Analysis of Erythroxylum coca Leaves by Imaging Mass Spectrometry (MALDI-FT-ICR IMS).

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can determine the chemical identity and spatial distribution of several molecules in a single analysis, conserving its natural histology. However, there are no specific studies on the spatial distribution of alkaloids in Erythroxylum coca leaves by MALDI IMS, preserving the histology of the monitored compounds. Therefore, in this work, positive-ion mode MALDI Fourier-transform ion cyclotron resonance imaging mass spectrometry (MALDI(+)FT-ICR IMS) was applied to identify and analyze the distribution of alkaloids on the surface of coca leaves, evaluating the ionization efficiency of three matrices (α-cyano-4-hydroxycinnamic acid (CHCA), 2-mercaptobenzothiazole (MBT), and 2,5-dihydroxybenzoic acid (DHB)). The last was chosen as the best matrix in this study, and it was studied in five concentrations (0.5, 1.0, 2.0, 4.0, and 8.0 mg·mL-1), where 2 mg·mL-1 was the most efficient. The washing of coca leaves with the organic solvents (acetonitrile, methanol, toluene, and dichloromethane) tested did not improve the performance of the ionization process. Finally, a tissue section, 50 µm thick, was used to study the inner part of the leaf tissue, where alkaloids and flavonoid molecules were detected.

A new sensor based on an amino-montmorillonite-modified inkjet-printed graphene electrode for the voltammetric determination of gentisic acid.

An amperometric sensor based on an inkjet-printed graphene electrode (IPGE) modified with amine-functionalized montmorillonite (Mt-NH2) for the electroanalysis and quantification of gentisic acid (GA) has been developed. The organoclay used as IPGE modifier was prepared and characterized by infrared spectroscopy, X-ray diffraction, scanning electron microscopy, CHN elemental analysis, and thermogravimetry. The electrochemical features of the Mt-NH2/IPGE sensor were investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The sensor exhibited charge selectivity ability which was exploited for the electrochemical oxidation of GA. The GA amperometric response was high in acidic medium (Brinton-Robinson buffer, pH 2) due to favorable interactions between the protonated amine groups and the negatively charged GA. Kinetic studies were also performed by cyclic voltammetry, and the obtained electron transfer rate constant of 11.3 s-1 indicated a fast direct electron transfer rate of GA to the electrode. An approach using differential pulse voltammetry was then developed for the determination of GA (at + 0.233 V vs. a pseudo Ag/Ag+ reference electrode), and under optimized conditions, the sensor showed high sensitivity, a wide working linear range from 1 to 21 µM (R2 = 0.999), and a low detection limit of 0.33 µM (0.051 ± 0.01 mg L-1). The proposed sensor was applied to quantify GA in a commercial red wine sample. The simple and rapid method developed using a cheap clay material could be employed for the determination of various phenolic acids.

Choline Salicylate Analysis: Chemical Stability and Degradation Product Identification.

Choline salicylate (CS) as a derivative of acetylsalicylic acid is commonly used in different drug forms. In medicine, it is applied topically to inflammation of the oral cavity mucosa and in laryngology. However, this substance in the form of an ionic liquid has not been investigated enough. There are no literature studies on stability tests constituting a stage of pre-formulation research. HPLC (Nucleosil C18, 4.6 × 150 mm, 5 µm; methanol-water-acetic acid 60:40:1, 230 nm or 270 nm) and UV (276 nm) methods for the determination of CS in 2% (g/mL) aqueous solutions were developed. Under stress conditions, CS susceptibility to hydrolytic degradation in aqueous medium, hydrochloric acid, sodium hydroxide, and hydrogen peroxide, and the effect of light on the stability of CS solutions were studied with HPLC analysis. The degradation degree of CS and the purity of the solutions were also tested. Choline salicylate has been qualified as practically stable in neutral and acid media, stable in an alkaline medium, very stable in an oxidizing environment, and photolabile in solution. The HPLC-MS/MS method was used to identify 2,3- and 2,5-dihydroxybenzoic acids as degradation products of CS under the tested conditions.

3D ToF-SIMS Analysis of Peptide Incorporation into MALDI Matrix Crystals with Sub-micrometer Resolution.

The analytical sensitivity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is largely affected by the specific analyte-matrix interaction, in particular by the possible incorporation of the analytes into crystalline MALDI matrices. Here we used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the incorporation of three peptides with different hydrophobicities, bradykinin, Substance P, and vasopressin, into two classic MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA). For depth profiling, an Ar cluster ion beam was used to gradually sputter through the matrix crystals without causing significant degradation of matrix or biomolecules. A pulsed Bi3 ion cluster beam was used to image the lateral analyte distribution in the center of the sputter crater. Using this dual beam technique, the 3D distribution of the analytes and spatial segregation effects within the matrix crystals were imaged with sub-µm resolution. The technique could in the future enable matrix-enhanced (ME)-ToF-SIMS imaging of peptides in tissue slices at ultra-high resolution. Graphical Abstract ᅟ.

Graphene modified glassy carbon sensor for the determination of aspirin metabolites in human biological samples.

A graphene modified glassy carbon (GR/GCE) sensor has been developed for the determination of aspirin metabolites 2,3- and 2,5-dihydroxybenzoic acids (2,3- and 2,5-DHB). The modified sensor was characterized by Field Emission Scanning Electron Microscopy and Electrochemical Impedance Spectroscopy. The electrochemical behavior of 2,3- and 2,5-DHB was investigated by cyclic and square wave voltammetry. The modified sensor exhibited excellent electrocatalytic activity for the oxidation of 2,3- and 2,5-DHB, leading to a remarkable enhancement in the peak current as compared to the bare sensor. The results were attributed to the enhanced surface area and high conductivity of GR. The anodic peak currents of 2,3- and 2,5-DHB were found to be linear in the concentration range of 1-150 µM and 1-200 µM with the detection limits of 47 nM and 51 nM, respectively. The sensor was capable to determine 2,5-DHB effectively without any interference from the uric acid and other metabolites present in the urine samples. The practical utility of GR/GCE has been successfully demonstrated for the determination of 2,5-DHB in the urine samples of persons undergoing treatment with aspirin.

Application of time-resolved fluorescence to the determination of metabolites.

A simple fluorescent methodology for the simultaneous determination of two major metabolites of acetylsalicylic acid--salicylic and gentisic acids--in pharmaceutical preparations and human urine is proposed. Due to the overlapping between the fluorescence spectra of both analytes, the use of the more selective fluorescence decay curves is proposed. Values of dependent instrumental variables affecting the signal-to-noise ratio were fixed with a simplex optimization procedure. A calibration matrix of thirteen standards plus two blank samples was processed using a partial least-squares (PLS) analysis. To assess the goodness of the proposed method, a prediction set of nine synthetic samples was analyzed, obtaining recovery percentages between 95% and 106%. Limits of detection, calculated by means of a new criterion, were 3.49 µg L(-1) and 1.66 µg L(-1) for salicylic and gentisic acids, respectively. The method was also tested in three pharmaceutical preparations containing salicylic acid, obtaining recovery percentages close to 100%. Finally, the simultaneous determination of both analytes in human urine samples was successfully carried out by the PLS-analysis of a matrix of thirteen standards plus five analyte blanks. Although spectra of analytes and urine overlap strongly, no extraction method neither prior separation of the analytes were needed.

Antioxidant and anti-inflammatory activities of methanol extracts of Tremella fuciformis and its major phenolic acids.

Methanol extract subfractions of the edible white jelly mushroom (Tremella fuciformis), were assessed for the following antioxidant properties: ABTS(+) radical scavenging activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, and inhibitory activity of human low-density lipoprotein (LDL) oxidation. Among the subfractions tested, the chloroform subfraction exhibited the strongest antioxidant activity, with the highest total phenolic content (66.31 µg CAE/mg extract) and flavonoids content (5.12 µg QE/mg extract). The ABTS(+) radical scavenging activity of the chloroform subfraction was 7.89 µmol trolox/mg extract, which was the highest among all subfractions. This subfraction also showed the highest DPPH radical scavenging activity and inhibitory activity of LDL oxidation. In addition, the chloroform subfraction demonstrated anti-inflammatory activity through inhibition of nitric oxide production and inducible nitric oxide synthase expression in RAW 264.7 cells. Major phenolic acids from the mushroom extract were identified as 4-hydroxybenzoic acid (323 mg/kg dry weight of mushroom), gentisic acid (174 mg/kg dry weight of mushroom), and 4-coumaric acid (30 mg/kg dry weight of mushroom).

Simultaneous determination of gallic acid and gentisic acid in organic anion transporter expressing cells by liquid chromatography-tandem mass spectrometry.

In order to elucidate the role of organic anion transporters (OATs) in the renal elimination of gallic acid and gentisic acid, a new, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of gallic acid and gentisic acid in cell lysate, using Danshensu as the internal standard (IS). After a simple liquid-liquid extraction, the analytes were detected in negative ESI mode using selected reaction monitoring. The precursor-to-product ion transitions (m/z) were 169.0→125.0, 153.1→108.0, and 196.8→135.2 for gallic acid, gentisic acid, and the IS, respectively. Chromatographic separation was achieved on a C18 column using mobile phases consisting of water with 0.1% acetic acid (A) and acetonitrile with 0.05% formic acid. (B) The total run time was 3min and calibration curves were linear over the concentrations of 0.33-2400ng/mL for both compounds (r(2)>0.995). Good precision (between 3.11% and 14.1% RSD) and accuracy (between -12.7% and 11% bias) was observed for quality controls at concentrations of 0.33 (lower limit of quantification), 1, 50, and 2000ng/mL. The mean extraction recovery of gallic acid and gentisic acid was 80.7% and 83.5%, respectively. Results from post-column infusion and post-extraction methods indicated that the analytical method exhibited negligible matrix effects. Finally, this validated assay was successfully applied in a cellular uptake study to determine the intracellular concentrations of gallic acid and gentisic acid in OAT expressing cells.

[Chemical constituents from flower buds of Lonicera japonica].

Eighteen compounds were isolated by a combination of various chromatographic techniques including column chromatography over macroporous resin, MCI gel, silica gel, and sephadex LH-20 and reversed-phase HPLC. Their structures were elucidated by spectroscopic data analysis as adinoside A (1), stryspinoside (2), benzyl alcohol beta-glucopyranoside (3), benzyl 2-o-beta-D-glucopyranosyl-2,6-dihydroxybenzoate (4) , gentisic acid 2-O-beta-D-glucopyranoside (5), eugenyl beta-D-glucopyranoside (6) , eugenyl-P-xylopyranosyl-(1-->6)-beta-glucopyranoside (7), (-)-lyoniresinol 9-O-fP-D-glucopyranoside (8) , (+)-lyoniresinol 9-O-beta-D-glucopyranoside (9) , apigenin-7-O-L-rhamnopyranoside (10), luteolin-3 '-O-L-rhamnoside (11) , ursolic acid (12) , beta-sitosteryl-3beta-glucopyranoside-6'-O-palmitate (13), abscisic acid (14), guanosine (15), 5-methyluracil (16), trans-cinnamic acid (17), and 4-hydroxybenzaldehyde(18). These compounds were obtained from this plant for the first time.

Compressed matrix thin film (CMTF)-assisted laser desorption ionization mass spectrometric analysis.

The inhomogeneous re-crystallization process of matrix materials is the major concerns associated with matrix assisted laser desorption/ionization (MALDI) analysis. We describe here the approach termed compressed matrix thin film (CMTF) in order to make a uniform matrix deposition. In this approach, solid matrix particles are compressed under 10 MPa of pressure by a compressor that is regularly used in infrared spectroscopic analysis. Then aqueous samples can be deposited on the surface of the matrix film. Major advantages of the CMTF approach are summarized as follows. (1) Reproducible sample preparation procedure. Size and thickness of matrix thin films can be controlled by using a fixed mold.force and known amount of matrix materials. (2) Significantly decreased shot-to-shot variations and enhanced reproducibility. (3) Tolerance for in situ salt washing. Because matrix materials are hydrophobic, salts can be washed away while proteins or peptides are retained on the surface of matrix thin films through hydrophobic interactions. (4) Improved sensitivity. The hydrophobic coating of MALDI sample plate by matrix thin films prevents the spreading of samples across the plate and confines analytes to a small area, leading to increased local concentration. (5) A new means for tissue analysis. Tissue sections can be directly transferred to the uniform surface of matrix materials for reproducible and quantitative comparison of different molecules in different localization. The proposed CMTF should be an enabling technique for mass spectrometric analysis with improved correlations between signal intensities and sample quantities.

Enhancement of soluble dietary fibre, polyphenols and antioxidant properties of chapatis prepared from whole wheat flour dough treated with amylases and xylanase.

BACKGROUND: Chapati preparation involves various processing steps such as mixing the flour into dough, sheeting and baking. During these processing steps, flour components are likely to undergo changes in their nutrient and polyphenol composition and their antioxidant properties due to phenol-mediated crosslinking of proteins and carbohydrates. Therefore, in the present study, changes in nutritional, nutraceutical and antioxidant properties of chapatis prepared from doughs treated with amylases and xylanase were determined. RESULTS: An increase in insoluble dietary fibre content and a decrease in soluble polyphenol content were observed during preparation of control chapatis from whole wheat flours. However, significant increases in soluble dietary fibre and soluble polyphenol contents were observed in chapatis prepared from amylase-treated doughs compared with control chapatis. Extracts of chapatis prepared from amylase- and xylanase-treated doughs showed better antioxidant properties than extracts of control chapatis. Among these enzyme treatments, chapatis prepared from amylase-treated doughs showed better antioxidant properties than chapatis prepared from xylanase-treated doughs. High-performance liquid chromatography analysis of extracts of chapatis prepared from doughs treated with amylases showed the presence of potential antioxidant phenolic acids such as caffeic, gentisic and syringic acids in addition to the phenolic acids present in control chapatis. CONCLUSION: Treatment of doughs with amylases increased the contents of soluble dietary fibre and soluble polyphenols as well as improving the antioxidant properties of chapatis.

Far infrared-assisted extraction followed by capillary electrophoresis for the determination of bioactive constituents in the leaves of Lycium barbarum Linn.

In this work, a method based on capillary electrophoresis with amperometric detection and far infrared-assisted extraction has been developed for the determination of rutin, gentisic acid, and quercetin in the leaves of Lycium barbarum Linn. The effects of detection potential, irradiation time, and the voltage applied on the infrared generator were investigated to acquire the optimum analysis conditions. The detection electrode was a 300-microm-diameter carbon disc electrode at a detection potential of +0.90 V. The three analytes could be well separated within 12 min in a 40 cm length fused-silica capillary at a separation voltage of 12 kV in a 50 mM borate buffer (pH 9.2). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with the detection limits (S/N = 3) of 0.31, 0.48, and 0.78 microM for rutin, gentisic acid, and quercetin, respectively. The proposed method has been applied to determine the three bioactive constituents in real plant samples.

Significant sensitivity improvements by matrix optimization: a MALDI-TOF mass spectrometric study of lipids from hen egg yolk.

Due to its sensitivity, the tolerance of impurities and the simplicity of performance, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is increasingly used to analyze lipids from biological sources. Although its detailed role is not understood so far, the applied matrix has a pronounced effect on the achievable spectrum quality and particularly how sensitive the individual lipid classes are detectable. Different matrix compounds were recently established in the lipid field including 2,5-dihydroxybenzoic acid (DHB), 9-aminoacridine (9-AA), para-nitroaniline (PNA), 2-mercaptobenzothiazole (MBT), and 2-(2-aminoethylamino)-5-nitropyridine (AAN). It is the aim of this paper to compare the properties of these matrices with the newly synthesized matrix, alpha-cyano-2,4-difluorocinnamic acid (Di-FCCA). An organic extract from hen egg yolk was used as a simple and easily available test system. It will be shown that Di-FCCA is the matrix of choice to detect lipids in the positive-ion mode due to an achievable sensitivity gain of more than one order of magnitude compared to alternative matrices. In contrast, Di-FCCA is not suitable for negative-ion detection of phospholipids. Here, 9-AA is unequivocally the matrix of choice.

The intact muscle lipid composition of bulls: an investigation by MALDI-TOF MS and 31P NMR.

The analysis of beef lipids is normally based on chromatographic techniques and/or gas chromatography in combination with mass spectrometry (GC/MS). Modern techniques of soft-ionization MS were so far scarcely used to investigate the intact lipids in muscle tissues of beef. The objective of the study was to investigate whether matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry and (31)P nuclear magnetic resonance (NMR) spectroscopy are useful tools to study the intact lipid composition of beef. For the MALDI-TOF MS and (31)P NMR investigations muscle samples were selected from a feeding experiment with German Simmental bulls fed different diets. Beside the triacylglycerols (TAGs), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI) species the MALDI-TOF mass spectra of total muscle lipids gave also intense signals of cardiolipin (CL) species. The application of different matrix compounds, 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9-AA), leads to completely different mass spectra: 9-AA is particularly useful for the detection of (polar) phospholipids, whereas apolar lipids, such as cholesterol and triacylglycerols, are exclusively detected if DHB is used. Finally, the quality of the negative ion mass spectra is much higher if 9-AA is used.

Metabolic changes of salicylic acid-elicited Catharanthus roseus cell suspension cultures monitored by NMR-based metabolomics.

The effect of salicylic acid (SA) on the metabolic profile of Catharanthus roseus suspension cells throughout a time course (0, 6, 12, 24, 48 and 72 h after treatment) was investigated using NMR spectroscopy and multivariate data analysis. When compared to control cell lines, SA-treated cells showed a high level of sugars (glucose and sucrose) up to 48 h after treatment, followed by a dynamic change in amino acids, phenylpropanoids, and tryptamine. Additionally, one compound--2,5-dihydroxybenzoic-5-O-glucoside--was detected solely in SA-treated cells.

A binary matrix of 2,5-dihydroxybenzoic acid and glycerol produces homogenous sample preparations for matrix-assisted laser desorption/ionization mass spectrometry.

We introduce a two-component matrix for ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI-MS) that consists of 2,5-dihydroxybenzoic acid (DHB) and glycerol. Upon slow evaporation of residual water/methanol solvents in a pre-vacuum chamber sample preparations are obtained that exhibit a homogeneous morphology with analyte-matrix crystals evenly distributed over the whole sample spot. At a molar DHB/glycerol ratio of approximately 1:5, the crystals range in length from approximately 100 to 300 microm and are about 15-30 microm wide. Mass spectra of peptides, proteins, and an oligosaccharide are presented and compared with those recorded from standard dried-droplet DHB matrix. The ion signals show a reproducibility of the order of 10-15% when scanning the surface of an individual sample or even different samples that contain the same amount of peptide, A close to linear relationship between peptide concentration and the corresponding peptide ion signal is found over three orders of magnitude of sample prepared. However, when a fixed position is irradiated with a large number of laser pulses, a monotonous decay of peptide ion signal with time is observed. Potentially, the binary matrix will be especially useful for the analysis of samples that are stabilized in buffered aqueous glycerol solution and preliminary results addressing this aspect are shown.

Simultaneous determination of active ingredients in ethnomedicine Gaultheria leucocarpa var. yunnanensis and its medicinal preparation by capillary electrophoresis with electrochemical detection.

A simple and rapid capillary electrophoresis (CE) with electrcochemical detection (ED) method has been established for the simultaneous determination of seven active ingredients in the stems and roots of Gaultheria leucocarpa var. yunnanensis and its medicinal preparation, including (+)-catechin, rutin, gentisic acid, vallinic acid, salicylic acid, quercetin, and protocatechuic acid. The effects of working potential, pH, and concentration of running buffer, separation voltage, and injection time on CE-ED are systematically investigated. Under the optimum conditions, the seven analytes could be completely separated within 23 min in a borax running buffer (pH 8.7). A good linear relationship is obtained over three orders of magnitude with detection limits (signal-to-noise ratio=3) ranging from 5x10(-8) g/mL to 3x10(-7) g/mL for the analytes. The proposed method is successfully used in the analysis of real samples after a relatively simple extraction procedure, and the assay results are satisfactory.

Rapid fingerprinting of red wines by MALDI mass spectrometry.

Here we report a simple and fast method for wine fingerprinting based on direct matrix-assisted laser desorption/ionization (MALDI) mass spectrometry analysis of different red wine samples, useful for batch-to-batch analysis and for the detection of key compounds even in trace amounts which may vary from vintage to vintage, and from one treatment to another one. A series of 20 samples from different wines were subjected to MALDI mass spectrometry. We found that 2,5-dihydroxybenzoic acid is far superior with respect to all the matrices tested To the best of our knowledge this is the first application of an effective wine profiling not limited to detection of anthocyanins. More than 80 molecular species were detected. Moreover, qualitative and quantitative differences were observed, owing to the nature and relative abundance of different chemical compounds among the wines.

Analysis of adenosine triphosphate and glutathione through gold nanoparticles assisted laser desorption/ionization mass spectrometry.

This paper describes the use of aptamer-modified gold nanoparticles (Apt-AuNPs) as selective probes and AuNPs as the surface-assisted laser desorption/ionization (SALDI) matrixes for the determination of adenosine triphosphate (ATP) by mass spectrometry (MS). The aptamers were covalently attached to the surface of AuNPs to form Apt-AuNPs that provided selectivity toward ATP. However, Apt-AuNPs are less efficient laser desorption/ionization (LDI) matrixes when compared to AuNPs. By using Apt-AuNPs as selective probes and AuNPs as LDI matrixes, the MS approach provided the limit of detection (LOD) for ATP at a signal-to-noise ratio of 3 of 0.48 microM. When compared to conventional organic matrixes (e.g., 2,5-dihydroxybenzoic acid), AuNPs as LDI matrixes provide a number of advantages, including ease of preparation, selectivity, sensitivity, and repeatability. Sequential analysis of ATP and GSH in human cell lysates by SALDI with negative and positive MS modes, respectively, using Apt-AuNPs and AuNPs has been demonstrated. The present results demonstrate the practicality of the approach for monitoring the bioactivity of cells through determinations of the concentrations of ATP and GSH.

Dissipation of charge on MALDI-TOF polymeric chips using an electron-acceptor: analysis of proteins.

Disposable polymeric devices used for direct sample pretreatment/signal enhancement and MALDI-MS analyses of biomolecules suffer from the accumulation of electric charge and related positive mass shifts due to photoelectric effects on non-conductive surfaces after irradiation with lasers. The effects of surface charging on the mass shift of protein spectra during MALDI-TOF/MS measurements on copolymeric sample array chips were studied. Methyl viologen is used to monitor the development of charge and is demonstrated to be an effective electron scavenger. The use of such reducible species leads to increased accuracy, signal homogeneity, and resolution for mass spectral measurements of proteins in mixtures with sinapinic and 2,5-dihydroxybenzoic (DHB) acids. This approach can be used on a wide range of nonconductive support materials.