Determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection

A modified preparation of sample was developed for the determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection, using on-line pre-column derivatization. Ice-cold deoxygenated methanol was used to deactivate the oxidation enzymes in juices or wines and keep the glutathione stable. The optimum recovery of glutathione content in grape juice and wine was obtained when either the sample of grape juice or wine was mixed in ice-cold deoxygenated methanol in the ratio 10:90 (v:v) and further diluted in sodium acetate buffer in the ratio 1:1 (v:v). The optimized method was validated for linearity, limit of detection, limit of quantification, precision and uncertainty. According to the validation data the method is appropriate for the determination of glutathione content in grape juice and wine. Glutathione contents in grape juices made from White Muscat grapes and Sauvignon Blanc wines were analysed. The average glutathione content in 28 young Sauvignon Blanc wines was 12.5 mg L⁻¹.     

Differentiation of Vitis vinifera varieties by MALDI-MS analysis of the grape seed proteins

Until now the study of pathogenic related proteins in grape juice and wine, performed by ESI-MS, LC/ESI-MS, and MALDI/MS, has been proposed for differentiation of varieties. In fact, chitinases and thaumatin-like proteins persist through the vinification process and cause hazes and sediments in bottled wines. An additional instrument, potentially suitable for the grape varieties differentiation, has been developed by MALDI/MS for the grape seed protein analysis. The hydrosoluble protein profiles of seeds extract from three different Vitis vinifera grape (red and white) varieties were analyzed and compared. In order to evaluate the environmental conditions and harvest effects, the seed protein profiles of one grape variety from different locations and harvests were studied.     

One step purification of the grape vacuolar invertase

Invertase is a major protein of grape juice and wine. Accordingly, in order to study the biochemical and structural characteristics of this protein and for a better understanding of its physico-chemical properties, large amounts of the pure protein are needed. A simple method for the purification of the grape vacuolar invertase in a preparative-scale is described in this work. The grape protein was isolated and purified from must by ultrafiltration and anion exchange chromatography. The identification and purity determination of the grape invertase fraction were assessed by SDS-PAGE, and were then confirmed using nanoLC-chip-MS/MS analysis. The laboratory fractionation procedure presented in this work generated large quantities of pure grape vacuolar invertase from must.     

A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 microg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.     

High-performance liquid chromatography coupled with fluorescence detection for the determination of trans-astringin in wine.

In this study a high-performance liquid chromatography (HPLC) method was developed for the determination of trans-astringin in wine using fluorescence detection. This is the first time the occurrence of trans-astringin has been reported in wine. The method allows analysis of both red and white wine samples with no prior treatment. The quantification threshold is 0.03 mg/l. Levels of trans-astringin in the French wines analyzed ranged from 0.09 mg/l to 0.29 mg/l. The reproducibility of the method was measured and the CV was less than 4.8% for both red and white wines.     

Improving the Phloroglucinolysis Protocol and Characterization of Sagrantino Wines Proanthocyanidins

Proanthocyanidins are key metabolites that explain wine sensorial character (bitterness and astringency) and red wine color changes during aging. Therefore, a fast and accurate method to evaluate the degree of polymerization and the structural composition of the polymeric proanthocyanidins is a crucial analytical tool. Phloroglucinolysis is the most used method for this analysis but, unfortunately, the phloroglucinol adducts of the monomeric flavan-3-ols are not commercially available, making the results less accurate. The aim of this work was the isolation by semi-preparative high performance liquid chromatography (HPLC) of these non-commercial compounds and their use for the development of an accurate UHPLC-MS/MS protocol. The purity of each adduct was established via quantitative ¹H-nuclear magnetic resonance (NMR) measurements with 3-trimethylsilyl-propionic-d4 acid sodium salt as the calibration standard. The developed method was applied to evaluate the proanthocyanidins profile of Sagrantino di Montefalco wines in comparison to other well-known tannic wines. Commercial, 6–8 years old Sagrantino wines were demonstrated to be very rich in epicatechin type B procyanidins, to have low galloylation %, and to have a high mean degree of polymerization of the proanthocyanidins with respect to the other analyzed wines.     

A new sensitive and selective method for analysis of ochratoxin A in grape and wine by direct liquid chromatography/surface-activated chemical ionization tandem mass spectrometry

A new sensitive and selective analytical method for the analysis of ochratoxin A (OTA) in grape and wine was developed by coupling liquid chromatography and surface activated chemical ionization and mass spectrometry with multistage fragmentation (LC/SACI-MS(3)). A high flow gradient was used to strongly reduce the matrix effect phenomenon, and the wine sample was directly injected onto the chromatographic column without sample pre-concentration or purification steps. The amount of OTA was determined for two grape extracts and the amount of OTA, percent accuracy error and percent precision error were analyzed for 15 wine samples. An excellent limit of detection of 0.02 ng/mL was achieved, and the limit of quantification was at least 20-fold lower than the maximum legal limit for OTA (2 ppb). Due to the low limit of quantification, this novel method is a potential tool for official OTA screening purposes.     

Determination of the biogenic amines in musts and wines before and after malolactic fermentation using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as the derivatizing agent

An accurate, rapid and sensitive high-performance liquid chromatographic (HPLC) method is reported for the determination of four biogenic amines in red and white wine samples. The method involves derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) followed by liquid chromatography gradient elution analysis without any other sample pretreatment. The procedure developed permitted the determination of the four amines in 40 min. The relative standard deviation (RSD) values ranged from 3.1% to 6.1% in peak areas and the response was linear (R2>0.994) in a range from 0.1 to 4 mg/l. Leaving aside putrescine in Tempranillo wines, the malolactic fermentation did not exert any effect on the concentration of these compounds. On the contrary, the influence of the grape variety was important, and Tempranillo wines showed higher levels of tyramine and histamine and lesser of putrescine and cadaverine (p<0.05) than wines made with Cabernet Sauvignon.     

高效液相色谱-串联质谱法检测红葡萄酒中功效成分

建立了高效液相色谱-串联质谱法快速测定葡萄酒中白藜芦醇、黄酮类、多酚类功效成分的分析方法。葡萄酒样品直接稀释后进样,用C18柱进行分离,以乙腈-0.1%(体积分数)甲酸水溶液为流动相进行梯度洗脱,通过多反应监测(MRM)模式进行检测。13种功效成分在各自线性范围内呈良好的线性关系,相关系数均大于0.99。除表没食子儿茶素、没食子儿茶素、儿茶素没食子酸酯、花旗松素的检出限为1.0、1.0、3.0、3.0μg/L外,其他9种化合物的检出限均小于1.0μg/L。回收率为80.9%~112.3%,相对标准偏差小于10%。该方法快速、准确、灵敏度高,适用于葡萄酒中功效成分的快速分析。对实际样品的检测表明,所测葡萄酒样品中均含有儿茶素、表儿茶素、表没食子儿茶素、没食子儿茶素、表儿茶素没食子酸酯/儿茶素没食子酸酯、白藜芦醇、大豆黄素等功效成分,不同品种葡萄酒中这些功效成分含量差异显著。     

Comparison of carboxymethyl-cellulose cation-exchange chromatography and high-performance liquid chromatography in the purification of guinea-pig insulin from pancreatic extracts

Guinea-pig insulin was purified from pancreatic extracts either by carboxymethyl-cellulose cation-exchange chromatography with a sodium chloride gradient or by high-performance liquid chromatography (HPLC) on octadecyl silica with mixtures of acetonitrile and phosphate buffer. HPLC proved to be superior to ion-exchange chromatography in the purification of insulin with respect both to time saving and to the purity of the product.     

Determination of proanthocyanidins in fresh grapes and grape products using liquid chromatography with mass spectrometric detection

Fresh grapes and grape products, such as grape wine and grape juice, were analyzed for proanthocyanidins (PACs) using liquid chromatography with electrospray ionization mass spectrometric (MS) detection. PACs were successfully separated and analyzed on the basis of their protonated molecules, allowing the identification of PACs in different degrees of polymerization from monomers to oligomers (up to 7 units), and in various isomeric forms. Using reversed-phase high-performance liquid chromatography (HPLC) combined with MS detection, the PAC monomers, (+)-catechin (C), (-)-epicatechin (EC), (-)-catechin gallate (CG), and (-)-epicatechin gallate (ECG), were successfully quantified using selected ion monitoring (SIM) mode. Standard curves were fitted for each PAC ranging from 43.8 to 5600 ng/mL for C, from 42.2 to 5400 ng/mL for EC, from 36.7 to 4700 ng/mL for CG, and from 39.8 to 5100 ng/mL for ECG. Good linearity (r2>0.999) was achieved for each analyte. The accuracy and precision (RSD) were within 10% (n=8) at the limit of detection. This method allows direct quantification of monomeric PACs in fresh grapes and grape-derived products. Additionally, flow injection analysis (FIA) was applied to estimate the concentration levels of PAC oligomers by comparing their FIA-MS peak areas, which were well correlated (r2=0.936) to the total concentrations of PAC monomers.     

Comparison of volatile constituents extracted from model grape juice and model wine by stir bar sorptive extraction-gas chromatography-mass spectrometry

A stir bar sorptive extraction (SBSE) method coupled with gas chromatography-mass spectrometry was optimised for the analysis of volatile components of a model wine, based on a previously optimised method used for analysis of the same components in model grape juice. The presence of ethanol in the model wine sample matrix resulted in decreased sensitivity of the method toward most of the volatile constituents. Mean percent relative recoveries and reproducibilities (%CV) were 22.8% and 7.1%, respectively, compared with 28.4% and 8.5% for model grape juice. The mean limit of detection (LoD) ratio (juice:wine) was 0.25. Similar sensitivities for the two sample matrices using this method were achieved by changing the split ratio from 20:1 (grape juice) to 5:1 (wine), giving a mean limit of detection ratio (juice:wine) of 1.0, thus allowing direct comparison of chromatograms of volatile components in the two matrices. This enabled direct comparisons of grape juices and the wines derived from them by alcoholic yeast fermentation. The influence of ethanol concentration in the range 9-15% on method sensitivity is discussed, using an overlay of the total ion chromatograms. The use of a gas saver device for the 5:1 split ratio analysis of desorbed model wine aroma compounds is discussed in terms of preventing extraneous reaction of sorbent and stationary phases with air during analysis.     

Comparison of preparative HPLC/MS and preparative SFC techniques for the high-throughput purification of compound libraries

A diverse set of 16 high-throughput organic synthesis libraries, consisting of 48 samples per library, has been purified by both preparative supercritical fluid chromatography (SFC) and preparative high-performance liquid chromatography (HPLC). This paper details the relative effectiveness of these two purification techniques in terms of success, yield, and purity of final product.     

Phenolic Composition and Total Antioxidant Capacity by Electrochemical,Spectrophotometric and HPLC‐EC Evaluation in Portuguese Red and White Wines

Phenolic composition and in vitro total antioxidant capacity have been evaluated, in red and white Portuguese wines, by electrochemical methods, spectrophotometric methods, and reverse‐phase high performance liquid chromatography with electrochemical detection (HPLC‐EC). The different phenolic compounds present in seventeen red and white wines, with different grape varieties, and from different geographical locations, were successfully identified and detected. The wines in vitro total antioxidant capacity, using the method of capture of diphenilpicrilhydrazil (DPPH^.) free radical “efficient concentration” (EC₅₀), and the electrochemical quantitative index (EI), was determined. The wine with the highest total antioxidant capacity exhibited the lowest antioxidant power, and the EI and EC₅₀ assays had a very good correlation.     

Phenolic profiles of Serbian autochthonous variety ‘Prokupac’ and monovarietal international wines from the Central Serbia wine region

Phenolic profiles of young monovarietal white and red wines from the Central Serbia were investigated. Quantification of phenolics was done using ultra-high-performance liquid chromatography (UHPLC) coupled with a diode array detector and a triple-quadrupole mass spectrometer. Generally, red wines were characterised with higher phenolics content when compared to the white wines. This applies also for the total phenolics content (TPC) and radical scavenging activity (RSA) values. Among all the samples, the red wine from autochthonous Serbian variety ‘Prokupac’ stood out with unique composition, showing the highest TPC, RSA and the highest concentration of the individual phenolics. Finally, based on a specific phenolic pattern, principal component analysis (PCA) indicated clustering of red and white wines, with the ‘Prokupac’ as a prominent outlier.     

Quantification of major grape polysaccharides (Tempranillo v.) released by maceration enzymes during the fermentation process

The influence of commercial enzymes on wine polysaccharide content was studied. Tempranillo wines were made using commercial maceration enzyme preparations along with controls. The analytical method for the quantification of wine polysaccharides was carried out by a multistep procedure. Wine-soluble polysaccharides were isolated by wine concentration polysaccharides precipitation with an acid-alcohol medium and separation of each polysaccharide family by high resolution size-exclusion chromatography on a Superdex-75 HR column. The glycosyl-residue compositions of the fractions obtained were determined by gas chromatography with flame ionisation and mass spectrometry of their trimethylsilyl-ester O-methyl glycosides after acidic methanolysis and derivatization. The content of each fraction was estimated from the concentration of individual glycosyl residues that are characteristic of well-defined wine polysaccharides. The analytical method proposed had good sensitivity, repeatability, reproducibility and accuracy. Soluble polysaccharides in wine were essentially composed of grape cell wall polysaccharides: arabinogalactans and arabinogalactan-proteins (38-41%), and rhamnogalacturonans-II (38-46%). Yeast mannans and mannoproteins were also present but in smaller proportions (14-19%). Wines treated with commercial enzymes had larger concentrations of arabinogalactans, arabinogalactan-proteins and rhamnogalacturonans-II than control wines, but the content of mannans and mannoproteins was similar in both wines. This indicated that the commercial enzymes hydrolysed grape pectic polysaccharides during the maceration-fermentation stage but had no influence on yeast parietal polysaccharides.     

Study of biologically active amines in grapes and wines by HPLC

Summary The biologically active amines agmatine, cadaverine, histamine, phenethylamine, putrescine, spermidine, and tyramine have been determined in different varieties of grape, aszu grape, wine and aszu wine from the Tokaj region of Hungary. Ion pairs formed between the amines and octanesulphonic acid were separated by liquid chromatography on a μBondapak C₁₈ reversed-phase column, and spectrofluorimetric detection was performed after post-column derivatization witho-phthalaldehyde and 2-mercaptoethanol. The method was linear for the amines between 0.1 and 10 mg L⁻¹, and for spermidine between 1 and 30 mg L⁻¹. Comparison of the results revealed that the qualitative and quantitative content of biologically active amines was mostly determined by the vintage of the wine and the technology used for wine-making. The biogenic amine content of Tokaj wines is well below suggested limits for any of the amines, showing that the wine-making technology of the Tokaj region is of high quality. The levels of biologically active amines (identified and quantified by HPLC) in grapes, wines and aszu wines can provide useful information about the weather, growth ofBotrytis cinerea in Tokaj, and aspects of the methods used for wine-making. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Separation of proteins by consecutive sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography

A procedure for the preparative separation of proteins was developed by using consecutively sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed phase high performance liquid chromatography (HPLC). The proteins were separated by SDS-PAGE and afterwards extracted from the gel. The extracted proteins were separated from SDS and other small molecular weight contaminants on a Fractogel TSK HW-40 (F) column in acidic aqueous acetonitrile. The proteins eluted from the Fractogel column were fractionated by HPLC. The identity and purity of the recovered proteins was confirmed by SDS-PAGE analysis.     

Analysis of the diastereoisomers of the cysteinylated aroma precursor of 3-sulfanylhexanol in Vitis vinifera grape must by gas chromatography coupled with ion trap tandem mass spectrometry

The diastereoisomeric distribution of S-3-(hexan-1-ol)cysteine (P-3SH), the cysteinylated precursor of 3-sulfanylhexan-1-ol (3SH) in Vitis vinifera grape juice, was determined by a new method. This procedure is based on the purification of P-3SH in a small volume of must (500 microL) by affinity chromatography, followed by the separation of chiral molecules in derivative forms by gas chromatography coupled with ion trap tandem mass spectrometry (GC-MS/MS). The diastereoisomers were easily separated using heptafluorobutyric anhydride and heptafluorobutanol (HFBA and HFOH) as derivatization reagents. Method validation was conducted using samples of grape juice, synthetic must, fermenting must, and wine that were fortified with P-3SH at concentrations of 0.6 and 2.5 microM. The relative standard deviation (RSD) and limit of detection (LOD) of the GC-MS/MS method were 4.6% and 1.5 nM, respectively. P-3SH assays in Bordeaux white grape juice affected by Botrytis cinerea showed an unusually increased proportion of the RS form of the precursor (approximately RR:RS=30:70) as compared to a diastereoisomer ratio (in the vicinity of 50:50) in healthy grape juice.     

Development and validation of a UHPLC method for the determination of flavonoids in red wine

A simple and fast ultra-high-performance liquid chromatography (UHPLC) method was developed for the identification and quantification of the following flavonoids in red wine: (+/-)-catechin, (-)-epicatechin, rutin, quercitrin, hesperidin, neohesperidin, (+/-)-naringenin, hesperetin, and chrysin. Chromatographic separation of the flavonoids was performed on a Chromolith Fast Gradient C18e column. A gradient elution was used with mobile phases consisting of 0.1% formic acid in water and acetonitrile. UV detection was performed at 280 nm. A complete separation of flavonoids was possible within 6 min. The calibration curves showed good linearity (R2 > or = 0.9990) in the selected range of each analyte; the LOD ranged between 0.06 and 0.19 microg/mL. An optimized sample preparation method utilized SPE. The Oasis HLB column with the highest recoveries was selected for the preconcentration step. This method was successfully applied to the determination of these flavonoids in the red wine samples with excellent results.