Catechins HPLC separation

An RP-HPLC separation of CF together with catechins in green tea was developed by Goto et al. (280) and carried out on a Develosil ODS-HG column with a gradient water-ACN-phosphoric acid elution. The DLs were about 0.2 ng for the nine analytes. 

Coelution of procyanidins with cinnamics and flavonoids presented a major problem in quantitating the procyanidins. (Cinnamics and flavonoids could be measured in the presence of procyanidins by monitoring at 320 nm where procyanidins are nonabsorbing.) This was overcome with preliminary separation of compound classes with mini column gel filtration (4). Figure 4B shows an HPLC separation of apple juice procyanidins. Compounds absorbing at 320 nm which coeluted with catechin, epicatechin and the procyanidins have been eliminated by the cleanup procedure. The HPLC conditions (column, solvent gradient, etc.) are identical to those used for separation of cinnamics and flavonoids, the separation taking 60 min. 

Pear Phenolics. The same HPLC analytical system gave good resolution of pear juice phenolics and enabled peak assignments and quantitation to be made in a similar manner to that described for apple phenolics (3,7). It was also necessary to isolate the procyanidins and catechins using the Sephadex clean-up procedure (4) in order to measure their concentration. Figure 6A shows the HPLC separation of cinnamics, flavonols, and arbutin in pear juice while Figure 6B is a HPLC chromatogram of the pear juice procyanidin fraction. 

A slightly different RP-HPLC technology has been developed and applied for the determination of catechins in tea infusions. Tea infusions were prepared by the traditional method, — filtered and used for HPLC analysis. Separation was carried out in an ODS column... 

Another experiment was carried out by adjusting the pH of the water and then they were employed for the preparation of the tea infusion. HPLC resulted in the separation of more than 50 components in the broken black tea infusion as demonstrated in Fig. 2.69. Unfortunately, the majority of peaks have not been identified and the chromatographic profiles only indicate the presence of many compounds in the infusions. Some results are presented in Table 2.66. The measurement indicated that theaflavins and tea catechins play a decisive role in the formation of cream particles and tea colour [184],... 

Another study employed a similar RP-HPLC method for the determination of trails- and d.v-rcsvcratrol, catechin, epicatechin, quercetin and rutin in wines and musts. Wine samples were filtered and diluted when necessary and used for analysis without any other pretreatment. Separation was performed in an ODS column (150 X 4 mm i.d. paricle size 5 71m) at ambient temperature. The gradient began with ACN-5 per cent aqueous acetic acid (9 91, v/v) for 0-10 min to 25 75 in 1 min hold for 11 min to 70 30 in 1 min, hold for 5 min. The flow rate was 1 ml/min. Analytes were detected by DAD. Fluorescence detection used 280/315 nm (excitation/emission) for catechin and epicatechin 314/370 nm for fims-resveratrol and 260/370 nm for d.v-rcsvcratrol. Chromatograms of a red wine sample obtained at different... 

The technique of CPC was also employed as a key step in the purification of 26 phenolic compounds from the needles of Norway spruce (Picea abies, Pinaceae). An aqueous extract of needles (5.45 g) was separated with the solvent system CHCl3-Me0H-i-Pr0H-H20 (5 6 1 4), initially with the lower phase as mobile phase and then subsequently switching to the upper phase as mobile phase. Final purification of the constituent flavonol glycosides, stilbenes, and catechins was by gel filtration and semipreparative HPLC. °... 

The HPLC-FTIR technique has recently been used to identify six catechins and two methyl-xanthines present in green tea extracts." " A reversed-phase separation of the compounds was performed on a C-18 column equilibrated at 30°C using an isocratic mobile phase of acetonitrile-0.1% formic acid (15 85), prior to introduction to the deposition interface linked to the FTIR detector. The solvent was evaporated at 130°C and spectra were collected every 6 sec during the run. Two distinct designs for HPLC-FTIR interfaces have been developed flow cells and solvent elimination systems. Flow cell systems acquired spectra of the eluent in the solvent matrix through IR transparent, nonhydroscopic windows. The... 

For the red wines (82-84), which were injected directly into the HPLC without sample preparation, a ternary-gradient system using aqueous acetic acid (1% and 5% or 6%), and acidified acetonitrile (acetonitrile-acetic acid-water, 30 5 6) was used for cinnamic acid derivatives, catechins, flavonols, flavonol glycosides, and proanthocyanidins. Due to the large number of peaks, the gradient was extended to 150 min for the resolution of many peaks of important phenolics. This direct injection method was able to separate phenolic acids and esters, catechins, proanthocyanidins, flavonols, flavonol glycosides, and other compounds (such as tyrosol, and rrans-resveratrol) in wine in a single analysis. However, use of acetic acid did not permit the detector (PDA) to be used to record the UV spectra of phenolics below 240 nm (84). 

There are several caveats associated with this assay that may affect accuracy and precision. (+)-Catechin is the natural form in proanthocyanidins. Part of (+)-catechin epimerizes at the C2 position to form (+)-epicatechin during depolymerization. Similarly, part of (—)-epicatechin epimerizes to form (—)-catechin as an artifact. (+)-Catechin and (+)-epicatechin are an epimer pair in solution (similar to a- and 3-glucose in solution), i.e. they are chiral isomers that cannot be separated on a common reversed-phase HPLC column. The degree of epimerization increases with reaction temperature and time. Depolymerization at room temperature for 10 h caused less than 10% of flavan-3-ols to undergo epimerization. Toluene-a-thiol also causes the heterocyclic ring fission of flavan-3-ols to form adducts that... 

HPLC analysis can be performed by direct injection of the tartrate buffer extract. The chromatographic peaks of catechin and epicatechin are well resolved, but an overlapping of procyanidins and epicatechin gallate with other compounds, may occur. To improve the separation among them, a fractionation of the sample on a C18 cartridge can be performed 5 mL of extract are added of 15 mL H2S04 5 x 10 3M and passed... 

SPE on a CLX cartridge was applied to separate acidic phenols such as chlorogenic acid (95) from neutral phenols such as (—)-epicatechin (2), (+)-catechin (3), phloridzin (96) and quercitrin (100). The neutral phenols were determined in apple juice by capillary LC with UVD at 280 nm, as an alternative to conventional HPLC. LOD were from 9 pg for 96 to 97 pg for 3. HPLC analysis with MS and DA-UVD showed that apple pomace is a good potential source for phenoUcs. The usefulness of arbutin (9) as specific marker for pear products was placed in doubt (see Section II.A °). 

Figure 6.28. Fragmentation spectra of (epi)catechin-Pn-3-glu (M+ at m/z 751), Mv-3-glu-8-ethyl-(epi)catechin (M+ at m/z 809) and Mv-3-glu-8-vinyl(epi)catechin (M+ at m/z 805). (Reprinted from Journal of Mass Spectrometry, 41, Pati et al., Simultaneous separation and identification of oligomeric procyanidins and anthocyanin-derived pigments in raw red wine by HPLC-UV-ESI-MSn, p. 867, Copyright 2006, with permission from John Wiley Sons, Ltd.)...

Since the 1950s, many efforts have been made to isolate polyphenols directly from black tea, and the structures of the major phenolic compounds characteristic of fermented tea have been elucidated." ° However, the composition of black tea polyphenols is so complex that the minor phenolic substances, which cumulatively account for a substantial portion of black tea polyphenols, are difficult to separate even by high-performance liquid chromatography (HPLC). This difficulty associated with the purification is mainly due to the presence of uncharacterized substances that are detected as a broad hump on the baseline during HPLC analysis but do not produce any clear spots on thin-layer chromatography. These snbstances are probably a complex mixture of catechin oxidation products with higher molecular sizes, and usually account for the majority of black tea polyphenols. 

---