Characterization and identification of isomeric flavonoid O-diglycosides from genus Citrus in negative electrospray ionization by ion trap mass spectrometry and time-of-flight mass spectrometry

Flavonoid O-diglycosides are important bioactive compounds from genus Citrus. They often occur as isomers, which makes the structural elucidation difficult. In the present study, the fragmentation behavior of six flavonoid O-diglycosides from genus Citrus was investigated using ion trap mass spectrometry in negative electrospray ionization (ESI) with loop injection. For the flavonoid O-rutinosides, [M − H − 308]⁻ ion was typically observed in the MS² spectrum, suggesting the loss of a rutinose. The fragmentation patterns of flavonoid O-neohesperidosides were more complicated in comparison with their rutinoside analogues. A major difference was found in the [M − H − 120]⁻ ion in the MS² spectrum, which was a common feature of all the flavonoid O-neohesperidosides. The previous literature for naringin located the loss of 120 Da to the glycan part, whereas the present study for naringin had shown that the [M − H − 120]⁻ ion was produced by a retro-Diels-Alder reaction in ring C, and this fragmentation pattern was confirmed by the accurate mass measurement using an orthogonal time-of-flight mass spectrometer. Combined with high performance liquid chromatography (HPLC) and diode array detection (DAD), the established approach to the structural identification of flavonoid O-diglycosides by ion trap mass spectrometry was applied to the analysis of extracts of two Chinese medicines derived from genus Citrus, namely Fructus aurantii and F. aurantii immaturus. According to the HPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by multistage mass spectrometry (MSⁿ) spectra, 13 flavonoid O-glycosides in F. aurantii and 12 flavonoid O-glycosides in F. a. immaturus were identified rapidly.     

Structural characterization and identification of dibenzocyclooctadiene lignans in Fructus Schisandrae using electrospray ionization ion trap multiple-stage tandem mass spectrometry and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry

The electrospray ionization ion trap multiple-stage tandem mass spectrometry (ESI-MSⁿ) and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-ICR-MSⁿ) have been applied successfully to the direct investigation of a number of dibenzocyclooctadiene lignan constituents from the methanol extracts of the Fructus Schisandrae in the positive ion mode. The detailed structural characterization of the same skeleton and different peripheral substituents had been studied and the precise elemental compositions of ions at high mass resolution had been obtained. So the fragmentation mechanisms could be clarified. And the lignan components in Schisandra chinensis (Turcz.) Baill. fruits (SCF) and Schisandra sphenanthera Rehd. et Wils. fruits (SSF) were identified by comparing the structural information and fragmentation mechanisms. Then a pair of isobaric compounds was differentiated. Meanwhile these two similar fruits were distinguished. The research results demonstrated that ESI-MSⁿ technique is a sensitive, selective and effective tool for the direct analysis and rapid determination of constituents in complex mixtures from nature products. And these should be useful for the identification of similar compounds and differentiation of similar species from Chinese herbs.     

Rapid authentication of Gastrodiae rhizoma by direct ionization mass spectrometry

In this study, direct ionization mass spectrometry (DI-MS) for rapid authentication of Gastrodiae rhizoma (known as Tianma in Chinese), a popular herbal medicine, has been developed. This method is rapid, simple and allows direct generation of characteristic mass spectra from the raw herbal medicines with the application of some solvents and a high voltage. The acquired DI-MS spectra showed that gastrodin, parishin B/parishin C and parishin, the major active components of Gastrodiae rhizoma, could be found only in genuine Gastrodiae rhizoma samples, but not in counterfeit samples, thus allowing rapid authentication of Gastrodiae rhizoma. Moreover, wild and cultivated Gastrodiae rhizoma could be classified and Gastrodiae rhizoma from different geographical locations could be differentiated based on their different intensity ratios of characteristic ions or principal component analysis (PCA). This method is simple, rapid, reproducible, and can be extended to analyze other herbal medicines.     

Characterization of peptides by liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a post-column complexant

Two model peptides, des-Arg1-bradykinin (DAB) and bradykinin (B), were cationized by Ag+ after their separation by reversed-phase liquid chromatography (RPLC) prior to mass spectrometry (MS). Silver nitrate solution was used as a post-column reagent. The RPLC and MS experimental conditions were optimized using flow injection in order to obtain sufficiently abundant silver adducts to permit MS/MS experiments. The use of water-methanol with 0.1% formic acid as mobile phase allowed a good chromatographic separation of the two peptides with a polymeric stationary phase and sufficiently abundant silver-containing adducts, [M + Ag + H]2+ and [M + 2Ag]2+. The gas-phase dissociation of [DAB + Ag + H]2+ and [DAB + 2Ag]2+ led to interpretable mass spectra during the on-line cationization experiment. Most of the ions obtained by dissociating [DAB + Ag + H]2+ and [DAB + 2Ag]2+ species are silver-containing ions but the ions produced depend on the parent. The ions coming from the dissociation of the doubly charged silver adducts [DAB + Ag + H]2+ or [DAB + 2Ag]2+ are of interest compared with those coming from the singly charged silver species or doubly charged protonated species. The fragmentation of the doubly charged silver adducts provides ions over the entire mass range. Although the presence of several prolines in des-Arg1-bradykinin prevents the formation of some expected ions, the observation of triplets [an-H + Ag]+, [bn-H + Ag]+ and [bn + OH + Ag]+ produced by the dissociation of on-line Ag(+)-cationized peptides could contribute to greater success of automatic sequencing of peptides.     

Direct analysis of herbal powders by pipette-tip electrospray ionization mass spectrometry

Conventional electrospray ionization mass spectrometry (ESI-MS) is widely used for analysis of solution samples. The development of solid-substrate ESI-MS allows direct ionization analysis of bulky solid samples. In this study, we developed pipette-tip ESI-MS, a technique that combines pipette tips with syringe and syringe pump, for direct analysis of herbal powders, another common form of samples. We demonstrated that various herbal powder samples, including herbal medicines and food samples, could be readily online extracted and analyzed using this technique. Various powder samples, such as Rhizoma coptidis, lotus plumule, great burdock achene, black pepper, Panax ginseng, roasted coffee beans, Fructus Schisandrae Chinensis and Fructus Schisandrae Sphenantherae, were analyzed using pipette-tip ESI-MS and quality mass spectra with stable and durable signals could be obtained. Both positive and negative ion modes were attempted and various compounds including amino acids, oligosaccharides, glycosides, alkaloids, organic acids, ginosensides, flavonoids and lignans could be detected. Principal component analysis (PCA) based on the acquired mass spectra allowed rapid differentiation of closely related herbal species.     

Separation of phospholipid classes by hydrophilic interaction chromatography detected by electrospray ionization mass spectrometry

A new isocratic separation method was developed for separation of phospholipid (PL) classes based on a silica hydrophilic interaction liquid chromatography (HILIC) column with electrospray ionization (ESI) mass spectrometric detection. Although HILIC is typically used for polar compounds, also amphiphilic molecules like phospholipids can be separated very well. Compared to normal-phase (NP) chromatography, which is usually used for PL class separation, HILIC has the advantage to use on-line ESI-MS detection because its eluents are ESI compatible. Furthermore, this HILIC method is isocratic and hence less time consuming than most (gradient) NP HPLC methods. A chromatographic baseline separation of a standard mixture containing phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM) and lysophosphatidylcholine (LPC) was achieved within a total run time of 17 min using a mobile phase consisting of acetonitrile, methanol and ammonium acetate 10 mM. The new method was subsequently tested on phospholipid fractions of a body fluid (human blood plasma) and a tissue extract (swine brain) whereby it achieved nearly the same baseline separation of the PL classes. The detected classes in both cases were PE, PC, SM and LPC.     

Simple and selective determination of arsenite and arsenate by electrospray ionization mass spectrometry

Arsenic pollution of public water supplies has been reported in various regions of the world. Recently, some cancer patients are treated with arsenite (As^III); most Japanese people consume seafoods containing large amounts of negligibly toxic arsenic compounds. Some of these arsenic species are metabolized, but some remain intact. For the determination of toxic As^III, a simple, rapid and sensitive method has been developed using electrospray ionization mass spectrometry (ESI-MS). As^III was reacted with a chelating agent, pyrrolidinedithiocarbamate (PDC, C₄H₈NCSS^-) and tripyrrolidinedithiocarbamate-arsine, As(PDC)₃, extracted with methyl isobutyl ketone (MIBK). A 1 μL aliquot of MIBK layer was directly injected into ESI-MS instrument without chromatographic separation, and was detected within 1 min. Arsenate (As^V) was reduced to As^III with thiosulfate, and then the total inorganic As was quantified as As^III. This method was validated for the analysis of urine samples. The limit of detection of As was 0.22 μg L⁻¹ using 10 μL of sample solution, and it is far below the permissible limit of As in drinking water, 10 μg L⁻¹, recommended by the WHO. Results were obtained in < 10 min with a linear calibration range of 1-100 μg L⁻¹. Several organic arsenic compounds in urine did not interfere with As^III detection, and the inorganic As in the reference materials SRM 2670a and 1643e were quantified after the reduction of As^V to As^III.     

合成产物PEPAMP及其副产物的电喷雾串联质谱分析

PEPAMP是应用于工业循环水中的新一代高效阻垢剂.采用电喷雾电离质谱和质谱/质谱技术分析了合成产物,注射泵直接进样,正离子方式检测,方法简便、快速.通过EI-MS技术获得了目标产物PEPAMP及主要副产物的准分子离子峰,通过ESI-MS-MS技术获得了其碎片信息,并对目标化合物及主要副产物进行了精确质量测定,确定了目标化合物及主要副产物的结构.     

Ambient imaging mass spectrometry by electrospray ionization using solid needle as sampling probe

Although being an atmospheric pressure ion source, electrospray ionization (ESI) has rarely been used directly for ambient imaging mass spectrometry because the sample has to be introduced as liquid solution through the capillary. Instead of capillary, probe electrospray ionization (PESI), which has been developed recently, uses a solid needle as the sampling probe, as well as the electrospray emitter, and has been applied not only for liquid solutions but also for the direct sampling on wet samples. Biological tissues are composed of cells that contain 70–90% water, and when the surface is probed by the needle tip, the biological fluid adhering to the needle can be electrosprayed directly or assisted by additional solvent added onto the needle surface. Here, we demonstrate ambient imaging mass spectrometry of mouse brain section using PESI, incorporated with an auxiliary heated capillary sprayer. The solvent vapor generated from the sprayer condensed on the needle tip, re‐dissolving the adhered sample, and at the same time, providing an indirect means for needle cleaning. The histological sections were prepared by fixation using paraformaldehyde, and the spatial analysis was automated by maintaining an equal sampling depth into the sample in addition to raster scan. Phospholipids and galactosylceramides were readily detected from the mouse brain section in the positive ion mode, and were mapped with 60 µm lateral resolution to form mass spectrometric images. Copyright © 2009 John Wiley & Sons, Ltd.     

High-throughput detection of drugs binding to proteins using desorption electrospray ionization mass spectrometry

In this paper, we present a strategy for screening drugs that bind to proteins by combining centrifugal filtration with desorption electrospray ionization mass spectrometry (DESI-MS). Membrane filtration was used to remove any unbound drugs. Then, drug–protein complexes deposited on the DESI substrate were dissociated during the DESI-MS analytical process, and the liberated drugs were measured. To validate the methodology, the screening of a series of drugs against two types of proteins was performed. Three DNA topoisomerase I (Topo I) inhibitors (camptothecin (CPT), hydroxycamptothecin (OHCPT) and 7-ethyl-10-hydroxycamptothecin (SN-38)) were screened against Topo I and the DNA-Topo I complex using DESI-MS. The results indicated that none of the inhibitors bound to Topo I, because the inhibitors had binding affinities only to the DNA-Topo I complex. Among the three drugs that bound to the DNA-Topo I complex, SN-38 had the strongest relative binding affinity, and CPT had the weakest relative binding affinity. The impact of the DESI spray solvent composition on the analysis of drug–protein complex binding was evaluated. Seven alkaloid drugs were also screened against Topo I using DESI-MS. Berberine and palmatine had good binding affinities. A screening of 21 types of drugs was carried out to determine whether the drugs bound to human serum albumin (HSA). The DESI-MS screening process could be achieved within 1.75 min. The study provides a method to qualitatively detect compounds that bind to proteins, showing great potential in drug design and screening.     

Metabolic profiling of Actaea species extracts using high performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry

Despite persistent questions about the safety of black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa L.), its products continue to be one of the most popular botanical supplements in the United States market. Black cohosh products have been associated with cases of liver toxicity, but subsequent evaluation found some products to be adulterated with other related plants from the same genus. US FDA regulations require that black cohosh products be unadulterated, and correct identification of different species of Actaea is a key first step for their good manufacturing practice. We have developed a phytochemical method to distinguish four different groups of Actaea, including: species other than A. racemosa, Asian species, A. racemosa, and North American species other than A. racemosa. Using HPLC-TOF-ESI-MS technique and principal component analysis, we identified 15 chemical markers (1-3, 5-6, 8-10, 12, 16-21). Three marker compounds were unambiguously identified using authentic standards, and 12 marker compounds were tentatively identified by comparison of fragmentation patterns with previously reported data. The presence of these marker compounds is critical for discrimination among the four groups of closely related plants. The use of metabolic profiling to distinguish black cohosh from related species of Actaea has broader implications in the identification of markers to help authenticate other important medicinal plants.     

Hydrolysis products of water treatment chemical aluminium sulfate octadecahydrate by electrospray ionization mass spectrometry

Hydrolysis and speciation of aluminium sulfate octadecahydrate Al₂(SO₄₎₃·18H₂O${\text{Al}}_2({\text{SO}}_4{)}_3·18{\text{H}}_2\text{O}$ was studied by electrospray time of flight mass spectrometry (ESI TOF MS). Several novel polymeric species were determined. Highly charged polymers, characterized by other methods, such as the Keggin cation [Al₁₃O₄(OH)₂₄(H₂O)₁₂]⁷⁺ and the octameric aluminium hydroxide cluster [Al₈(OH)₁₄(H₂O)₁₈](SO₄)₅ 16H₂O, were found using ESI-MS as the anions [Al₁₃O₄(OH)₂₅(SO₄)₄]²⁻ and [Al₈O(OH)₁₄(SO₄)₅(H₂O)₄]²⁻. All the main species identified contained sulfate or hydrogen sulfate. The compositions of the determined ions mimicked those of several stable mineral forms.     

Factors that affect molecular weight distribution of Suwannee river fulvic acid as determined by electrospray ionization/mass spectrometry

Effects of methylation, molar response, multiple charging, solvents, and positive and negative ionization on molecular weight distributions of aquatic fulvic acid were investigated by electrospray ionization/mass spectrometry. After preliminary analysis by positive and negative modes, samples and mixtures of standards were derivatized by methylation to minimize ionization sites and reanalyzed.Positive ionization was less effective and produced more complex spectra than negative ionization. Ionization in methanol/water produced greater response than in acetonitrile/water. Molar response varied widely for the selected free acid standards when analyzed individually and in a mixture, but after methylation this range decreased. After methylation, the number average molecular weight of the Suwannee River fulvic acid remained the same while the weight average molecular weight decreased. These differences are probably indicative of disaggregation of large aggregated ions during methylation. Since the weight average molecular weight decreased, it is likely that aggregate formation in the fulvic acid was present prior to derivatization, rather than multiple charging in the mass spectra.     

Structural characterization of alachlor complexes with transition metal ions by electrospray ionization tandem mass spectrometry

Electrospray ionization mass (ESI-MS) spectrometry was used to investigate the nature of metal complexes of alachlor and their dissociations on activation. Ions of the first row transition metal series were employed to react with alachlor and the products were subjected to collision-induced dissociation (CID) for further structural characterization. The formation of diverse complex ions including doubly charged metal/alachlor complexes; [3L + M]²⁺ and [4L + M]²⁺ (L: alachlor and M: transition metal ions) were observed depending on the experimental conditions including the tube lens offset voltage (TLOV) and relative concentrations of alachlor and transition metal ions. It is clear that complexation with transition metal ions alters the reactive site of alachlor, promoting the loss of chlorine over the loss of CH₃OH that is the major reaction pathway in uncomplexed system. Direct elimination of chlorine from alachlor molecule was confirmed by the use of MnBr₂ instead of MnCl₂. These evidences clearly illustrate the catalytic activities of the metal ions through insertion mechanism. The function of transition metal ions in complexation was emphasized comparing the fragmentation patterns with those of protonated molecule. A change in the oxidation state of copper from + 2 to + 1 during the dissociation of metal complex was observed in company with elimination of radicals which is specific for the copper complex ions.     

Probing of arginine residues in peptides and proteins using selective tagging and electrospray ionization mass spectrometry

A general labelling method is presented which allows the determination of the number of guanidine groups (related to arginine and homoarginine in peptides and proteins) by means of mass spectrometry. It implies a guanidine-selective derivatization step with 2,3-butanedione and an arylboronic acid under aqueous, alkaline conditions (pH 8-10). The reaction mixture is then directly analysed by electrospray ionization mass spectrometry without further sample pretreatment. Other amino acids are not affected by this reaction although it is demonstrated that lysine side-chains may be unambiguously identified when they are converted to homoarginine prior to derivatization. Guanidine functionalities, as e.g. in the amino acid arginine, are easily identified by the characteristic mass shift between underivatized and derivatized analyte. The tagging procedure is straightforward and selective for guanidine groups. The influence of several experimental parameters, especially the pH of the solution and the choice of reagents, is examined and the method is applied to various arginine-containing peptides and to lysozyme as a representative protein. Possible applications of this technique and its limitations are discussed.     

Simultaneous measurement of proline and related compounds in oak leaves by high-performance ligand-exchange chromatography and electrospray ionization mass spectrometry for environmental stress studies

A mass spectrometer was coupled to high-performance ligand-exchange liquid chromatography (HPLEC) for simultaneous analysis of stress associated solutes such as proline, hydroxyproline, methylproline, glycine betaine and trigonelline extracted from leaves of drought stressed oaks and an internal standard namely N-acetylproline. Methanol/chloroform/water extracts were analyzed using an Aminex HPX-87C column and specifically quantified by the positive ion mode of an electrospray ionisation-mass spectrometry (ESI-MS) in single ion monitoring (SIM) mode. The recovery of N-acetyl proline added to oak leaf extracts ranged from 85.2 to 122.1% for an intra-day study. Standard calibration curves showed good linearity in the measured range from 0.3125 to 10 μmol L⁻¹ with the lowest correlation coefficient of 0.99961 for trigonelline. The advantages of this alternative procedure, compared to previously published methods using fluorescence or amperometric detections, are the simultaneous and direct detection of osmoprotectants in a single chromatographic run, a minimal sample preparation, a good specificity and reduced limits of quantification, ranging from 0.1 to 0.6 μmol L⁻¹. Fifty-six days of water deficit exposure resulted in increased foliar free proline levels (2.4-fold, P < 0.001, 155 μmol g⁻¹ FW) and glycine betaine contents (2.5-fold, P < 0.05, 175 μmol g⁻¹ FW) of drought stressed oak compared to control.     

Field-induced wooden-tip electrospray ionization mass spectrometry for high-throughput analysis of herbal medicines

This study demonstrates the first application of field-induced wooden-tip electrospray ionization (ESI) mass spectrometry (MS) for high-throughput analysis of herbal medicines. By application of an opposite and sample-contactless high voltage on the MS inlet rather than wooden tips, a high-throughput analysis device is easily set up, and a relatively fast analysis speed of 6 s per sample was successfully achieved. In addition, fast polarity switching between positive and negative ion detection mode is readily accomplished, which provides more complete chemical information for quality assessment and control of herbal medicines. By using the proposed method, various active ingredients present in different herbal medicines were rapidly detected, and the obtained mass spectra were served as the samples' fingerprints for tracing the origins, establishing the authenticity, and assessing the quality consistency and stability of herbal medicines. Our experimental results demonstrated that field-induced wooden-tip ESI-MS is a desirable method for high-throughput analysis of herbal medicines, with promising prospects for rapidly differentiating the origin, determining the authenticity, and assessing the overall quality of pharmaceuticals.     

Analysis of microcystins by capillary zone electrophoresis coupling with electrospray ionization mass spectrometry

In this paper, a rapid and effective method based on capillary zone electrophoresis (CZE) coupled with electrospray ionization mass spectrometry (ESI-MS) was established for the trace analysis of microcystin (MC) isomers in crude algae sample. The experimental conditions including the composition, acidity and concentration of buffer, separation voltage, injection time, and MS detection parameters were investigated in detail. A capillary separation system was as follows: a uncoated fused-silica capillary tube (50 μm i.d. × 90 cm), 40 mmol L⁻¹ ammonium acetate solution (pH 9.86) as running buffer, 25 kV as separation voltage, 20 kV × 3 s water first and 20 kV × 20 s for sample injection. Mass analysis was performed in ESI source, with sheath gas temperature 150 °C, sheath gas pressure 10 psi, and sheath gas flow 6 L min⁻¹. And sheath liquid was 7.5 mmol L⁻¹ acetic acid in 50% isopropanol-water (3 μL min⁻¹). Protonation and ammonium adduct molecular ions m/z 506.9 (MC-LR) and 532.0 (MC-YR) were used for the quantification of MCs. Under these conditions, two MCs were baseline separated within 9 min, the calibration curves were obtained in the range of 0.11-10.0 μg mL⁻¹ and 0.16-10.5 μg mL⁻¹ for MC-LR and MC-YR, respectively. Meanwhile, limits of detection were 0.05 and 0.08 μg mL⁻¹ for MC-LR and MC-YR, respectively. The recoveries for the two MCs were in the range of 95.8-108%. The developed approach had been successfully applied to the analysis of MCs in crude algae samples.     

Probing the mechanism of direct Mannich-type alpha-methylenation of ketoesters via electrospray ionization mass spectrometry

Reactions promoting direct Mannich-type alpha-methylenation of alpha, beta and gamma-ketoesters have been monitored via electrospray ionization mass and tandem mass spectrometric experiments. Key intermediates of the catalytical cycle of this synthetically useful reaction have been intercepted and characterized. The mechanistic information provided by electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS) guided the optimization of reaction conditions, allowing alpha-methyleneketoesters to be prepared in high yields (80-95%) and in high-enough purity for immediate further manipulation.