Molecular structure of fulvic acids by electrospray with quadrupole time-of-flight mass spectrometry

Characterisation of the molecular structure of aquatic fulvic acids (FA) has been performed using a quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an electrospray ionisation interface. Molecular masses centred around 450 Da and sinusoidal spectral distributions have been obtained for all fulvic acids. Tandem mass spectrometry (MS/MS) experiments showed losses of 18 Da (H(2)O) and 44 Da (CO(2)), and possible molecular structures were determined for the first time to our knowledge. A methodology is reported for evaluating the average elemental composition of FA from high-resolution mass spectra by processing post-acquisition data calculations using molecular size distributions and atomic compositions of ions. The results are found to be consistent with elemental analysis data.     

On-line size-exclusion chromatography/electrospray ionisation mass spectrometry of aquatic humic and fulvic acids

Isolated aquatic humic and fulvic acids were analysed with on-line size exclusion chromatography/electrospray ionisation mass spectrometry (SEC/ESI-MS). An eluent composition which enabled electrospray ionisation was identified. The SEC separation improved interpretability of mass spectra and may open up new possibilities for molecular weight determination of humic substances. A linear dose-response relationship over a factor of 20 was obtained and the limit of detection was 50ng/uL for humic and fulvic acids. Spectral changes due to different ionisation conditions (pH and cone voltage) were investigated. A natural water sample from a Swedish lake was analysed. Copyright 2000 John Wiley & Sons, Ltd.     

Enzymatic strategies for the characterization of nucleic acids by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is a powerful technique used for the identification and characterization of DNA polymorphisms. Continual improvement in instrument design assures high mass measurement accuracy, sensitivity, and resolving power. This work describes an eclectic array of enzymatic strategies we have invoked in order to detect single-nucleotide polymorphisms by ESI-MS, although other applications may be envisioned. One strategy combines the use of two enzymes, exonuclease III and lambda exonuclease, to provide a ladder of single-stranded DNA fragments for straightforward sequence identification by mass spectrometry. A second strategy combines restriction enzymes to screen for polymorphisms present within specific amplicons. Finally, we describe the use of stable-isotope-labeled nucleotides for the determination of length and base composition of a PCR product.     

Characterization of fulvic acids by liquid chromatography-quadrupole time-of-flight mass spectrometry

Fulvic acid standards from Suwannee River, Pony Lake, Elliot Soil, Waskish Peat, and Nordic Reservoir were characterized by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) operating in negative electrospray ionization mode. The method employed a commercially available stationary phase that resulted in a distinctive chromatographic peak for each of the fulvic acid samples that differed in width and retention time at peak maximum. The QTOF-MS, operating in TOF mode, revealed that the unique chromatographic peak shapes were the result of the relative fraction of hydrogen and oxygen contained in various fulvic acid components. Those species that contained larger amount of hydrogen displayed a larger mass defect and were retained longer on the LC column, indicating reduced polarity. This is supported by a reduction in the degree of fragmentation related to polar functional groups as the mass defect and retention time increased. Lastly, the analysis of even and odd mass (at m/z 1 greater) ion intensity ratios revealed a correlation to the percent nitrogen of the various standards.     

Identification of compound classes in soil and peat fulvic acids as observed by electrospray ionization tandem mass spectrometry

Soil and peat fulvic acids obtained from the International Humic Substances Society were fractionated by their solubility in methanol and analyzed by electrospray ionization tandem mass spectrometry. Precursor and product ion experiments produced mass spectra that indicated the presence of benzene, phenol, dihydroxy benzene, furan and thiophene carboxylic acids. Standards were used to substantiate the fragmentation patterns observed in the product ion spectra of the fulvic acid samples. This study makes significant progress into the direct identification of individual compounds in humic substances using a non-degradation technique.     

Induced in-source fragmentation for the selective detection of organic bound iodine by liquid chromatography/electrospray mass spectrometry

The detection of unknown organic compounds, e.g., transformation products of known trace pollutants in environmental samples, is a difficult task which can be simplified if the compounds of interest are characterized by a functional group or a heteroatom which can selectively be detected. For the detection of iodinated benzene derivatives, used as X-ray contrast media, we have studied whether such compounds can be selectively detected by negative ion (NI) electrospray ionization via iodide as a product ion of in-source fragmentation, generated by applying a high cone voltage. It was found that selective detection of iodinated benzene derivatives is possible by liquid chromatography/negative ion electrospray ionization (LC/NI-ESI) if the compounds are destroyed before entering the mass spectrometer. To our knowledge this is the first report of the use of NI in-source fragmentation MS for analyses traditionally performed with inductively coupled plasma (ICP)-MS. The novel application of NI in-source fragmentation MS is a tool for those who do not possess the facilities to perform ICP-MS. Furthermore, NI-ESI at varying cone voltages provides both structural information and elemental detection, whereas ICP-MS can only provide elemental detection.     

Structural characterization of glycoporphyrins by electrospray tandem mass spectrometry

Porphyrin derivatives having a galactose or a bis(isopropylidene)galactose structural unit, linked by ester or ether bonds, were characterized by electrospray tandem mass spectrometry (ES-MS/MS). The electrospray mass spectra of these glycoporphyrins show the corresponding [M + H](+) ions. For the glycoporphyrins with pyridyl substituents and those having a tetrafluorophenyl spacer, the doubly charged ions [M + 2H](2+) were also observed in ES-MS with high relative abundance. The fragmentation of both [M + H](+) and [M + 2H](2+) ions exhibited common fragmentation pathways for porphyrins with the same sugar residue, independently of the porphyrin structural unit and type of linkage. ES-MS/MS of the [M + H](+) ions of the galactose-substituted porphyrins gave the fragment ions [M + H - C(2)H(4)O(2)](+), [M + H - C(3)H(6)O(3)](+), [M + H - C(4)H(8)O(4)](+) and [M + H - galactose residue](+). The fragmentation of the [M + 2H](2+) ions of the porphyrins with galactose shows the common doubly charged fragment ions [porphyrin + H](2+), [M + 2H - C(2)H(4)O(2)](2+), [M + 2H - C(4)H(8)O(4)](2+), [M + 2H - galactose residue](2+) and the singly charged fragment ions [M + H - C(3)H(6)O(3)](+) and [M + H - galactose residue](+). The fragmentation of the [M + H](+) ions of glycoporphyrins with a protected galactosyl residue leads mainly to the ions [M + H - CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2) - CO](+), [M + H - C(10)H(16)O(4)](+) and [M + H - protected galactose](+). The doubly charged ions [M + 2H](2+) fragment to give the doubly charged ions [porphyrin + H](2+) and the singly charged ions [M + H - protected galactose residue](+) and [M + H - CO(CH(3))(2)](+). For the porphyrins where the sugar structural unit is linked by an ester bond, [M + 2H](2+), ES-MS/MS showed a major and typical fragmentation corresponding to combined loss of a sugar structural unit and further loss of water, leading to the ion [M + 2H - sugar residue - H(2)O](2+), independently of the structure of the sugar structural unit. These results show that ES-MS/MS can be a powerful tool for the characterization of the sugar structural unit of glycoporphyrins, without the need for chemical hydrolysis.     

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.     

Relative basicities toward metal triflates Lewis acids by electrospray mass spectrometry

The relative Lewis basicity of a series of phosphoryl compounds toward cations M(n+)(OTf)(n-1) derived from triflate salts, M(OTf)(n), is ranked using electrospray mass spectrometry according to competitive adduct formation.     

Self-esterification of fulvic acid model compounds in methanolic solvents as observed by electrospray ionization mass spectrometry

The self-esterification of two fulvic acid model compounds in methanolic solvents was studied by electrospray ionization mass spectrometry (ESI-MS). The strongly acidic tetrahydrofurantetracarboxylic acid rapidly self-esterified to form mono- and dimethyl esters when stored in methanol, even at reduced temperatures. The weakly acidic analogue, cyclopentanetetracarboxylic acid, reacted minimally under the same conditions. The use of 50:50 methanol/water as a solvent reduced self-esterification of the strong acid. However, the presence of water promoted the formation of multiply charged ions in the ESI mass spectra. The use of water and 50:50 acetonitrile/water as solvents eliminated self-esterification but the mass spectra still contained multiply charged ions. This study implies that the use of methanolic solvents with humic substances may compromise analytical data through the formation of methyl esters.     

Structural characterization of fatty acids cationized with copper by electrospray ionization mass spectrometry under low-energy collision-induced dissociation

Fatty acids have for many years been characterized by mass spectrometry using electron ionization after chemical derivatization. When fatty acids are ionized using desorption/ionization methods such as electrospray ionization or fast atom bombardment, structural information is usually obtained through high-energy collision-induced dissociation (CID) using sector instruments. It has been shown that copper displays very interesting properties in the gas phase during CID. In this study, the reactivity of saturated and unsaturated fatty acid-copper [M-H+Cu(II)]+ complex and the role of the copper ion in promoting fragmentations were investigated under low-energy collisional activation conditions. The decomposition of these species in an ion trap instrument led to diagnostic ion series that reflect C--C bond cleavage, which involves Cu(II) reduction followed by the release of an alkyl radical. It was demonstrated that in this way the localization of one or two homoconjugated double bonds is possible using low-energy CID. Moreover, the distinction of cis and trans isomers is possible through characteristic product ions related to a specific loss of CO2. When these experiments are repeated using a triple-quadrupole instrument with argon as collision gas, a different behavior is observed as in this case, in addition to the product ion distributions observed in the ion trap, other distributions are observed that reflect the influence of the different kinetic shifts and the occurrence of consecutive decompositions. Different examples are presented with various saturated and unsaturated fatty acid chains. Mechanisms are proposed in order to rationalize the experimental observations.     

Structural characterization of acetylpyridinium-ethyl pyruvate adducts by electrospray ionization mass spectrometry

The reactions of ethyl pyruvate with acetic anhydride and pyridine were studied by electrospray ionization mass spectrometry (ESI-MS). Ethyl 2-acetoxy-2-pyridiniumpropionate (1) (m/z 238) resulting from the reaction of the acetylpyridinium cation with ethyl pyruvate, and the adduct of ethyl 2-acetoxyacrylate with a pyridinium cation (2), bound together by non-covalent interactions (m/z 238), were identified by ESI-MS for the first time. Structures 1 and 2 cannot be distinguished, probably because one may be converted into the other and vice versa.     

Identification of self-assembly products from N-phosphoamino acids by electrospray ionization mass spectrometry

The self-assembly products of N-(O,O'-diisopropyl)-L-alpha-amino acids in aqueous media, N-(O,O'-diisopropyl)phosphoryl dipeptides and tripeptides, were identified by electrospray ionization mass spectrometry. A stepwise fragmentation of the [M + H](+) ions from the C-terminus of N-phosphopeptides was observed. Copyright 2000 John Wiley & Sons, Ltd.     

Characterization of synthetic nucleic acids by electrospray ionization quadrupole time-of-flight mass spectrometry

The potential of electrospray ionization quadrupole-quadrupole-time-of-flight mass spectrometry (ESI-QqTOF-MS) for the characterization of synthetic nucleic acids was evaluated. Oligonucleotides ranging in size from 12 up to 51 nucleotides were analyzed via direct infusion MS as well as via liquid chromatography (LC) online hyphenated to MS. These experiments proved the outstanding mass spectrometric performance of the TOF mass analyzer in regard of accuracy, reproducibility, resolution, and sensitivity. During a 1-min run, the monoisotopic mass of (dT)(24) was measured with a maximum relative mass deviation of 7.64 ppm proving the high mass accuracy of the TOF analyzer. Over a period of 1 h, mean deviations were determined in the range between -3.58 ppm and 3.06 ppm demonstrating the high stability of the applied external calibration. The molecular mass of a 51-mer was measured with a deviation smaller than 3.23 ppm from the theoretical value. The resolution exceeded a value of m/Deltam = 20 000 (m is the measured mass and Deltam the full peak width at half-maximum), which enabled the separation of the isotopic peaks of all investigated oligonucleotides. Because of the outstanding transmission and detection efficiency of the TOF mass analyzer, detection limits in the amol/microl to low fmol/microl range were reached. The usability of LC-ESI-QqTOF-MS for the qualitative and quantitative analysis of synthetic oligonucleotide mixtures was demonstrated.     

Determination of molecular formulas and structural regularities of low molecular weight fulvic acids by size-exclusion chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry

Size-exclusion chromatography was coupled to a quadrupole time-of-flight mass spectrometer using electrospray ionization in the negative ion mode to analyze Suwannee River fulvic acid. About 220 exact masses of low molecular weight fulvic acids (FAs) were measured in the range from m/z 190 to 350, and molecular formulas could be deduced for all these molecular masses. All molecular formulas can be described by the superimposition of three homologous series in the FA mixture: (a) a series of hydrogen homologs constituted of five to six members for a given number of carbons and oxygens; (b) a series of alkyl chain homologs with stepwise addition of one methylene group; and (c) isobaric compounds that differ by the formal replacement of an oxygen atom by a methane group. Product ion spectra of the FA species reflect the repeated parallel losses of carbon dioxide and water. The minimum number of carboxylate groups and the maximum number of hydroxy moieties could be determined from the product ion spectra. Furthermore, it was obvious that the structural differences between homologs in the three series are located in the carbon backbone of the FA rather than in its extremities that are expelled as neutral fragments. These structural regularities reduce the complexity of FA mixtures to a certain set of yet unknown basic structures.     

Analysis of amino acids as formamidene butyl esters by electrospray ionization tandem mass spectrometry.

Amino acid formamidene butyl esters are optimally prepared by heating amino acids with dimethylformamide dimethylacetal (DMF-DMA) for 2 minutes at 65 degrees C and then with n-butanol/hydrogen chloride for 15 minutes at 65 degrees C. The formamidene butyl esters of simple alpha-amino acids and beta-amino acids afford between 1.1 and 20 times the signal intensity of the corresponding butyl esters during electrospray ionization tandem mass spectral analysis. Complex alpha-amino acids, such as ornithine, arginine and citrulline, and gamma-amino acids are better analyzed as butyl esters. Secondary alpha-amino acids, such as proline and sarcosine, give a mixture of two iminium salts with the DMF-DMA derivatization method. A derivative incorporating two molecules of the amino acid predominates at higher derivatization temperatures. Contrary to a previous report, N-formylamino acids were not detected. The presence of secondary amino acids in amino acid mixtures, derivatized as formamidene butyl esters, affords derivatives that incorporate two different amino acids. The new formamidene butylation method is unlikely to replace the butylation procedure used by neonatal blood spot screening programs for amino acid disorders, since a much poorer response was obtained with formamidene butyl esters of arginine and citrulline, important in the diagnosis of arginase deficiency and citrullinaemia.     

Direct detection of chloramphenicol in honey by neutral desorption-extractive electrospray ionization mass spectrometry

Herein, we constructed a platform of neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) for direct and rapid detection of chloramphenicol (CAP) in honey samples diluted with methanol. Under the optimized working conditions, the quantitative information of CAP residues was acquired effectively by EESI-Ion Trap MS ⁿ . Using heated methanol-N₂ as spray reagent, we reduced the limit of determination (LOD) from 73.3 ng/mL to 0.3 ng/mL, and the CAP detection is linear in the range of 1–5000 ng/mL (R?=?0.9947). For the honey samples with CAP of 10, 100, and 1000 ng/mL, the recoveries were 133.0, 80.6, and 101.1 %, and the relative standard deviations were 5.96, 8.82, and 8.71 %, respectively. The reproducibility assays showed the stability of this method. Therefore, this ND-EESI-MS method is powerful for direct, rapid, and quantitative CAP analysis in honey samples with high sensitivity, precision, and specificity. Graphical Abstract

In the current neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) method, N₂ is inlet into samples to desorb chloramphenicol (CAP). We tried to use some organic solvents as the spray reagent to dissolve CAP, and then the best neutral desorption efficiency was obtained when using methanol. We applied this modified ND-EESI-MS method to detect CAP in honey samples only with sample dilution. The limit of CAP detection was then reduced from 73.3 to 0.3 ng/mL, reaching the current EU standard. Therefore, this is a powerful method for direct, rapid, and quantitative CAP analysis in honey samples.     

直接电喷雾离子阱质谱法快速表征烟叶

建立了一种快速、简单的同时检测烟叶中多酚类和非挥发性有机酸的电喷雾离子阱质谱方法。该方法采用水溶液萃取烟叶,萃取液无需色谱柱分离提纯,直接进入质谱检测,得到烟草质谱轮廓图。采用该方法对25种不同产地、不同等级的烟叶样品进行了测量,并结合模式识别的方法,对这25种烟叶进行了聚类分析,其结果基本符合烟草专家的等级划分。最后对各类烟叶进行了统计分析,得到了表征各类烟叶的平均质谱轮廓和各特征组分对区分烟叶贡献大小。表明该方法可以用于辅助烟叶等级划分及卷烟配方设计。     

Sensitivity improvement of amino acids by N-terminal diisopropyloxyphosphorylation in electrospray ionization mass spectrometry

With the introduction of a diisopropyloxyphosphoryl (DIPP) group at the N-terminus of amino acids, the signal response in electrospray ionization mass spectrometry can be enhanced by 10-100-fold in positive mode and even more in negative mode. An enhancement of proton affinity due to the N-terminal DIPP derivatization was considered to be responsible for the sensitivity improvement in positive mode, which was supported by kinetic method studies. Two possible mechanisms, i.e., an increase in gas-phase acidity due to the introduction of the electron-withdrawing DIPP group, and the effect of pH on the formation of anions of amino acids and their phosphorylated derivatives, were considered for the signal response enhancement in negative mode.