Identification of procyanidins in cocoa (Theobroma cacao) and chocolate using high-performance liquid chromatography/mass spectrometry

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.     

Prediction of Soil Cadmium Bioavailability to Cacao (Theobroma cacao L.) using Single-Step Extraction Procedures

In this study, complexation extractants ammonium bicarbonate diethylene triamine pentaacetic acid (AB-DTPA), diethylene triamine pentaacetic acid (DTPA), and ethylene diamine tetraacetic acid (EDTA) and mild cation-exchange extractants calcium chloride (CaCl₂) and ammonium nitrate (NH₄NO₃) were used to evaluate the bioavailability of soil cadmium (Cd) to cacao in the field. Among the five extractants, the extractable Cd generally followed the order EDTA > DTPA > AB-DTPA > CaCl₂ > NH₄NO₃. Correlation analysis was done between the extractable Cd in soil and total Cd content of cacao tissues (nibs, shells, leaves, and pods). The Cd extracted by CaCl₂ and NH₄NO₃ was significantly (P < 0.05) correlated with some of the tissues but their Pearson correlation coefficients were weak. In contrast, extractants AB-DTPA, DTPA, and EDTA showed stronger, significant correlations to the Cd concentration in all four tissues. Overall, regression analysis demonstrated that AB-DTPA, DTPA, or EDTA can be used to predict bioavailable Cd in soils for cacao. Of these, AB-DTPA and DTPA both showed the strongest correlations compared to EDTA. However, the ease of preparation and the superior shelf-life of DTPA over AB-DPTA make it the preferred reagent for Cd bioavailability extractions from cacao soils and is currently being used to develop cost-effective soil treatments to reduce bioavailable Cd to cacao plants.     

Allelic Size Standards and Reference Genotypes to Unify International Cocoa (Theobroma cacao L.) Microsatellite Data

Standardisation of microsatellite allele profiles between laboratories is of fundamental importance to the transferability of genetic fingerprint data and the identification of clonal individuals held at multiple sites. Here we describe two methods of standardisation applied to the microsatellite fingerprinting of 429 Theobroma cacao L. trees representing 345 accessions held in the worlds largest Cocoa Intermediate Quarantine facility: the use of a partial allelic ladder through the production of 46 cloned and sequenced allelic standards (AJ748464 to AJ48509), and the use of standard genotypes selected to display a diverse allelic range. Until now a lack of accurate and transferable identification information has impeded efforts to genetically improve the cocoa crop. To address this need, a global initiative to fingerprint all international cocoa germplasm collections using a common set of 15 microsatellite markers is in progress. Data reported here have been deposited with the International Cocoa Germplasm Database and form the basis of a searchable resource for clonal identification. To our knowledge, this is the first quarantine facility to be completely genotyped using microsatellite markers for the purpose of quality control and clonal identification. Implications of the results for retrospective tracking of labelling errors are briefly explored.     

Conservation and use of genetic resources of cacao (Theobroma cacao L.) by gene banks and nurseries in six Latin American countries

Genetic Resources and Crop Evolution - Cacao (Theobroma cacao L.) is among the most important cash crops in tropical countries. The existing cacao genetic diversity represents a key resource to...     

Effect of fertilizer and endomycorrhizal inoculum on growth and nutrient uptake of cocoa (Theobroma cacao L.) seedlings

Summary A greenhouse experiment was carried out to evaluate the influence of vesicular-arbuscular mycorrhiza (VAM) on growth and nutrient uptake of cocoa seedlings treated with five levels of palm oil mill effluent, in an unsterilized Oxisol and an Ultisol, either with or without addition of the VAM fungus Scutellospora calospora (Nicol. & Gred.) Walker and Sanders. Inoculation with the VAM fungi significantly increased nutrient uptake and plant growth in both soils. The dry matter yield, and the tissue N and K concentration in the plant tops increased significantly with increasing levels of palm oil mill effluent applied to both the Oxisol and the Ultisol. The maximum tissue P concentration, however, was obtained from plants grown in the Ultisol that was given 50.0 g palm oil mill effluent per kg while the maximum P recovery of 26% was obtained from plants given only 16.7 g effluent per kg. Overall, the percentage of P recovery decreased with the addition of increasing levels of palm oil mill effluent. In the Oxisol, the tissue P concentration increased with the addition of increasing levels of palm oil mill effluent, but the maximum recovery of P was recorded from plants given only 0.3 g effluent per kg. The percentage P recovery decreased with subsequent additions of the effluent.     

Identification of procyanidins and anthocyanins in blueberries and cranberries (Vaccinium spp.) using high-performance liquid chromatography/mass spectrometry

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.     

Genetic diversity and parentage in farmer varieties of cacao (Theobroma cacao L.) from Honduras and Nicaragua as revealed by single nucleotide polymorphism (SNP) markers

Cacao (Theobroma cacao L.) is the main source for chocolate with an annual production of four million tons worldwide. This Neotropical tree crop was domesticated in Mesoamerica as far back as 3,000 years ago. Knowledge of genetic diversity and population structure in farmer varieties of cacao in the center of domestication is essential for sustainable production of fine-flavored cacao beans and contributes to in situ/on-farm conservation of farmer varieties. Based on 70 single nucleotide polymorphism markers, we analyzed 84 fine-flavored farmer varieties collected from traditional cacao farms in Honduras and Nicaragua. The study also included 31 clones from the international cacao collections to serve as references. The SNP based multilocus matching identified six synonymous groups, including 14 Criollo and two Amelonado varieties. A moderately high level of genetic diversity was observed in these farmer varieties, indicating the possibility to further explore intra-population variation and breed for fine-flavored cocoa. Multivariate analysis showed clustering of the 84 farmer accessions in five genetic groups: ancient Criollo, Amelonado, Trinitario (including Nicaragua Trinitario and Honduras Trinitario) and Upper Amazon Forastero (only one accession). The Honduras Trinitario differed from the Nicaragua Trinitario group. The clustering results largely supported the perceived classification of cacao by local farmers and researchers, which was mainly based on morphological traits. However, the well known traditional variety “Indio” in this region was identified as synonymous with Amelonado. Parentage analysis showed that the variety “Indio” (or Amelonado) contributed more to the Trinitario type farmer varieties, whereas ancient Criollo had less influence. The present study demonstrates the efficacy of using a small set of SNP makers for cacao germplasm characterization, and further depicts the diverse origins and parentage in farmer varieties from Mesoamerica. This information thus will be highly useful for conservation and utilization of cacao germplasm from this region.     

High-performance liquid chromatography analysis of black currant (Ribes nigrum L.) fruit phenolics grown either conventionally or organically

Black currants (Ribes nigrum L.) contain a diverse range of phenolics and possess a high antioxidant activity, which makes them an interesting target for the functional food industry. In this study, phenolic profiles of organically and conventionally grown black currant fruits, collected from commercial farms within a climatically similar area, were compared. Compounds were identified using UV/vis and mass spectroscopy techniques and quantified with high-performance liquid chromatography equipped with UV/vis detection. Several different conjugates of hydroxycinnamic acids, flavonols, and anthocyanins were quantified. Statistically significant differences between farms were found for almost all compounds. Differences between the highest and the lowest measured values of major phenolic compounds of different phenolic classes ranged from 24 to 77%. Principal component analysis quite effectively separated farms from each other but did not cluster them according to cultivation technique. Thus, it was concluded that the biochemical quality of organically grown black currant fruits does not differ from those grown conventionally.     

The Criollo cacao tree (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.): a review

In commercial terms, Criollo cacao trees (Theobroma cacao L.) are reputed to be the source of the commercial product (fermented and dried cocoa beans), which sells for the best price on the market. Nevertheless, the term “Criollo” has numerous meanings and interpretations depending on if it is used by commercial users or botanists, growers or breeders. Our review aims to specify which cocoas can justifiably carry the Criollo name. “Criollo” is a botanical subspecies of Theobroma cacao, i.e. Theobroma cacao subsp. cacao; however, the true Criollos form just one of the ten currently accepted genetic groups in the species. We thus provide an overview of genetic studies on the subject (published or not), along with what is currently known about “True Criollo” or “Ancient Criollo” cacao trees. In fact, there are few representatives in collections that are duly acknowledged to be true Criollos, particularly in the two International Cocoa Genebanks, where only seven clones are available. It is nonetheless certain that some true Criollos do exist in other collections but have not been formally identified (by genetic studies) as members of the Criollo genetic group. Likewise, some true Criollos, be they cultivated or subspontaneous, exist in Mexico and Central and South America (Venezuela and Colombia). However, certain clones called “modern Criollos”, which are closely related to the true Criollos but arise from hybridization with other genetic groups, are more common.     

Effect of zinc application on the dry matter yield uptake and distribution of zinc and other micronutrients in cocoa (Theobroma cacao. L.)

Abstract

The effects of application of zinc fertilizer on dry matter yield, uptake and distribution of zinc and other nutrients by Amazon and Amelonado cocoa cultivars grown in a soil of low zinc content in the greenhouse were investigated.

There was a differential response to zinc fertilization by the two cocoa cultivars. Maximum dry matter yields of Amazon and Amelonado were attained with 10 ppm Zn and 50 ppm Zn, respectively. Under similar experimental conditions Amelonado seedlings expressed zinc deficiency symptoms whereas Amazon did not.

Zinc concentrations in the leaves, stem and roots of both cuitivars did not give a good index of the zinc status of the crops. This was because of the existence of the “Piper‐Steenbjerg”; effect in that nil zinc rates often gave higher leaf concentration of the zinc than next higher rate. In general, the relative content of zinc followed the pattern; leaves > roots > stem with the Amazon cultivar containing more zinc than the Amelonado.

The distribution of absorbed Cu in the leaves, stem and roots did not differ in both varieties. Whereas Fe uptake was mostly concentrated in the roots, Mn absorbed was largely concentrated in the leaves of both varieties and only Mn uptake in the leaves of Amazon consistently increased with Zn application.

The differences in the uptake and distribution of nutrients between the two cocoa cultivars was attributed to differences in their ability to extract nutrients from the soil and in their requirements for metabolic processes.     

Quantitative analysis of polymeric procyanidins (Tannins) from grape (Vitis vinifera) seeds by reverse phase high-performance liquid chromatography

A reverse phase C(18) HPLC method with potential for high automated throughput has been developed for the quantitative analysis of polymeric procyanidins (tannins) in grape seed extracts. Chromatography gave rise to 13 distinct UV-absorbing peaks with good baseline separation. The UV-absorbing peak eluting last is distinct and therefore easily quantified. Biochemical analyses including ultrafiltration, protein precipitation, and Sephadex LH20 chromatography combined with electrospray mass spectrometric analyses establish that this peak predominantly contains polymeric procyanidins. The polymers, which appear to be galloylated to various degrees and seem to fragment in a characteristic manner during electrospray mass spectrometry, are well separated from catechins and procyanidin oligomers of up to 4 units. The recovery of polymeric grape seed tannins with this HPLC method was 86%, which is similar to the 89% recovery achieved with commercial quebracho tannins. The concentration of tannins in seeds from ripe Vitis vinifera cv. Shiraz grapes ranged from 1360 to 2830 mg/kg of berries.     

Resolution and analysis of enantiomers of amphetamines by liquid chromatography on a chiral stationary phase: collaborative study

A rapid, accurate method for separating and determining the enantiomeric composition of amphetamine bulk drug and commercial preparations was developed and subjected to collaborative study. Amide derivatives of the amphetamine enantiomers are formed by using achiral 2-naphthoyl chloride. The resulting enantiomeric amides are then chromatographed on a commercially available chiral stationary phase with hexane-isopropyl alcohol-acetonitrile (97 + 3 + 0.5) mobile phase, with detection at 254 nm. Seven collaborators received bulk drug and commercial samples of amphetamine. The collaborators and authors determined the mean percent l- and d-amphetamine from 2 injections of each sample. The method can detect the presence of as little as 0.5% of the l-enantiomer in d-amphetamine, with reproducibility between laboratories of +/- 71.3%. The method has been adopted official first action for determination of the enantiomeric composition of amphetamine bulk drug and preparations.     

Analysis of nitrate ion in nettle (Urtica dioica L.) by ion-pair chromatographic method on a C30 stationary phase

Nitrate ion is a frequent pollutant not only in soil and natural water resources but in vegetables and foods as well. In our study we focused on nettle due to its increased ability to accumulate nitrate ions. A new, simple method for the separation and determination of nitrate ion based on reversed-phase ion-pair chromatography has been elaborated. A new four-step sample pretreatment method enables the precipitation of proteins and oxidative degradation of compounds that may disturb the identification of the nitrate ion: (1) extraction of the total nitrate content, (2) precipitation of proteins with acetonitrile, (3) oxidative degradation of the organic contaminants with H2O2, (4) evaporation of the solvent and taking up of the residue in water. The chromatographic separations were carried out on a high-density C30 stationary phase under isocratic conditions. The optimal mobile-phase composition was 10% (v/v) acetonitrile and 90% (v/v) 20 mmol L(-1) phosphate buffer, containing 2 mmol of tetrabutylammonium hydroxide at pH 6.0. The method could also be used for the separation of IO3(-), SeO3(2-), BrO3(-), NO2(-), Br-, SeO4(2-), and I- ions. The validated method is sensitive (the detection limit is 0.18 ng of nitrate ion). The method is linear in a high concentration range (0.031-30.66 microg mL(-1)). Recoveries varied between 98% and 103%. Reproducibility of the elaborated sample pretreatment method showed 1.54%. The method can be used for the determination of nitrate ion from different plants.     

Analysis of Flavan-3-ols and procyanidins in food samples by reversed phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS)

Concentrations of the main dimeric and trimeric procyanidins (PC) and their monomeric constitutive units catechin (CT) and epicatechin (EC) were determined in food samples by using reversed phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS). In a first step, 12 PCs (PC B1, B2, B3, B4, B5, B6, B7, B8, C1, C2, and A2 and cinnamtannin B1), of which most are not commercially available, were isolated from plant materials or synthesized and purified by a combination of column chromatographic separation techniques with different stationary phases. These PCs in combination with CT and EC were used as standard substances for identification and quantification during the following screening of food samples by RP-HPLC-ESI-MS/MS analysis. The main focus of the newly developed RP-HPLC-ESI-MS/MS method is the compensation of matrix effects by using the echo-peak technique simulating internal standard injection. The suitability of this new method was demonstrated by the determination of recovery rates being 90% or higher. Use of this method allowed the determination of patterns and concentrations of PCs in 55 food samples.     

Combined use of supercritical fluid extraction, micellar electrokinetic chromatography, and reverse phase high performance liquid chromatography for the analysis of antioxidants from rosemary (Rosmarinus officinalis L.)

Antioxidants from rosemary were determined by the combined use of supercritical fluid extraction (SFE) prior to reverse-phase high-performance liquid chromatography (RP-HPLC) or micellar electrokinetic chromatography (MEKC). The separation of antioxidants found in the SFE fractions was achieved by using a new MEKC method and a published HPLC procedure, both with diode array detection. The characterization of the different antioxidants was further done by HPLC-mass spectrometry. Advantages and drawbacks of HPLC and MEKC for analyzing the antioxidants found in the different extracts are discussed. From the results it is concluded that HPLC renders higher peak area and is better in its reproducibility than MEKC; both techniques provide similar analysis time reproducibility. The main advantage of MEKC is its much higher separation speed, which is demonstrated to be useful for the quick adjustment of SFE conditions, allowing rosemary fractions of higher antioxidative power to be obtained quickly. Moreover, the possibilities of this approach for following the degradation of antioxidants are discussed.     

Acid-induced phase separation of anionic surfactants for the extraction of 1,4-dichlorobenzene from honey prior to liquid chromatography

The acid-induced liquid-liquid phase separation of anionic surfactants in aqueous solutions and its applicability to cloud point extraction methodology were applied as a tool for the extraction of 1,4-dichlorobenzene (p-DCB) from aqueous samples. p-DCB is extracted into the micelles of sodium dodecane sulfonate (SDSA) in a 4.2 M HCl solution. The micellar phase is separated from the bulk aqueous solution after centrifugation and collected from the surface of the suspension. The micellar extracts are injected into a high-performance liquid chromatographic apparatus and quantified at 225 nm with a reference wavelength of 280 nm. Following the proposed methodology, a preconcentration factor of ca. 160 is achieved (starting from 50 mL solutions) allowing for detection limits at the low microg/L level. Application to honey samples produced detection limits of 2.5 microg/kg with quantification limits of 7.5 microg/kg, while the recoveries of the method ranged from 85% at high concentrations to 95% at lower concentrations of p-DCB. The combined uncertainty of the entire analytical procedure was 4.5% at the concentration level of 30 microg/kg allowing for reliable and reproducible results for the determination of p-DCB at the concentration levels considered as thresholds for EU and U.S. legislation (10 microg/kg).     

Separation and purification of sulforaphane from broccoli seeds by solid phase extraction and preparative high-performance liquid chromatography

A novel, rapid, and economical method to isolate and purify natural sulforaphane from broccoli seeds is described. The procedure involves solvent extraction of autolyzed seed meal, followed by separation by solid phase extraction (SPE) and purification by preparative high-performance liquid chromatography (HPLC). The SPE method provides higher yield of sulforaphane from crude extracts compared to conventional liquid-liquid extraction. High purity and recovery of sulforaphane product can be obtained by preparative HPLC with a C 18 column and 30% methanol in water as the mobile phase. The purified compound was characterized by MS and (1)H and (13)C NMR. The techniques described here are useful tools in the preparative-scale isolation of sulforaphane in a fast, cost-effective, and waste-conscious manner.