Cordycedipeptide A, a new cyclodipeptide from the culture liquid of Cordyceps sinensis (Berk.) Sacc

A new cyclodipeptide named as cordycedipeptide A, a new natural compound and two known compound were isolated from the culture liquid of Cordyceps sinensis (BERK.) SACC. Their structures were elucidated as 3-acetamino-6-isobutyl-2,5-dioxopiperazine (1), 3-isopropyl-6-isobutyl-2,5-dioxopiperazine (2) and 3,6-di(4-hydroxy)benzyl-2,5-dioxopiperazine (3) by 1D and 2D-NMR techniques. The cytotoxic assay showed compound 1 had the cytotoxic activities to L-929, A375, and Hela.     

High-performance liquid chromatography quantification of principal antioxidants in black seed (Nigella sativa L.) phytopharmaceuticals

A new, simple, sensitive, rapid, and accurate isocratic RP-HPLC method was developed and validated for simultaneous analysis of the principal antioxidants of Nigella sativa, i.e., thymoquinone (TQ), carvacrol (CR), and its isomer thymol (THY), in different phytopharmaceuticals. The mobile phase was water-methanol (40 + 60, v/v) at a flow rate of 1.5 mL/min. Quantification was achieved with UV detection at 254 nm, based on peak area. The method was validated for linearity, accuracy, precision, selectivity, and robustness. The proposed method is stability-indicating for determination of TQ in the presence of its degradants. The LOD and LOQ (microg/mL) were, respectively, 0.006 and 0.021 for TQ, 0.002 and 0.006 for CR, and 0.027 and 0.090 for THY. The mean recoveries measured at three concentrations were higher than 99%, with RSD <2%. This analytical method is suitable for quality control of the marker substances in this widely used natural protective and curative remedy.     

High-performance liquid chromatography of sulphonamides extracted from bovine and porcine muscle by solid-phase dispersion.

A method has been developed for the analysis of sulphonamides in bovine and porcine muscle, based on solid-phase dispersion. Muscle tissue was blended with pre-washed C18 coated silica (55-105 microns), and the resulting homogeneous solid packed into a polypropylene syringe barrel. Fatty material was washed from the sample using hexane, and the sulphonamide analytes eluted with dichloromethane. The collected fraction was dried under nitrogen and reconstituted in 20% methanol in 0.01 M sodium acetate/acetic acid (pH 5) buffer. After sonication and filtration, the sample was analysed by high-performance liquid chromatography on a C18 column using UV diode array detection. Individual sulphonamides could be detected down to 0.01 ppm, whilst analyte identity could be confirmed by diode array spectrum down to 0.02 ppm.     

High-performance liquid chromatography of amino acids, peptides and proteins. CXV. Thermodynamic behaviour of peptides in reversed-phase chromatography.

The thermodynamic behaviour of three peptides, bombesin, beta-endorphin and glucagon, was studied under reversed-phase high-performance liquid chromatographic conditions. Experimental data related to the interactive surface contact area (S values) and solute affinity (log k0) were derived over a range of temperatures between 5 and 85 degrees C. These experimental conditions allowed changes in the secondary structure of the solute to be monitored. The influence of the nature of the stationary phase ligand on the relative conformational stability of the three peptides was analysed by acquiring data with n-octadecyl silica (C18) and n-butyl silica (C4) sorbents. Values for the relative changes in entropy and enthalpy associated with the interactive process were also determined. The results provide further insight into the factors involved with the stabilization of secondary structure and the mechanism of the interaction of peptides with hydrophobic surfaces.     

High-performance liquid chromatographic method for the simultaneous purification of cathepsins B, H and L from human liver.

A high-performance liquid chromatographic procedure for the isolation of the three cysteine proteinases, namely cathepsins B, H and L, is described. The method is based on the following four steps. (1) A classical AcA 44 gel permeation separation with a 30-70% ammonium sulphate fraction from the human liver homogenate is used to remove the non-enzymic high-molecular-mass components. (2) Preparative cation-exchange chromatography on a CM-SW TSK column can separate the three proteinases. (3) An anion-exchange step on a semi-preparative DEAE-SW TSK column for the cathepsin H fraction is used to remove a small amount of cathepsins B and L activities. (4) The three separated enzymes are purified on an analytical TSK gel 2000 SW column. The purity of each enzyme is assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrofocusing on polyacrylamide gels. To check the activities of the purified proteinases, the kinetic constants [Michaelis constant (KM) and catalytic constant (Kcat)] and the ratio Kcat/KM against the fluorigenic substrates Arg-NH-Mec, Z-Arg-Arg-NH-Mec and Z-Phe-Arg-NH-Mec after active-site titration using E-64, were determined. Z-Phe-Phe-CNH2 was also used as a specific inhibitor of cathepsin L. This method requires only 6 g of human liver, and gives a high yield of the three lysosomal cysteine-proteinases: thus, about 150 micrograms of cathepsin B and 50 micrograms each of cathepsins L and H are obtained in a single run.     

High-performance liquid affinity chromatography for the purification of immunoglobulin A from human serum using jacalin

A high-performance liquid affinity chromatographic method for the purification of serum immunoglobulin A (IgA) using a jacalin column is described. The automated procedure takes about 2 with minimal manipulation. The yields of the isolated IgA and of its IgG and IgM contamination were studied by enzyme-linked immunosorbent assay (ELISA) of 30 sera. Purity was assured by immunoelectrophoresis. The ratio of IgA1 to total IgA was unchanged after purification, as verified by ELISA. The results showed that greater than 90% IgA could be recovered with less than 0.5% total IgG and greater than 2.0% total IgM remaining in the fractions containing purified IgA.     

High-performance liquid chromatography coupled with fluorescence detection for the determination of trans-astringin in wine.

In this study a high-performance liquid chromatography (HPLC) method was developed for the determination of trans-astringin in wine using fluorescence detection. This is the first time the occurrence of trans-astringin has been reported in wine. The method allows analysis of both red and white wine samples with no prior treatment. The quantification threshold is 0.03 mg/l. Levels of trans-astringin in the French wines analyzed ranged from 0.09 mg/l to 0.29 mg/l. The reproducibility of the method was measured and the CV was less than 4.8% for both red and white wines.     

High-performance liquid chromatography: purification and chromatographic behaviour of molecular variants of pepsinogen A from human urine.

By combining conventional DEAE chromatography with high-performance liquid chromatography on Sephacryl S-200 HR and Mono-Q columns, we have been able to isolate and fractionate human pepsinogen A (PGA) isozymogens from large amounts of urine. This method of fractionation is simple and allows one to obtain pepsinogen in a native non-denatured conformation. The isozymogens are homogeneous by electrophoretic and chromatographic criteria; this was confirmed by N-terminal amino acid sequencing. Purified PGA-3 and PGA-5 can be converted into an additional, more anionic, isoform on incubation at 37 degrees C. This isoform exists not only in vitro but also in vivo. The net negative charge of the PGA isozymogens is in the order PGA-5 less than deamidated PGA-5 less than PGA-3 less than deamidated PGA-3. Surprisingly, the elution order on the Mono-Q column was PGA-5/PGA-3/deamidated PGA-5/deamidated PGA-3. We have performed molecular modelling on PGA to investigate this phenomenon in terms of surface charge (not net charge) of the proteins. The model provides evidence that (1) only a fraction of the protein surface interacts with the support and (2) regions of localized charge at the protein surface may allow portions of the external surface to dominate chromatographic behaviour, resulting in a steering of the proteins with respect to the oppositely charged matrix. Pepsinogens may serve as model proteins for elucidating some of the variables that determine the chromatographic behaviour of proteins on ion-exchange columns.     

High-performance liquid chromatography of biogenic amines in the corpus cardiacum of the American cockroach, Periplaneta americana

The simultaneous determination of biogenic amines in the corpus cardiacum of the American cockroach, Periplaneta americana, was carried out using high-performance liquid chromatography with a Neurochem neurochemical analyser. Vanillic acid, dopamine, octopamine and tyramine were detected. Tyrosine and tryptophan were also detected at high levels. Octopamine levels in the corpus cardiacum were increased on injection of an acetone solution. The biological function of the biogenic amines detected is discussed.     

High-performance liquid chromatography for the characterization of carotenoids in the new sweet orange (Earlygold) grown in Florida, USA

High-performance liquid chromatography with photodiode array detection was developed for the separation and identification of carotenoids from a new sweet orange, Earlygold. Carotenoid pigments were extracted using hexane-acetone-ethanol and saponified using 10% methanolic potassium hydroxide. More than 25 carotenoid pigments were separated within 40 min using a ternary gradient (acetonitrile-methanol, methyl tert-butyl ether and water) elution on a C30 reversed-phase column. The carotenoid pattern of Earlygold was generally similar to the early season Hamlin but with some quantitative differences, especially with violaxanthin. Major carotenoids including violaxanthin, lutein, beta-cryptoxanthin, antheraxanthin, luteoxanthin, zeaxanthin, beta-carotene, and alpha-carotene were identified based on on-line spectral data obtained by a photodiode array detector, and comparison to the spectra of the standards and reported values. A numerical notation, the ratio of the peak heights between absorption bands, was also calculated to compare to the literature values.     

High-performance liquid chromatography of arachidonic acid metabolites and its application to the determination of leukotriene B4 in stimulated leukocytes

A highly sensitive high-performance liquid chromatography with fluorescence detection for the determination of arachidonic acid metabolites is described. The metabolites are converted into corresponding fluorescent derivatives by reaction with 3-bromomethyl-6,7-methylenedioxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated on a reversed-phase column (Inertsil ODS) with aqueous acetonitrile and detected fluorimetrically. The detection limits are 5-15 fmol at a signal-to-noise ratio of 3 in a 10-microliter injection volume. The method is applied to the determination of leukotriene B4 produced in stimulated leukocytes.     

The isolation and structure of a toxic metabolite from Diplodia maydis (Berk.) Sacc.

Diplodiatoxin is a toxic metabolite of Diplodia maydis (Berk.) Sacc. Studies are described which have led to the structure as shown in 1a.     

A liquid chromatography method for determination of selected amino acids, coenzymes, growth regulators, and vitamins from Cicer arietinum (L.) and Solanum lycopersicum (L.)

A bottleneck in crosstalk and QC research has been the quantification of diverse chemotypes in small amounts of tissue. An LC-UV method for estimating 28 selected metabolites of the regulatory network underlying growth, development, maintenance, vital functions, defense reactions, and food quality is reported. The method was based on binary gradient resolutions of the analytes in an RP C18 column. The mobile phase comprised solvent A [water+0.1% trifluoroacetic acid (TFA)] and B (acetonitrile + 0.085% TFA at a flow rate of 1 ml/min. Twenty-three metabolites (selected amino acids, coenzymes, growth regulators, phenolic antioxidant, and water-soluble vitamins) were detected at 254 nm, and four fat-soluble vitamins at 280 nm. Jasmonic acid was quantified at 210 nm. The RSDs of peak area and retention time for each metabolite were <5.8%. The calibration graphs were linear with R2 values ranging from 0.98 to 0.99. The LODs (microg/mL) were about 0.01-1.0 for 23 metabolites quantified at 254 nm, 0.1-0.2 for fat-soluble vitamins, and 0.1 for jasmonic acid. The recoveries ranged from 80 to 105%, with RSDs of 2.8 to 11.2%. The method has been satisfactorily applied for determination of 28 metabolites from Cicer arietinum (L.) and Solanum lycopersicum (L.). It could be an alternative and competitive method of choice that can cheaply and easily perform routine analysis for food quality and targeted metabolomics of chickpea and tomato in response to stressors.     

High-performance liquid chromatography of substituted p-benzoquinones and p-hydroquinones. II. Retention behavior, quantitative structure-retention relationships and octanol-water partition coefficients

The retention behavior of methoxy-substituted p-benzoquinones and the corresponding hydroquinones in reversed-phase chromatography was examined on octylsilica and two octadecylsilica stationary phases and with five hydroorganic mobile phases containing acetonitrile, methanol or tetrahydrofuran and additionally in most cases (NH3OH)3PO4 used as a reducing and buffering agent. The retention order of benzoquinones and hydroquinones was the same on each stationary phase with either methanol or acetonitrile as the organic modifier. On the other hand, minor differences in the retention order were observed with the various stationary phases. In all cases, satisfactory quantitative structure-retention relationships (QSRRs) were found and the data suggest that the differences in the retention behaviour of octadecylsilicas used in this study are silanophilic interactions which, together with solvophobic interaction contribute to the retention of these eluites. Further analysis showed that QSRRs of sterically crowded molecules must take into account reduced surface area available for binding. The retention data obtained with use of aqueous tetrahydrofuran as mobile phase failed to give rise to satisfactory QSRRs. This was attributed to selective solvation of eluite by tetrahydrofuran and/or nearly equipotent binding of eluite and tetrahydrofuran to stationary phase.     

High-performance liquid chromatography with electrochemical detection for the simultaneous determination of vitamin A, D3 and E in milk.

A high-performance liquid chromatographic electrochemical detection for the rapid and simultaneous determination of the vitamin A, D3 and E is described. The separation is carried out by using a C18 reversed-phase column and 0.1 M LiClO4 in methanol-water (99:1, v/v) as the mobile phase. The compounds are eluted with good resolution in the above order within about 15 min and are determined by amperometric detection with a glassy carbon electrode at +1050 mV (vs. Ag/AgCl). The method gave reproducible results and the detection limits were of the order of 0.07, 4 and 0.2 ng of vitamin A, D3 and E, respectively. The method was successfully applied to the determination of vitamin A, D3 and E in liquid cow milk and milk powder samples. After saponification, fat-soluble vitamins were extracted with hexane and a methanolic solution of the dried extract was injected directly into the chromatographic system, avoiding the clean-up step that is necessary for vitamin D3 when electrochemical detection is not used. Good recoveries were obtained.