Polarographic (DCP & DPP) Determination of Ellagic Acid in Strawberries & Pharmaceutical Formulations

Direct Current (DC) and Differential Pulse (DP) Polarographic methods have been developed for the qualitative as well as quantitative analysis of ellagic acid in plant products (i.e. strawberries) and pharmaceutical formulations (Ellagic insurance formula). Ellagic acid produces a well‐defined polarographic wave/peak in pyridine hydrochloride at pH 6.8 ± 0.1 with E_1/2 /Ep = ?1.298 V vs. SCE. The wave/peak height is found to be proportional to the concentration of ellagic acid. The developed procedure was used for the analysis of ellagic acid in an extract of strawberries (Fragaria × ananassa) and pharmaceutical formulations. Statistical treatment of the observed polarographic data revealed high accuracy and good precision of determination. The work has been supplemented by FTIR screening of the sample. The percentage of ellagic acid in pharmaceutical formulations was determined by the developed method, which is in close agreement with the labeled amount.     

How to distinguish between cinnamoylshikimate esters and chlorogenic acid lactones by liquid chromatography-tandem mass spectrometry

In this study, liquid chromatography–tandem mass spectrometry (LC–MSⁿ; n = 2–3) has been used to characterize and distinguish chlorogenic acid lactones from cinnamoylshikimate esters. This is the first time when an LC–MSⁿ method has been developed to distinguish between these two isomeric classes of compounds formed in particular in food processing from chlorogenic acids at elevated temperature through loss of water. The structures of regioisomeric chlorogenic acid lactones and shikimate esters have been assigned on the basis of LC–MSⁿ patterns of fragmentation, relative hydrophobicity, and fragmentation analogy with the synthetic standards of dimethoxycinnamic, ferulic, and caffeic acid containing monoacyl chlorogenic acid lactones and shikimate esters. Copyright © 2011 John Wiley & Sons, Ltd.     

反相高效液相色谱法同时分离测定杜仲雄花及其产品中的 京尼平苷酸和绿原酸

建立了利用反相高效液相色谱法（RP-HPLC）同时测定杜仲雄花及其产品中京尼平苷酸和绿原酸的方法。所用的色谱柱为Shim-pack VP-ODS(150 mm×4.6 mm,5 μm),流动相组成为甲醇-水-乙酸(体积比为24∶75∶1),检测波长为240 nm。在该色谱条件下,京尼平苷酸的含量在0.025~0.400 g/L、绿原酸的含量在0.075~1.200 g/L范围内线性关系良好,相关系数分别为0.9997和0.9999;加标回收率分别为100.2%和100.5%。该法适用于杜仲雄花及其产品中这2种成分的含量分析。     

Analysis of phenolic acids and flavonoids in leaves of Lycium barbarum from different habitats by ultra‐high‐performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry

The leaves of Lycium barbarum (LLB) have been utilized as crude drugs and functional tea for human health in China and Southeast Asia for thousands of years. To control its quality, a rapid and sensitive ultra‐high‐performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry method was established and validated for the first time for simultaneous determination of 10 phenolic acids and flavonoids (including neochlorogenic acid , protocatechuic aldehyde, p‐hydroxybenzoic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, p‐coumaric acid, ferulic acid, rutin and kaempferol‐3‐O‐rutinoside) in LLB. The separation was performed on an Acquity UPLC C₁₈ chromatographic column (100 × 2.1 mm internal diameter, 1.7 μm particle size) with 0.1% formic acid in water (A)–acetonitrile (B) as the mobile phase under gradient elution. Multiple reaction monitoring mode was adopted to simultaneously monitor the target components. The developed method was fully validated in terms of linearity (r² ≥ 0.9860), precision (RSD ≤ 6.58%), repeatability (RSD ≤ 6.60%), stability (RSD ≤ 6.17%), recovery (95.56–108.06%, RSD ≤ 4.64%) and limit of detection (0.021–0.664 ng/mL) and limit of quantitation (0.069–2.210 ng/mL), and then successfully applied to evaluate the quality of 64 batches of LLB collected from 41 producing areas in four different provinces of China. The results showed that the LLB, especially collected from Inner Mongolia regions, were rich in the phenolic acids and flavonoids. Rutin, kaempferol‐3‐O‐rutinoside and chlorogenic acid are the predominant compounds contained in LLB. The above findings will provide helpful information for the effective utilization of LLB.     

Determination of Chlorogenic Acid in a Herbal Medicinal Product with Preconcentration by Ionic Liquid-Modified Magnetic Nanoparticles and High-Performance Liquid Chromatography

Magnetic nanoparticles (MNPs) have been widely used in herbal analysis, and potential ionic liquid-based MNPs are expected in similar applications. In this study, the new functional Fe₃O₄ nanoparticles combined with a previously unreported ionic liquid were synthesized by three methods and used to enrich chlorogenic acid from aqueous solution and crude herbal extracts for the first time. The combination of pyrrole cation and proline anion immobilized on Fe₃O₄ nanoparticles had higher immobilized concentrations and ideal morphology, which can be easily adsorbed and recovered by an external magnetic field from the nanosuspension. When the mobile phase composed of acetonitrile and 0.5% phosphate aqueous solution (3:22, v:v) was used on a C₁₈ chromatographic column (4.6?×?250?mm, 5?µm) at 25°C, the concentration of chlorogenic acid in herbal product was determined to be 7.01?mg/g. The validation indicated the method had good linearity, recovery, repeatability, and precision. These results show that the ionic liquid has been chemically bonded to the magnetic nanoparticles through the developed procedures and may be effectively used for the high-performance liquid chromatographic determination of chlorogenic acid in herbal products.     

Quantitative determination of saturated oligogalacturonic acids in enzymatic digests of polygalacturonic acid,pectin and carrot pomace by on-line LC-ESI-MS

A method for the quantitative determination of oligogalacturonic acids (OGAs) by on-line high-performance liquid chromatographic (HPLC) separation and mass spectrometric detection via an electrospray interface (ESI-MS) without additional desalting steps was developed. Saturated OGAs up to a degree of polymerization (dp) of 3 were quantified by comparison with reference compounds. The calibration plots showed high linearity (R²>0.99), and the detection limits for dp 1, 2, and 3 were 11, 28, and 6 ng per injection, respectively. Non- and partially methyl-esterified OGAs with a dp of 3 and 4 were calculated semi-quantitatively as dp 3. The analytical system was used for the quantification of OGAs of digests obtained by incubation of polygalacturonic acid, pectin, and carrot pomace with commercial enzyme preparations. Furthermore, methyl-esterified OGAs up to a dp of 12 containing up to 4 methyl esters were detected in a pectin digest.     

基于纳米Nb_2O_5/石墨烯复合材料的增强效应电化学测定绿原酸

制备了Nb_2O_5/石墨烯修饰玻碳电极(Nb_2O_5/RGO/GCE),建立了一种简便、灵敏检测绿原酸的电化学方法。用氧化石墨烯(GO)和五氯化铌(Nb Cl5)一步溶剂热法制备Nb_2O_5/RGO复合材料,并用扫描电子显微镜(SEM)对其进行形貌表征。采用循环伏安法(CV)和方波伏安法(SWV)研究了绿原酸在Nb_2O_5/RGO/GCE上的电化学行为。结果发现,Nb_2O_5/RGO复合材料能显著增强绿原酸的电化学活性。对实验条件(如pH值、扫描速率与富集时间等)进行了优化。在最佳条件下,绿原酸的氧化峰电流与浓度在5.0×10~(-7)~1.2×10~(-5)mol/L范围内呈良好的线性关系,检出限为2.0×10~(-7)mol/L。采用修饰电极测定各种药物中绿原酸的含量,得到加标回收率为96.6%~101.5%。该方法具有良好的灵敏度和稳定性,已成功应用于药物中绿原酸含量的测定。     

Simultaneous determination of <Emphasis Type="Italic">p</Emphasis>-hydroxyacetophenone,chlorogenic acid,and caffeic acid in Herba Artemisiae Scopariae by capillary electrophoresis with electrochemical detection

Capillary electrophoresis with electrochemical detection has been employed for the determination of p-hydroxyacetophenone, chlorogenic acid, and caffeic acid in Herba Artemisiae Scopariae (the dried sprout of Artemisia scoparia Waldst. et Kit.). The effects of several important factors, such as the concentration and the acidity of the running buffer, separation voltage, injection time, and detection potential, were investigated to acquire the optimum conditions. The detection electrode was a 300-μm-diameter carbon disc electrode at a working potential of +0.90 V (relative to the saturated calomel electrode). The three analytes can be well separated within 11 min in a 40-cm-long fused-silica capillary at a separation voltage of 15 kV in 50 mM borate buffer (pH 9.2). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude, with detection limits (signal-to-noise ratio of 3) of 0.31, 0.39, and 0.50 μM for p-hydroxyacetophenone, chlorogenic acid, and caffeic acid, respectively. The proposed method has been successfully applied to monitor the three bioactive constituents in real plant samples and to differentiate between different herbal drugs with satisfactory assay results.     

茶叶样品中绿原酸含量的高效液相色谱法测定

建立了茶叶绿原酸含量测定的择相高效液相色谱法,采用PecosphereC18柱,以甲醇－水（体积比18：20,水中含φ＝1／61的甲酸）淋洗,用紫外检测器于326nm处测定,各组分的色谱峰达到基线分离。绿原酸在0．02－4．02μg范围内线性关系良好,线性相关系数 r＝0．9996。在安徽黄山产贡菊和安徽产金银花中加标的回收率分别为86．8％、102．0％,该法已用于十多种茶趺实际样品的测定。     

UHPLC‐ESI‐MS/MS determination and pharmacokinetics of pinoresinol glucoside and chlorogenic acid in rat plasma after oral administration of Eucommia ulmoides Oliv extract

This study aimed to develop a specific UHPLC‐ESI‐MS/MS method for simultaneous determination and pharmacokinetics of pinoresinol glucoside and chlorogenic acid in rat plasma after oral administration of Eucommia ulmoides . The chromatographic separation was achieved on a Hypersil GOLD column with gradient elution by using a mixture of 0.1% formic acid aqueous solution and acetonitrile as the mobile phase at a flow rate of 200 μL/min. A tandem mass spectrometric detection was conducted using multiple‐reaction monitoring via an electrospray ionization source in negative ionization mode. Samples were pre‐treated by a single‐step protein precipitation with acetonitrile, and bergenin was used as internal standard. After oral administration of 3 mL/kg E. ulmoides extract in rats, the maximum plasma concentrations of pinoresinol glucoside and chlorogenic acid were 57.44 and 61.04 ng/mL, respectively. The times to reach the maximum plasma concentration were 40.00 and 23.33 min for pinoresinol glucoside and chlorogenic acid, respectively. The intra‐ and inter‐day precision (RSD) values for the two analytes were <2.46 and 5.15%, respectively, and the accuracy (RE) values ranged from −12.76 to 0.00. This is the first study on pharmacokinetics of bioactive compounds in rat plasma after oral administration of E. ulmoides extract.     

Qualitative and quantitative analysis of phenolic acid glycosides in Ginkgo biloba L. leaf,G. biloba leaf extract and its injection

Ginkgo biloba L. leaf (GBL) is one of the most commonly used medicinal plants in the world. Phenolic acids with biological activities have a relatively high content in G. biloba leaf extracts (GBE); therefore they are of great significance for the quality control of GBL, GBE and its preparations. However, there have been few studies focused on their analysis. In this work, 12 phenolic acids, including 11 phenolic acid glycosides, were identified by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC–Q-TOF/MS). Then, a method combining enzymolysis with HPLC was established for quantification of phenolic acid glycosides. It was found that the aglycones of phenolic acid glycosides mainly comprised five phenolic acids: 2,4,6-trihydroxybenzoic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid and p-coumaric acid. The quantitative method was validated, and the correlation coefficient (0.9993–0.9999), recovery (≥88.4%), repeatability (≤0.8%), and inter-day precision (≤5.5%) were satisfactory. Finally, the contents of glycosides of five phenolic acids in GBL, GBE and GBE injection from different sources were determined by the developed method. The method was accurate, repeatable and practicable, which could be helpful for the quantification of phenolic acid glycosides in other products containing GBL or GBE.     

Simultaneous determination of corosolic acid and euscaphic acid in the plasma of normal and diabetic rat after oral administration of extract of Potentilla discolor Bunge by high‐performance liquid chromatography/electrospray ionization mass spectrometry

Potentilla discolor Bunge has been used for diabetes in China for a long time. Corosolic acid (CA) and euscaphic acid (EA), with significant anti‐diabetic activity, are two major triterpenoids in P. discolor. In this study, a specific, sensitive and convenient LC‐MS method has been developed for simultaneous determination of CA and EA in the plasma of normal and diabetic rats after oral administration of the extract of P. discolor. The chromatographic separation was achieved using an Alltima C₁₈ column (53 × 7.0 mm, i.d., 3 µm) with a mobile phase composed of 0.1% formic acid water and 0.1% formic acid acetonitrile at a flow rate of 1.0 mL/min. The detection was performed by MS with electrospray ionization interface in negative selected ion monitoring mode. All the validation data, such as specificity, linearity (r² > 0.9991 within 0.025–10.0 µg/mL), lower limit of quantitation (2.5 ng/mL), precision (intra‐ and inter‐day <14.7%), accuracy (<15.0%), recovery (85.7–110.8%) and stability were determined and all of them were within the required limits. This method was successfully applied for the evaluation of the pharmacokinetic behaviors of these two compounds in the plasma of normal and diabetic rats. Copyright © 2013 John Wiley & Sons, Ltd.     

A novel thiolysis-high-performance liquid chromatography method for the determination of proanthocyanidins in grape seeds

A novel thiolysis-high-performance liquid chromatography method for the quantitative determination of total proanthocyanidins and the mean degree of polymerization in grape seeds has been developed. Following thiolysis with formic acid and benzyl mercaptan, reaction products were separated and purified. Three proanthocyanidin monomers and three derivatives were obtained and their structures were identified by liquid chromatography-mass spectrometry, FTIR spectroscopy, and NMR spectroscopy. A decomposition model of the thiolysis products and a correction formula for proanthocyanidins concentration were established. This thiolysis-high-performance liquid chromatography method displayed good calibration linearity (R² > 0.999 over the concentration range 0.01 to 10 mg/mL), excellent accuracy (recoveries of 97.9–99.6%), and precision (repeatability relative standard deviations of 0.45–0.75%). This method is suitable for the quantitative analysis of proanthocyanidins in grape seed products.     

Separation of chlorogenic acid and concentration of trace caffeic acid from natural products by pH‐zone‐refining countercurrent chromatography

Chlorogenic acid and caffeic acid were selected as test samples for separation by the pH‐zone‐refining countercurrent chromatography (CCC). The separation of these test samples was performed with a two‐phase solvent system composed of methyl‐tert‐butyl‐ether/acetonitrile/water at a volume ratio of 4:1:5 v/v/v where trifluoroacetic acid (TFA; 8 mM) was added to the organic stationary phase as a retainer and NH₄OH (10 mM) to the aqueous mobile phase as an eluter. Chlorogenic acid was successfully separated from Flaveria bidentis (L.) Kuntze (F. bidentis) and Lonicerae Flos by pH‐zone‐refining CCC, a slightly polar two‐phase solvent system composed of methyl‐tert‐butyl‐ether/acetonitrile/n‐butanol/water at a volume ratio of 4:1:1:5 v/v/v/v was selected where TFA (3 mM) was added to the organic stationary phase as a retainer and NH₄OH (3 mM) to the aqueous mobile phase as an eluter. A 16.2 mg amount of chlorogenic acid with the purity of 92% from 1.4 g of F. bidentis, and 134 mg of chlorogenic acid at the purity of 99% from 1.3 g of crude extract of Lonicerae Flos have been obtained. These results suggest that pH‐zone‐refining CCC is suitable for the isolation of the chlorogenic acid from the crude extracts of F. bidentis and Lonicerae Flos.     

Quantitative determination of seven chemical constituents and chemo‐type differentiation of chamomiles using high‐performance thin‐layer chromatography

A simple and rapid high‐performance thin‐layer chromatographic method was developed for the separation and determination of six flavonoids (rutin, luteolin‐7‐O‐β‐glucoside, chamaemeloside, apigenin‐7‐O‐β‐glucoside, luteolin, apigenin) and one coumarin, umbelliferone from chamomile plant samples and dietary supplements. The separation was achieved on amino silica stationary phase using dichloromethane/acetonitrile/ethyl formate/glacial acetic acid/formic acid (11:2.5:3:1.25:1.25 v/v/v/v/v) as the mobile phase. The quantitation of each compound was carried out using densitometric reflection/absorption mode at their respective absorbance maxima after postchromatographic derivatization using natural products reagent (1% w/v methanolic solution of diphenylboric acid‐β‐ethylamino ester). The method was validated for specificity, limits of detection and quantification, precision (intra‐ and interday) and accuracy. The limits of detection and quantification were found to be in the range from 6–18 and 16–55 ng/band for six flavonoids and one coumarin, respectively. The intra‐ and interday precision was found to be <5% RSD and recovery of all the compounds was >90%. The data acquired from high‐performance thin‐layer chromatography was processed by principal component analysis using XLSTAT statistical software. Application of principal component analysis and agglomerative hierarchial clustering was successfully able to differentiate two chamomiles (German and Roman) and Chrysanthemum.     

UHPLC–MS/MS quantification combined with chemometrics for the comparative analysis of different batches of raw and wine‐processed Dipsacus asper

A rapid and sensitive ultra‐high performance liquid chromatography with tandem mass spectrometry approach was established for the simultaneous determination of 4‐caffeoylquinic acid, loganic acid, chlorogenic acid, loganin, 3,5‐dicaffeoylquinic acid, dipsacoside B, asperosaponin VI, and sweroside in raw and wine‐processed Dipsacus asper . Chloramphenicol and glycyrrhetinic acid were employed as internal standards. The proposed approach was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. Intra‐ and interassay variability for all analytes were 2.8–4.9 and 1.7–4.8%, respectively. The standard addition method determined recovery rates for each analytes (96.8–104.6%). In addition, the developed approach was applied to 20 batches of raw and wine‐processed samples of Dipsacus asper . Principle component analysis and partial least squares‐discriminate analysis revealed a clear separation between the raw group and wine‐processed group. After wine‐processing, the contents of loganic acid, chlorogenic acid, dipsacoside B, and asperosaponin VI were upregulated, while the contents of 3,5‐dicaffeoylquinic acid, 4‐caffeoylquinic acid, loganin, and sweroside were downregulated. Our results demonstrated that ultra‐high performance liquid chromatography with tandem mass spectrometry quantification combined with chemometrics is a viable method for quality evaluation of the raw Dipsacus asper and its wine‐processed products.     

Development and application of UHPLC-MS/MS method for the determination of phenolic compounds in Chamomile flowers and Chamomile tea extracts

UHPLC-MS/MS method using BEH C18 analytical column was developed for the separation and quantitation of 12 phenolic compounds of Chamomile (Matricaria recutita L.). The separation was accomplished using gradient elution with mobile phase consisting of methanol and formic acid 0.1%. ESI in both positive and negative ion mode was optimized with the aim to reach high sensitivity and selectivity for quantitation using SRM experiment. ESI in negative ion mode was found to be more convenient for quantitative analysis of all phenolics except of chlorogenic acid and kaempherol, which demonstrated better results of linearity, accuracy and precision in ESI positive ion mode. The results of method validation confirmed, that developed UHPLC-MS/MS method was convenient and reliable for the determination of phenolic compounds in Chamomile extracts with linearity >0.9982, accuracy within 76.7-126.7% and precision within 2.2-12.7% at three spiked concentration levels. Method sensitivity expressed as LOQ was typically 5-20 nmol/l.Extracts of Chamomile flowers and Chamomile tea were subjected to UHPLC-MS/MS analysis. The most abundant phenolic compounds in both Chamomile flowers and Chamomile tea extracts were chlorogenic acid, umbelliferone, apigenin and apigenin-7-glucoside. In Chamomile tea extracts there was greater abundance of flavonoid glycosides such as rutin or quercitrin, while the aglycone apigenin and its glycoside were present in lower amount.     

Phytochemical investigation of Gynura bicolor leaves and cytotoxicity evaluation of the chemical constituents against HCT 116 cells

Gynura bicolor (Compositae) is a popular vegetable in Asia and believed to confer a wide range of benefits including anti-cancer. Our previous findings showed that the ethyl acetate extract of G. bicolor possessed cytotoxicity and induced apoptotic and necrotic cell death in human colon carcinoma cells (HCT 116). A combination of column chromatography had been used to purify chemical constituents from the ethyl acetate and water extract of G. bicolor leaves. Eight chemical constituents 5-p-trans-coumaroylquinic acid (I), 4-hydroxybenzoic acid (II), rutin (III), kampferol-3-O-rutinoside (IV), 3,5-dicaffeoylquinic acid (V), kampferol-3-O-glucoside (VI), guanosine (VII) and chlorogenic acid (VIII) were isolated from G. bicolor grown in Malaysia. To our best knowledge, all chemical constituents were isolated for the first time from G. bicolor leaves except rutin (III). 3,5-dicaffeoylquinic acid (V), guanosine (VII) and chlorogenic acid (VIII) demonstrated selective cytotoxicity (selective index>3) against HCT 116 cancer cells compared to CCD-18Co human normal colon cells.     

Thermal decomposition of solid state poly(β-L-malic acid)

Thermal decomposition process of solid state poly(β -L-malic acid) was traced by DSC combined with FT-IR. Melting temperature of this partially crystallized polymer was detected at 46-60°C. The thermal decomposition initiated at ca 185°C accompanied by an evolution of gaseous products. In contrast to the cleavage reaction in the aqueous polymer solutions which gives L-malic acid and corresponding dimer of L-malic acid, the solid state poly(β -L-malic acid) decomposed at above the decomposition temperature giving not the constituent L-malic acid but fumaric acid at the first stage of the reaction then, maleic and maleic anhydride. This revised version was published online in July 2006 with corrections to the Cover Date.     

HPLC determination and pharmacokinetics of chlorogenic acid in rabbit plasma after an oral dose of Flos Lonicerae extract

A simple and sensitive high-performance liquid chromatography (HPLC) method has been developed for the determination of chlorogenic acid (3-O-caffeoyl-D-quinic acid) in plasma and applied to its pharmacokinetic study in rabbits after administration of Flos Lonicerae extract. Plasma samples are extracted with methanol. HPLC analysis of the extracts is performed on a C(18) reversed-phase column using acetonitrile-0.2% phosphate buffer (11:89, v/v) as the mobile phase. The UV detector is set at 327 nm. The standard curves are linear in the range 0.0500-1.00 microg/mL (r = 0.9987). The mean extraction recovery of 85.1% is obtained for chlorogenic acid. The interday precision (relative standard deviation) ranges from 5.0% to 7.5%, and the intraday precision is better than 9.0%. The limit of quantitation is 0.0500 microg/mL. The plasma concentration of chlorogenic acid shows a C(max) of 0.839 +/- 0.35 microg/mL at 34.7 +/- 1.1 min and a second one of 0.367 +/- 0.16 microg/mL at 273.4 +/- 39.6 min.