quality compounds in saffron quantified by HPLC, are shown. The extractant solvents used in the investigations (Table 1) are polar and are in agreement with the descriptions by Rahaiee et al. (2015), who suggested that solvents such as water, ethanol, and pure methanol can be used but that mixtures would be more appropriate for the extractions of bioactive compounds [110]. For many authors, ethanol is the most suitable solvent (compared to methanol, ethyl acetate, diethyl ether, hexane, and/or water) for extracting metabolites from saffron stamens [111]. Meanwhile, Rahaiee et al. (2015) showed that an ethanolic extract obtained higher yields compared to water and methanol [66]. Similarly, this solvent was better than methanol for obtaining qualitative and quantitative data from saffron extracts. Meanwhile, Kyriakoudi et al. (2012) recommended the mixture of methanol: water (1:1, v/v) as a suitable solvent for industrial and analytical applications of saffron apocarotenoids [112]. Crocin isolation by solubility in a water–organic solvent mixture was tested by Zhang et al. (2004), who showed better results for methanol–water > ethanol–water > acetone–water extract [113]. Crocins are the most determined compound, followed by picrocrocin and safranal. In crocins, the ratios determined from highest to lowest were trans-4-GG, trans-3-Gg, cis-4-GG, trans-2-G, and trans-2-gg, respectively. An exception was Moratalla-López et al. [109], whose results did follow this relationship because the saffron samples used in their research were only of quality grade III. In general, ISO 3632 is used by researchers as a preliminary test. However, to perform the true quantification of saffron’s commercial-quality compounds, more precise spectroscopic techniques are used (HPLC, GC-MS, etc.).