High-performance liquid chromatography separation and purification of cacao (Theobroma cacao L.) procyanidins according to degree of polymerization using a diol stationary phase

A new chromatographic approach for separating cacao procyanidins according to their degree of polymerization has been developed. It 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 their degree of polymerization. The new separation approach was developed on an analytical scale but further extended to the preparative scale. These newly developed analytical and preparative high-performance liquid chromatography procedures were successfully applied to the separation, as well as isolation, of cacao procyanidins from unfermented cacao seeds. The degree of polymerization associated with each molecular weight fraction was determined by mass spectrometry.     

Isolation and structure elucidation of procyanidin oligomers from Saskatoon berries (Amelanchier alnifolia)

Proanthocyanidin oligomers with different degrees of polymerization were isolated from Saskatoon berries (Amelanchier alnifolia) by means of gel adsorption and normal-phase liquid chromatography. The proanthocyanidins were identified using electrospray ionization mass spectrometry, nuclear magnetic resonance spectroscopy, and thiolytic degradation coupled with reversed-phase liquid chromatography. The results established that Saskatoon berries contain proanthocyanidins from dimers through heptamers and higher polymers. Saskatoon proanthocyanidins are essentially of procyanidin type, consisting mainly of epicatechin units linked by B-type bonds. The simple procyanidin profile of Saskatoon berries allowed the procyanidins to be separated precisely according to their degrees of polymerization. In the future they can be used as standard compounds for qualitative and quantitative analysis of procyanidins as well as for elucidation of the biological activities of proanthocyanidins.     

Comparative study of polyphenol scavenging activities assessed by different methods

The effect of procyanidin solutions on superoxide anion radicals was studied with an enzymatic method and their EC(50) values were determined. A comparative study of the results suggested that the free radical scavenger potential of these phenolic compounds closely depends on their chemical and stereochemical structures. Oligomeric procyanidins were isolated in different fractions from grapes and wines by low- and high-pressure liquid chromatography. These compounds were found to be efficient free radical scavengers even for the weak concentrations in wines. Their activity in grapes or wines was much stronger than that of other commercially available natural antioxidants (such as ascorbic acid and gallic acid). The effect of tannins isolated from grapes on different radicals was analyzed according to three distinct methods: an enzymatic method for superoxide anion radicals (O(2)(*)(-)), a chemical method for the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH(*)), and an immunochemical method to study the scavenging activity of seed procyanidins on DNA lesions induced by the radical HO(*).     

Improved cadmium sorption isotherms by the determination of initial contents using the radioisotope 109Cd

Cadmium (Cd) sorption isotherms were estimated by two different analytical approaches to assess the influence of initial Cd concentrations of soil matrix on the sorption of added Cd. For the laboratory experiments a heterogeneous set of samples was collected to include a wide range of different initial Cd concentrations. Comparison of both analytical methods (conventional analysis, radioanalysis) resulted in a strong conformity of Cd contents in solution at equilibrium. The calculated Cd concentrations in the soil solid phase differ according to the analytical approach for considering the initial contents. The determination of the initial contents by the proposed radioanalytical method with ¹⁰⁹Cd resulted in long linear Freundlich‐isotherms, even in the low concentration range. Thus, radioanalysis seems to be the most suitable method to recognise the initial contents of Cd in soil. EDTA extractable Cd represent the initial concentrations, which are averaged over solid and liquid phase. However, depending on the sorption characteristics of the soil these rates vary. In the investigated set of soil samples 52.3 to 99.3% of Cd must be added to the solid phase.     

Quantitative fractionation of grape proanthocyanidins according to their degree of polymerization.

A method was developed for the fractionation of grape (seed or skin) proanthocyanidins according to their degree of polymerization. After precipitation in chloroform/methanol (75:25, v/v), the grape proanthocyanidins were deposited onto an inert glass powder column and sequentially dissolved in several fractions by increasing proportions of methanol in the solvent. Each fraction from each proanthocyanidin source was quantified and characterized after acidic degradation with phenylmethanethiol (i.e., thiolysis). The comparison of data from total extract and successive fractions showed that a quantitative separation was achieved so that estimation of polymer size distribution in relation to other compositional characteristics (proportions of prodelphinidin units, galloylation rate) was thus possible. Mean degree of polymerization of separated proanthocyanidins ranged increasingly from 4.7 to 17.4 in seed (8.1 for total extract) and from 9.3 to 73.8 in skin (34.9 for total extract). The method proposed is very interesting for the study of grape proanthocyanidins according to their degree of polymerization because it gives both qualitative and quantitative information especially on the highly polymerized forms, which were not fractionated by previous techniques.     

应用Real time PCR方法测定瘤胃液功能菌群数量

摘要：【目的】本文拟采用real time PCR技术对瘤胃液中三种功能菌即脂解厌氧弧杆菌（Anaerovibrio lipolytica,AL）,琥珀酸酸丝状杆菌(Fibrobacter succinogene,FS)和黄色瘤胃球菌(Ruminococcus flavefaciens,RF)的16S rDNA基因拷贝数进行测定,并建立起反映瘤胃液中三种功能菌的数量的方法。【方法】在相同日粮的情况下,不同个体牛在同一时间点、以及相同个体牛在不同时间点时采集瘤胃液微生物样品,并总DNA。根据三种菌16SrDNA保守序列设计特异性引物,利用Real time PCR技术考察三种功能菌的数量差异和变化。【结果】通过数据统计分析发现,不同个体动物间的三种功能菌的数量在相同时间点的差异不显著;而在不同时间点相同个体动物瘤胃中三种功能菌数量差异显著（P＜0.05）。     

Solanidine hydrolytic extraction and separation from the potato (Solanum tuberosum L.) vines by using solid-liquid-liquid systems

Solanidine is a steroidal aglycon of potato (Solanum tuberosum L.) glycoalkaloids and a very important precursor for the synthesis of hormones and some pharmacologically active compounds. Glycoalkaloids are hydrolyzed by mineral acid, yielding solanidine. This paper deals with the kinetics of solanidine hydrolytic extraction in different solid-liquid-liquid systems. The dried and milled potato (S. tuberosum L.) vines were used as a source of glycoalkaloids and as the solid phase. The solutions of hydrochloric acid in 2 and 10% (w/v) aqueous acetic acid, in 50% (volume) aqueous methanol, and in 50% (volume) aqueous ethanol were first liquid phase, and the medium for glycoalkaloid extraction from potato vines and their hydrolysis to solanidine. The chloroform, trichloroethylene, or carbon tetrachloride were the second, organic, liquid phase and the medium for solanidine extraction. This procedure combines three different processes: extraction of glycoalkaloids from potato vines, their hydrolysis to solanidine, and the extraction of solanidine, in a single step. The term hydrolytic extraction of solanidine was used for these processes. The purpose of the paper was to choose an optimal solid-liquid-liquid system for solanidine extraction and to define the procedure for its isolation from the organic liquid phase. The best degree of solanidine hydrolytic extraction (DHE) of more than 98% was achieved when 10% (w/v) hydrochloric acid in 50% (volume) methanol were the first liquid phase and chloroform was the second liquid phase, after 90 min. The yield of solanidine (q(S)) under these conditions is calculated to be 0.24 g/100 g of potato vines. Approximately 78% of the maximal possible yield of solanidine was isolated from chlorofom liquid phase. The IR and MS spectra of isolated solanidine were recorded.     

HPLC determination of extractable and unextractable proanthocyanidins in plant materials

This study developed a method for the determination of extractable and unextractable proanthocyanidins. Extractable proanthocyanidins were separated according to their degree of polymerization using normal phase HPLC. Unextractable proanthocyanidins were measured after acid-catalyzed depolymerization as flavan-3-ols (terminal units) and benzylthioethers (external units). Electrospray ionization mass spectrometry (ESI-MS) was used for the identification of proanthocyanidins in the samples. Hubaux-Vos detection limits were 0.01-0.15 ng/injection for extractable proanthocyanidins, with recovery rates from 69 to 91%. Detection limits for unextractable proanthocyanidin derivatives were 0.002-0.035 ng/injection with 80% recovery. The developed method was applied to the analysis of several fruit and berry samples. Results showed great variation in the proportion of unextractable proanthocyanidins in total proanthocyanidin content between samples, being highest in the green variety of table grape (63%) and lowest in the apple cultivar 'Valkeakuulas' (4.1%). The method reported herein is reliable and gives valuable information on the nature of proanthocyanidins in plant-derived foods.     

Apple procyanidin oligomers absorption in rats after oral administration: analysis of procyanidins in plasma using the porter method and high-performance liquid chromatography/tandem mass spectrometry

In this study, we investigated the absorption of apple procyanidins, namely, apple condensed tannins (ACTs), in rats using the Porter method and high-performance liquid chromatography/tandem mass spectrometry. The apple procyanidin concentrations in the rat plasma reached a maximum 2 h after administration and decreased thereafter. To investigate the limits of the absorption of apple procyanidins in the polymerization degree, we administered the procyanidin oligomer fraction, which was separated from ACT using normal-phase chromatography according to the degree of polymerization. Procyanidins from each dimer to pentamer group were detected in the plasma by the Porter method. Moreover, by the study using reconstituted procyanidins, polymeric procyanidins influenced the absorption of procyanidin oligomers. These results suggest that ACTs are absorbed and directly involved in physiological functions in the rats.     

Characterization of pigments from different high speed countercurrent chromatography wine fractions

A red wine, made from Cabernet Sauvignon (60%) and Tannat (40%) cultivars, was fractionated by high speed countercurrent chromatography (HSCCC). The biphasic solvent system consisting of tert-butyl methyl ether/n-butanol/acetonitrile/water (2/2/1/5, acidified with 0.1% trifluoroacetic acid) was chosen for its demonstrated efficiency in separating anthocyanins. The different native and derived anthocyanins were identified on the basis of their UV-visible spectra, their elution time on reversed-phase high-performance liquid chromatography (HPLC), and their mass spectra, before and after thiolysis. The HSCCC method allowed the separation of different families of anthocyanin-derived pigments that were eluted in different fractions according to their structures. The hydrosoluble fraction was almost devoid of native anthocyanins. Further characterization (glucose quantification, UV-visible absorbance measurements) indicated that it contained flavanol and anthocyanin copolymers in which parts of the anthocyanin units were in colorless forms. Pigments in the hydrosoluble fraction showed increased resistance to sulfite bleaching and to the nucleophilic attack of water.     

Rapid preparation of procyanidins B2 and C1 from Granny Smith apples by using low pressure column chromatography and identification of their oligomeric procyanidins

Research in the field of procyanidins is always hindered by the lack of procyanidin standards, and the preparation of procyanidins, especially in large scale, remains difficult and time-consuming. Commercial sources of procyanidin standards are scarce. In this study, a rapid preparation method of procyanidins by using low-pressure column chromatography was developed. Procyanidins in Granny Smith apples were extracted with boiled water and purified on an ADS-17 macroporous resin column to obtain a Granny Smith apple procyanidin extract (GSE). GSE was fractionated according to its degree of polymerization on a Toyopearl TSK HW-40s column. Procyanidins B2 (epicatechin-(4beta-8)-epicatechin) and C1 (epicatechin-(4beta-8)-epicatechin-(4beta-8)-epicatechin) were prepared without HPLC separation. Oligomeric procyanidins from Granny Smith apples were also identified by liquid chromatography-electrospray ionization-mass spectrometry.     

Development and Application of a Methodology to Determine Free Ferulic Acid and Ferulic Acid Ester‐Linked to Different Types of Carbohydrates in Cereal Products

Ferulic acid bioavailability is dependent on its form present in food. This necessitates a methodology to quantify different groups of ferulic acid derivatives in food products, especially cereal‐based products. The aim of the proposed methodology is to separate and quantify ferulic acid ester‐linked to mono‐ and/or oligosaccharides (OF), to soluble polysaccharides (SPF), and to insoluble polysaccharides (IPF) as well as in its free form. Development and partial validation of this method, which was widely based on liquid/liquid extraction and precipitation steps, was performed using characterized standard materials isolated from corn bran. As the determination of OF was one of the major goals of this methodology, three different feruloylated mono‐ and oligosaccharides were used for method development and validation. To determine the accuracy of the method, ferulic acid–containing standard materials added to a starch matrix were extracted and separated according to the developed protocol. The separated ferulic acid esters were saponified before ferulic acid was analyzed by reversed phase HPLC. Recovery rates were generally between 70 and 103%, with the lowest recovery rates for SPF and highest recovery rates for IPF and OF. Finally, the applicability of the method to unprocessed and processed wheat bran samples was demonstrated.     

Quantification of uronic acids in tea polysaccharide conjugates and their antioxidant properties

A technique of high-performance liquid chromatography (HPLC) was described for the measurement of total uronic acids in tea polysaccharide conjugates. This method was applied to polysaccharide conjugate extracts obtained from green tea after most of the components that produce interference were removed. The preliminary extraction process was according to the procedure of isolation of polysaccharide conjugates. The uronic acid content of different polysaccharide conjugate fractions was quantified by HPLC on a Sugar-Pak I column with a 1.0 x 10(-)(4) mol x L(-)(1) calcium disodium ethylenediaminetetraacetic acid solution as the mobile phase and refractive index detection. The validation study showed high recoveries (>97.0%) and low coefficients of variance (<3.0%). The minimum detectable limit concentration of uronic acid was 10 microg x mL(-)(1). The analysis of a standard range of galacturonic acid concentrations (100-4000 microg x mL(-)(1)) yielded linear results. The use of the method on different polysaccharide conjugate fraction samples confirmed its effectiveness. With the high content of uronic acids in polysaccharide conjugates, the stronger reactive oxygen species scavenging activities were found.     

Pressurized liquid extraction of capsaicinoids from peppers

A method has been developed for the extraction of capsaicinoids from peppers by pressurized liquid extraction (PLE); these compounds are determined by reverse phase high-performance liquid chromatography (HPLC), with detection by fluorescence spectrophotometry and mass spectrometry (MS). The stability of capsaicin and dihydrocapsaicin has been studied at different temperatures (50-200 degrees C), and several extraction variables have been assayed: solvent (methanol, ethanol, and water), different percentages of water in the methanol (0-20%) and in the ethanol (0-20%), and the number of extraction cycles. The study has evaluated the repeatability (RSD < 7%) and the reproducibility (RSD < 7%) of the method. Finally, the PLE method developed has been applied to quantify the capsaicinoids present in three varieties of hot peppers cultivated in Spain, quantifying five capsaicinoids: nordihydrocapsaicin, capsaicin, dihydrocapsaicin, an isomer of dihydrocapsaicin, and homodihydrocapsaicin.     

Polyphenolic profiles of Basque cider apple cultivars and their technological properties

The polyphenolic compositions of 31 Basque cider apple cultivars were determined in pulp, peel, and juice by high-performance liquid chromatography with diode array detection analysis of crude extracts and after thiolysis. Total polyphenols are distributed in a wide concentration range depending on the cultivar. Procyanidins are the class of polyphenols that present major concentrations in apple. Their average degrees of polymerization range from 4 to 8 depending on the cultivar. Apple cultivars were technologically classified into bitter and nonbitter categories using different classification systems obtained by applying several pattern recognition techniques, such as principal component analysis, K-nearest neighbors, soft independent modeling of class analogy, partial least-squares, and multilayer feed-forward-artificial neural networks, to apple pulp, peel, or juice data (individual polyphenol concentrations, total procyanidin content, and the average degree of polymerization of procyanidins). Bitter apple cultivars present higher contents of flavan-3-ols and/or dihydrochalcones than nonbitter cultivars. Detailed knowledge of the polyphenolic profile of each apple cultivar affords information about their susceptibility to oxidation, their sensory properties (bitterness, astringency), and their possible influence on the characteristics and quality of the final product (juice, cider) when apples are processed.     

Direct enzyme-linked immunosorbent assay for determining aflatoxin M1 at picogram levels in dairy products

Protocols for detecting picogram quantities of aflatoxin M1 in dairy products were established. Milk samples were subjected to a reverse phase Sep-Pak C18 cartridge treatment before analysis by an enzyme-linked immunosorbent assay (ELISA) according to previously published procedures. M1 in yogurt, brick cheddar, and ripened Brie cheese was extracted by a modified Pons method, subjected to a normal phase silica cartridge treatment, and analyzed by ELISA. The detection limits for M1 in milk, yogurt, cheddar, and Brie were 10, 10, 50, and 25 ppt (ng/kg), respectively. Recovery for M1 added to these products was in the range 70-110%. Good agreement was found for M1 levels in several naturally contaminated milk samples analyzed by both ELISA and liquid chromatography.     

Analysis of fat-soluble vitamins. XXVIII. High performance liquid chromatographic determination of vitamin D in pet foods and feeds: collaborative study

A high performance liquid chromatographic (HPLC) method for vitamin D in pet foods and feeds at low concentrations (2-8 IU/g = 50-200 ppb) was studied collaboratively. The procedure consists of the following purification steps: saponification, extraction of the unsaponifiable fraction, chromatography on alumina, cleanup on reverse phase HPLC, and quantitation with straight phase HPLC. The original method, developed by Knapstein, was simplified by deleting the quantitative TLC step. Six coded samples were distributed to 31 laboratories, along with a known sample containing 15 IU/g to allow practice of the rather complicated procedure. Eighteen collaborators returned their results. Results for the spiked samples show good recovery. The estimates of repeatability and reproducibility are 0.96 and 2.2 IU/g for spiked samples and 1.5 and 3.1 IU/g for commercial samples, respectively, which are considered acceptable for these low concentrations. The method has been adopted official first action.     

Extraction of polyphenols from vine shoots of Vitis vinifera by superheated ethanol-water mixtures

A study of the nonvolatile fraction of extracts from vine shoots obtained by superheated ethanol-water mixtures is presented. The influence of the temperature, extraction time, and percentage of ethanol on extraction was investigated by a multivariate experimental design to maximize the yield of total phenolic compounds, measured by using the Folin-Ciocalteu method. The best values found for these variables were 80% (v/v) ethanol, 240 degrees C, and 60 min. Under these conditions, the effect of pH was also investigated, and a strong improvement of yield was observed by decreasing the pH. The extracts were subject to liquid-liquid extraction with n-hexane. The remaining polar phase was dried in a rotary evaporator and then reconstituted in 10 mL of water. The insoluble residue was dissolved in 10 mL of methanol. Both fractions (aqueous and methanolic) were analyzed by HPLC, and the differences in composition according to the extraction conditions were studied. Compounds usually present in commercial wood extracts were identified (mainly benzoic and hydroxycinnamic acids and aldehydes); the most abundant were quantified, and the stability of the identified phenolic families under different extraction conditions was also investigated. Finally, the superiority of the superheated liquid extraction over conventional solid-liquid extraction was demonstrated.     

Liquid chromatographic determination of pentaerythritol tetranitrate in pharmaceuticals: collaborative study

A liquid chromatographic (LC) method for the determination of pentaerythritol tetranitrate (PETN) in pharmaceutical formulations and the bulk drug triturate was evaluated in an interlaboratory study that included 12 participating laboratories. The procedure involves extraction of the active ingredient with mobile phase, followed by filtration of the extract and reverse-phase liquid chromatography using an octadecylsiliane bonded phase column and UV detection at 230 nm. The mobile phase composition is 35% water in acetonitrile (v/v). Three bulk drug samples (20, 20, and 35% PETN), 2 commercial tablet formulations (20 and 80 mg PETN/tablet), and 1 commercial capsule formulation (45 mg PETN/capsule) were analyzed in duplicate by the proposed method. Repeatability (sr, RSDr) and reproducibility (SR, RSDR) based on peak height measurement for these samples ranged from 0.0066 to 0.1806 (0.53-3.36%) and 0.0165 to 0.2075 (0.76-3.86%), respectively. Results for peak area measurements ranged from 0.0145 to 0.2011 (0.93-3.74%) and 0.0231 to 0.2091 (1.28-3.89%), respectively. The method has been approved interim official first action by AOAC.     

Evidence of the formation of light polycyclic aromatic hydrocarbons during the oxidation of edible oils in closed containers at room temperature

Solid phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS) of the headspace composition of two sunflower oil samples was carried out; both samples were taken from the same original oil, stored for a prolonged time (112 months) in closed containers at room temperature under different air/oil volume ratios. Great differences in the headspace compositions of both samples were found due to the different oxidation levels reached. One of the most significant findings is that both contain monocyclic and light polycyclic aromatic hydrocarbons, the proportions of which are in line with the oxidation level of the sample. The determination of polycyclic aromatic compounds in the oil liquid matrix of both oil samples, carried out by means of a classical scheme of isolation, cleanup, separation, and quantification, showed that the concentrations of these compounds in the oil liquid phase also follow the oxidation degree reached by each sample, proving that this oxidation process at room temperature leads to the formation of these compounds.