Determination of catechins and caffeine in proposed green tea standard reference materials by liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS)

Presented here is the quantitative analysis of green tea NIST standard reference materials (SRMs) via liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS). Three different NIST green tea standard reference materials (SRM 3254 Camellia sinesis Leaves, SRM 3255 C. sinesis Extract and SRM 3256 Green Tea-containing Oral Dosage Form) are characterized for the content of caffeine and a series of catechin species (gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate (EGCG)). The absolute limits of detection for caffeine and the catechin species were determined to be on the nanogram level. A reversed-phase chromatographic separation of the green tea reference materials was carried out on a commercial C₁₈ column using a gradient of water (containing 0.1% TFA) and 2:1 methanol:acetonitrile (containing 0.1%TFA) at 0.9 mL min⁻¹ and an analysis time of 50 min. Quantification of caffeine and the catechin species was carried out using the standard addition and internal standard methods, with the latter providing appreciable improvements in precision and recovery.     

Optimization and validation of a method for the direct determination of catechin and epicatechin in red wines by HPLC/fluorescence

The present paper describes a direct procedure for the determination of catechin and epicatechin concentrations in red wines employing reverse-phase high performance liquid chromatography (RP HPLC) and detection by fluorescence. The method was performed using a sample volume of 10 µL without dilution. The separation process employed a Chromolith performance RP-18e (100 mm × 4.6 mm) column, and the mobile phase was composed of solvent A: methanol-acetic acid-water (90:8:2) and solvent B: water-acetic acid-methanol (10:2:88) at a flow rate of 1.0 mL min^− 1. Linearity was observed in the range of 1 to 30 mg L^− 1, with limits of detection and quantification of 0.27 and 0.89 mg L^− 1, respectively, for catechin and 0.33 and 1.01 mg L^− 1, respectively, for epicatechin. The precisions estimated by the relative standard deviation were 3.34 and 1.09% for catechin concentrations of 0.5 and 20 mg L^− 1 respectively and 2.82 and 0.49% for epicatechin concentrations of 0.5 and 20 mg L^− 1, respectively. The evaluation of the accuracy was done using an addition/recovery assay. Four wine samples were used, and the recoveries varied from 105 to 108% for catechin and from 97 to 119% for epicatechin. The method was applied to the analysis of red wine samples collected from the São Francisco region, Bahia State, Brazil. Nine samples were analyzed, and the catechin and epicatechin concentrations varied from 7.51 to 73.20 and from 5.08 to 43.32 mg L^− 1, respectively. The concentrations found agree with data reported in the literature.     

Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins,three purine alkaloids,and gallic acid in tea by high-performance liquid chromatography with diode array detection

Monomers of (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCG), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) and (−)-3-O-methyl epicatechin gallate (ECG3′Me) (purity, >97%) were successfully prepared from extract of green tea by two-time separation with Toyopearl HW-40S column chromatography eluted by 80% ethanol. In addition, monomers of (−)-catechin (C), (−)-gallocatechin (GC), (−)-gallocatechin gallate (GCG), and (−)-catechin gallate (CG) (purity, >98%) were prepared from EC, EGC, EGCG, and ECG by heat-epimerization and semi-preparative HPLC chromatography. With the prepared catechin standards, an effective and simultaneous HPLC method for the analysis of gallic acid, tea catechins, and purine alkaloids in tea was developed in the present study. Using an ODS-100Z C₁₈ reversed-phase column, fourteen compounds were rapidly separated within 15 min by a linear gradient elution of formic acid solution (pH 2.5) and methanol. A 2.5–7-fold reduction in HPLC analysis time was obtained from existing analytical methods (40–105 min) for gallic acid, tea catechins including O-methylated catechins and epimers of epicatechins, as well as purine alkaloids. Detection limits were generally on the order of 0.1–1.0 ng for most components at the applied wavelength of 280 nm. Method replication generally resulted in intraday and interday peak area variation of <6% for most tested components in green, Oolong, black, and pu-erh teas. Recovery rates were generally within the range of 92–106% with RSDs less than 4.39%. Therefore, advancement has been readily achievable with commonly used chromatography equipments in the present study, which will facilitate the analytical, clinical, and other studies of tea catechins.     

Extraction of Catechin Compounds from Green Tea with a New Green Solvent

An emerging green solvent called a deep eutectic solvent（DES） was applied to the extraction and determination of catechin（C）,（＋）epicatechin gailate（ECG） and （-）epigallocatechin gallate（EGCG） from Chinese green tea.After evaluating different combinations of them by extraction methods and DESs,a DES-based extraction method was established and optimized by a systematic investigation of the influencing factors.As a result,a total of 3.629,35.25 or 114.2 mg/g catechin,（＋）epicatechin gallate or （-）epigaliocatechin gallate were extracted respectively under optimal conditions with extraction efficiencies of 82.7％,92.3％ and 97.0％,respectively.By comparing with other common used solvents for extracting catechin compounds,DESs were proved to be potential extraction solvents for bioactive ingredients.     

高效液相色谱-质谱法及模式识别法用于鉴别普洱生茶与熟茶

采用高效液相色谱-质谱联用技术及高效液相色谱法对生熟普洱茶中的主要成分进行定性和定量分析。鉴定出普洱茶水溶液中8种主要成分,分别为没食子酸(GA)、没食子酸儿茶素(GC)、表没食子酸儿茶素(EGC)、儿茶素(C)、咖啡因(CAF)、表儿茶素(EC)、表没食子酸儿茶素没食子酸酯(EGCG)和表儿茶素没食子酸酯(ECG)。以这8种成分的含量为指标,对普洱生茶和熟茶各20批进行主成分分析、聚类分析和判别分析,能准确地区分普洱生茶与熟茶。     

Mesoporous silica SBA-15, a new adsorbent for bioactive polyphenols from red wine

The aim of this paper is to evaluate the ability of the mesoporous silica SBA-15 to adsorb polyphenols from red wine. The mesoporous molecular sieve silica SBA-15 was hydrothermally synthesized in acidic media and characterized by SAXRD, BET, EDX and SEM. The adsorption behavior of mesoporous silica SBA-15 was investigated at 5 °C for 24 h using an adsorbent dose of 8 g SBA-15 L⁻¹ red wine. The total polyphenols content expressed as mg of gallic acid equivalents (GAE L⁻¹) was estimated from the standard curve of gallic acid (absorbance at 280 nm). HPLC chromatograms of methanolic extract from mesoporous SBA-15 at 256, 280, 324, and 365 nm exhibits the strong retention of quercetin and cis-resveratrol and a reasonable retention of trans-resveratrol, catechin, epicatechin, rutin, and phenolic acids (meta- and para-hydroxybenzoic, vanillic, caffeic, syringic, salicylic and para-coumaric acids).     

Oligomeric proanthocyanidin glycosides ofRhodiola pamiroalaica

Two oligomeric proanthocyanidin glycosides have been isolated from the roots ofRhodiola pamiroalaica and their structures and relative configurations have been established: — RP-1 - and — RP-2. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 484–491, July–August, 1998.     

半制备型液相色谱法分离纯化茶叶鲜叶中7种儿茶素类化合物

建立了从茶叶鲜叶中分离纯化7种儿茶素类化合物(没食子儿茶素(GC)、表没食子儿茶素(EGC)、儿茶素(C)、表没食子儿茶素没食子酸酯(EGCG)、表儿茶素(EC)、表没食子儿茶素3-O-(3-O-甲基)没食子酸酯(EGCG3"Me)和表儿茶素没食子酸酯(ECG))的半制备色谱法。铁观音鲜叶经甲醇超声浸提、浓缩、氯仿萃取后,向水相中加入碱式醋酸铅沉淀,得到茶多酚粗品。分别以甲醇-水、乙腈-水作为流动相,采用半制备色谱法纯化7种儿茶素类化合物,纯度均达到90%。此外,利用同样的方法分离纯化另外两种茶叶鲜叶中的7种儿茶素类化合物,得到相似的结果。该方法以溶剂提取、离子沉淀结合半制备色谱,适于简单、高效地同时分离制备多种儿茶素类化合物。     

Bubble-point measurements for the system CO2 + aqueous ethanol solutions of boldo leaf antioxidant components (boldine and catechin) at high pressures

The bubble-points pressures of solute(s) + ethanol + water + CO₂ mixtures were determined visually using a synthetic method in an experimental apparatus that included a variable-volume equilibrium cell. Tested solutes included boldo leaf tincture, a boldine + catechin mixture, pure boldine, and pure catechin. Uncertainties in bubble-point pressures were estimated to be <5%, based on comparisons with literature values and replicate measurements. The largest effect we observed was an average increase of 205% in the bubble-point pressure when decreasing the ethanol-to-water ratio from 63:37 to 37:63 (w/w). The bubble-point pressure increased 11% when increasing the temperature from 313 to 343 K, and decreased 8.2% when increasing the concentration of solids from 400 to 1500 ppm. The bubble-point pressure was higher for boldo leaf tincture than for a boldine + catechin mixture having the same boldine-to-catechin weight ratio, but this was partially due to a lower content of solids in the tincture. On the other hand, bubble-point pressures of the boldine + catechin mixture were marginally (0.33%) higher than the weighed average of the bubble-point pressures for pure boldine and pure catechin.     

Solid-phase microextraction on-fiber derivatization for the analysis of some polyphenols in wine and grapes using gas chromatography–mass spectrometry

The present study describes a new environmentally friendly sample pretreatment system based on solid-phase microextraction (SPME) for the sensitive determination of polyphenols. A derivatization process was necessary to convert the polar non-volatile compounds into volatile derivatives. Direct immersion (DI) SPME was used for the adsorption of polyphenols, and then the fiber was placed in the headspace of the derivatizing reagent, bis(trimethylsilyl)trifluoroacetamide (BSTFA). The separation was carried out by coupling gas chromatography with mass spectrometry in the selected ion monitoring mode, after silylation. Optimal extraction conditions were 25 °C for 10 min under continuous stirring using DI and a polyacrylate fiber. After extraction, the fiber was inserted into the headspace of BSTFA (10 μL) and the polyphenols were derivatized for 15 min at 50 °C. Desorption was carried out at 280 °C for 5 min. The method allowed the determination of both isomers cis- and trans-resveratrol, piceatannol, catechin and epicatechin in wine and grapes, and it was validated for linearity, detection and quantitation limits, selectivity, accuracy and precision. Detection limits ranged from 0.05 to 0.9 ng mL⁻¹ at a signal-to-noise ratio of 3, depending on the compound. Recoveries obtained for spiked samples were satisfactory for all compounds.     

Identifying Chemical Composition,Safety and Bioactivity of Thai Rice Grass Extract Drink in Cells and Animals

Rice grass has been reported to contain bioactive compounds that possess antioxidant and free-radical scavenging activities. We aimed to assess rice grass extract (RGE) drink by determining catechin content, free-radical scavenging and iron-binding properties, as well as toxicity in cells and animals. Young rice grass (Sukhothai-1 strain) was dried, extracted with hot water and lyophilized in a vacuum chamber. The resulting extract was reconstituted with deionized water (260 mg/40 mL) and served as Sukhothai-1 rice grass extract drink (ST1-RGE). HPLC results revealed at least eight phenolic compounds, for which the major catechins were catechin, epicatechin and epigallocatechin-3-gallate (EGCG) (2.71–3.57, 0.98–1.85 and 25.47–27.55 mg/40 mL serving, respectively). Elements (As, Cu, Pb, Sn and Zn) and aflatoxin (B1, B2, G1 and G2) contents did not exceed the relevant limits when compared with WHO guideline values. Importantly, ST1-RGE drink exerted radical-scavenging, iron-chelating and anti-lipid peroxidation properties in aqueous and biological environments in a concentration-dependent manner. The drink was not toxic to cells and animals. Thus, Sukhothai-1 rice grass product is an edible drink that is rich in catechins, particularly EGCG, and exhibited antioxidant, free radical scavenging and iron-binding/chelating properties. The product represents a functional drink that is capable of alleviating conditions of oxidative stress and iron overload.     

Polychlorinated dibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxins,dibenzofurans and biphenyls in fish from the Netherlands: concentrations,profiles and comparison with DR CALUX® bioassay results

A liquid chromatography–particle-beam mass spectrometer (LC–PB/MS) with interchangeable electron-impact (EI) and glow-discharge (GD) ion sources was evaluated for future application in analysis of botanical extracts. In this work a green tea tincture was characterized for a series of catechin components (catechin, epicatechin, epigallocatechin, and epigallocatechin gallate (EGCG)) and caffeine. Special emphasis was given to EGCG and caffeine, because they are important in determining the possible health effects of the green tea. The effects of instrument operating conditions were evaluated for the EI and GD ionization sources to determine their effect on analyte intensities and fragmentation patterns. These studies furnished information about the effects of these conditions in determining possible ionization pathways in the two ion sources. The mass spectra of these compounds obtained with the GD ion source are EI-like in appearance, with clearly identified molecular ions and fragmentation patterns that are easily rationalized. The absolute limits of detection for EGCG and caffeine were, respectively, 11 ng and 0.77 ng for the EI source and 3.2 ng and 0.61 ng for the GD source. The PB/EIMS and PB/GDMS combinations can be operated in a flow-injection mode, wherein the analyte is injected directly into the mobile phase, or coupled to high-performance liquid chromatography (HPLC), enabling LC–MS analysis of complex mixtures. A reversed-phase chromatographic separation of the green tea tincture was performed on a commercial C₁₈ column using a gradient of water (containing 0.1% TFA) and ACN. Quantification of EGCG and caffeine was performed by the standard addition method. The amounts of EGCG and caffeine in the tested green tea tincture were each ∼14 mg mL⁻¹.     

Determination of catechins and catechin gallates in biological fluids by HPLC with coulometric array detection and solid phase extraction

Tea polyphenols are well known for their beneficial health effects that involve their anti-carcinogenic, anti-mutagenic, anti-pathogenic and anti-oxidative properties. The main polyphenols of green tea are favan-3-ols (catechins) and their corresponding gallate compounds, which constitute about one-third of the dry weight of tea leaves. Their main ingredients are (+)-catechin (C), (−)-epicatechin (EC), (−)-gallocatechin (GC), (−)-epigallocatechin (EGC), (−)-catechin gallate (CG), (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG) and (−)-epigallocatechin gallate (EGCG). Each has slightly different biological properties. We have developed a method to simultaneously analyze all these compounds in plasma and urine. The samples were first incubated with β-d-glucuronidase and sulfatase to release the catechin residues from their corresponding conjugates for subsequent extraction by selective solid phase column, Waters Oasis HLB extraction cartridges. The extracted molecules were resolved by reversed phase HPLC and monitored by coulometric chemical detection on a CoulArray detector. All eight catechin compounds were analyzed in a single chromatogram within 25 min. For plasma and urine analyses, good linearity (>0.9950) was validated in the range 10-2000 and 10-5000 ng/ml, respectively. The coefficients of variance (CV) were less than 5%. Absolute recovery was greater than 85% and detection limit was 5 ng/ml. The chromatogram exhibited minimal interference as a result of the highly selective solid phase extraction and CoulArray detection.     

Selective and sensitive hydroxypropyl-beta-cyclodextrin based sensor for simple monitoring of (+)-catechin in some commercial drinks and biological fluids

(+)-Catechin (CAT) was considered as a polyphenolic compound abundantly contained in plants. It exerts protective effect against cancer, inflammatory and cardiovascular diseases. These protective effects are mainly attributed to its antioxidative activity by scavenging free radicals. Therefore, the need of simple, selective and sensitive monitoring of (+)-catechin in commercial drinks and biological fluids is crucial. A new selective and sensitive voltammetric quantification of (+)-catechin was investigated at low cost hydroxypropyl-beta-cyclodextrin modified carbon paste sensor in acidic solutions. The constructed sensor was treated in simple and fast manner to increase its stability for catechin determination. The effect of solution and instrumental parameters was investigated by using osteryoung square-wave anodic voltammetry (OSWAV) at pH 2.20 and differential pulse cathodic voltammetry (DPCV) at pH 4.40 in 0.10 M Britton-Robinson buffer. Acidic solutions were chosen to increase the stability of (+)-catechin, reduce its adsorption on the sensor surface and increase the selectivity of proposed method. Cyclic voltammetry (CV) was used to elucidate the electrochemical mechanism of catechin at the modified electrochemical sensor. A linear range up to 7.20 and 4.20 μg mL⁻¹ of catechin was achieved in anodic and cathodic voltammetry, respectively. The method gave reproducible and reliable results with 1.50 (g mL⁻¹ catechin (S.D. 0.062). Limit of detection of 0.12 and 0.30 ng mL⁻¹ and limit of quantification (LOQ) of 1.10 and 2.80 ng mL⁻¹ were easily achieved using anodic and cathodic voltammetry, respectively. Selectivity of the proposed procedure was estimated by testing recovery and adding the most interfering metal ions and/or organic compounds. The proposed method was applied successfully to selective determination of catechin in some commercial drinks like tea, cocoa and coffee with acceptable recovery range (98-102%). The extraction of catechin was rather simple, making it suitable for studies with a large number of commercial samples. Furthermore, the application to urine samples without pretreatment was achieved and statistically confirmed at 95% confidence level. It was easy to analyze catechin in urine down to 0.55 ng mL⁻¹.     

Microwave‐assisted extraction followed by CE for determination of catechin and epicatechin in green tea

In this work, for the first time, microwave‐assisted extraction (MAE) followed by CE was developed for the fast analysis of catechin and epicatechin in green tea. In the proposed method, catechin and epicatechin in green tea samples were rapidly extracted by MAE technique, and then analyzed by CE. The MAE conditions and the method's validation were studied. It is found that the extraction time of 1 min with 400 W microwave irradiation is enough to completely extract catechin and epicatechin in green tea sample, whereas the conventional ultrasonic extraction (USE) technique needs long extraction time of 60 min. The method validations were also studied in this work. The calibration curve shows good linearity in 0.01–3 mg/mL for catechin (R²=0.993), and 0.005–3 mg/mL for epicatechin (R²=0.996), respectively. The RSD values for catechin and epicatechin are 0.65 and 2.58%, respectively. This shows that the proposed method has good reproducibility. The proposed method has good recoveries, which are 118% for catechin and 120% for epicatechin. The proposed method was successfully applied to determination of the catechin and epicatechin in different green tea samples. The experiment results have demonstrated that the MAE following CE is a simple, fast and reliable method for the determination of catechin and epicatechin in green tea.     

茶叶及茶多酚中儿茶素的高效液相色谱分析方法研究

 筛选出HypersilBDSC18和ZorbaxSBC18两种适合同时分离茶叶和茶多酚中 7种儿茶素和咖啡因的反相柱。采用甲醇 水 醋酸 (或三氟醋酸 )作流动相 ,分别以等强度洗脱和梯度洗脱 (均在 30min内 )分离测定了我国 6种不同产地茶叶样品和 3种茶多酚样品中 7种儿茶素的含量。考察了 7种儿茶素和咖啡因的保留值与流动相组成及柱温的关系 ,优化了色谱条件及样品前处理方法。用电喷雾电离质谱 (ESI MS)定性确认没食子儿茶素没食子酸酯(GCG)和儿茶素没食子酸酯 (CG)两组分 ,并用高效液相色谱制备两对照品用于定量分析。     

Effect of Water Hardness on Catechin and Caffeine Content in Green Tea Infusions

The health benefits of green tea are associated with its high catechin content. In scientific studies, green tea is often prepared with deionized water. However, casual consumers will simply use their local tap water, which differs in alkalinity and mineral content depending on the region. To assess the effect of water hardness on catechin and caffeine content, green tea infusions were prepared with synthetic freshwater in five different hardness levels, a sodium bicarbonate solution, a mineral salt solution, and deionized water. HPLC analysis was performed with a superficially porous pentafluorophenyl column. As water hardness increased, total catechin yield decreased. This was mostly due to the autoxidation of epigallocatechin (EGC) and epigallocatechin gallate (EGCG). Epicatechin (EC), epicatechin gallate (ECG), and caffeine showed greater chemical stability. Autoxidation was promoted by alkaline conditions and resulted in the browning of the green tea infusions. High levels of alkaline sodium bicarbonate found in hard water can render some tap waters unsuitable for green tea preparation.     

Self-assembled monolayer of nickel(II) complex and thiol on gold electrode for the determination of catechin

Self-assembled monolayers of a nickel(II) complex and 3-mercaptopropionic acid on a gold electrode were obtained for determination of catechin by square wave voltammetry. The complex [Ni^IIL] with L = [N-(methyl)-N′-(2-pyridylmethyl)-N,N′-bis(3,5-di-tert-butyl-2-hydroxybenzyl)-1,3-propanediamine[nickel(II)] was synthesized and characterized by ¹H NMR, IR, and electronic spectroscopies and electrochemical methods. The optimized conditions obtained for the electrodes were 0.1 mol L⁻¹ phosphate buffer solution (pH 7.0), frequency of 80.0 Hz, pulse amplitude of 60.0 mV and scan increment of 10.0 mV. Under these optimum conditions, the resultant peak current on square wave voltammograms increases linearly with the concentration of catechin in the range of 3.31 × 10⁻⁶ to 2.53 × 10⁻⁵ mol L⁻¹ with detection limits of 8.26 × 10⁻⁷ mol L⁻¹. The relative standard deviation for a solution containing 1.61 × 10⁻⁵ mol L⁻¹ catechin solution was 2.45% for eight successive assays. The lifetime of the Ni(II) complex-SAM-Au electrode was investigated through testing every day over 4 weeks. The results showed apparent loss of activity after 20 days. The results obtained for catechin in green tea samples using the proposed sensor and those obtained by electrophoresis are in agreement at the 95% confidence level.     

Stability of phenolic compounds during extraction with superheated solvents

The stability of nine phenolic compounds in the extraction with superheated methanol at different temperatures (40, 50, 100 and 150 degrees C) has been tested. The evolution of the same compounds in boiling methanol (65 degrees C) in contact with air was also determined. All the assayed phenolic compounds were stable under the extraction conditions with the exception of catechin and epicatechin (recoveries: 87.4% for catechin and 86.0% for epicatechin at 150 degrees C and 94.1% for epicatechin at 100 degrees C). Phenolic compounds kept at the boiling point of methanol (65 degrees C) showed lower recoveries: gentisic acid (85.5%), syringic aldehyde (92.8%), catechin (63.7%) and epicatechin (63.4%). Extraction with superheated solvents was also applied to the extraction of phenolic compounds from solid wastes of the winemaking process.     

Determination of metabolites in Uncaria sinensis by HPLC and GC-MS after green solvent microwave-assisted extraction

Uncaria sinensis (Oliv.) Havil (Rubiaceae) has been used as an important Traditional Chinese Medicine (TCM) herb for the treatment of fevers and various nervous disorders. The major bioactive secondary metabolites from different classes of chemical compounds, i.e. organic acid, flavonoid and alkaloid, present in this TCM herb, namely catechin, caffeic acid, epicatechin and rhynchophylline, were extracted by microwave-assisted extraction (MAE) method with ultra-pure water as the extraction solvent. The optimal extraction conditions for this green solvent MAE method were found to be 100 °C for 20 min. The recoveries of the compounds were found to be comparable to that of heating under reflux using ultra-pure water for 60 min. The method precision (RSD, n = 6) was found to vary from 0.19% to 5.60% for the proposed method on different days for the secondary metabolites. Simultaneously, the key primary metabolites such as sucrose and phenylalanine for the biosynthesis of bioactive secondary metabolites were successfully characterized by GC-MS. Furthermore, an approach using the combination of primary and secondary metabolite profiling based on their chemical fingerprints with Principal Component Analysis (PCA) was successfully developed to evaluate the quality of U. sinensis obtained from different sources. This approach was shown to be feasible in discriminating U. sinensis from different origins and thus a potential application for the quality control of other medicinal herbs.