Identification of flavonoids and chlorogenic acids in elm fruits from the genus Ulmus and their antioxidant activity

Elm fruits were once an important food source in the years of famine. Research on the functional compounds in elm fruits was almost unavailable. In this study, we established an efficient high‐performance liquid chromatography method for the simultaneous separation of eight chlorogenic acids and 28 flavonoids in elm fruits for the first time. Total flavonoid contents ranged from 286 mg/100 g (Ulmus laciniata) to 1228 mg/100 g (U. pumila). High concentrations of rutin, quercetin 3‐O‐glucoside, and kaempferol derivatives were present in U. laevis, U. castaneifolia, and U. pumila, respectively. Furthermore, the fruit extracts of U. americana, U. castaneifolia, U. davidiana, and U. pumila showed higher antioxidant activity. These results suggest that fruits of these species can be used as bioresources for the extraction of the corresponding functional compounds. This work provides informative data and can be an important reference for future research on elm fruits as a renewed food resource.     

Determination of oroxylin A,oroxylin A 7‐O‐glucuronide,and oroxylin A sodium sulfonate in beagle dogs by using UHPLC MS/MS Application in a pharmacokinetic study

Oroxylin A, obtained from the root of Scutellaria baicalensis Georgi, is a flavonoid with antitumor and other pharmacological activities. Our previous studies showed for the first time that it is mainly metabolized to oroxylin A sodium sulfonate by sulfotransferase enzymes in beagle dogs. In this study, rapid, universal, selective, and robust ultra‐high‐performance liquid chromatography–tandem mass spectrometry methods were established and fully validated to quantitatively detect oroxylin A, oroxylin A 7‐O‐glucuronide, and oroxylin A sodium sulfonate in beagle dog plasma. The quantitative analysis for oroxylin A sodium sulfonate was reported for the first time. Plasma samples were processed with acetonitrile, a universal protein precipitant. Gradient elution was performed to resolve carryover effects and to achieve separation efficiency and sufficient chromatographic retention. The linear relationships of oroxylin A, oroxylin A 7‐O‐glucuronide, and oroxylin A sodium sulfonate in plasma were in the range of 2.0–500.0, 5.0–500.0, and 1.881–940.5 ng/mL, respectively. The assay method was successfully applied to pharmacokinetic study. This is the first paper that reveals the pharmacokinetic profile of oroxylin A, oroxylin A 7‐O‐glucuronide, and oroxylin A sodium sulfonate after single‐dose intravenous and oral administration of Oroxylin A in beagle dogs.     

Development and validation of a UHPLC–MS/MS method for the simultaneous determination of five bioactive flavonoids in rat plasma and comparative pharmacokinetic study after oral administration of Xiaochaihu Tang and three compatibilities

An accurate, rapid, and reliable ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of baicalin, wogonoside, baicalein, wogonin, and oroxylin A in rat plasma. Then, the stability of baicalin and baicalein in the preparation of plasma sample was systematically investigated. The Waters BEH C₁₈ column was used with a gradient mobile phase system of acetonitrile and water containing 0.1% formic acid. The analytes were detected in the multiple reaction monitoring mode with positive electrospray ionization. 100 μL fresh plasma was added with 50 μL antioxidant reagent (1 mol/L HCl containing 0.5% Vitamin C), and liquid–liquid extraction with ethyl acetate was used to extract the analytes from plasma. Lower limits of quantification of baicalin, wogonoside, baicalein, wogonin, and oroxylin A were 21.9, 4.80, 1.20, 0.848, and 0.800 ng/mL, respectively. The mean extract recoveries of five flavonoids were 69.1∼89.2%, and the precision and accuracy were within the acceptable limits. This method was further successfully applied to the comparative pharmacokinetic study of these five flavonoids in rats after oral administration of Xiaochaihutang and three compatibilities. The obtained results may be helpful to reveal the mechanism of Xiaochaihutang formula compatibility.     

Determination of 18 phenolic acids in tobacco and rhizosphere soil by ultra high performance liquid chromatography combined with triple quadrupole mass spectrometry

An ultra high performance liquid chromatography with triple quadrupole mass spectrometry method for the determination of free and bound phenolic acids in tobacco plant and soil was developed. A simple solid‐phase extraction, which used Polar Enhanced Polymer column as stationary phase and methanol as mobile phase, was used for the clean‐up of bound phenolic acids, and a liquid‐phase extraction using chloroform as solvent was used to purify free phenolic acids. With our method, 18 phenolic acids in rhizosphere soil of continuous cropping flue‐cured cultivar k326 were separated and determined within 6 min with recoveries of 82–107% and relative standard deviations (n = 5) of 1.1–4.8%. Results showed that free phenolic acids accounted for 0–9, 92–100, and 69–100% of total phenolic acids in rhizosphere soil, cultivar k326 roots and leaves, respectively. Results also revealed that p‐hydroxybenzoic acid, p‐coumaric acid, vanillic acid, ferulic acid, and syringic acid were the predominant phenolic acids in rhizosphere soil of cultivar k326, and continuous cropping of cultivar k326 in the same farmland could lead to the accumulation of these phenolic acids in soil except syringic acid. The determination of phenolic acids provided detailed information for evaluating their source and characteristics in continuous cropping tobacco plant and soil.     

A rapid,sensitive, and widely applicable method for quantitative analysis of underivatized amino acids in different biological matrices by UHPLC‐MS/MS

A rapid, sensitive, and widely applicable method for the simultaneous quantitative analysis of 20 underivatized amino acids in different biological matrices, including serum, plasma, and tissue homogenates, using ultra high performance liquid chromatography with tandem mass spectrometry was developed and validated. Only 4 µL of serum, plasma, or tissue homogenate was extracted with 996 µL of solution (1.7 mM ammonium formate in 85% acetonitrile containing 0.1% formic acid) containing 100 ng/mL phenylalanine‐d5 as an internal standard without any further derivatization step. In addition, the matrix effects were small because a large volume of extraction solution was used. The total run time including reequilibration was 13 min. The results of linearity, accuracy, repeatability, precision, limits of detection, limits of quantification, and sample stability were sufficient to allow the measurement of the amino acids in different biological matrices. We conclude that our method is rapid, sensitive, and widely applicable and represents an improvement over other currently available technologies.     

Fast determination of virgin olive oil phenolic metabolites in human high‐density lipoproteins

In recent years it has been confirmed that the consumption of olive oil prevents the oxidation of biomolecules owing to its monounsaturated fatty acids (MUFA) and phenolic content. The main objective of the study was to develop an ultra‐high‐performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method for the determination of phenolic compounds in human high‐density lipoprotein (HDL) samples. At the same time, the influence of olive oil consumption on the phenolic metabolite levels was evaluated in a European population. The participants were 51 healthy men, aged 20–60. They were randomized to two consecutive intervention periods with the administration of raw olive oil with low and high polyphenolic content. The UHPLC‐MS/MS analytical method has been validated for hydroxytyrosol and homovanillic acid in terms of linearity (r² = 0.99 and 1.00), repeatability (5.7 and 6.5%) reproducibility (6.2 and 7%), recovery (98 to 97%), limits of detection (1.7 to 1.8 ppb) and quantification (5.8 and 6.3 ppb).The levels of the studied metabolites increased significantly after high polyphenolic content virgin olive oil ingestion (p <0.05) compared with lowpolyphenolic content olive oil. Virgin olive oil consumption increases the levels of phenolic metabolites in HDL and thus provides human HDL with more efficient antioxidant protection. Copyright © 2014 John Wiley & Sons, Ltd.     

Screening for potential quality markers of Callerya nitida var. hirsutissima. Z.Wei based on components profile,pharmacokinetics, and anti-inflammatory study

Callerya nitida var. hirsutissima. Z.Wei is a classical, traditional Chinese herbal medicine mostly used to treat rheumatoid arthritis. Recent reports suggest that inconsistent and poor-quality control levels have primarily affected the therapeutic efficacy. Therefore, the aim of the current study was to investigate the active chemical ingredients, stability of components in blood, pharmacokinetics, and pharmacodynamics to specify the potential markers for quality control and quality evaluation of Callerya nitida. The active components in vitro and in vivo were obtained by ultra-high-performance liquid chromatography-mass spectrometry. Moreover, the changes of the bioactive components in the blood were monitored over time (0–24 h) in order to identify stable active components. On this basis, the pharmacokinetic characteristics of these ingredients combined with the anti-inflammatory activity were determined to screen out the potential markers for ensuring the quality control of Callerya nitida. The identified four components, such as calycosin, daidzein, formononetin, and 5-hydroxymethylfurfural, have the characteristics of intrinsic components, clearly defined structures, high exposure values, and in vivo stability, which are important for the therapeutic activity of pharmacologically active materials. Therefore, they can be used as potential markers to control the quality levels of Callerya nitida.     

Mass spectrometry in the study of anthocyanins and their derivatives: differentiation of Vitis vinifera and hybrid grapes by liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

A mass spectrometric-based procedure for anthocyanin profiling was set up to distinguish authentic Vitis vinifera from hybrid red grapevine cultivars. 3-O-Monoglucoside and the related acetyl-, p-coumaryl- and caffeoyl-monoglucoside anthocyanins occurred only in Vitis vinifera, whereas 3,5-O-diglucoside and the substituted acetyl-, p-coumaryl-, feruloyl- and caffeoyl-diglucoside anthocyanins were the additional pigments in hybrid grapevines. The procedure was applied expressly to identify red grape cultivars based on the anthocyanin chemo-type determination. In particular, a red grape cultivar, having 3,5-O-diglucoside anthocyanins and a novel class of anthocyanin monoglucosides, such as cyanidin-3-O-, cyanidin-3-O-(6-O-acetyl)- and cyanidin-3-O-(6-O-p-coumaryl)pentoside, was classified as hybrid. A second vine cultivar, characterized exclusively by 3-O-monoglucoside anthocyanins, was included among the Vitis vinifera species. Anthocyanin profiling by mass spectrometry could represent the core of a chemotaxonomic procedure for distinguishing American and European grapevines based on the identification of post-synthetic anthocyanidin modification.     

Multidimensional preparative liquid chromatography to isolate flavonoids from bergamot juice and evaluation of their anti‐inflammatory potential

Important objectives of a high‐performance liquid chromatography preparative process are: purity of products isolated, yield, and throughput. The multidimensional preparative liquid chromatography method used in this work was developed mainly to increase the throughput; moreover purity and yield are increased thanks to the automated collection of the molecules based on the intensity of a signal generated from the mass spectrometer detector, in this way only a specific product can be targeted. This preparative system allowed, in few analyses both in the first and second dimensions, the isolation of eight pure compounds present at very different concentration in the original sample with high purity (>95%) and yield, which showed how the system is efficient and versatile. Pure molecules were used to validate the analytical method and to test the anti‐inflammatory and antiproliferative potential of flavonoids. The contemporary presence, in bergamot juice, of all the flavonoids together increases the anti‐inflammatory effect with respect to the single compound alone.     

Determination of carbapenems in water samples by UHPLC–MS/MS

A rapid and reliable method for the detection of five carbapenems (biapenem, imipenem, doripenem, meropenem, and faropenem) in water was developed and validated. After acidification of water samples with acetic acid, carbapenems were isolated using a Bond Elut PPL cartridge. The target compounds were separated using ultra high performance liquid chromatography with a chromatographic run time of 5 min and detected on a triple quadrupole mass spectrometer operated in positive electrospray ionization and multiple reaction monitoring mode. Mean recoveries were in the range of 76.6–106.5%, with satisfactory intraday and interday relative standard deviations lower than 10.0 and 10.8%, respectively. The limits of detection and quantification were in the ranges of 0.05–0.2 µg/L and 0.1–0.5 µg/L, respectively, depending on the analyte. The proposed method was applied to the analysis of river samples and wastewater samples from swine farms, and no carbapenems were detected in the collected samples.     

Development and validation of an analytical method for multiresidue determination of pesticides in lettuce using QuEChERS–UHPLC–MS/MS

A new analytical method for multiresidue determination of 16 multiclass pesticides in lettuce was developed using ultra‐high performance liquid chromatography with tandem mass spectrometry with a triple quadrupole mass analyzer and positive mode electrospray ionization, using a previously optimized quick, easy, cheap, effective, rugged, and safe method for sample preparation. Validation studies, according to document SANTE/11945/2015, demonstrated that the developed method is selective, accurate, and precise, providing recoveries of 70–120%, relative standard deviations ≤20% and quantification limits from 3 μg/kg. The method was compared with one based on high‐performance liquid chromatography with tandem mass spectrometry, in terms of chromatographic performance, detectability and matrix effect for five varieties of lettuce. The new method provided a reduction in the time for the chromatographic analysis of 50%, from 30 to 15 min, using a lower mobile phase flow rate (0.147 mL/min), which reduced the consumption of mobile phase by 25%, and injection of smaller amounts of sample (1.7 μL). Lower limits of quantification were obtained for almost all pesticides studied for green‐leaf lettuce. However, in relation to the matrix effect, four of the five types of lettuce studied presented higher matrix effects.     

Fast and efficient immunoaffinity column cleanup and liquid chromatography–tandem mass spectrometry method for the quantitative analysis of aflatoxins in baby food and feeds

An ultra high performance liquid chromatography‐tandem mass spectrometric method has been developed for the highly sensitive and selective determination of regulated aflatoxins. The extraction of aflatoxins from baby food matrices were performed using liquid–liquid extraction procedure followed by immunoaffinity column cleanup. The higher sensitivity for the determination of target aflatoxins was fulfilled by applying a preconcentration step with immunoaffinity columns after acetonitrile–water extraction. The enhanced selectivity was attained with the triple quadrupole mass analyzer operated in electrospray positive ionization mode. Method validation was tested in five different baby food matrices by recovery experiments. Satisfactory recoveries, between 92 and 103%, with relative standard deviations lower than 8% were achieved in all the tested matrices. The proposed method was found to be specific as no interference peaks were observed for blank samples. The limit of detection of the method was found to be in the range of 0.003–0.008 ng/mL. The validated method was fruitfully applied to the screening of aflatoxins in baby foods and feeds sample retailed in local markets of Riyadh, Saudi Arabia. The obtained levels of all analyzed aflatoxins were below the regulation limits set by European Agency.     

超高效液相色谱-串联质谱法测定甜樱桃果皮中花青苷类物质

建立了甜樱桃果皮中7种花青苷类物质的超高效液相色谱-串联质谱(UPLC-MS/MS)定性和定量检测方法。样品经0.1%(v/v)盐酸甲醇提取液提取,AB-8型大孔吸附树脂净化,在电喷雾离子源正离子(ESI⁺)多反应监测(MRM)模式下对目标物质进行定性和定量分析。结果表明:7种目标化合物的定量限为0.26~1.42 μg/kg;在0~100 μg/L范围内呈现很好的线性关系,相关系数(r²)为0.9964~0.9993;方法的加标回收率为97.2%~105.4%,相对标准偏差(RSD)为1.9%~5.8%。采用该方法对甜樱桃红色品种"美早"和黄色品种"13-33"成熟果实样品进行了分析,发现主要花青苷类物质的成分和含量存在显著性差异。该方法操作简单,灵敏度高,重复性好,花青苷类物质的分析覆盖面广,可用于甜樱桃中花青苷类成分快速鉴定及含量测定。     

Determination of flavonoids from Gynostemma pentaphyllum using ultra-performance liquid chromatography with triple quadrupole tandem mass spectrometry and an evaluation of their antioxidant activity in vitro

Gynostemma pentaphyllum, a traditional herbal tea, and its flavonoids possess antioxidant activities. This study was carried out to isolate and identify these flavonoids from G. pentaphyllum and determinate the contents of the main flavonoids and their antioxidant activities. Nine flavonoids were determinated rapidly from G. pentaphyllum using medium-pressure liquid chromatography combined with other simple chromatographic methods. And nine flavonoids were identified as rutin (1), 4′-O-methyl-kaempferol-3-O-rutinoside (2), ombuoside (3), kaempferol-3-β-D-O-rutinoside (4), isorhamnetin-3-O-β-D-rutinoside (5), quercetin-3-O-β-D-glucoside (6), isorhamnetin (7), kaempferol (8), and quercetin (9) by liquid chromatography mass spectrometry-ion trap time of flight and ¹³C nuclear magnetic resonance spectroscopy, respectively. All of them displayed various potent antioxidant effects against the DPPH radical and A549 cell injury by using H₂O₂-generate peroxyl radicals in vitro . A sensitive and specific ultra-performance liquid chromatography coupled with multiple-reaction monitoring mode was utilized to rapidly analyze the main six flavonoids of the ethanol extract of G. pentaphyllum within less than 8?min using a Waters Acquity UPLC BEH C₁₈ column. The contents of them were from 57.11 to 12907.74?µg/g in G. pentaphyllum.     

高效液相色谱法测定茶饮料中的黄酮类化合物

建立了茶饮料中六种黄酮类化合物的高效液相色谱检测方法。流动相采用梯度洗脱。结果表明芦丁、漆黄素、木犀草素、芹菜素在0.025~20 mg·L-1范围内呈现良好线性关系,桑色素、槲皮素在0.050~20 mg·L-1范围内呈现良好线性关系。加标回收率在84.5%~107.5%。该方法精密度良好(六种黄酮类化合物的相对标准偏差均不大于3.2%),检测限为6.2~34μg·L-1。该方法简便,灵敏、重现性好,可用于同时分析茶饮料中的黄酮化合物。     

Identification and determination of chemical constituents of Citrus reticulata semen through ultra high performance liquid chromatography combined with Q Exactive Orbitrap tandem mass spectrometry

Citrus reticulata semen, a traditional Chinese medicinal material, has desirable medicinal and dietary properties. In this study, a method combining ultra high performance liquid chromatography with Q Exactive Orbitrap tandem mass spectrometry was established and validated for the identification and analysis of the chemical components of C. reticulata semen for the first time. The evaluation of different retention times and fragmentation characteristics, as well as comparative analysis with the literature, resulted in the identification of 35 chemical constituents, including 21 flavonoids and 14 other compounds. The 21 flavonoids derived from C. reticulata semen were reported for the first time. Seven of the chemical components of C. reticulata semen were quantitatively analyzed using the developed method under the optimal conditions. The results showed that the content of limonin, hesperidin, nobiletin, synephrine, tangeretin, 3,5,6,7,8,3′,4′‐heptamethoxyflavone and 5‐hydroxide‐6,7,8,3′,4′‐pentamethoxyflavone in C. reticulata semen was 11.1666, 0.0404, 0.0092, 0.0255, 0.0087, 0.0010, and 0.0008 mg/g, respectively. This study demonstrated that the ultra high performance liquid chromatography Q Exactive Orbitrap mass spectrometry based method can be used to rapidly and reliably analyze the chemical constituents of C. reticulata semen. These results provide a scientific basis for further studies of C. reticulata semen.     

Capillary electrophoresis,high‐performance liquid chromatography,and thin‐layer chromatography analyses of phenolic compounds from rapeseed plants and evaluation of their antioxidant activity

Rapeseed plants, known for oil production, are also known to contain phenolic compounds such as phenolic acids and flavonoids, with potential antioxidant and anticancer activities. The separation and identification of 11 phenolic acids in rapeseed extracts (including leaves, flowers, Chinese seeds, Belgian seeds, and cake) by capillary electrophoresis were investigated. The results were compared with those obtained with high‐performance liquid chromatography and thin‐layer chromatography and showed that the capillary electrophoresis technique offers several advantages for the identification of phenolic compounds in various rapeseed extracts. The antioxidant activity of rapeseed extracts and reference compounds was evaluated using four different approaches, namely, 2,2′‐azinobis‐ (3‐ethylbenzohiazoline‐6‐sulfonic acid assay, free radical 2,2‐diphenyl‐1‐picrylhydrazyl assay, electron paramagnetic resonance spectroscopy and the measurement of the total polyphenol content. The contents of total polyphenols in the tested extracts were ranging between 5.4 and 21.1% m/m and ranked as follows: Chinese seeds ? Belgian seeds ? Flowers ? Cake ? Leaves.     

Nevirapine quantification in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Application to bioequivalence study

A rapid, sensitive and specific method to quantify nevirapine in human plasma using dibenzepine as the internal standard (IS) was developed and validated. The method employed a liquid-liquid extraction. The analyte and the IS were chromatographed on a C(18) analytical column, (150 x 4.6 mm i.d. 4 microm) and analyzed by tandem mass spectrometry in the multiple reaction monitoring mode. The method had a chromatographic run time of 5.0 min and a linear calibration curve over the range 10-5000 ng ml(-1) (r(2) > 0.9970). The between-run precision, based on the relative standard deviation for replicate quality controls was 1.3% (30 ng ml(-1)), 2.8% (300 ng ml(-1)) and 3.6% (3000 ng ml(-1)). The between-run accuracy was 4.0, 7.0 and 6.2% for the above-mentioned concentrations, respectively. This method was employed in a bioequivalence study of two nevirapine tablet formulations (Nevirapina from Far-Manguinhos, Brazil, as a test formulation, and Viramune from Boehringer Ingelheim do Brasil Química e Farmacêutica, as a reference formulation) in 25 healthy volunteers of both sexes who received a single 200 mg dose of each formulation. The study was conducted using an open, randomized, two-period crossover design with a 3 week washout interval. The 90% confidence interval (CI) of the individual ratio geometric mean for Nevirapina/Viramune was 96.4-104.5% for AUC((0-last)), 91.4-105.1% for AUC((0-infinity)) and 95.3-111.6% for C(max) (AUC = area under the curve; C(max) = peak plasma concentration). Since both 90% CI for AUC((0-last)) and AUC((0-infinity)) and C(max) were included in the 80-125% interval proposed by the US Food and Drug Administration, Nevirapina was considered bioequivalent to Viramune according to both the rate and extent of absorption.     

中药复方六神丸中多类成分的多维液质系统筛查和鉴定

中药复方中成分复杂,通常包含不同类别的多种化合物。因此,建立无靶标的筛查和鉴定中药复方的分析模式是十分必要的。本研究采用液相色谱/飞行时间质谱(HPLC/TOF-MS)和液相色谱/离子阱多级质谱(HPLC/IonTrapMSn)的多维液质联用系统筛查和鉴定中药复方六神丸中的多类化学成分。在优化的液质条件下,飞行时间质谱对六神丸质谱信息中的每个色谱峰进行了精确分子量测定,并匹配出可能的化学组成;再结合离子阱质谱提供的多级碎片结构信息对其进行结构解析,最后采用相应的标准品进行确证。本研究共鉴定出六神丸中分别来源于蟾酥、麝香和牛黄三味药材的25种化合物,为复杂体系的多类别成分分析提供了一种有效、可靠的新模式。     

高效液相色谱-质谱法鉴定总合草苔虫中的草苔虫内酯成分

采用高效液相色谱分离与质谱检测的联用技术,对总合草苔虫中的草苔虫内酯成分进行定性分析。在以体积比为80∶20的甲醇-水溶液为流动相、采用ODS柱进行分离的色谱条件下,10种草苔虫内酯成分均可获得良好的分离。分析结果表明：深圳大亚湾产地的总合草苔虫中共含有9种已知和1种未知的草苔虫内酯成分。已知成分分别是bryostatin 4,5,6(9),7,8,10,16,17,18,其中bryostatin 7和17是首次从中国海域总合草苔虫中发现的2种痕量草苔虫内酯。该方法简便、准确、灵敏度高,可以很好的对总合草