Analysis of grape and wine anthocyanins by HPLC-MS

The development and application of valuable analytical tools suitable for the varietal authentication of premium red wines are matters of interest in order to avoid fraud. In this study, an HPLC-MS procedure has been developed using trifluoroacetic acid as an acid modifier in the mobile phase. This method may be used as a routine method using UV-vis detection and allows the simultaneous analysis of the structural features of anthocyanins by MS under the same chromatographic conditions. Twenty different anthocyanins have been detected in 19 different samples of both grape extracts and wines. Cis and trans isomers of p-coumaryl derivatives have been identified for the first time. Important qualitative and quantitative differences among cultivars have been detected.     

土壤中微塑料的分离及检测方法研究进展

微塑料一般是指粒径小于5 mm的塑料颗粒,具有稳定性高、粒径小及迁移性强等特性,能长期存在于土壤环境中,并且充当各种污染物迁移的载体,甚至通过植物富集等方式经食物链逐级传递,对环境和人体健康造成严重危害。然而,由于土壤基质的复杂性和分析技术的限制,关于土壤微塑料的研究尚存很多空白。开展土壤微塑料分析技术的研究是探索微塑料在土壤中的迁移转化规律和评估微塑料生态风险的基础。本文综述了国内外环境样品中微塑料的分离提取和识别定量技术的研究进展,探讨了各方法的优缺点及其对土壤样品的适用性,并对未来分析技术的发展方向提出展望。文章认为,密度分离法作为最常用的分离方法,操作简单且分离效果良好,但存在无法有效去除有机物和分离小塑料颗粒（<50μm）的问题;新兴的加压流体萃取技术会对微塑料结构造成一定破坏,但由于其自动化程度高、成本低、效率高,仍具有良好的应用前景;其他替代方法（如油提法、磁性分离法等）的应用相对有限,其对土壤样品的适用性还有待研究。不同强度的消解方法均会对微塑料结构造成不同程度的破坏,且增强有机质消解效率往往以牺牲微塑料回收率为代价。现阶段的识别定量方法主要包括借助显微镜的目视鉴...     

Characterization, quantification, and bioactivities of anthocyanins in Cornus species

Cornus mas, Cornus officinalis, Cornus controversa, and Cornus kousa (Cornaceae) bear edible fruits that are consumed in parts of Europe and Asia. This study undertook the investigation of the presence and levels of anthocyanins in the fruits of these Cornus species by HPLC. The anthocyanins present in Cornelian cherries, C. mas, are delphinidin 3-O-beta-galactopyranoside (1), cyanidin 3-O-beta-galactopyranoside (2), and pelargonidin 3-O-beta-galactopyranoside (3). C. officinalis contains only anthocyanins 1-3, similar to C. mas, but in different proportions. However, C. controversa contains anthocyanins 1-3 among other anthocyanins, but Chinese dogwood, C. kousa, did not contain 1-3. The contents of pure anthocyanins 1, 2, and 3 in 1 kg of fresh fruits of C. mas, C. officinalis, and C. controversa were 280, 1079, and 710 ppm; 11, 77, and 230 ppm; and 600, 1000, and 700 ppm, respectively. In cyclooxygenase (COX)-I and -II enzyme inhibitory assays, anthocyanins 1-3 (all 40 microM) showed activities of 9.2 and 11.7%; 7.6 and 12.4%; and 5.3 and 7.8%, respectively, compared to Naproxen (54.3 and 41.3%; 10 microM), ibuprofen (47.5 and 39.8%; 10 microM), Celebrex (46.2 and 66.3%; 1.67 ppm), and Vioxx (23.8 and 88.1%, 1.67 ppm). In the antioxidant assay, anthocyanins 1-3 (all 40 microM) showed activities of 70.2, 60.1, and 40.3%, respectively. At 10 microM concentration, commercial synthetic antioxidants tert-butylhydroquinone, butylated hydroxytoluene, butylated hydroxyanisole, and vitamin E gave 83.2, 79.7, 82.1, and 10.2% of antioxidant activity, respectively.     

Determination of anthocyanins from camu-camu (Myrciaria dubia) by HPLC-PDA, HPLC-MS, and NMR

Camu-camu [Myrciaria dubia (HBK) McVaugh] is a small fruit native to the Amazonian rain forest. Its anthocyanin profile has now been investigated for the first time. Fruits from two different regions of the S?o Paulo state, Brazil, were analyzed. The major anthocyanins were isolated by high-speed countercurrent chromatography. HPLC-PDA, HPLC-MS/MS, and 1H NMR were used to confirm the identity of the main anthocyanins of camu-camu. Cyanidin-3-glucoside was identified as the major pigment in the fruits from both regions, representing 89.5% in the fruits produced in Iguape and 88.0% in those from Mirandópolis, followed by the delphinidin-3-glucoside, ranging between 4.2 and 5.1%, respectively. Higher total anthocyanin contents were detected in the fruits from Iguape (54.0 +/- 25.9 mg/100 g) compared to those from Mirandópolis (30.3 +/- 6.8 mg/100 g), most likely because of the lower temperatures in the Iguape region.     

Characterization and quantification of phenolic compounds in olive oils by solid-phase extraction, HPLC-DAD, and HPLC-MS/MS

A simple and reproducible method for qualitative and quantitative analysis of phenolic compounds in virgin olive oils by solid-phase extraction (SPE), high performance liquid chromatography with diode array detector (HPLC-DAD), and HPLC-mass spectrometry (MS) in tandem mode was developed. The polar fraction was obtained from samples of three different virgin olive oils. Detection and quantification were performed at 280, 240, and 320 nm. For identification purposes, HPLC-MS/MS was equipped with turbo ion spray source in the negative-ion mode. Twenty compounds of twenty-three detected and quantified were characterized. The method showed satisfactory linearity (r > 0.99), good recovery, satisfactory precision, and appropriate limits of detection (LOD) and quantification (LOQ).     

Separation and HPLC-MS identification of phenolic antioxidants from agricultural residues: almond hulls and grape pomace

Almond hulls and grape pomace are residues abundantly generated by agricultural industries, which could be processed to obtain bioactive products. To this purpose, crude ethanol extracts from both agricultural byproducts were attained and subsequently fractionated in order to obtain an organic/water fraction (FOW). Extracts and fractions were analyzed for antioxidant power and their phenolic components tentatively identified by HPLC-MS. Chromatographic peaks of almond hull extracts showed the occurrence of hydroxybenzoic and cinnamic acid derivatives, with minor presence of flavan-3-ols (ECG, EGCG), whereas the FOW fraction offered the additional presence of epicatechin (EC) and glycosylated flavonols. In the composition for extracts of white and red grape pomace several of these compounds were also detected but basically consisted of glycosylated flavonols (quercetin, kaempferol). As a difference between both grape pomaces, myricetin glycosyde was found in that from the red variety, whereas flavan-3-ols (EC, afzelechin) were only identified in white pomace. When their FOW fractions were analyzed, gallic acid and some hydroxybenzoic acids were additionally detected. Antioxidant activity was assessed by DPPH and TBARS assays. Almond hulls showed inhibition percentages lower than 50% in both assays, while the inhibition percentage ranged from 80% to 90% in pomace extracts. Red grape pomace extract was the most efficient antioxidant, with an EC50 value of 0.91 g/L for TBARS and 0.20 g/L for DPPH. Even appearing as two quite different vegetal matrixes, the composition of phenolics in grape pomace and almond hulls is quite similar, the main difference being the major occurrence of flavonols in grape pomace. This fact could presumably explain the lower antiradical activity of hull extracts.     

Extraction,semi-quantification,and fast on-line identification of oligopeptides in Grana Padano cheese by HPLC-MS

Sixteen samples of Grana Padano cheese aged from 2 to 33 months were analyzed by HPLC-MS. The extraction process involved the use of diluted HCl, thus avoiding a strong deproteinizing agent (TCA), and allowing to maintain in solution also very lipophilic peptides. The molecular mass of the most abundant peptides were determined by electrospray ionization (ESI) mass spectrometry. A new method was developed based on the small fragmentation peaks arising from in-source fragmentation and from software analysis of the known casein sequences, which in many cases allowed the direct on-line identification of the oligopeptide sequences. Several new peptides never previously reported were identified, some of which containing bioactive sequences, consistently with what was described in the literature. Semiquantification of peptides at the different stages of aging was also performed by using a suitable internal standard, providing new insights into the evolution of the oligopeptide fraction during aging.     

Structural profiling and quantification of sphingomyelin in human breast milk by HPLC-MS/MS

The sphingolipid composition of food as well as of physiological samples has received considerable interest due to their positive biological activities. This study quantified the total amount of sphingomyelin (SM) in 20 human breast milk samples from healthy volunteers and determined the structures of SM by detailed mass spectrometric studies in combination with enzymatic cleavage. The quantification of SM was performed by hydrophilic interaction liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (HILIC-HPLC-ESI-MS/MS) measuring the characteristic fragment ion of the phosphorylcholine group at m/z 184.2 and by using hexanoylsphingomyelin (C6-SM) and heptadecanoylsphingomyelin (C17-SM) as internal standards. The structures of SM species were identified after enzymatic cleavage with alkaline sphingomyelinase (SMase) to the corresponding ceramides. Structure elucidation of the sphingoid base and fatty acid backbone was performed by reversed-phase HPLC-ESI-MS/MS. The method includes the sphingoid bases dihydrosphingosine (d18:0), sphingosine (d18:1(Δ4)), 4,8-sphingadienine (d18:2(Δ4,8)), 4-hydroxysphinganine (phytosphingosine (t18:0)), and 4-hydroxy-8-sphingenine (t18:1(Δ8)) and fatty acids with even-numbered carbon atoms (C12-C26) as well as their (poly)unsaturated and monohydroxylated analogues. The total amount of SM in human breast milk varied from 3.87 to 9.07 mg/100 g fresh weight. Sphingosine (d18:1) was the predominant sphingoid base, with 83.6 ± 3.5% in human breast milk, followed by 4,8-sphingadienine (d18:2) (7.2 ± 1.9%) and 4-hydroxysphinganine (t18:0) (5.7 ± 0.7%). The main SM species contained sphingosine and palmitic acid (14.9 ± 2.2%), stearic acid (12.7 ± 1.5%), docosanoic acid (16.2 ± 3.6%), and tetracosenoic acid (15.0 ± 3.1%). Interestingly, the fatty acid composition of SM species in this study differs from the total fatty acids in human breast milk, and the fatty acids are not consistently distributed among the different sphingoid bases.     

Identification by HPLC-DAD and HPLC-MS analyses and quantification of constituents of fennel teas and decoctions

Qualitative and quantitative differences among the constituents in various fennel (Foeniculum vulgare Mill., family Apiaceae) teas prepared by classical infusion, microwave decoction, and dissolution are reported. Different commercial starting materials, such as fruit (unbroken and crushed), four herbal teas, and two instant herbal teas were evaluated. Chlorogenic acid (1), quercetin-3-O-beta-D-glucuronide (2), p-anisaldehyde (3), and trans-anethole (4) were identified by HPLC-DAD and HPLC-MS as constituents of fennel teas. No coumarins, which are characteristic constituents of plants of Apiaceae family, were found. Trans-anethole (4), the main constituent of the essential oil, was present in all teas. In addition p-anisaldehyde (3), a degradation product of trans-anethole, was also identified in all teas with the exception of two samples. Chlorogenic acid (1) and quercetin-3-O-beta-D-glucuronide (2) were also present in all teas. In addition, minor unidentified flavonol constituents were found in two teas. Quality, activity, and safety of the content of the investigated preparations are also discussed.     

Podophyllotoxin and deoxypodophyllotoxin in Juniperus bermudiana and 12 other Juniperus species: optimization of extraction, method validation, and quantification

The lignans podophyllotoxin and deoxypodophyllotoxin are secondary metabolites with potent pharmaceutical applications in cancer therapy. However, the supply of podophyllotoxin from its current natural source, Podophyllum hexandrum, is becoming increasingly problematic, and alternative sources are therefore urgently needed. So far, podophyllotoxin and deoxypodophyllotoxin have been found in some Juniperus species, although at low levels in most cases. Moreover, extraction protocols deserve optimization. This study aimed at developing and validating an efficient extraction protocol of podophyllotoxin and deoxypodophyllotoxin from Juniperus species and applying it to 13 Juniperus species, among which some had never been previously analyzed. Juniperus bermudiana was used for the development and validation of an extraction protocol for podophyllotoxin and deoxypodophyllotoxin allowing extraction yields of up to 22.6 mg/g DW of podophyllotoxin and 4.4 mg/g DW deoxypodophyllotoxin, the highest values found in leaf extract of Juniperus. The optimized extraction protocol and HPLC separation from DAD or MS detections were established and validated to investigate podophyllotoxin and deoxypodophyllotoxin contents in aerial parts of 12 other Juniperus species. This allowed either higher yields to be obtained in some species reported to contain these two compounds or the occurrence of these compounds in some other species to be reported for the first time. This efficient protocol allows effective extraction of podophyllotoxin and deoxypodophyllotoxin from aerial parts of Juniperus species, which could therefore constitute interesting alternative sources of these valuable metabolites.     

Development and validation of a spectrophotometric method for quantification of total glucosinolates in cruciferous vegetables

Given their putative role in chemoprevention, validated methods are needed for quantification of total glucosinolates. Based on the colorimetric reaction of ferricyanide with 1-thioglucose, released by alkaline hydrolysis of glucosinolates, we developed a simple and sensitive method for spectrophotometric quantification of total glucosinolates in cruciferous vegetables. Lyophilized and ground vegetables are extracted with 80% boiling methanol. Extracted glucosinolates are isolated using a strong anion exchange column and then hydrolyzed with 2 N NaOH to release 1-thioglucose. Ferricyanide is added, and the decrease in absorbance is measured at 420 nm, with final values adjusted for background. Recovery of internal standard (sinigrin) was 107%. Intra- and interassay coefficients of variation were 5.4% and 15.8%, respectively. Dose response was linear with sinigrin and amount of plant material extracted (R(2) ≥ 0.99). Using sinigrin, the lower limit of quantification was 0.6 mg. This straightforward method may be an alternative to time-consuming and costly chromatographic methods.     

Separation and purification of anthocyanins by high-speed countercurrent chromatography and screening for antioxidant activity

The all-liquid chromatographic technique of high-speed countercurrent chromatography (HSCCC) has been applied for separations of anthocyanins. The biphasic mixture of tert-butyl methyl ether/n-butanol/acetonitrile/water (2:2:1:5) acidified with trifluoroacetic acid was found to be a suitable solvent system for anthocyanin separation. In some cases, enrichment of the pigments on Amberlite XAD-7 resin prior to HSCCC has been carried out. The anthocyanin mixtures from red cabbage, black currant, black chokeberry, and roselle were successfully fractionated using HSCCC. Peak purity control was done by nuclear magnetic resonance spectroscopy as well as electrospray ionization ion trap multiple mass spectrometry. Finally, antioxidant activity of the purified pigments was determined using the Trolox equivalent antioxidant capacity test.     

Fast and versatile multiresidue method for the analysis of botanical insecticides on fruits and vegetables by HPLC/DAD/MS

A simple multiresidue method for screening analysis of 12 botanical insecticides used by organic farmers has been developed. The method involves a rapid and small-scale extraction procedure with acetonitrile. For all fruit and vegetable samples, there was no need for clean up. Rotenone, azadirachtin, ryanodines, and pyrethrins can be separated by high-performance liquid chromatography, quantified, and confirmed with a diode array detector (DAD) and atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in the select ion-monitoring mode (SIM). The majority of pesticide recoveries for various fruits and vegetables were >70% in the concentration range from 0.01 to 5 mg/kg. The limit of quantitation for most of the pesticides was 0.01 mg/kg, with the majority of relative standard deviations (RSD) mostly below 10%.     

Identification of the botanical origin of raw spirits produced from rye, potato, and corn based on volatile compounds analysis using a SPME-MS method

Determination of the botanical origin of raw spirit used for alcoholic beverage production is of great importance for rectifying units, control laboratories, and proper product labeling. Raw spirit samples (138) produced from rye, corn, and potato were analyzed using a solid phase microextraction-mass spectrometry (SPME-MS) method, which involved volatiles preconcentration by SPME with subsequent volatile fraction characterization by MS without particular compounds separation by GC. Obtained data were treated using principal component analysis and linear discriminant analysis (LDA) to test the possibility of sample classification. SPME sampling conditions allowed rapid extraction in 2 min at 50 °C using a carboxen/divinylbenzene/polydimethylsiloxane fiber, followed by rapid MS analysis. Use of LDA made possible the classification of raw spirits based on the material they were produced from. The classification ability of the developed SPME-MS method was 100%, whereas its prediction ability was 96%.     

Comparison of the HPLC method and FT-NIR analysis for quantification of glucose, fructose, and sucrose in intact apple fruits

A rapid quantification method was developed and validated for simultaneous and nondestructive quantifying the constituent sugar concentrations of intact apples using Fourier transform near-infrared (FT-NIR) spectroscopy in diffuse reflectance mode. Multiplicative scatter correction (MSC), the second derivative of Savitsky-Golay, and mean centering were used as spectral preprocessing options. Calibration models were established by the partial least squares (PLS) regression analysis, and validation of the method was performed according to the high-performance liquid chromatography (HPLC) chromatographic method. Spectral range and the number of PLS factors were optimized for the lowest root-mean-square error of prediction (RMSEP) and correlation coefficient of determination (r). The best models showed satisfactory predictions as measured by the RMSEP and r values: glucose, 0.201 and 0.950; fructose, 0.298 and 0.968; sucrose, 0.335 and 0.969, respectively. FT-NIR analysis of constituent sugar concentrations in the intact apple form was found to be more flexible and much faster than performed with the HPLC method.     

Separation, characterization and quantification of phenolic compounds in blueberries and red and black currants by HPLC-DAD-ESI-MSn

The phenolic profile of four blueberry varieties (Vaccinium corymbosum L., cv. Toro, Legacy, Duke and Bluecrop) and two varieties (Rosenthal and Rovada) of red currants (Ribes rubrum L.) and black currants (Ribes nigrum L.) cultivated in Macedonia have been analyzed using HPLC coupled to diode-array detection and tandem mass spectrometry with electrospray ionization. A complex profile of anthocyanins, flavonols, flavan-3-ols and hydroxycinnamic acid derivatives has been assayed in acetone-acetic acid (99:1, v/v) extracts. Anthocyanins comprised the highest content of total phenolic compounds in currants (>85%) and lower and variety dependent in blueberries (35-74%). Hydroxycinnamic acid derivatives comprised 23-56% of total phenolics in blueberries and 1-6% in currants. Chlorogenic acid was the major hydroxycinnamic acid in blueberries, only in the Legacy variety, two malonyl-caffeoylquinic acid isomers were major components. Flavonols, mainly quercetin and myricetin glycosides, were a minor group, but glucosides of laricitrin and syringetin were also detected in the blueberry varieties counting for 10-34% of total flavonols. From flavan-3-ols, catechin was detected in most samples; the dimer B2 was specific for blueberries whereas epigallocatechin was detected in currants.     

Separation, characterization, and quantitation of phenolic acids in a little-known blueberry (Vaccinium arctostaphylos L.) fruit by HPLC-MS

The aim of this study was the qualitative and quantitative determination of free, ester, glycoside, and ester-bound phenolic acids in the blueberry (Vaccinium arctostaphylos L.) fruit. A method for the determination of the profile of phenolic acids of four different phenolic fractions in the fruit was developed using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Thirteen compounds (gallic, protocatechuic, p-hydroxybenzoic, m-hydroxybenzoic, gentisic, chlorogenic, p-coumaric, caffeic, ferulic, syringic, sinapic, salicylic, and trans-cinnamic acids) were identified and quantified in the berry. These experimental results showed that the predominant phenolic acid in the fruit of V. arctostaphylos is caffeic acid in free and insoluble ester-bound forms and p-coumaric acid in soluble ester and glycoside forms. Seven phenolic acids were identified as hydroxybenzoic acid derivatives (HBAs) and four as hydroxycinnamic acid derivatives (HCAs). Total content of HBAs and HCAs in the four phenolic fractions constituted 30.1 and 69.9% of the free, 27.9 and 72.1% of the ester, 24.7 and 75.3% of the glycoside, and 51.7 and 48.3% of the ester-bound forms, respectively. Total phenolics as the sum of individual phenolic acids identified is 698.5 ng/g of fresh weight (fw) for the free, 3399.2 ng/g of fw for the ester, 3522.1 ng/g of fw for the glycoside, and 3671.6 ng/g of fw for the ester-bound phenolic fractions. The present results were compared with reported levels of phenolic acids in the fruits of different Vaccinium species. These data suggest that the fruit can be considered as a potentially good dietary source of phenolic acids.     

Assessment of screening methods for the identification of genetically modified potatoes in raw materials and finished products

Qualitative polymerase chain reaction methods for the detection of genetically modified potatoes have been investigated that can be used for screening purposes and identification of insect-resistant and virus-resistant potatoes in food. The presence of the nos terminator from Agrobacterium tumefaciens and the antibiotic marker gene nptII (neomycin-phosphotransferase II) was demonstrated in three commercialized Bt-potato lines (Monsanto Co., St. Louis, MO, USA) and one noncommercial GM-potato product (high amylopectin starch, AVEBE, Veendam, The Netherlands) and allows for general screening in foods. For further identification, specific primers for the FMV promoter derived from the figwort mosaic virus, the CryIIIA gene (delta-endotoxin from Bacillus thuringiensis subsp. tenebrionis), potato leafroll virus replicase gene, and the potato virus Y coat protein gene, were designed. The methods described were successfully applied to processed potato raw materials (dehydrated potato powders and flakes), starch samples, and finished products.     

Optimisation of amino sugar quantification by HPLC in soil and plant hydrolysates

Amino sugars are increasingly used as indicators for the accumulation of microbial residues in soil and plant material. A reverse-phase high-performance liquid chromatography method was improved for the simultaneous determination of muramic acid, mannosamine, glucosamine and galactosamine in soil and plant hydrolysates via ortho-phthaldialdehyde (OPA) pre-column derivatisation and fluorescence detection. The retention time was reduced, and the separation of muramic acid and mannosamine was optimised by modifying the mobile phase. The effects of excitation wavelength, OPA reaction time, tetrahydrofuran concentration and pH value of the mobile phase on the amino sugar separation were tested. Quantification limits were in the range of 0.13 to 0.90 μg ml⁻¹. No interferences exist from amino acids or other primary amines, occurring in soil and plant hydrolysates.     

刺葡萄皮中花色苷的分离纯化与结构鉴定

为研究刺葡萄花色苷的结构及其纯化分离的柱层析法,将刺葡萄色素粗提液依次经大孔树脂HP-20、聚酰胺树脂、葡聚糖凝胶Sephadex LH-20吸附纯化,利用超高效液相色谱三重四级杆飞行时间质谱联用技术对分离所得花色苷进行结构鉴定,并运用荧光光度法探索荧光图谱与花色苷结构的关系。研究发现,聚酰胺树脂对部分花色苷产生了吸附作用,而Sephadex LH-20凝胶能起到较好的分离作用,最终得到3种色素,经鉴定,确定色素I为锦葵素-3,5-O-双葡萄糖苷,通过质谱信息初步确定色素III可能为锦葵素-3,5-O-双葡萄糖苷-香豆酰,色素IV可能为飞燕草素-3-O-芸香糖苷和锦葵素-3-O-芸香糖苷的混合物,经高效液相色谱以归一法计算峰面积,色素I和色素III的纯度分别达到了98.64%、98.33%,得率分别为0.114%和0.076%。研究结果为花色苷的分离及鉴定提供参考。