Comprehensive characterization of C‐glycosyl flavones in wheat (Triticum aestivum L.) germ using UPLC‐PDA‐ESI/HRMSn and mass defect filtering

A comprehensive characterization of C‐glycosyl flavones in wheat germ has been conducted using multi‐stage high resolution mass spectrometry (HRMSⁿ) in combination with a mass defect filtering (MDF) technique. MDF performed the initial search of raw data with defined C‐glycosyl flavone mass windows and mass defect windows to generate the noise‐reduced data focusing on targeted flavonoids. The high specificity of the exact mass measurement permits the unambiguous discrimination of acyl groups (nominal masses of 146, 162 and 176.) from sugar moieties (rhamnose, glucose or galactose and glucuronic acid). A total of 72 flavone C‐glycosyl derivatives, including 2 mono‐C‐glycosides, 34 di‐C‐glycosides, 15 tri‐glycosides, 14 acyl di‐C‐glycosides and 7 acyl tri‐glycosides, were characterized in wheat germ, some of which were considered to be important marker compounds for differentiation of whole grain and refined wheat products. The 7 acylated mono‐O‐glycosyl‐di‐C‐glycosyl flavones and some acylated di‐C‐glycosyl flavones are reported in wheat for the first time. The frequent occurrence of numerous isomers is a remarkable feature of wheat germ flavones. Both UV and mass spectra are needed to maximize the structure information obtained for data interpretation. Copyright © 2016 John Wiley & Sons, Ltd.     

Flavonoid profiling in three citrus varieties native to the Republic of Korea using liquid chromatography coupled with tandem mass spectrometry: contribution to overall antioxidant activity

A mixture of flavonoid components was isolated from the fruit peel of three varieties of citrus native to Republic of Korea, Citrus leiocarpa Hort. ex Tanaka (CLHT), Citrus aurantium L. (CAL) and Citrus erythrosa Hort. (CEH), via 70% methanol extraction followed by ethyl acetate elution over a silica gel cartridge. The flavonoid components of the mixture were analyzed via high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) in positive‐ion mode and a comparison of the reported data. Among 17 characterized components, two flavanones, four flavones and two coumarin derivatives in the fruit peel of the three varieties were identified for the first time. The individual characterized components were quantified via HPLC‐UV. The flavanones dominated in CAL, whereas the flavones prevailed in CLHT and CEH. The antioxidant activity of the flavonoid mixture of the fruit peel was determined via DPPH^?, ABTS^?+ and reducing power assays. The antioxidant activity of CEH and CAL was greater than that of CLHT. Copyright © 2011 John Wiley & Sons, Ltd.     

Sensitive determination of glucose in Dulbecco's modified Eagle medium by high‐performance liquid chromatography with 1‐phenyl‐3‐methyl‐5‐pyrazolone derivatization: application to gluconeogenesis studies

A new pre‐column derivative high‐performance liquid chromatography (HPLC) method for determination of d ‐glucose with 3‐O‐methyl‐d ‐glucose (3‐OMG) as the internal standard was developed and validated in order to study the gluconeogenesis in HepG2 cells. Samples were derivatized with 1‐phenyl‐3‐methy‐5‐pyrazolone at 70°C for 50 min. Glucose and 3‐OMG were extracted by liquid–liquid extraction and separated on a YMC‐Triart C₁₈ column, with a gradient mobile phase composed of acetonitrile and 20 mm ammonium acetate solution containing 0.09% tri‐ethylamine at a flow rate of 1.0 mL/min. The eluate were detected using a UV detector at 250 nm. The assay was linear over the range 0.39–25 μm (R² = 0.9997, n = 5) and the lower limit of quantitation was 0.39 μm (0.070 mg/mL). Intra‐ and inter‐day precision and accuracy were <15% and within ±3%, respectively. After validation, the HPLC method was applied to investigate the gluconeogenesis in Dulbecco's modified Eagle medium (DMEM) cultured HepG2 cells. Glucose concentration was determined to be about 1–2.5 μm in this gluconeogenesis assay. In conclusion, this method has been shown to determine small amounts of glucose in DMEM successfully, with lower limit of quantitation and better sensitivity when compared with common commercial glucose assay kits. Copyright © 2015 John Wiley & Sons, Ltd.     

Preparative isolation of flavonoid compounds from Oroxylum indicum by high‐speed counter‐current chromatography by using ionic liquids as the modifier of two‐phase solvent system

A preparative high‐speed counter‐current chromatography method for isolation and purification of flavonoid compounds from Oroxylum indicum was successfully established by using ionic liquids as the modifier of the two‐phase solvent system. Two flavonoid compounds including baicalein‐7‐O‐diglucoside and baicalein‐7‐O‐glucoside were purified from the crude extract of O. indicum by using ethyl acetate–water–[C₄mim][PF₆] (5:5:0.2, v/v) as two‐phase solvent system. 36.4 mg of baicalein‐7‐O‐diglucoside and 60.5 mg of baicalein‐7‐O‐glucoside were obtained from 120 mg of the crude extract. Their purities were 98.7 and 99.1%, respectively, as determined by HPLC area normalization method. The chemical structures of the isolated compounds were identified by ¹H‐NMR and ¹³C‐NMR.     

Combined mass spectrometric and chromatographic methods for in‐depth analysis of phenolic secondary metabolites in barley leaves

Structural analysis via HPLC‐ESI‐MSn, UPLC‐HESI‐MS/MS and NMR reported 152 phenolic secondary metabolites in spring barley seedlings (Hordeum vulgare L.). Flavonoids with various patterns of glycosylation and acylation, as well as hydroxycinnamic acid glycosides, esters and amides, were identified in methanolic extracts from leaves of nine varieties of barley originating from different regions of the world. Hordatines derivatives, flavones acylated directly on the aglycone, and hydroxyferulic acid derivatives deserve special attention. Preparative chromatography enabled characterization of a number of compounds at trace levels with the 6‐C‐[6″‐O‐glycosyl]‐glycosides and the 6‐C‐[2″,6″‐di‐O‐glycosides]‐glucoside structure of flavones. Derivatives of flavonols, quercetin and isorhamnetin were observed only in Syrian varieties. The ultra performance liquid chromatography profiles of UV‐absorbing secondary metabolites were used for chemotaxonomic comparison between nine varieties of barley from different climatic conditions. The hierarchical clustering of bred lines from the Fertile Crescent and European and American varieties indicates a great diversity of chemical phenotypes within barley species. Copyright © 2015 John Wiley & Sons, Ltd.     

Efficient analysis of phytochemical constituents in the peel of Chinese wild citrus Mangshanju (Citrus reticulata Blanco) by ultra high performance liquid chromatography–quadrupole time‐of‐flight‐mass spectrometry

An efficient ultra high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was developed for separation and profiling of phytochemical constituents of Chinese wild mandarin Mangshanju (Citrus reticulata Blanco). All constituents were well separated within 16 min. Based on retention times, accurate mass, MS^E fragments, and/or reference standards as well as databases, a total of 81 compounds were unambiguously identified or tentatively assigned including flavonoid glycosides, acylated flavonoid glycosides, flavones, polymethoxylated flavonoids, and limonoids as well as four other compounds. Among them, 22 polymethoxylated flavones and ten polymethoxylated flavanones/chalcones were identified in Mangshanju, more types than other citrus reported before. A basic procedure for identifying flavonoid‐O‐glycosides and the aglycones including polymethoxylated flavonoids was proposed. In addition, this method was successfully used to analyze another four mandarin germplasms, Cenxi suan ju, Xipi gousi gan, Nanfeng miju, and Or, showing that Mangshanju contained two characteristic compounds distinct from the other four citrus species. This study systematically profiled phytochemical constituents of Mangshanju, which was helpful for further utilization of Mangshanju owing to its abundant bioactive compounds.     

Determination and comparison of flavonoids and anthocyanins in Chinese sugarcane tips,stems, roots and leaves

The flavonoids and anthocyanins in Chinese sugarcane (Saccharum sinensis Roxb.) tips, stems, roots and leaves were separately analyzed by HPLC‐UV diode array detector, respectively. The results indicated that the content of flavonoids in sugarcane leaves was considerably high in comparison with previous reports in Brazil sugarcane (S. officinarum L.) leaves. Moreover, the content of flavonoids in sugarcane tips and roots was also high in comparison with sugarcane stems. For another, the content of anthocyanins in sugarcane roots was higher than that in other parts of the sugarcane, such as leaves, tips and stems. In addition, two anthocyanins, named petunidin 3‐O‐(6″‐succinyl)‐rhamnoside and cyanidin‐3‐O‐glucoside, were first identified from S. sinensis by HPLC‐UV diode array detector and HPLC‐MS/MS.     

Flavone 8‐C‐Glycosides from Haberlea rhodopensis Friv. (Gesneriaceae)

Phytochemical profiling of a MeOH extract from Haberlea rhodopensis by a combination of liquid/liquid extraction, and preparative and semi‐preparative HPLC afforded three new flavone C‐glycosides, hispidulin‐8‐C‐(2″‐O‐syringoyl)‐β‐glucopyranoside ( 3 ), hispidulin 8‐C‐(6‐O‐acetyl‐β‐glucopyranoside) ( 4 ), and hispidulin 8‐C‐(6‐O‐acetyl‐2‐O‐syringoyl‐β‐glucopyranoside) ( 5 ), along with two known phenolic glycosides, myconoside ( 1 ) and paucifloside ( 2 ). The structures were established by extensive spectroscopic analyses including 1D‐ and 2D‐NMR (COSY, HSQC, and HMBC), and HR‐ESI‐TOF‐MS, and by comparison with published data.     

Dietary‐flavonoid‐rich flowers of Rumex nervosus Vahl: Liquid chromatography with electrospray ionization tandem mass spectrometry profiling and in vitro anti‐inflammatory effects

Rumex nervosus is a plant species found widely in Eastern Africa and the Arabian Peninsula. In addition to its uses in traditional medicinal, the plant shows various biological activities, such as antiviral, antibacterial, and antioxidant activity. In this study, nine flavonols, six flavones, three flavanones, and one flavanol were characterized from the flowers of R. nervosus using liquid chromatography with electrospray ionization tandem mass spectrometry and literature data. Validation data indicated that the determination coefficients (R²) were ≥ 0.9914. The limits of detection and quantification were in the ranges of 0.15–1.24 and 0.50–4.13 mg/L, respectively. Recoveries at 10 and 50 mg/L were 71.1–110.2 and 65.4–115.1%, with relative standard deviations of 7.4–40.1 and 2.1–13.0%, respectively. Quercetin 3‐O‐rhamnoside ( 10 ) was the dominant component, contributing 30.8% of total flavonoids (1003.0 ± 26.2 mg/kg fresh flower sample), whereas luteolin 6‐C‐glucoside (3) was the lowest yielding compound (0.1%). The 19 flavonoids identified were characterized for the first time. In vitro anti‐inflammatory studies showed that this mixture can suppress the production of inflammatory mediators, including inducible nitric oxide synthase, cyclooxygenase‐2, kappa B inhibitor, and interleukin‐1β, by down‐regulating the nuclear factor‐kappa B and mitogen‐activated protein kinases pathways. The results of this study may provide information for processing R. nervosus as a potential source of functional food.     

Three New C19‐Diterpenoid Alkaloids from Aconitum forrestii

A further study of the alkaloid constituents of Aconitum forrestii led to the isolation of three new C₁₉‐diterpenoid alkaloids, named 14‐acetoxy‐8‐O‐methylsachaconitine ( 1 ), 14‐acetoxyscaconine ( 2 ), and 8‐O‐ethylcammaconine ( 3 ). Their structures were determined by UV, IR, and MS, 1D‐ and 2D‐NMR analyses.     

Structural characterization and identification of flavonoid aglycones in three Glycyrrhiza species by liquid chromatography with photodiode array detection and quadrupole time‐of‐flight mass spectrometry

Flavonoids, including flavones, isoflavones, flavanones, chalcones, and isoflavans, have long been recognized as the main active ingredients in licorice. A method combining liquid chromatography with photodiode array detection and quadrupole time‐of‐flight mass spectrometry was developed to characterize components in three Glycyrrhiza species, and to expound the characteristic fragmentation behaviors in the positive ion mode. Based on the fragmentation patterns of reference compounds, a total of 39 compounds, including 37 flavonoid aglycones and two coumestans, were identified or tentatively identified. Besides, some common features, such as H₂O, CO, and CH₂O₂ losses, together with retro‐Diels–Alder fragmentation, were observed in these compounds. Furthermore, diagnostic fragmentations of C‐ring cleavages and UV absorption on the skeleton groups were observed to structurally characterize flavonoid aglycones. In addition, typical losses of different substituent groups were detected: Neutral losses of 56 (C₄H₈) and 68 Da (C₅H₈) were yielded from a prenyl chain; neutral losses of 42 (C₃H₆), 54 (C₄H₆), and 70 Da (C₄H₆O) were generated by a pyran ring. Particularly, neutral losses of 18 (H₂O), 16 (CH₄), 112 (C₈H₁₆), and 98 Da (C₇H₁₄) predicted a hydroxyl, a methoxyl, double prenyl chains, and a prenyl chain with a pyran ring, respectively.     

Characterization of polyphenol compounds from the roots and stems of Parthenocissus laetevirens by high‐performance liquid chromatography/tandem mass spectrometry

A facile method based on high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC/(–)ESI‐MSⁿ) has been established for the analyses of polyphenol compounds in the root and stems of Parthenocissus laetevirens. Two characteristic fragments [C₃O₂ (68 Da) and C₂H₂O (42 Da)] were utilized for the structural identification of polyphenols. Based on the reference standards, the fragment C₃O₂ was presented when the compound possessed a 2,3‐dihydro‐1H‐indene‐4, 6‐diol moiety. Meanwhile, the C₂H₂O fragment (42 Da) yielded from the resorcinol ring was confirmed by resveratrol and three synthesized compounds identified as (E)‐5‐styrylbenzene‐1,3‐diol, (E)‐4‐styrylphenol and (E)‐4‐(3,4,5‐trimethoxystyryl)phenol. FTICR‐MSⁿ was performed to further confirm the structures of the fragments. Overall, 15 polyphenol compounds were characterized. Three polyphenol compounds were initially and tentatively characterized from P. laetevirens for the first time, and one was proposed as a novel compound. Furthermore, a pair of stereoisomers was readily distinguished by breakdown curves, and the trans‐, cis‐isomers could be identified by HPLC/DAD‐UV spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

Monitoring photo‐induced transformations in crystals of 2,6‐difluorocinnamic acid under ambient conditions

Several conditions need to be fulfilled for a photochemical reaction to proceed in crystals. Some of these conditions, for example, geometrical conditions, depend on the particular type of photochemical reaction, but the rest are common for all reactions. The mutual directionality of two neighbouring molecules determines the kind of product obtained. The influence of temperature on the probability of a photochemical reaction occurring varies for different types of photochemical reaction and different compounds. High pressure imposed on crystals also has a big influence on the free space and the reaction cavity. The wavelength of the applied UV light is another factor which can initiate a reaction and sometimes determine the structure of a product. It is possible, to a certain degree, to control the packing of molecules in stacks by using fluoro substituents on benzene rings. The crystal and molecular structure of 2,6‐difluorocinnamic acid [systematic name: 3‐(2,6‐difluorophenyl)prop‐2‐enoic acid], C₉H₆F₂O₂, (I), was determined and analysed in terms of a photochemical [2 + 2] dimerization. The molecules are arranged in stacks along the a axis and the values of the intermolecular geometrical parameters indicate that they may undergo this photochemical reaction. The reaction was carried out in situ and the changes of the unit‐cell parameters during crystal irradiation by a UV beam were monitored. The values of the unit‐cell parameters change in a different manner, viz. cell length a after an initial increase starts to decrease, b after a decrease starts to increase, c increases and the unit‐cell volume V after a certain increase starts to decrease. The structure of a partially reacted crystal, i.e. containing both the reactant and the product, namely 2,6‐difluorocinnamic acid–3,4‐bis(2,6‐difluorophenyl)cyclobutane‐1,2‐dicarboxylic acid (0.858/0.071), 0.858C₉H₆F₂O₂·0.071C₁₈H₁₂F₄O₄, obtained in situ, is also presented. The powder of compound (I) was irradiated with UV light and afterwards crystallized [as 3,4‐bis(2,6‐difluorophenyl)cyclobutane‐1,2‐dicarboxylic acid toluene hemisolvate, C₁₈H₁₂F₄O₄·0.5C₇H₈] in a space group different from that of the crystal containing the in‐situ dimer.     

Pharmacokinetics of luteolin and tetra‐acetyl‐luteolin assayed by HPLC in rats after oral administration

Accurate and reproducible HPLC methods were developed and validated for the determination of concentrations of luteolin (LT) and tetra‐acetyl‐luteolin (TALT) in rat plasma. HPLC analyses were performed on an Agilent TC‐C₁₈ column protected by a guard Agilent Zorbax Eclipse Plus. The mobile phase for LT was a binary mixture of acetonitrile–water (40:60, v/v) containing 0.5% phosphoric acid at a flow rate of 1.0 mL/min, and that for TALT was a binary mixture of methanol–water (70 : 30, v/v) containing 0.5% glacial acetic acid at the same flow rate. The UV detection wavelength for both analytes was set at 350 nm. The calibration curve was linear over the range of 40–1800 ng/mL, the lower limit of quantitation was 40 ng/mL and the lower limit of detection was 20 ng/mL for both LT and TALT. The intra‐ and inter‐day precision (RSD) values for all samples were within 7.9%. The concentration–time curves of LT and TALT after oral administration (30 mg/kg) were both fitted to a two‐compartment model. The pharmacokinetic characteristics of TALT were better than that of LT in the maximum plasma concentration (C_max) and the area under the concentration–time curve (AUC). Copyright © 2010 John Wiley & Sons, Ltd.     

Effect of pH on the charge‐assisted hydrogen‐bonded assembly of the anionic [Cu(oxalate)2]2− building unit and N,N′‐ditopic cations

By the solvothermal reaction under acidic conditions of Cu(NO₃)₂·3H₂O, Na₂C₂O₄ and the N,N′‐ditopic organic coligands 1‐(pyridin‐4‐yl)piperazine (ppz) and 1,2‐bis(pyridin‐4‐yl)ethane (bpa), two novel anionic copper(II) coordination compounds were obtained, namely the one‐dimensional coordination polymer catena‐poly[4‐(pyridin‐1‐ium‐4‐yl)piperazin‐1‐ium [[(oxalato‐κ²O¹,O²)copper(II)]‐μ‐oxalato‐κ³O¹,O²:O^1′]], {(C₉H₁₅N₃)[Cu(C₂O₄)₂)]}_n or {(H₂ppz)[Cu(C₂O₄)₂]}_n, (I), and the discrete ionic complex 4,4′‐(ethane‐1,2‐diyl)dipyridinium bis(oxalato‐κ²O¹,O²)copper(II), (C₁₂H₁₄N₂)[Cu(C₂O₄)₂] or (H₂bpa)[Cu(C₂O₄)₂], (II). The products were characterized by single‐crystal X‐ray diffraction, elemental analysis, powder X‐ray diffraction, thermogravimetric analyses and UV and IR spectroscopic techniques. The [Cu(C₂O₄)₂]²⁻ units for (I) and (II) are stabilized by H₂ppz²⁺ and H₂bpa²⁺ cations, respectively, via charge‐assisted hydrogen bonds. Also, a study of the pH‐controlled synthesis of this system shows that (I) was obtained at pH values of 2–4. When using bpa, a two‐dimensional square‐grid network of [Cu(C₂O₄)(bpa)]_n was obtained at a pH of 4. This indicates that the pH of the reaction also plays a key role in the structural assembly and coordination abilities of oxalate and N,N′‐ditopic coligands.     

6‐Propyl‐2‐thiouracil versus 6‐methoxymethyl‐2‐thiouracil: enhancing the hydrogen‐bonded synthon motif by replacement of a methylene group with an O atom

The understanding of intermolecular interactions is a key objective of crystal engineering in order to exploit the derived knowledge for the rational design of new molecular solids with tailored physical and chemical properties. The tools and theories of crystal engineering are indispensable for the rational design of (pharmaceutical) cocrystals. The results of cocrystallization experiments of the antithyroid drug 6‐propyl‐2‐thiouracil (PTU) with 2,4‐diaminopyrimidine (DAPY), and of 6‐methoxymethyl‐2‐thiouracil (MOMTU) with DAPY and 2,4,6‐triaminopyrimidine (TAPY), respectively, are reported. PTU and MOMTU show a high structural similarity and differ only in the replacement of a methylene group (–CH₂–) with an O atom in the side chain, thus introducing an additional hydrogen‐bond acceptor in MOMTU. Both molecules contain an ADA hydrogen‐bonding site (A = acceptor and D = donor), while the coformers DAPY and TAPY both show complementary DAD sites and therefore should be capable of forming a mixed ADA/DAD synthon with each other, i.e. N—H…O, N—H…N and N—H…S hydrogen bonds. The experiments yielded one solvated cocrystal salt of PTU with DAPY, four different solvates of MOMTU, one ionic cocrystal of MOMTU with DAPY and one cocrystal salt of MOMTU with TAPY, namely 2,4‐diaminopyrimidinium 6‐propyl‐2‐thiouracilate–2,4‐diaminopyrimidine–N,N‐dimethylacetamide–water (1/1/1/1) (the systematic name for 6‐propyl‐2‐thiouracilate is 6‐oxo‐4‐propyl‐2‐sulfanylidene‐1,2,3,6‐tetrahydropyrimidin‐1‐ide), C₄H₇N₄⁺·C₇H₉N₂OS⁻·C₄H₆N₄·C₄H₉NO·H₂O, (I), 6‐methoxymethyl‐2‐thiouracil–N,N‐dimethylformamide (1/1), C₆H₈N₂O₂S·C₃H₇NO, (II), 6‐methoxymethyl‐2‐thiouracil–N,N‐dimethylacetamide (1/1), C₆H₈N₂O₂S·C₄H₉NO, (III), 6‐methoxymethyl‐2‐thiouracil–dimethyl sulfoxide (1/1), C₆H₈N₂O₂S·C₂H₆OS, (IV), 6‐methoxymethyl‐2‐thiouracil–1‐methylpyrrolidin‐2‐one (1/1), C₆H₈N₂O₂S·C₅H₉NO, (V), 2,4‐diaminopyrimidinium 6‐methoxymethyl‐2‐thiouracilate (the systematic name for 6‐methoxymethyl‐2‐thiouracilate is 4‐methoxymethyl‐6‐oxo‐2‐sulfanylidene‐1,2,3,6‐tetrahydropyrimidin‐1‐ide), C₄H₇N₄⁺·C₆H₇N₂O₂S⁻, (VI), and 2,4,6‐triaminopyrimidinium 6‐methoxymethyl‐2‐thiouracilate–6‐methoxymethyl‐2‐thiouracil (1/1), C₄H₈N₅⁺·C₆H₇N₂O₂S⁻·C₆H₈N₂O₂S, (VII). Whereas in (I) only an AA/DD hydrogen‐bonding interaction was formed, the structures of (VI) and (VII) both display the desired ADA/DAD synthon. Conformational studies on the side chains of PTU and MOMTU also revealed a significant deviation for cocrystals (VI) and (VII), leading to the desired enhancement of the hydrogen‐bond pattern within the crystal.     

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

On‐line high‐performance liquid chromatography coupled with biochemical detection method for screening of α‐glucosidase inhibitors in green tea

An on‐line high‐performance liquid chromatography–biochemical detection (HPLC‐BCD) method, in which compounds separated by HPLC were on‐line reacted with enzyme and substrate solutions delivered by flow injection and the enzyme inhibition signal was collected by UV detection, was developed to rapidly screen α‐glucosidase inhibitors from green tea extracts in this study. The chromatographic fingerprints and enzyme inhibition profiles of the different brands of green tea could be simultaneously detected by the on‐line HPLC‐BCD method. Enzyme inhibition profiles were detected by the UV detector at 415 nm based on the reaction of α‐glucosidase and p‐nitrophenyl α‐d ‐glucopyranoside (PNPG). PNPG (1.25 mm ), α‐glucosidase (0.4 U/mL) and the flow rate 0.07 mL/min were applied as optimized parameters to detect α‐glucosidase inhibitors in green tea. Four components in green tea showed α‐glucosidase inhibition action and three of them were identified as HHDP‐galloyl glucose, (−)‐epigallocatechin‐3‐gallate and (−)‐epicatechin‐3‐gallate by HPLC–fourier‐transform mass spectrometry (HPLC‐FTMS). Two brands of green tea derived from Mengding and Enshi mountainous areas might be superior to the other samples in the prevention and treatment of diabetes owing to their stronger activities of enzyme inhibitors. The proposed on‐line HPLC‐BCD method could be used to rapidly identify the potential enzyme inhibitors in complex matrixes.     

Rapid high‐performance liquid chromatography–tandem mass spectrometry method for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma and its application in comparative bioavailability study

A rapid high‐performance liquid chromatography–tandem mass spectrometry method has been developed and validated for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma using fluoxetine as an internal standard. In the liquid–liquid extraction method, compounds and internal standard were extracted from plasma using methyl tertiary butyl ether as an extraction solvent. The HPLC separation of the analytes was performed on a Zorbax SB‐C₁₈, 50 × 4.6 mm, 5 µm column, using a isocratic elution program using a mobile phase consisting of HPLC‐grade methanol: 5 mm ammonium acetate (80:20 v/v) at a flow‐rate of 1.0 mL/min with a total runtime of 3.0 min. The proposed method has been validated with a linear range of 4–400 ng/mL for venlafaxine and 5–500 ng/mL for O‐desmethyl venlafaxine. The method was applied for a bio‐equivalence study of 75 mg tablets formulation in 32 Indian male healthy subjects under fasting conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Approach to the study of flavone di‐C‐glycosides by high performance liquid chromatography‐tandem ion trap mass spectrometry and its application to characterization of flavonoid composition in Viola yedoensis

The mass spectrometric (MS) analysis of flavone di‐C‐glycosides has been a difficult task due to pure standards being unavailable commercially and to that the reported relative intensities of some diagnostic ions varied with MS instruments. In this study, five flavone di‐C‐glycoside standards from Viola yedoensis have been systematically studied by high performance liquid chromatography‐electrospray ionization‐tandem ion trap mass spectrometry (HPLC‐ESI‐IT‐MSⁿ) in the negative ion mode to analyze their fragmentation patterns. A new MS² and MS³ hierarchical fragmentation for the identification of the sugar nature (hexoses or pentoses) at C‐6 and C‐8 is presented based on previously established rules of fragmentation. Here, for the first time, we report that the MS² and MS³ structure‐diagnostic fragments about the glycosylation types and positions are highly dependent on the configuration of the sugars at C‐6 and C‐8. The base peak (^0,2X₁^0,2X₂^? ion) in MS³ spectra of di‐C‐glycosides could be used as a diagnostic ion for flavone aglycones. These newly proposed fragmentation behaviors have been successfully applied to the characterization of flavone di‐C‐glycosides found in V. yedoensis. A total of 35 flavonoid glycosides, including 1 flavone mono‐C‐hexoside, 2 flavone 6,8‐di‐C‐hexosides, 11 flavone 6,8‐di‐C‐pentosides, 13 flavone 6,8‐C‐hexosyl‐C‐pentosides, 5 acetylated flavone C‐glycosides and 3 flavonol O‐glycosides, were identified or tentatively identified on the base of their UV profiles, MS and MSⁿ (n = 5) data, or by comparing with reference substances. Among these, the acetylated flavone C‐glycosides were reported from V. yedoensis for the first time. Copyright © 2014 John Wiley & Sons, Ltd.