Procyanidins sample extraction

Reversed-phase HPLC is used for the analysis of the different groups of phenols, phenolic acids, hydroxycinnamic acids, flavonoids, and procyanidins in grapes and wines (22,46,47,77-80). However, due to the presence of a large quantities of various compounds, wine analysis is difficult. Thus, different sample preparation procedures, including fractionation and extraction, are often applied when various groups of phenolic compounds are studied together. 

Subjects suffering from coronary artery disease were given two capsules containing 300 mg of grape procyanidin extracts (Leucoselect-phytosome) or placebo daily for 5 days. TAC of their blood serum was reported to increase on day 5 from 408.1 22.9 to 453.3 453.3 /u.M) however, samples were taken 1 hr postdose and most probably the results indicate a transient rather than a permanent effect (N10). In a 6-month randomized controlled intervention study, subjects with moderately increased cardiovascular risk factors (1) adhered to an advised diet, (2) performed controlled moderate exercise, (3) were subjected to both diet and exercise regime, or (4) were not subjected to any intervention. No significant alteration in blood serum TAC was observed in any group tested (R22). 

Figure 8.8 Chromatograms of procyanidins extracted from cranberries (A) and Hi-tannin sorghum (B). Excitation and emission wavelength were 210 nm and 321 nm using fluorescent detector. Sample was run on a Develosil Diol 100 A column (250 x 4.6 mm, 5 pm particle size) using gradient in Table 8.3. Numbers above the peak denote the DP values of procyanidins in the peaks.

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... 

Figure 2.20 Chromatogram relative to analysis of dimer procyanidins in the skins extract (fraction ethyl acetate from C18 cartridge) (sample volume injected 10p,L). 1. procyanidin Bl, 2. procyanidin B3, 3. procyanidin B2...

The analysis of procyanidins starts at the point of sample collection. Care should be taken not to damage the plant material when collecting it. Any disruption of membrane integrity increases the risk of oxidation reactions and the formation of complexes with biomolecules such as proteins. Consequently an alteration in extraction efficiency and chemical composition of the samples is inevitable [45-46],... 

One of the most critical steps in the analysis of procyanidins is their extraction. Preservation procedures and storage conditions of the crude samples as well as procedures and conditions during homogenization and extraction have a tremendous impact on the amount and composition of the extractable procyanidins. In view of a good reproducibility a complete standardization of all procedures and experimental conditions from the time of sample collection to the storage of the final extract must be emphasized. It is recommended to analyze fresh samples. If this is not possible, lyophilization is the drying procedure of choice. Samples should not be stored at all. If this can not be avoided care must be taken to exclude... 

The oligomeric procyanidin content of samples using the DAC protocol averaged 0.89 % (triplicate determinations). HPLC analyses of samples revealed only trace amounts of cyanidin although solutions were intensely red-colored (contents in the plant material calculated as cyanidin amounted to 0.0015 %). Addition of cyanidin to the extract solutions (spike levels 2.5 and 5 times delected concentrations) and subsequent conductance of the assay led to recoveries of 48 % and 85 %, respectively. HPLC conditions Hypersil ODS, 5 pm, 100 x 4.0 mm 70 % methanol in 0.5 % aqueous orr/to-phosphoric acid flow 1.0 ml/min temperature 25.0 °C injection volume 5 pi detection 540 nm and 280 nm retention time of cyanidin 4.2 minutes. 

Calibration factors are most often used for the quantitation of results. For the original method (see Tab. (3.1)) a specific absorbance Al% of 150 is proposed, if the degree of polymerization of the procyanidins under analysis is not known [130]. For the Porter s reagent (see Tab. (3.1)) the use of a specific absorbance A10/0 of 270 for dimers and 470 for higher oligomers has become common practice [55,129]. These values are averages of measured absorbance data of purified procyanidin standards originally determined by Porter et al. [113]. In pharmaceutical standardization procedures "plant-specific" calibration factors are also used [120]. These factors are determined for a specified plant extract and may only be used for quantitation of samples of this same matrix [131]. 

Solid phase extraction is the method of choice for sample clean up in the analysis of procyanidins. Using this technology procyanidins are selectively and reversibly bound to a solid stationary phase. Because of their highly polar nature and their ability to form multiple hydrogen bonds... 

Reversed-phase HPLC is the method of choice for the quantitative analysis of procyanidins. In view of the known instability of procyanidins and the problem of developing suitable sample clean up procedures direct analysis of crude extracts is probably the best approach for quantitation. However, the separation capacity of HPLC in combination with the most commonly used UV detection at 280 nm is generally insufficient to generate useful quantitative results. Direct chromatographic determination of procyanidins in qualitative analysis has been frequently performed for example in the analysis of wine [168,252], beer [32], grape seeds [28], rhizomes of tormentil (Potentilla tormentilla) [253], Sesbania sesban... 

In order to determine the procyanidins and alkaloids in cocoa and their metabo-Ules in biological samples, a sample has to be pretreated prior to chromatographic analysis to extract the target compounds and clean up the sample matrix. For the analysis of biological samples, it is very important to concentrate the generated procyanidin and alkaloid metaboUles, as these are present at low concentrations in these complex matrices. In addition, cleaning up the cocoa and biological samples is essential in order to ranove the interference compounds, as these could suppress the ionization. 

For the analysis of cocoa samples, the beans were ground using a mortar and pestle [27] or a laboratory mill [28,29] to obtain a homogeneous material. Then, to analyze procyanidins and alkaloids, these were first defatted between three and four times with n-hexane to remove the fat from the cocoa matrix. Second, an off-line liquid-liquid extraction (LLE) was performed to extract the procyanidins and alkaloids from this matrix. Various solutions formed by acetone were used as the extraction solutions. These were acetone/water (70/30, v/v) [27], acetone/water/acetic acid (70/28/2, v/v/v) [18], and acetone/water/acetic acid (70/29.5/0.5, v/v/v) [28,29] (see Table 14.1). Afterwards, the extracts were sonicated [18] or vortexed [28,29] and centrifuged. The supernatants were combined from the centrifuged tubes and filtered with glass wool [28,27]. Finally, the organic solvent of these extracts was removed by rotary evaporation at a temperature between 30°C and 40°C. 

This sample pretreated is the only one reported in the literature for determining procyanidins in cocoa samples. This is tedious and uses a large volume of solvent, both n-hexane and acetone, and takes a long time to extract these target compounds. This time required for defatting and extracting is much higher than in UHPLC separations. Normally, the use of this cocoa pretreatment requires 1 or 2 h or more to extract the compounds, while the UHPLC separations only require a few minutes. Further studies should be carried out in order to improve the cocoa sample pretreatment for the analysis of procyanidins. 

The sample pretreatment used to determine procyanidins and their metabolites in plasma samples (from rats) is off-line solid-phase extraction (SPE) with hydrophilic-lipophilic balanced (HLB) copolymer. Both cartridges (60 mg) [31,32] and microelution SPE plates (pSPEs) (2 mg) [33,36,37] have been used as the device format. For the analysis of tissues (also from rats), such as the liver, brain, aorta artery, and adipose tissue, these were first freeze-dried and then pretreated by off-line Ll.F. (with the solution made up of methanol, water, and phosphoric acid) followed by pSPE [35-37]. The use of two sample pretreatments (LLE and pSPE) is due to the complexity of the tissue sample compared with the biofluid plasma sample. The extraction recoveries (%Rs) for determining the procyanidins catechin, epicatechin, dimer B2, and trimer in the plasma samples by pSPE [33] and SPE [31] were higher... 

This sample pretreatment dilution with ultracentrifugation devices [40] and pSPE [33] allows the procyanidins and alkaloids to be extracted from the biological matrix in a short analysis time of less than 15 min. Until now, these two sample pretreatments have only been reported in UHPLC. 

The following sections examine the chromatographic UHPLC separations and MS conditions used to determine procyanidins and alkaloids in cocoa samples and also procyanidins, alkaloids, and their metabolites in biological samples obtained after procyanidin-rich or alkaloid-rich extract consumptions. Tables 14.1 and 14.2 show a... 

Procyanidin Extract Grape pomace Dose (g/kg) 1 Biological Sample Rat adipose tissue Time after Consumption 4h Sample Pretreatment Off-line LLE-pSPE... 

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