Characterization of a new anthocyanin in black raspberries (Rubus occidentalis) by liquid chromatography electrospray ionization tandem mass spectrometry

Anthocyanin composition of black raspberry (Rubus occidentalis) was studied using high-performance liquid chromatography coupled to photodiode array (PDA) detection and electrospray ionization mass spectrometry (LC-ESI/MS) and tandem mass spectrometry (MS/MS). Pelargonidin 3-rutinoside was isolated and identified in black raspberries using HPLC, UV–Vis spectroscopy, MS, and NMR spectroscopy. No pelargonidin derivative had been previously found in Rubus occidentalis. In addition, the presence and identities of four previously reported anthocyanins (cyanidin 3-glucoside, cyanidin 3-sambubioside, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside) were confirmed by HPLC/MS and MS/MS analyses.     

‘紫娟’茶花色苷的分离鉴定

依次以MCI gel CPH 20P(75~150μm)树脂和Sephadex~(TM) LH-20葡聚糖凝胶为层析柱填料,对‘紫娟’茶花色苷进行分离纯化,采用5%乙酸-甲醇溶液和5%乙酸溶液对花色苷提取液梯度洗脱,得到6种花色苷组分。采用薄层层析、高效液相色谱及高效液相色谱-电喷雾-串联质谱对‘紫娟’茶花色苷组成成分进行研究。结果表明:从‘紫娟’茶鲜叶中分离出的花色苷为飞燕草素-3-O-半乳糖苷、矢车菊素-3-O-半乳糖苷、飞燕草素-3-O-(6-(Z)对香豆酸)吡喃半乳糖苷、矢车菊素-3-O-(6-(Z)对香豆酸)吡喃半乳糖苷、飞燕草素-3-O-(6-(E)对香豆酸)吡喃半乳糖苷、矢车菊素-3-O-(6-(E)对香豆酸)吡喃半乳糖苷。     

Determination of Anthocyanins, Total Phenolic Content, and Antioxidant Activity in Andes Berry (Rubus glaucus Benth)

ABSTRACT:  Anthocyanins, total phenolic content, ascorbic acid content, and the antioxidant activity were determined in extracts of Andes berry fruit ( Rubus glaucus Benth). Anthocyanis (ACNs) were isolated and characterized by means of high-performance liquid chromatography (HPLC) with photodiode array detection and electro spray ionization/mass spectrometry (PDA-ESI/MS/MS) analysis. The anthocyanin (ACN) content was 45 mg/100 g FW. The isolated anthocyanins were characterized as cyanidin 3-sambubioside, cyanidin 3-glucoside, cyanidin 3-xylorutinoside, cyanidin 3-rutinoside, pelargonidin 3-glucoside, and pelargonidin 3-rutinoside. The ascorbic acid content was 10.1 mg/100 g FW. The total phenolic content as determined by the Folin–Ciocalteau method was 294 mg GAE/100 g FW while the antioxidant activity as measured by ABTS ^{· +} radical scavenging capacity and ferric reducing antioxidant power (FRAP) was 2.01 and 4.50 mmol TE/100 g FW or 8.22 mmoles ferric iron reduced/100 g FW, respectively. The high phenolic content and antioxidant capacity of Andes berry suggest that this fruit could be a rich source of natural pigments, nutraceuticals, and natural antioxidants.     

Identification of anthocyanin pigments in strawberry (cv Camarosa) by LC using DAD and ESI-MS detection

New high performance liquid chromatography (HPLC) conditions were developed for the separation of strawberry anthocyanins that provided a good resolution of peaks at a low flow rate compatible with the requirements of the mass spectrometry (MS) detector. A strawberry extract was fractionated by column chromatography and simple fractions containing basically anthocyanins were obtained, making their analysis by HPLC possible using on-line photodiode array detection and MS. Information on the identity of the major and some secondary anthocyanins in strawberry was obtained from their retention characteristics, UV-visible spectra and mass spectra. The presence in strawberry of the previously reported cyanidin 3-glucoside, pelargonidin 3-glucoside, pelargonidin 3-rutinoside and pelargonidin 3-acetylglucoside was confirmed and cyanidin 3-rutinoside was identified in strawberry for the first time. Furthermore, cyanidin 3-malonyldiglucoside, pelargonidin 3-malylglucoside, a pelargonidin bioside and two possible pelargonidin 3-biosides acylated with acetic acid were also tentatively assigned.     

Characterization and application of isotope-substituted (13C15)-deoxynivalenol (DON) as an internal standard for the determination of DON

The powerful combination of liquid chromatography and mass spectrometry (MS) is often limited by matrix effects during ionization in the MS ion source. The use of fully isotope-substituted (¹³C₁₅)-deoxynivalenol ((¹³C₁₅)-DON) as an internal standard (IS) corrects matrix effects and improves the accuracy of analytical methods using mass spectrometry for the quantitative determination of the Fusarium mycotoxin deoxynivalenol (DON). The IS was characterized with respect to its chromatographic purity by liquid chromatography-ultraviolet light and its isotope distribution by time-of-flight mass spectrometry. Its low-energy collision-induced dissociation behaviour was compared with DON. Moreover, this work describes the successful application of (¹³C₁₅)-DON as IS for the determination of DON in maize using high-performance liquid chromatography (HPLC) electrospray (ESI) with tandem mass spectrometry. The results demonstrate that the IS can successfully correct for fluctuations during extraction and clean-up of the sample as well as the ionization of DON in the MS ion source. Random variations in ionization affect the IS in the same way as the analyte. Recoveries for DON in maize of 76% ± 1.9% (external calibration) or 101% ± 2.4% (internal calibration) were reached, respectively, after sample clean-up.     

Characterisation of anthocyanins in red cabbage using high resolution liquid chromatography coupled with photodiode array detection and electrospray ionization-linear ion trap mass spectrometry

The aim of this work was to analyse and tentatively identify anthocyanin species in red cabbage using HPLC/DAD-ESI/Qtrap MS. The extraction was realized by using a pressurized liquid technique and the separation of the pigments was achieved by a high resolution liquid chromatography system with a 1.8 μm particles C-18 column. Photodiode array detection was employed to determine the UV/Vis spectral characteristic of the pigments. Electrospray ionization-linear ion trap mass spectrometry allowed the specific determination of the fragmentation patterns of the anthocyanins, by performing different ion scan modes. Twenty four anthocyanins were separated and identified, all having cyanidin as aglycon, represented as mono- and/or di-glycoside, and acylated, or not, with aromatic and aliphatic acids. Nine anthocyanins were identified for the first time in red cabbage.     

Determination of anthocyanins in four Croatian cultivars of sour cherries (Prunus cerasus)

The anthocyanins were characterised and quantified in four cultivars of sour cherries by means of high-performance liquid chromatography and mass spectrometry. Column chromatography on XAD-2 Amberlite was the method used for isolation of anthocyanins. A diode-array detector was employed. For anthocyanins determination was chosen wavelength of 518 nm. Cyanidin-3-glucosylrutinoside and cyanidin-3-rutinoside were major anthocyanins. The total anthocyanin content in sour cherries, (expressed as cyanidin-3-glucosylrutinoside) varied from 2.7 to 28.0 mg/100 g of fresh weight, with the highest content being observed in Oblainska cultivar.     

紫薯花色苷在贮藏过程中的降解特性

本研究通过高效液相色谱-串联质谱（high performance liquid chromatography-tandem mass spectrometry,HPLC-MS/MS）法分析鉴定‘鄂薯12号’紫薯的花色苷成分,采用pH示差法和HPLC法研究该紫薯花色苷提取液在4、20、35 ℃贮藏98 d期间总花色苷和各单体花色苷的变化规律,并在此基础上研究了花色苷的降解动力学以及褐变指数和聚合物颜色指数。结果表明：从‘鄂薯12号’紫薯提取物中鉴定出13 种花色苷,主要为矢车菊素-3-槐糖苷-5-葡糖苷和芍药素-3-槐糖苷-5-葡糖苷与对羟基苯甲酸、阿魏酸或咖啡酸形成的酰基化花色苷;贮藏期间总花色苷和各单体花色苷的含量呈下降趋势,花色苷的降解符合一级动力学模型;4、20 ℃和35 ℃贮藏条件下总花色苷半衰期分别为228.8、48.1 d和32.6 d;在相同糖苷配体情况下,矢车菊素类花色苷的半衰期要短于芍药素;在相同花色素配体情况下,酰基化花色苷的半衰期要长于未酰基化花色苷,且二酰化花色苷的半衰期长于单酰化花色苷;褐变指数和聚合物颜色指数随贮藏时间的延长和贮藏温度的升高而增大,并且聚合物颜色指数与花色苷含量之间呈指数关系。     

利用HPLC-DAD-ESI/IT-TOF/MSn 分析不同品种蓝莓果实的花青素结构

以6种不同品种的蓝莓果实(埃利奥特、爱国者、北蓝、北陆、杜克、蓝丰)为原料,利用甲醇水溶液提取蓝莓中的花青素。用二极管阵列-高效液相色谱-电喷雾离子阱飞行时间串联多级质谱仪(High performance liquid chromatography-electric spray four pole flight time series multipolar mass spectrometer,HPLC-DAD-ESI/IT-TOF/MSn)及紫外可见光谱及多级质谱数据对得到的6种不同品种的蓝莓花青素结构进行鉴定和对比。结果表明,6种蓝莓果实中共鉴定出13种花青素类化合物,苷元分别为锦葵色素、花翠素、矢车菊素、牵牛花色素、芍药素。其中埃利奥特蓝莓检测到11种花青素,爱国者10种,北蓝7种,北陆12种,杜克9种,蓝丰6种。从花青素结构上分析,6种蓝莓均可检测到带糖配基的锦葵色素-3-O-半乳糖、不连接糖苷的花翠素及矢车菊素;不同的蓝莓品种间所含有的花青素种类、个数、结构等均有明显差异。本研究为快速获取蓝莓中花青素的结构信息提供一定的理论支撑。     

LC‐MS identification of anthocyanins in boysenberry extract and anthocyanin metabolites in human urine following dosing

The anthocyanin composition of boysenberry (Rubusloganbaccus × baileyanus Britt) extract was determined by LC‐ESI‐MS. Four anthocyanins were identified, all comprising a cyanidin‐anthocyanidin‐type skeleton. The two major components were identified as the disaccharide cyanidin‐3‐O‐sophoroside and the monosaccharide cyanidin‐3‐O‐glucoside. The two less abundant components were identified as the rutinosides, cyanidin‐3‐O‐2^G‐glucosylrutinoside and cyanidin‐3‐O‐rutinoside, respectively. These same four anthocyanins were also detected in human urine following a dosing study with boysenberry extract indicating that glycosylated anthocyanins can be absorbed from the gut and excreted intact in the urine. Several anthocyanin metabolites were also detected in the urine and were identified by LC‐ESI‐MS as monoglucuronides of peonidin, cyanidin and pelargonidin. The results suggest that anthocyanins consumed as part of a diet are bioavailable and are present as intact or metabolized forms in the body. Copyright © 2004 Society of Chemical Industry     

Anthocyanin changes in the Korean purple-fleshed sweet potato,Shinzami, as affected by steaming and baking

As hydrophilic pigments, anthocyanins reduce the incidence of cancer and cardiovascular diseases. In this study, the anthocyanin content and composition following steaming and baking of the roots of the Korean purple-fleshed sweet potato variety “Shinzami” were evaluated using liquid chromatography with diode array detection and electrospray ionisation/mass spectrometry (LC-DAD-ESI/MS). Anthocyanins of Shinzami were composed of mono- or di-acylated forms of p-hydroxybenzoic acid, caffeic acid and ferulic acid with the basic structure of cyanidin 3-sophoroside-5-glucoside or peonidin 3-sophoroside-5-glucoside. A total of 15 individual anthocyanins were isolated and confirmed, one of which was presumed to be a newly identified compound, peonidin 3-feruloyl-p-hydroxybenzoyl sophoroside-5-glucoside. Additionally, the amounts of di-acylated cyanidin 3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside and peonidin 3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside were the highest (137.0 and 565.9 mg/100 g DW, respectively) among cyanidin and peonidin compounds. After steaming, the total anthocyanin content was reduced by nearly a half, while roasting only slightly reduced the total anthocyanin content.     

基于液相色谱-质谱联用技术定性定量分析黑豆种皮中可溶型和结合型花青素

以超声波辅助有机溶剂提取法获得黑豆种皮可溶型花青素提取物,再进一步对不含可溶型花青素的黑豆种皮残渣使用酸水解和碱水解以及酸碱/碱酸轮提水解,获得黑豆种皮结合型花青素提取物。采用超高效液相色谱-电喷雾-四极杆飞行时间质谱联用仪分析鉴定各黑豆种皮提取物中所含有的共17 种花青素成分,包括11 种花青素糖苷类：飞燕草素-3-O-葡萄糖苷、飞燕草素-3-O-半乳糖苷、矮牵牛花素-3-O-葡萄糖苷、矮牵牛花素-3-O-半乳糖苷、天竺葵素-3-O-芸香糖苷、矢车菊素-3-O-葡萄糖苷、矢车菊素-3-O-半乳糖苷、天竺葵素-3-O-己糖苷、芍药花素-3-O-己糖苷、天竺葵素-3-O-(6”-丙二酰葡萄糖苷)和芹菜定-3-O-(6”-丙二酰葡萄糖苷);6 种花青素苷元：飞燕草素、矢车菊素、矮牵牛花素、天竺葵素、芹菜定和芍药花素。采用高效液相色谱-电喷雾-三重四极杆质谱联用仪精确定量各类黑豆种皮提取物中的花青素含量,结果表明,酸性结合型花青素提取物中结合型花青素的总含量最高。此外,在黑豆种皮的可溶型花青素提取物中,花青素主要以花青素糖苷类形式存在,苷元含量相对极少;而在结合型花青素提取物中,则主要以花青素苷元为主,糖苷类化合物相对少见。     

鲜切紫甘蓝中花青苷和硫代葡萄糖苷在贮藏过程中的变化

采用高效液相色谱和液相色谱-质谱联用技术,对紫甘蓝（Brassica oleracea L. ssp. capitata f. rubra）中花青苷、硫代葡萄糖苷（简称硫苷）成分和含量进行鉴定和分析,并对低温（8±1）℃条件下经鲜切处理的紫甘蓝中花青苷和硫苷在货架期内（7 d）的变化情况进行研究。结果表明：紫甘蓝中存在11 种不同矢车菊花青苷,矢车菊-3-阿魏酰-槐糖苷-5-葡萄糖苷作为紫甘蓝中最主要的花青苷,在贮藏过程中的损失量占总花青苷损失量的55.51%,其占总量比例也在贮藏7 d后由35.12%下降至30.60%;紫甘蓝中存在10 种硫苷（由6 种脂肪族硫苷、3 种吲哚族硫苷和1 种芳香族硫苷组成）,以4-戊烯基硫苷和3-丁烯基硫苷为主,贮藏7 d后,二者损失率均为7%左右,损失量相加却达总花青苷损失量的50.07%,而所占比例相加也由71.68%上升到74.33%。     

Development of a green two‐dimensional HPLC‐DAD/ESI‐MS method for the determination of anthocyanins from Prunus cerasifera var. atropurpurea leaf and improvement of their stability in energy drinks

This study aimed to develop a green two‐dimensional HPLC‐DAD/ESI‐MS method for analysing anthocyanins from Prunus cerasifera var. atropurpurea leaf and improve their stability in energy drinks by the addition of phenolic acids. Ethanol and tartaric acid solutions were used as mobile phases for one‐dimensional HPLC‐DAD for quantitative analysis of anthocyanins, and the primary anthocyanins were identified as cyanidin‐3‐O‐galactoside, cyanidin‐3‐O‐glucoside and cyanidin‐3‐O‐rutinoside using two‐dimensional HPLC‐MS. Method validation showed that the developed method was accurate, stable and reliable for the analysis of P. cerasifera anthocyanins. The effects of gallic, ferulic and caffeic acid on the stability of cyanidin‐3‐O‐galactoside, cyanidin‐3‐O‐glucoside, cyanidin‐3‐O‐rutinoside and total anthocyanins from P. cerasifera leaf in energy drinks were evaluated, and the degradation of P. cerasifera anthocyanins ideally followed a first‐order model (R² > 0.98). Gallic acid showed stronger protective effects on P. cerasifera anthocyanins in energy drinks, and adding/increasing ferulic and caffeic acids accelerated the degradation reactions.     

红树莓果汁和桑椹果汁花色苷结构的鉴定及杀菌方式对果汁品质的影响

采用高效液相色谱-电喷雾质谱法和紫外-可见光谱法鉴定了红树莓及桑椹中主要花色苷及黄酮的组成,并 以红树莓果汁及桑椹果汁为原料,对比分析了经巴氏杀菌（pasteurization,PS）、煮沸杀菌（boiling sterilization, BS）、微波杀菌（microwave sterilization,MS）3 种杀菌方式处理前后其总花色苷、单个花色苷、总酚、主要黄 酮、H2O2的相对含量及其他理化性质（pH值、可溶性固形物含量、吸光度、褐变度、透光率）的变化。结果表 明：红树莓中的主要花色苷为矢车菊素-3-槐糖苷和矢车菊素-3-葡萄糖苷,桑椹中主要花色苷为矢车菊素-3-葡萄糖 苷和矢车菊素-3-芸香糖苷;与对照组相比,红树莓果汁和桑椹果汁总花色苷、单个花色苷及总酚的相对含量经3 种 杀菌方式处理后都有不同程度的降低,MS处理对其影响最小;3 种杀菌方式处理后的果汁中H2O2相对含量没有显 著性差别（P＞0.05）。MS处理可以较好地保持红树莓果汁和桑椹果汁的营养品质。     

LC–MS analysis of anthocyanins from purple corn cob

Liquid chromatography coupled with diode array spectrophotometry and mass spectrometry detection (LC–DAS–MS) has been applied to the study of the anthocyanin composition of a commercial extract from purple corn cob used as a colourant additive in the food industry. Nine different anthocyanins were isolated using semipreparative HPLC and identified by LC–MS and hydrolytic techniques. Useful information for the identification of compounds was also obtained from their fragmentation patterns (MS–MS spectra). Six of these anthocyanins seem to be present in the original cob, namely cyanidin‐3‐glucoside, pelargonidin‐3‐glucoside, peonidin‐3‐glucoside and their respective malonyl derivatives. The other three are produced during the industrial extraction process and have been identified as the corresponding ethylmalonyl derivatives. © 2002 Society of Chemical Industry     

Phenolic Analyses of Haskap Berries (Lonicera caerulea L.): Spectrophotometry Versus High Performance Liquid Chromatography

Haskap berries (Lonicera caerulea L.) are known for their high phenolics, anthocyanins, and antioxidant potential. The data on the phenolic profile of these fruits are lacking. In this study, the phenolic profiles of three haskap varieties; tundra, berry blue, and indigo gem grown in Nova Scotia, Canada were investigated for the first time using spectrophotometery and high-performance liquid chromatography. Berries were analyzed for total phenolic content/total reducing capacity, total anthocyanin content, and antioxidant potential (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity). The total reducing capacity, total anthocyanin content, and 2,2-diphenyl-1-picrylhydrazyl values were 6.17–8.42 mg gallic acid equivalents/gram fresh weight, 4.49–6.97 mg cyanidin-3-glucoside equivalents/gram fresh weight, and 78.70–89.55%, respectively. The extracts were analyzed by reversed-phase diode array detector–high-performance liquid chromatography through a gradient elution using Synergi 4 µm Max-RP C12 column and the chromatograms were acquired at 520, 360, and 320 nm for athocyanins, flavonoids, and free phenolic acids, respectively. The identified anthocyanins were cyanidin-3-glucoside (82.81–91.99% of the total anthocyanins), cyanidin 3,5-di-glucoside (2.31–4.27%), cyanidin-3-rutinoside (1.54–9.20%), peonidin-3-O-glucoside (0.75–3.44%), and pelargonidin-3-glucoside (0.77–2.98%). Other flavonoids (quercetin-3-β-D-glucoside and quercetin-3-rutinoside) and free phenolic acids (chlorogenic and neochlorogenic) were also quantified.     

野生黑枸杞果实中酚类物质的组成分析

以产自宁夏的野生黑枸杞鲜果为实验材料,采用高效液相色谱-三重四极杆串联质谱联用仪对黑枸杞果实中的酚类物质组成进行定性、定量分析。结果显示,黑枸杞果实富含酚类物质,共鉴定到花色苷类物质20 种,非花色苷酚类物质16 种。其中,最主要的花色苷为甲基花翠素-3,5-O-双葡萄糖苷和一些6-O-香豆酰化衍生的3-O-单糖苷花色苷,而最主要的非花色苷酚类物质为绿原酸等一系列羟基肉桂酸,与葡萄、蓝莓等富含酚类物质的深色水果有极大不同,具有典型的种属特异性。本研究将为黑枸杞的鲜果消费以及产品开发提供一定的理论基础。     

Changes in Radical-scavenging Activity and Components of Mulberry Fruit During Maturation

ABSTRACT Extracts of mulberry fruits (Morus sp.) were prepared from 8 cultivars harvested at 4 stages of maturity, and their radicalscavenging activity, anthocyanin content, and total phenolic content were measured. The radical‐scavenging activity was evaluated by a spectrophotometric assay using the 1,1‐diphenyl‐2‐picrylhydrazyl radical (DPPH) in a 96‐well microplate. Mulberry fruit extracts exhibited the DPPH‐scavenging activities, ranging from 2.5 to 20.3 μmol‐Trolox equiv/g‐FW. Their activities were variable during maturation, and the highest activity was observed in the fully mature mulberry fruit in all cultivars. Anthocyanin was scarcely present in the immature mulberry fruits; however, its content increased as the fruit matured in all cultivars. On the other hand, all immature mulberry fruits contained non‐anthocyanin phenolic compound. An on‐line high‐performance liquid chromatography (HPLC) method for the detection of DPPH‐scavenging compounds revealed the difference in predominant radical scavengers between the immature and fully mature stages in the Miran 5 cultivar. Four major radical scavengers in the Miran 5 cultivar were assigned to 2 caffeoylquinic acids (chlorogenic acid and its isomer) and 2 anthocyanins (cyanidin 3‐glucoside and cyanidin 3‐rutinoside) in the immature and fully mature stages, respectively, by LC‐ESI‐MS/MS analysis. The change in the content of 4 compounds in mulberry fruits during maturation demonstrated that the most likely contributors to the DPPH‐scavenging activity were caffeoylquinic acids in the immature mulberry and anthocyanins in the mature and fully mature mulberry.     

越橘提取物中花青素分析及其体外抗氧化活性

研究越橘提取物中花青素的组成与含量及其体外抗氧化活性。实验采用高效液相色谱与质谱联用法对越橘提取物中的花青素组成进行鉴定,并以矢车菊素-3-O-葡萄糖苷为标准品测定了花青素的含量。以V_C为对照,测定越橘提取物对DPPH自由基、ABTS自由基、羟基自由基的清除能力和还原力。结果表明,越橘提取物中含有14种花青素,包括飞燕草素、矢车菊素、矮牵牛素、芍药素和锦葵素类;越橘提取物中花青素的总含量为408.74 μg/mg,其中,矢车菊素类花青素含量最高,为159.93 μg/mg,占总含量的39.12%;体外抗氧化实验表明,越橘提取物的DPPH自由基清除能力、ABTS自由基清除能力和羟基自由基清除能力的IC₅₀分别为26.16、13.07、1.91 mg/mL,还原力在浓度为250 μg/mL时达到0.617。本研究为越橘的开发利用提供一定理论与数据基础。