Molecularly imprinted polymer‐based solid phase clean‐up for analysis of ochratoxin A in beer,red wine,and grape juice

A simple, reliable, and low‐cost method based on molecularly imprinted polymer as a selective sorbent of SPE was proposed for the determination of ochratoxin A (OTA) in beer, red wine, and grape juice by HPLC coupled with fluorescence detection (HPLC‐FLD). Samples were diluted with water and cleaned up with an AFFINIMIP® SPE OTA column. After washing and eluting, the analyte was analyzed by HPLC‐FLD. Under the optimized conditions, LOD and LOQ for OTA were 0.025 and 0.08 ng/mL, respectively. The recoveries of OTA from beer, red wine, and grape spiked at 0.1, 2, and 5 ng/mL ranged from 91.6 to 101.7%. Furthermore, after a simple regenerated procedure, the molecularly imprinted polymer based SPE column could be reused at least 14 times to achieve more than 80% recoveries of OTA in real samples. The developed method was applied to the detection of 30 beer, red wine, and grape juice samples and only four samples were contaminated by OTA with levels below the legal limits.     

Development and validation of a capillary zone electrophoresis method for the quantitative determination of anthocyanins in wine

A capillary zone electrophoresis (CZE) method is proposed for the quantitative determination of anthocyanins in wine as an alternative to high-performance liquid chromatography. The CZE separation was carried out using a 46 cm (effective length)×75 μm I.D. fused-silica capillary at 10 °C and a 50 mM sodium tetraborate buffer at pH 8.4 with 15% of methanol as modifier. A voltage of 25 kV and a hydrodynamic injection of 300 mbar s were used. The electropherograms were recorded at 599 nm. It was found that SO₂ (antibacterial and antioxidant agent added to wine during its production) increased the absorbance of anthocyanins at 599 nm in a basic medium. Therefore, a concentration of 250 mg/l of SO₂ was added to the samples and the calibration solution before the analysis in order to avoid errors by this matrix effect. The analytical response was linear (R=0.998) between 10 and 700 μg/ml of malvidin-3-O-glucoside. The limit of detection and the reproducibility (as a relative standard deviation, n=11) were 1 μg/ml and 1.5%, respectively. Finally, the CZE method was validated by the analysis of synthetic wine samples (errors less than 8%) and by the comparison of the results obtained in the analysis of different monovarietal wines by CZE with those obtained by the standard HPLC method. In this comparison, a good correlation (R=0.998) with a slope of 1.005±0.044 and an intercept of −0.752±6.690 was obtained for malvidin-3-O-glucoside.     

Simultaneous Determination of Quercetin and Luteolin in Dried Flowers by Multivariate HPLC-ECD Calibration

Two calibration models, partial least squares (PLS) and principal component regression (PCR), were applied to the simultaneous determination of quercetin and luteolin by high performance liquid chromatography (HPLC) with electrochemical detection (ECD). The proposed methods were successfully applied to the analysis of dried flower samples. It was found that the relative standard errors of prediction (RSEP) for the validation set of PLS of quercetin and luteolin was 1.31 and 2.23%, and the RSEP of PCR was found to be 3.56 and 4.32%, respectively. Several dried flower samples were analysed and the recoveries were in the range of 95.9–103.3%.     

毛细管区带电泳法测定葡萄籽中儿茶素类化合物

 采用毛细管区带电泳法测定了 10种中国产葡萄籽中的 4个主要儿茶素类化合物 :(+)儿茶素、(- )表儿茶素、(± )表没食子儿茶素、(± )表儿茶素没食子酸酯的含量。在 0 0 2mol/L硼砂和 0 0 0 5mol/L磷酸盐的混合缓冲体系 (pH 10 0 )的背景缓冲液中 ,4个化合物在 10min内取得了令人满意的分离。迁移时间的重现性(RSD)小于 2 % ,峰面积的重现性 (RSD)小于 5 %。在质量浓度为 0 0 0 5g/L～ 0 5 g/L时 ,线性相关系数大于0 995。检测限为 3mg/L～ 10mg/L。该方法简单、快速、准确 ,可作为葡萄籽分析和药用开发过程中分析儿茶素类化合物的有效方法推广使用。     

Anthocyanin Monitoring in Four Red Grape Skin Extract Varieties Using RP‐HPLC‐ED

Abstract

Anthocyanin compounds, cyanin, peonidin, myrtillin, malvin, kuromanin, oenin, petunidin and pelargonidin, present in four varieties of red grape skin extracts were quantified by reversed‐phase high‐performance liquid chromatography with electrochemical detection (RP‐HPLC‐ED). Several extraction procedures were evaluated for anthocyanin extraction from red grape skins of varieties of the Dão wine demarcated region in Portugal. The developed method was successfully applied to the analysis of the anthocyanins in skins extracts of red grapes during the 2005 harvest campaign, and monitoring these compounds can give great insight toward choosing the best time for the grape harvest for achieving a better wine quality.     

Pressurized liquid extraction of vitamin E from Brazilian grape seed oil

The goal of this paper is to optimize the pressurized liquid extraction (PLE) of vitamin E from grape seed oil from residues of the wine industry. For this purpose an experimental planning to optimize the extraction of Brazilian grape seed oil by means of PLE with hexane as solvent was applied and the results are compared with conventional methods (Soxhlet and mechanical press extraction). Vitamin E was separated and analyzed using HPLC with UV detection. This study demonstrates the ability of the PLE in extracting grape seed oil rich in vitamin E.     

Comparison of shikimic acid determination by capillary zone electrophoresis with direct and indirect detection with liquid chromatography for varietal differentiation of red wines

Two capillary zone electrophoretic (CZE) methods for determination of shikimic acid in Chilean red wine were developed and compared with a HPLC method. Both electrophoretic methods were carried out by using a reversed electroosmotic flow induced by trimethyl(tetradecyl)ammoniumbromide (TTAB) with indirect detection at 260 nm using p-aminobenzoic acid as a UV-absorbing co-ion or by direct detection at 213 nm. In both cases, the separation was carried out in a 50 microm I.D. uncoated capillary with an effective length of 48 cm, a negative power supply of 30 kV, using a buffer based on bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane (Bis-Tris), pH 7.0 or 7.5 and hydrodynamic injection. The chromatographic separations were carried out on a C-18 reversed phase column followed by a sulfonyl-styrene-divinylbenzene (S-DVB) ion exclusion column at 70 degrees C with H2SO4 0.02 M as isocratic mobile phase and a flow rate of 0.5 mL min(-1). The three methods allowed the quantification of shikimic acid with quantification limits between 1.0 and 12.0 mg L(-1) and precision between 7.3 and 10.1%, however, only the concentrations obtained by CZE with direct detection were statistically similar to those of HPLC. This parameter was evaluated as analytical tool to verify varietal authenticity of red wines. In all cases, the Cabernet Sauvignon wines presented higher concentrations of shikimic acid, compared with Merlot or Carmenère wines.     

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.     

Quantification of chitinase and thaumatin-like proteins in grape juices and wines

Chitinases and thaumatin-like proteins are important grape proteins as they have a great influence on wine quality. The quantification of these proteins in grape juices and wines, along with their purification, is therefore crucial to study their intrinsic characteristics and the exact role they play in wines. The main isoforms of these two proteins from Chardonnay grape juice were thus purified by liquid chromatography. Two fast protein liquid chromatography (FLPC) steps allowed the fractionation and purification of the juice proteins, using cation exchange and hydrophobic interaction media. A further high-performance liquid chromatography (HPLC) step was used to achieve higher purity levels. Fraction assessment was achieved by mass spectrometry. Fraction purity was determined by HPLC to detect the presence of protein contaminants, and by nuclear magnetic resonance (NMR) spectroscopy to detect the presence of organic contaminants. Once pure fractions of lyophilized chitinase and thaumatin-like protein were obtained, ultra-HPLC (UHPLC) and enzyme-linked immunosorbent assay (ELISA) calibration curves were constructed. The quantification of these proteins in different grape juice and wine samples was thus achieved for the first time with both techniques through comparison with the purified protein calibration curve. UHPLC and ELISA showed very consistent results (less than 16% deviation for both proteins) and either could be considered to provide an accurate and reliable quantification of proteins in the oenology field.     

Capillary electrophoresis for the analysis of paralytic shellfish poisoning toxins in shellfish: Comparison of detection methods

Paralytic shellfish toxins (PSTs) are produced by marine and freshwater microalgae and accumulate in shellfish including mussels, oysters, and scallops, causing possible fatalities when inadvertently consumed. Monitoring of PST content of shellfish is therefore important for food safety, with currently approved methods based on HPLC, using pre‐ or postcolumn oxidation for fluorescence detection (HPLC‐FLD). CE is an attractive alternative for screening and detection of PSTs as it is compatible with miniaturization and could be implemented in portable instrumentation for on‐site monitoring. In this study, CE methods were developed for C⁴D, FLD, UV absorption detection, and MS—making this first report of C⁴D and FLD for PSTs detection. Because most oxidized toxins are neutral, MEKC was used in combination with FLD. The developed CZE‐UV and CZE‐C⁴D methods provide better resolution, selectivity, and separation efficiency compared to CZE‐MS and MEKC‐FLD. The sensitivity of the CZE‐C⁴D and MEKC‐FLD methods was superior to UV and MS, with LOD values ranging from 140 to 715 ng/mL for CZE‐C⁴D and 60.9 to 104 ng/mL for MEKC‐FLD. With the regulatory limit for shellfish samples of 800 ng/mL, the CZE‐C⁴D and MEKC‐FLD methods were evaluated for the screening and detection of PSTs in shellfish samples. While the CZE‐C⁴D method suffered from significant interferences from the shellfish matrix, MEKC‐FLD was successfully used for PST screening of a periodate‐oxidized mussel sample, with results confirmed by HPLC‐FLD. This confirms the potential of MEKC‐FLD for screening of PSTs in shellfish samples.     

Flow nephelometric analysis of protein-tannin interactions

The interaction of tannins from different sources (grape skin, grape seed and wine) with bovine serum albumin (BSA) was studied using a continuous flow method with nephelometric detection. The tannin samples were mixed in a reaction chamber with increasing BSA concentration and the resulting tannin-BSA insoluble aggregates passed through a flow cell where the turbidity was monitored. Based on this technique, the tannin specific activity (TSA) of wine can be directly determined from different wine flows without previous dilutions or any other treatment. The maximum amount of insoluble aggregates formed with BSA and wine or grape skin tannins seemed to remain constant even with the addition of an excess of protein, whereas a decrease in the amount of insoluble aggregates with an excess of BSA was observed in the case of grape seed tannins. It should be noted that BSA was used in these experiments as a model protein and only as an analytical reagent since bovine additives are not allowed in wine.     

Supercritical fluid extraction of resveratrol from grape skin of Vitis vinifera and determination by HPLC

Supercritical fluid extraction of resveratrol from grape skin of Vitis vinifera was studied. Extraction variables such pressure, modifier concentration (ethanol), and extraction time were optimised. Final extraction conditions were: 40 degrees C, 150 bar, 7.5% ethanol and extraction time 15 min. Extraction recovery and precision (variation coefficient between 0.2 and 1.0%) were calculated. The resveratrol content in the ethanolic extract was determined by HPLC with UV detection at 306 nm. Acetic acid-methanol-water was used as the mobile phase, and C-18 and C-8 columns were tested, instrumental parameters were optimised, and analytical parameters were calculated (lineal interval 0-75 mg l(-1), detection limit 0.1 mg l(-1), sensitivity 125530 mg(-1) mg l(-1), coefficient variation 0.8-1.6%). Six different varieties of grape skin, from the same geographical area and representative of the wine elaboration, were analysed.     

Separation of polyphenols in Canary Islands wine by capillary zone electrophoresis without preconcentration

A method for separation and determination of polyphenols in wine by capillary zone electrophoresis (CZE) without any preconcentration step is described. The sensitivity and limits of detection for gentisic and p-coumaric acid are better than those previously published. The effect of a possible C18 solid-phase extraction prior to the CZE analysis was examined. The developed optimized method (without any extraction step) was applied to the analysis of wines from Tenerife, Canary Islands.     

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.     

液相微萃取-高效液相色谱法分析葡萄汁中多酚类化合物

建立了一种基于液相微萃取与高效液相色谱联用技术测定葡萄汁中鞣花酸、白藜芦醇和槲皮素的分析方法. 比较了单液滴液相微萃取和中空纤维液相微萃取两种萃取模式, 选择了单液滴液相微萃取作为3种多酚类化合物的液相微萃取模式. 考察了搅拌速度、萃取时间、料液相pH和料液相离子强度的影响. 鞣花酸、白藜芦醇和槲皮素的富集倍数分别为48.4、 79.4和155.8, 方法的线性范围为0.0050～5.0 μg/mL, 鞣花酸、白藜芦醇和槲皮素的检出限分别为0.015, 0.0020, 0.0080 μg/mL, 相对标准偏差分别为2.0%, 1.8%和1.7%. 用于实际样品葡萄汁的分析, 加标回收率在81.9%～102.3%之间.     

One‒pot green synthesized protein‒based silver nanocluster as prooxidant biosensor

In this study, silver nanoclusters as prooxidant biosensor were eco‒friendly synthesized using chicken egg white protein without any chemical reducing agents for measuring copper(II)-induced prooxidant activities of catechin, epicatechin, epigallocatechin gallate, resveratrol, gallic acid, chlorogenic acid, and rutin. The prooxidant activities were evaluated via measuring the absorption at 450 nm wavelength of the Cu(I)‒neocuproine chelate formed by extraction of protein-bound Cu(I) with neocuproine reagent. Accuracy was determined by evaluating recovery values of wine, grape and apple samples and the obtained values were between 97.2%‒98.9%. Intra-day precision and inter-day reproducibility experiments were studied with three different experiments in a day and three different days respectively. The obtained relative standard deviation values were 0.96% and 1.91%. The detection limit of the biosensor was found as 0.2 µM. The total prooxidant activities of fresh apple and grape fruits, apple and grape juices, and red wine were determined and the results obtained were compared with the findings of the carbonyl assay. In this study, a cheap, easily applicable, sensitive, and reproducible biosensor was developed. It was seen that it could be used in the measurement of the prooxidant activity of different food samples and give an idea about diet, healthy life, and nutrition.     

Capillary electrophoretic analysis of flavonoids in single-styled hawthorn (Crataegus monogyna Jacq.) ethanolic extracts

Flavonoids are an important group of natural compounds, which can prevent coronary heart disease and have antioxidant properties. Hawthorn is a well known and widely used medicinal plant due to its cardiotonic activity. Previous studies refer mostly to the HPLC analysis of the flavonoids: vitexin, quercetin, hyperoside, oligomeric procyanidins, which appear to be primarily responsible for the cardiac action of the plant. Aqueous ethanolic extracts of single-styled hawthorn (Crataegus monogyna Jacq., f.: Rosaceae Juss.) leaves and sprouts were analyzed by means of capillary zone electrophoresis (CZE). Influence of vegetation period on the extract qualitative composition and flavonoids quantities was evaluated. Sample preparation by extraction using different concentration of aqueous ethanol (40-96%, v/v) and the influence of extractant composition on the recovery of flavonoids are discussed in detail. The results obtained using CZE are compared to the results of spectrophotometric and HPLC analysis of the extracts. The effect of storage conditions of extracts (solar irradiation, temperature and duration) on degradation of flavonoids was investigated.     

Separation of flavonoids and phenolic acids from propolis by capillary zone electrophoresis

The simultaneous determination of twelve different flavonoids, pinocembrin, acacetin, chrysin, rutin, catechin, naringenin, galangin, luteolin, kaempferol, apigenin, myricetin, and quercetin, two phenolic acids, cinnamic acid and caffeic acid, and one stilbene derivative, resveratrol, in propolis extracts used in medicine has been investigated by capillary zone electrophoresis (CZE). With a buffer constituted by sodium tetraborate 30 mM, pH 9.0, and 15 kV applied voltage, the 15 polyphenols were separated on an uncoated fused-silica capillary within 40 min using normal polarity. Under the experimental conditions used, a linear relationship was calculated between the CZE migration times and the molecular weight of polyphenols' expression of the increasing amount of their hydroxyl groups and polarity. Regression equations revealed a linear relationship (correlation coefficients > 0.97) between the peak area of each polyphenol species and their concentration, from 6 to 120 ng. The levels of analytes in three different propolis extracts, ethanolic, aqueous-ethanolic and aqueous-glycolic, used to prepare various commercial medicinal products, were determined. The aqueous-ethanolic propolis extract showed a great percentage of caffeic acid, galangin, quercetin, and chrysin, whilst the ethanolic preparation was composed of a great amount of resveratrol, chrysin, and caffeic acid. On the contrary, the aqueous-glycolic propolis preparation was composed of approx. 11% of caffeic acid and a low amount of the other identified flavonoids due to the presence of approx. 85% of nonidentified compounds. CZE represents a valuable method for the qualitative and quantitative assay of the most relevant polyphenol components of propolis, representing an alternative to obtain typical fingerprints of propolis and a reliable identification of a large number of propolis polyphenolic species.     

Identification of Natural and Early Synthetic Textile Dyes with HPLC and UV/Vis-Spectroscopy by Diode Array Detection

Abstract

The separation and identification of complex mixtures of natural and synthetic textile dyes was investigated using HPLC with diode array detection. Separation was carried out on a reversed phase column with acetonitrile-phosphoric acid gradient elution. The results show that the anthraquinones from madder root and the insect dye cocheneal present in ancient red dyes can easily be distinguished from azo-dyes present in later textile fibres. They further show that for an analysis of the numerous flavones and flavonoles, constituting most of the yellow natural dyestuffs, the combination of HPLC with on-line optical spectroscopy is particularly useful. Even when retention times are identical - as for instance for quercetin and luteolin - the ratio of a mixture can still be estimated by evaluating the ratio-chromatogram.     

Automated solid-phase extraction hyphenated to voltammetry for the determination of quercetin using magnetic nanoparticles and sequential injection lab-on-valve approach

In this study, an automated sequential injection lab-on-valve (SI-LOV) system was designed for the on-line matrix removal and preconcentration of quercetin. Octadecyl functionalized magnetic silica nanoparticles were prepared and packed into the microcolumn of the LOV as adsorbents. After being adsorbed through hydrophobic interaction, the analyte was eluted and subsequently introduced into the electrochemical flow cell by voltammetric quantification. The main parameters affecting the performance of solid-phase extraction, such as sample pH and flow rate, eluent solution and volume, accumulation potential and accumulation time were investigated in detail. Under the optimum experimental conditions, a linear calibration curve was obtained in the range of 1.0 × 10(-8) to 1 × 10(-5) mol L(-1) with R(2) = 0.9979. The limit of detection (LOD) and limit of quantitation (LOQ) were 1.3 × 10(-9) and 4.3 × 10(-9) mol L(-1), respectively. The relative standard deviation (RSD) for the determination of 1.0 × 10(-6) mol L(-1) quercetin was found to be 2.9% (n = 11) along with a sampling frequency of 40 h(-1). The applicability and reliability of the automated method described here had been applied to the determination of quercetin in human urine and red wine samples through recovery experiments, and the obtained results were in good agreement with those obtained by the HPLC method.