Chenxu Ding

Detection of carbohydrates using new labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone by capillary zone electrophoresis with absorbance (UV).

A novel labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) coupled with capillary electrophoresis (CE) with DAD detection for the determination of carbohydrates has been developed. The chromophore in the 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent is replaced by naphthyl functional group, which results in a reagent with very high molar absorptivity (epsilon251 nm = 5.58 x 10(4) L mol(-1) cm(-1)). This permits NMP-labeled carbohydrates to be detected with UV absorbance in standard 50-mum-i.d. fused silica capillaries by zone electrophoresis. In this mode, nanomolar concentrations of detection limits are obtained. The method for the derivatization of carbohydrates with NMP is simplified. The derivatization reaction is rapid and mild in the presence of ammonia catalyst without further transfer steps. Nine monosaccharide derivatives such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose and fucose can successfully be detected in CE mode. Good reproducibility can be obtained with relative standard deviation (R.S.D.) values of the migration times and peak area, respectively, from 0.44 to 0.48 and from 3.2 to 4.8. Furthermore, the developed method has been successfully applied to the analysis of carbohydrates in the hydrolyzed rape bee pollen samples.

Determination of amines using 2-(11H-benzo[a]carbazol-11-yl) ethyl chloroformate (BCEC-Cl) as labeling reagent by HPLC with fluorescence detection and identification with APCI/MS

A pre-column derivatization method for the sensitive determination of amines using a labeling reagent 2-(11H-benzo[a]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by LC/APCI/MS in positive-ion mode. The chromophore of 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) reagent was replaced by 2-(11H-benzo[a]-carbazol-11-yl) ethyl functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEC-Cl. BCEC-Cl could easily and quickly label amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M+H](+) under APCI/MS in positive-ion mode. The collision-induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 261.8 and m/z 243.8 corresponding to the cleavages of CH(2)O-CO and CH(2)-OCO bonds. Studies on derivatization demonstrated excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% were observed with three- to four-fold molar reagent excess. In addition, the detection responses for BCEC-derivatives were compared to those obtained using 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) and 9-fluorenyl methylchloroformate (FMOC-Cl) as labeling reagents. The ratios I(BCEC)/I(BCEOC)=1.94-2.17 and I(BCEC)/I(FMOC)=1.04-2.19 for fluorescent (FL) responses (here, I was relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C(8) column. Detection limits calculated from 0.50pmol injection, at a signal-to-noise ratio of 3, were 1.77-14.4fmol. The relative standard deviations for within-day determination (n=11) were 1.84-2.89% for the tested amines. The mean intra- and inter-assay precision for all amines levels were <3.64% and 2.52%, respectively. The mean recoveries ranged from 96.6% to 107.1% with their standard deviations in the range of 0.8-2.7. Excellent linear responses were observed with coefficients of >0.9996.

Determination of Free Fatty Acids from Soil and Bryophyte by HPLC with Fluorescence Detection and Identification with Mass Spectrometry

Abstract A simple and sensitive method for the determination of free fatty acids using acridone-9-ethyl- p -toluenesulfonate (AETS) as tagging reagent by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed. Studies on derivatization conditions indicate that free fatty acids react rapidly and smoothly with AETS in the presence of K 2 CO 3 catalyst in N , N -dimethylformamide (DMF) solvent at 85°C to give the corresponding sensitive fluorescence derivatives. The maximal derivatization yields are close to 100% with a six fold molar reagent excess. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. Separation of the derivatized fatty acids exhibits a good baseline resolution in combination with a gradient elution on a reversed-phase Eclipse XDB-C 8 column. The identification of 19 fatty acid derivatives was carried out by online post-column mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion mode. The contents of 19 free fatty acids in soil and three kinds of bryophytes ( Tree Moss, Entodon macropodus, Dicranum caesium ) were determined. The results indicate that the bryophyte plants enrich an abundance of free fatty acids from soil. The fluorescence excitation and emission wavelengths of derivatives were 404nm of λ ex and 440 nm of λ em , respectively. Excellent linear responses were observed with the linear correlation coefficients over 0.9996, and detection limits (at signal-to-noise of 3:1) are 12.3–43.7 fmol. The proposed method is sensitive and reproducible for the determination of free fatty acids from real samples with satisfactory results.

In vitro and in vivo biological activities of anthocyanins from Nitraria tangutorun Bobr. fruits

Anthocyanins are the main compounds in Nitraria tangutorun Bobr. The enrichment and purification of anthocyanins on macroporous resins were investigated. Regarding anthocyanin purification, static adsorption and desorption were studied. The optimal experimental conditions were the following: resin type: X-5; static adsorption time: 6h; desorption solution: ethanol-water-HCl (80:19:1, V/V/V; pH 1); desorption time: 40min. Furthermore, the in vitro and in vivo biological activities of the anthocyanins were evaluated. The anthocyanins showed ideal scavenging effects on free radicals in vitro, especially on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl free radical (OH). In the animal experiment, blood lipid metabolism of hyperlipidemia rats was regulated by anthocyanin contents. The superoxide dismutase (SOD) activity and the total antioxidant capacity (TAC) of hyperlipidemia rats were also improved by anthocyanins. These results showed that anthocyanins from N. tangutorun Bobr. fruits had potential biological activities in vivo as well as in vitro.

Efficient Protocol for Isolation of Rhaponticin and Rhapontigenin with Consecutive Sample Injection from Fenugreek (Trigonella foenum-graecum L.) by HSCCC

High efficiency and less solvent consumption are the essential requirements of high-speed countercurrent chromatography (HSCCC), especially for the large-scale preparation. In this study, an efficient HSCCC strategy with consecutive sample injection was successfully developed to rapidly separate and purify rhaponticin and rhapontigenin from the seeds of the Chinese medicinal herb fenugreek (Trigonella foenum-graecum L.). The effective separation was achieved using n-hexane-ethyl acetate-methanol-water (1:4:2:6, v/v/v/v) as the two-phase solvent system, in which the mobile phase was eluted at an optimized flow rate of 2.2 mL/min and a revolution speed of 850 rpm. After consecutively loading four identical fenugreek samples, each containing 120 mg, HSCCC separation yielded 146.4 mg of rhaponticin and 174.8 mg of rhapontigenin with purities of 98.6 and 99.1%, respectively, as determined by high-performance liquid chromatography at 320 nm. Their chemical structures were identified using UV spectroscopy, (1)H-NMR and (13)C-NMR. The HSCCC method with consecutive sample injection allowed faster separation and produced less solvent waste, suggesting that it is an efficient way to rapidly separate and purify natural products on a large scale.

Application of 2-(11H-benzo[a]carbazol-11-yl) Ethyl Carbonochloridate as a Pro-column Derivatization Reagent of Amino Acid by High Performance Liquid Chromatography with Fluorescence Detection

On a reversed phase Hypersil BDS C-18 (200 mm x 4. 6 mm, 5 mu m) column, 20 amino acids, which were derivatized using 2-(11H-benzo [a] carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl) as pre-column derivatization reagent, were separated in conjunction with a gradient elution. Optimum derivatization was obtained by reacting of amino acids with BCEC-Cl at room temperature for 5 min in the presence of sodium borate catalyst in acetonitrile solvent. The fluorescence excitation and emission wavelengths were 279 nm and 380 nm respectively. The identification of amino acid derivatives from hydrolyzed bovine serum albumin and bee pollen was carried out by post-column mass spectrometry with electrospray ion source in positive ion mode. Linear correlation coefficients of the amino acid derivatives were > 0.9990, and detection limits (at signal to noise of 3:1) were 1.49 - 19.74 fmol for the labeled amino acids.

HPLC-APCI-MS determination of free fatty acids in Tibet folk medicine Lomatogonium rotatum with fluorescence detection and mass spectrometric identification

Abstract A simple and sensitive method for the determination of free fatty acids (FFAs) using acridone‐9‐ethyl‐p‐toluenesulfonate (AETS) as a fluorescence derivatization reagent by high performance liquid chromatography (HPLC) has been developed. Free fatty acid derivatives were separated on an Eclipse XDB‐C8 column with a good baseline resolution and detected with the fluorescence of which excitation and emission wavelengths of derivatives were set at λex=404 and λem=440 nm, respectively. Identification of 19 fatty acid derivatives was carried out by online post‐column mass spectrometry with an atmospheric pressure chemical ionization (APCI) source under positive‐ion detection mode. Nineteen FFAs from the extract of Lomatogonium rotatum are sensitively determined. The results indicate that the plant Lomatogonium rotatum is enriched with an abundance of FFAs and FFAs of higher contents, which mainly focus on even carbon atoms, C14, C16, and C18. The validation of the method including linearity, repeatability, and detection limits was examined. Most linear correlation coefficients for fatty acid derivatives are >0.9989, and detection limits (at signal‐to‐noise of 3:1) are 12.3–43.7 fmol. The relative standard deviations (RSDs) of the peak areas and retention times for 19 FFAs standards are <2.24% and 0.45%, respectively. The established method is rapid and reproducible for the separation determination of FFAs from the extract of Lomatogonium rotatum with satisfactory results.

Synthesis and properties of the rapeseed meal-grafted-poly(methyl methacrylate-co-butyl acrylate) oil-absorbents

Owing to the unique properties such as oil body (OB) membranes and cellulose components within the structures of rapeseed meals (RSMs), the novel rapeseed meal-grafted-poly(methyl methacrylate-co-butyl acrylate) (RSMs-g-P(MMA-co-BA)) oil-absorbents were prepared and used for oil/water separation in the present study. Specifically, the RSMs-g-P(MMA-co-BA) were successfully synthesized through free radical graft copolymerization from RSMs, methyl methacrylate (MMA) and butyl acrylate (BA) with benzoyl peroxide (BPO) as initiator and N,N′-methylene-bisacrylamide (MBA) as crosslinker. The synthetic mechanism and 3D network structures were confirmed by FTIR and SEM, and the effects of reaction conditions, swelling kinetics, thermodynamics as well as reusability were investigated in detail. The RSMs-g-P(MMA-co-BA) demonstrated good absorption capacity for both organic solvents and oils, and the change of oil absorbency after eight repeated cycles of swelling/deswelling was marginal. Furthermore, the RSMs-g-P(MMA-co-BA) could selectively remove oils from water rapidly, which is promising for their potential applications in oil spill cleanup.

Separation and purification of four oligostilbenes from Iris lactea Pall. var. chinensis (Fisch.) Koidz by high-speed counter-current chromatography.

A method of using high-speed counter-current chromatography (HSCCC) for preparative isolation and purification of oligostilbenes from the ethanol extracts of seed kernel of Iris lactea Pall. var. chinensis (Fisch.) Koidz was established in this study. Four oligostilbenes were successfully separated and purified by HSCCC with two sets of two-phase solvent system, n-hexane-ethyl acetate-methanol-water (3:6:4.2:5.5, v/v/v/v) in the head-to-tail elution mode for the first separation to mainly isolate vitisin A (58 mg), ɛ-viniferin (76 mg) and peak II (43 mg) from 300 mg of the crude ethanol extracts, and then light petroleum-ethyl acetate-methanol-water (5:5:3:6, v/v/v/v) in the tail-to-head elution mode for the second separation to isolate vitisin B (52 mg) and vitisin C (11 mg) from 100mg of peak II. The purities of the isolated four oligostilbenes were all over 95.0% as determined by HPLC. Vitisin A, vitisin B and vitisin C, resveratrol tetramers, were isolated from Iris lactea for the first time. The preparation of crude sample was simple and the HSCCC method for the isolation and purification of four oligostilbenes was rapid, efficient and economical.

Preparative separation and purification of four cis-trans isomers of coumaroylspermidine analogs from safflower by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was successfully applied for the first time to isolate and purify four cis-trans isomers of coumaroylspermidine analogs from Safflower. HSCCC separation was achieved with a two-phase solvent system composed of chloroform-methanol-water (1:1:1, v/v/v) with the upper phase as the mobile phase. In a single run, a total of 1.3mg of N(1), N(5), N(10)-(E)-tri-p-coumaroylspermidine (EEE), 4.4mg of N(1)(E)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (EZE), 7.2mg of N(1)(Z)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (ZZE), and 11.5mg of N(1),N(5),N(10)-(Z)-tri-p-coumaroylspermidine (ZZZ) were obtained from 100mg of crude sample. High Performance Liquid Chromatography (HPLC) analysis showed that the purities of these four components are 95.5%, 98.1%, 97.5% and 96.2%, respectively. The chemical structures were identified by ESI-MS, (1)H NMR and (13)C NMR.