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The quantitative parameters and method performance for a normal-phase HPLC separation of flavanols and procyanidins in chocolate and cocoa-containing food products were optimized and assessed. Single laboratory method performance was examined over three months using three separate secondary standards. RSD(r) ranged from 1.9%, 4.5% to 9.0% for cocoa powder, liquor and chocolate samples containing 74.39, 15.47 and 1.87 mg/g flavanols and procyanidins, respectively. Accuracy was determined by comparison to the NIST Standard Reference Material 2384. Inter-lab assessment indicated that variability was quite low for seven different cocoa-containing samples, with a RSD(R) of less than 10% for the range of samples analyzed.

There has been growing interest in the potential cardiovascular benefits associated with cocoa consumption. As a result of accurate analytical methodologies, there is evidence to support that the flavanols in cocoa can be absorbed, are bioactive, and may be responsible for the cardiovascular benefits associated with regular cocoa consumption. The flavanols in cocoa exist in a multitude of different stereochemical configurations, thus giving rise to a unique and complex mixture of compounds. Given this complexity, the quantitative analysis of cocoa flavanols in foods can be challenging. While there are published methods suitable for the analysis of these compounds, these methods require sophisticated instrumentation and can be challenging to set up. As such, simpler techniques that measure such things as total phenolic content or antioxidant potential have been used as indicators of flavanol content. However, as these simpler assays are prone to interferences and are not specific for flavanols, these methods are not appropriate for use in studies that aim to examine the physiological effects of cocoa flavanols. It is only through the use of methods that can accurately quantify these flavanols that it will be possible to make meaningful dietary recommendations regarding the consumption of cocoa flavanol containing foods.

A new chromatographic approach for separating cocoa procyanidins according to their degree of polymerization (dp) was developed. The technique utilizes diol stationary phase columns operating in normal phase mode with a binary gradient of acidified acetonitrile and methanol-water. Performance of the diol stationary phase was evaluated on an analytical scale utilizing classical chromatographic conditions for the normal phase separation of procyanidins according to dp. The new separation approach was developed on an analytical scale but further extended to the preparative scale. These newly developed analytical and preparative HPLC procedures were successfully applied to the separation and isolation of cacao procyanidins from unfermented cocoa beans. The dp associated with each mol. wt. fraction was determined by MS.

Flavanols and their related oligomeric compounds, the procyanidins, have received increased attention during the past decade due to their reported health benefits. On the basis of compelling data published during the past decade demonstrating that the consumption of certain flavanol-rich foods can improve markers of cardiovascular health, additional clinical, and epidemiological research is clearly warranted to establish appropriate public health recommendations. However, recommendations on the consumption of these foods appropriate for use by health professionals can only be made on the basis of clinical investigations that accurately identify and quantify--through proper analytical measurement systems--the flavanols in the foods used in these investigations. This manuscript provides an overview of the strengths, weaknesses, and limitations of commonly used analytical methods to characterize the content of flavanols in foods. Two nonspecific measurements widely used by investigators, the Folin-Ciocalteu assay and the Oxygen Radical Absorbance Capacity (ORAC) measurement, are discussed in this context, as is the use of various high-performance liquid chromatography methods that provide more specific data related to the content of flavanols in foods. A comparison of the data obtained from these analytical methods to those of the more rigorous high-performance liquid chromatography analyses demonstrates that these nonspecific methods are ill-suited for providing unequivocal data necessary to evaluate the importance of dietary flavanols in the context of improving cardiovascular health. Meaningful dietary recommendations for the consumption of flavanol-rich foods will only be made possible by additional well-designed clinical and epidemiological studies enabled by detailed compositional data obtained through use of appropriate analytical methods.

Evidence suggesting that dietary polyphenols, flavanols, and proanthocyanidins in particular offer significant cardiovascular health benefits is rapidly increasing. Accordingly, reliable and accurate methods are needed to provide qualitative and quantitative food composition data necessary for high quality epidemiological and clinical research. Measurements for flavonoids and proanthocyanidins have employed a range of analytical techniques, with various colorimetric assays still being popular for estimating total polyphenolic content in foods and other biological samples despite advances made with more sophisticated analyses. More crudely, estimations of polyphenol content as well as antioxidant activity are also reported with values relating to radical scavenging activity. High-performance liquid chromatography (HPLC) is the method of choice for quantitative analysis of individual polyphenols such as flavanols and proanthocyanidins. Qualitative information regarding proanthocyanidin structure has been determined by chemical methods such as thiolysis and by HPLC-mass spectrometry (MS) techniques at present. The lack of appropriate standards is the single most important factor that limits the aforementioned analyses. However, with ever expanding research in the arena of flavanols, proanthocyanidins, and health and the importance of their future inclusion in food composition databases, the need for standards becomes more critical. At present, sufficiently well-characterized standard material is available for selective flavanols and proanthocyanidins, and construction of at least a limited food composition database is feasible.

Proanthocyanidins (PAs) have been shown to have potential health benefits. However, no data exist concerning their dietary intake. Therefore, PAs in common and infant foods from the U.S. were analyzed. On the bases of our data and those from the USDA's Continuing Survey of Food Intakes by Individuals (CSFII) of 1994-1996, the mean daily intake of PAs in the U.S. population (>2 y old) was estimated to be 57.7 mg/person. Monomers, dimers, trimers, and those above trimers contribute 7.1, 11.2, 7.8, and 73.9% of total PAs, respectively. The major sources of PAs in the American diet are apples (32.0%), followed by chocolate (17.9%) and grapes (17.8%). The 2- to 5-y-old age group (68.2 mg/person) and men >60 y old (70.8 mg/person) consume more PAs daily than other groups because they consume more fruit. The daily intake of PAs for 4- to 6-mo-old and 6- to 10-mo-old infants was estimated to be 1.3 mg and 26.9 mg, respectively, based on the recommendations of the American Academy of Pediatrics. This study supports the concept that PAs account for a major fraction of the total flavonoids ingested in Western diets.

The proanthocyanidins in three foods (pinto beans, plums and cinnamon) were studied with electrospray ionization (ESI) mass spectrometry (MS) in the negative mode following separation by normal-phase high-performance liquid chromatography. The MS/MS analysis demonstrated that the major ions derived from heterocyclic ring fission and retro-Diels-Alder reaction of flavan-3-ol provided information about the hydroxylation pattern and type of interflavan bond. The connection sequence of the oligomers was identified through diagnostic ions derived from quinone methide (QM) cleavage of the interflavan bond. Novel heterogeneous B-type proanthocyanidins containing (epi)afzelechin as subunits were identified in pinto beans. Proanthocyanidins with interestingly different A-type linkages were identified in plums and cinnamon. In efforts aimed at extending the identification capacity of ESI-MS to polymers, we found that the polymeric procyanidins fragmented readily instead of forming multiply charged ions in the negative ESI mode. Fragmentation patterns were proposed based on our data obtained by ESI-MS/MS and ESI time-of-flight MS.

A normal-phase HPLC-MS/MS method was applied to screen for proanthocyanidins in 88 different kinds of foods. Thirty-nine foods were found to contain proanthocyanidins. These foods include 19 kinds of fruits, eight cereals/beans, seven nuts, two beverages, two spices, and one vegetable. Twenty-five kinds of foods were found to contain both oligomeric (DP </= 10) and polymeric proanthocyanidins (DP > 10), and the other 14 foods contained only oligomers. Procyanidins with B-type linkages were detected as the only components in 21 foods and also as principal components in the others. Propelargonidins were identified in pinto bean, raspberry, strawberry, and almond, etc. Plum, avocado, peanut, curry, and cinnamon were identified as potential sources of A-type proanthocyanidins in addition to cranberry. Thiolytic degradation and MS/MS analyses indicated that the A-type linkages are present as a terminal unit in plum or between the extension units in curry, cinnamon, and avocado, whereas A-type linkages exist at both positions in cranberry and peanut.

We report an improved synthesis of bis(5,7,3',4'-tetra-O-benzyl)epicatechin 4beta,8-dimer (3) from 5,7,3',4'-tetra-O-benzylepicatechin (1) and 5,7,3',4'-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin (2) by replacing the previously employed Lewis acid, titanium tetrachloride, with the clay mineral Bentonite K-10. Under the same conditions, the benzyl-protected all-4beta,8-trimer, -tetramer, and -pentamer were obtained regioselectively from their lower homologues, albeit in rapidly decreasing yields. Reaction of 2 with an organoaluminum thiolate generated from 2-mercaptobenzothiazole and trimethylaluminum followed by acetylation produced 3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3',4'-tetra-O-benzylepicatechin (12). Medium-sized protected oligomers with 4beta,8-interflavan linkages are obtained in improved yields by using this compound as the electrophile and silver tetrafluoroborate as activator and are isolated by reversed-phase HPLC. Their deprotection by ester saponification followed by hydrogenolysis yielded the free procyanidins, which were characterized as their peracetates. The synthetic procyanidins are identical by normal-phase HPLC with fractions isolated from cocoa. The principle of chain extension by two members was demonstrated using a dimeric electrophile obtained by self-condensation of compound 12. Both the synthetic and natural pentamer 32 inhibit the growth of several breast cancer cell lines. Using the MDA MB 231 line, it was established that this outcome is based on the induction of cell cycle arrest in the G0/G1 phase. Subsequent cell death is more likely necrotic rather than apoptotic. Control experiments demonstrate that the polyphenol itself, rather than hydrogen peroxide potentially formed by its autoxidation, is the causative agent.

The polymeric procyanidins were fractionated from lowbush blueberry on a Sephadex LH-20 column. The degree of polymerization (DP) for the polymers was determined by thiolysis to be in a range of 19.9 to 114.1. Normal-phase HPLC analysis indicated that the polymeric procyanidins did not contain oligomeric procyanidins with DP < 10. The polymers eluted as a single peak at the end of the chromatogram. The normal-phase HPLC gradient was modified to improve the separation of procyanidin monomers through decamers and to elute all the polymers beyond those as a distinct peak. Monomers through decamers were quantified individually. All the polymers (DP > 10) were quantified using a mixture of purified polymers as an external standard. Polymers were found to be the dominant procyanidins in brown sorghum bran, cranberry, and blueberry. Thiolysis of the polymer peaks indicated that epicatechin was present as extension units in these foods, however, the composition of terminal units varied considerably between catechin and epicatechin, or an A-type dimer linkage in the case of cranberry.

Blueberries and cranberries were analyzed for procyanidins using normal-phase HPLC/MS. Monomers, identified as (+)-catechin and (-)-epicatechin, and a series of oligomers were detected in blueberries, and MS data confirmed that the oligomers consisted of (epi)catechin units that were exclusively singly linked (B-type). The procyanidin "fingerprints" were similar for Tifblue and Rubel but higher than that for lowbush blueberries. In whole cranberries, (-)-epicatechin was present, along with a complex series of oligomers. Both A-type (contained only one double linkage per oligomer) and B-type oligomers were present. Two commercial cranberry juices exhibited similar procyanidin profiles, except that one contained increased quantities. There were processing effects on the procyanidin content of cranberry extract and juices when compared to those of the unprocessed fruits. Monomer, dimers, and A-type trimers were the primary procyanidins, with only trace levels of the B-type trimers and A-type tetramers and with an absence of the higher oligomers in cranberry extract and juices.

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The assignment of interflavan bond regio- and stereochemistry in oligomeric proanthocyanidins has in the past relied on empirical spectroscopic techniques which are influenced by the conformation of the C rings. Only recently was the 4,8-regiochemistry of procyanidin B2 (3b) firmly established by 2-dimensional NMR methods. We describe herein the proof of 4beta-stereochemistry in 3b by oxidative degradation of the derivative 3d bearing differential (O-benzyl and O-methyl) protecting groups in its "top" and "bottom" epicatechin moieties, to (R)-(-)-2,4-diphenylbutyric acid. The key elements of the degradative process are (1) removal of the C-3 alcohol functions through a modified Barton deoxygenation employing hypophosphorous acid as the reducing agent; (2) deprotection of the "top" unit by hydrogenolysis, followed by exhaustive aryl triflate formation with N,N-bis(trifluoromethanesulfonyl)aniline and DBU in DMF; (3) hydrogenolytic deoxygenation of the "top" unit over Pearlman's catalyst with concomitant scission of the O-C2 bond; (4) selective oxidation of the "bottom" unit with NaIO4/RuCl3. The hitherto unreported absolute configuration of (-)-2,4-diphenylbutyric acid was established as R by X-ray crystal structure analysis of the (R)-(+)-alpha-methylbenzylamine salt. As a corollary, the selectivity of hydrogenolytic and solvolytic reactions of epicatechin-derived tetrasulfonates has been investigated.

Procyanidins are a subclass of flavonoids found in commonly consumed foods that have attracted increasing attention due to their potential health benefits. However, little is known regarding their dietary intake levels because detailed quantitative information on the procyanidin profiles present in many food products is lacking. Therefore, the procyanidin content of red wine, chocolate, cranberry juice and four varieties of apples has been determined. On average, chocolate and apples contained the largest procyanidin content per serving (164.7 and 147.1 mg, respectively) compared with red wine and cranberry juice (22.0 and 31.9 mg, respectively). However, the procyanidin content varied greatly between apple samples (12.3-252.4 mg/serving) with the highest amounts on average observed for the Red Delicious (207.7 mg/serving) and Granny Smith (183.3 mg/serving) varieties and the lowest amounts in the Golden Delicious (92.5 mg/serving) and McIntosh (105.0 mg/serving) varieties. The compositional data reported herein are important for the initial understanding of which foods contribute most to the dietary intake of procyanidins and may be used to compile a database necessary to infer epidemiological relationships to health and disease.

Monomeric and oligomeric procyanidins present in cocoa liquors and chocolates were separated and quantified in four different laboratories using a normal-phase high-performance liquid chromatography (HPLC) method with fluorescence detection. Procyanidin standards through decamers were obtained by extraction from cocoa beans, enrichment by Sephadex LH-20 gel permeation chromatography, and final purification by preparative normal-phase HPLC. The purity of each oligomeric fraction was assessed using HPLC coupled to mass spectrometry. A composite standard was then prepared, and calibration curves were generated for each oligomeric class using a quadratic fit of area sum versus concentration. Results obtained by each of the laboratories were in close agreement, which suggests this method is reliable and reproducible for quantification of procyanidins. Furthermore, the procyanidin content of the samples was correlated to the antioxidant capacity measured using the ORAC assay as an indicator for potential biological activity.

Monomeric and oligomeric proanthocyanidins present in a range of plant-derived foods and beverages were separated by degree of polymerization and identified using a modified normal-phase high-performance liquid chromatography (HPLC) method coupled with on-line mass spectrometry (MS) analysis using an atmospheric pressure ionization electrospray chamber. In addition, ultraviolet (UV) and fluorescence detection were used to monitor the separation of proanthocyanidins, with fluorescence detection demonstrating both increased sensitivity and the ability to reduce interfering signals from other components present in the food and beverage matrices as compared to UV detection. This qualitative study demonstrates the ability of this HPLC/MS technique to separate singly and doubly linked procyanidins, prodelphinidins, and copolymer oligomers, including their galloylated derivatives, present in a range of food and beverage samples.

Monomeric and oligomeric procyanidins present in cocoa and chocolate were separated and identified using a modified normal-phase high-performance liquid chromatography (HPLC) method coupled with on-line mass spectrometry (MS) analysis using an atmospheric pressure ionization electrospray chamber. The chromatographic separation was achieved using a silica stationary phase in combination with a gradient ascending in polarity. This qualitative report confirms the presence of a complex series of procyanidins in raw cocoa and certain chocolates using HPLC/MS techniques. Although both cocoa and chocolate contained monomeric and oligomeric procyanidin units 2-10, only use of negative mode provided MS data for the higher oligomers (i.e., >pentamer). Application of this method for qualitative analysis of proanthocyanidins in other food products and confirmation of this method as a reliable quantitative tool for determining levels of procyanidins in cocoa, chocolate, and other food products are currently being investigated.