Anthocyanidins conditions

Although chromatographic conditions are extremely variable and widely discussed in the literature, broad trends may nonetheless be perceived. A binary solvent such as acidified water/acidified methanol or acidified water/acidified acetonitrile is primarily used because acidification of the solvent converts nearly all anthocyanidins... 

Proanthocyanidins are so named because under oxidative and acidic conditions they are converted to anthocyanidins, a subclass of flavonoid (for general structure, see Fig. 11.3.4). Historically, they have also been referred to as leucoanthocyanidins, condensed tannins, or simply tannins because of their ability to fix or tan leather hides. 

Condensed tannins are also referred to as proanthocyanidins. They are oligomeric or polymeric flavonoids consisting of flavan-3-ol (catechin) units. Hydrolysis under harsh conditions, such as heating in acid, yields anthocyanidins. An example of a condensed tannin is procyanidin B2 (epicatechin-(4 3—>8 )-epicatechin 1.90). In this case the interflavanyl linkage is between C4 of the lower unit, and C8 of the upper unit. The linkage can also be between C4 of one unit and C6 of the second unit. 

Anthocyanins are generally more stable at an acidic pH. Therefore, anthocyanins are commonly extracted under cold conditions using either acidic methanol or ethanol to avoid degradation1 5169 (Table 3.4). In comparison, acetone allows more reproducible extraction and avoids problems with pectins. However, it is limited by the coextraction of proanthocyanins.39 In general, ethanol is preferable as an extraction solvent, although it can require an additional step for the removal of lipid-soluble substances. SPE using Ci8, polyamide, HLB (hydrophilic lipophilic balanced stationary phases), or Amberlite has been employed for the purification of anthocyanidins prior to HPLC analysis.39-51 66 69... 

Flavanol oligomers and polymers are also called condensed tannins or proan-thocyanidins. The term tannin refers to their capacity to interact or react with proteins and precipitate them out. When heated under acidic conditions, these molecules release red anthocyanidin pigments, hence the term proanthocyanidins. The term leucoanthocyanidin, also referring to this particular property, is sometimes encountered in the literature. However, this should be restricted to another group of compounds, flavan 3,4-diols, which are intermediates in the biosynthetic pathway leading to flavanols and anthocyanins (Stafford and Lester 1984 Nakajima et al. 2001 Abrahams et al. 2003) but have never been isolated from grapes, presumably due to their instability. 

A detailed study of the effects of pH on anthocyanin stability has been presented by Cabrita et al. (2000). Buffered solutions in the range of pH 1-12 of the 3-glucosides of the six common anthocyanidins were stored in the dark over a 60-day period at 10°C and 23°C. Under strong acidic conditions (pH 1-3), more than 70% of the initial concentration remained after 60 days at 10°C for all anthocyanins, while considerable losses (>90%) occurred at pH 5-6 even after 8 days. Similar stability patterns occurred at the higher temperature of 23°C, although the rates of anthocyanin degradation were higher, and only 40% of the initial anthocyanins were detectable after 60 days. 

Figure 6.14. Positive-ion mode fragmentation patterns of trimeric procyanidins. The ESI-MS conditions spray voltage 4.5 kV sheath gas nitrogen 0.9L/min capillary voltage 35 V capillary temperature 200 °C tube lens offset voltage 15 V. (Reprinted from Pati et al., 2006, Simultaneous separation and identification of oligomeric procyanidins and anthocyanidins-derived pigments in raw red wine by HPLC-UV-ESI-MSn, Journal of Mass Spectrometry, 41, p. 869, with permission from John Wiley Sons, Ltd.)...

Treatment of procyanidin dimers with acid gives the appropriate catechin unit corresponding to the lower half of the molecule and a carbocation that normally collapses to cyanidin (an anthocyanidin) under the reaction conditions. 

Anthocyanins and anthocyanidins are stable as chloride salts in solid state preferably stored at frozen conditions. Berry anthocyanins are excellent soluble in water and polar organic solvents. In case of anthocyanidins, water solubility is limited therefore polar organic solvents (ethanol, methanol or acetone) are preferred solvents for anthocyanidins and anthocyanins. 

Under routine conditions, enrichment and isolation of single anthocyanins followed by classical structural elucidation (mass spectrometry, elemental analysis, UVA IS absorbance evaluation using shift reagents, NMR, thin layer chromatography of sugars and anthocyanidins) is rarely necessary, hence will not be discussed in detail. Routine analysis focuses on the quantification and the distribution of known anthocyanins to confirm the content and source of anthocyanins. 

Treatment of anthocyanins imder alkaline conditions (10 % KOH for few minutes at room temperature) as described for the selective removal of acyl-groups and of sugars (positions 7 and 4") is suitable for structural elucidation but not for quantification of anthocyanidins [90]. 

---