Identification of phosphoramide mustard/DNA adducts using tandem mass spectrometry

The reaction pathway of alkylating agents is often exploited in the design of bifunctional anti-cancer drugs. These drugs form mono-DNA adducts as well as inter- and intra-strand cross-linked adducts, notably by reaction at DNA bases, including the N-7-position of guanine (G). A positive-ion fast-atom bombardment (FAB) mass spectrum of an in vitro preparation of DNA alkylated with phosphoramide mustard (the active metabolite of the anti-cancer drug cyclophosphamide) indicated the presence of the two mono-DNA adducts N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl] amine, designated NOR-G, and N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl] amine, designated NOR-G-OH, (MH+ 257/259 and 239, respectively) but not the presence of the cross-linked adduct N,N-bis-[2-(7-guaninyl)ethyl] amine, designated G-NOR-G (MH+ 372). Using synthetic standards, daughter-ion spectra of NOR-G, NOR-G-OH and G-NOR-G were obtained (matrix 0.2 M p-toluene sulphonic acid in glycerol) by positive-ion FAB tandem mass spectrometry (FAB-MS/MS). The daughter-ion spectra of both mono-DNA adducts NOR-G and NOR-G-OH contained a fragment ion at m/z 152 [G + H]+, whereas the cross-linked adduct, G-NOR-G, showed an ion at m/z 221, [MH-G]+. Evidence for the presence of NOR-G, NOR-G-OH and G-NOR-G in the in vitro preparation was obtained by performing a double parent-ion scan on m/z 152 and 221. The presence of G-NOR-G was further supported by performing a single parent-ion scan on m/z 221.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrospray and atmospheric pressure chemical ionization tandem mass spectrometric behavior of eight anabolic steroid glucuronides

Mass spectrometric and tandem mass spectrometric behavior of eight anabolic steroid glucuronides were examined using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in negative and positive ion mode. The objective was to elucidate the most suitable ionization method to produce intense structure specific product ions and to examine the possibilities of distinguishing between isomeric steroid glucuronides. The analytes were glucuronide conjugates of testosterone (TG), epitestosterone (ETG), nandrolone (NG), androsterone (AG), 5alpha-estran-3alpha-ol-17-one (5alpha-NG), 5beta-estran-3alpha-ol-17-one (5beta-NG), 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol (5alpha-MTG), and 17alpha-methyl-5beta-androstane-3alpha,17beta-diol (5beta-MTG), the last four being new compounds synthesized with enzyme-assisted method in our laboratory. High proton affinity of the 4-ene-3-one system in the steroid structure favored the formation of protonated molecule [M + H]+ in positive ion mode mass spectrometry (MS), whereas the steroid glucuronides with lower proton affinities were detected mainly as ammonium adducts [M + NH4]+. The only ion produced in negative ion mode mass spectrometry was a very intense and stable deprotonated molecule [M - H]- . Positive ion ESI and APCI MS/MS spectra showed abundant and structure specific product ions [M + H - Glu]+, [M + H - Glu - H2O]+, and [M + H - Glu - 2H2O]+ of protonated molecules and corresponding ions of the ammonium adduct ions. The ratio of the relative abundances of these ions and the stability of the precursor ion provided distinction of 5alpha-NG and 5beta-NG isomers and TG and ETG isomers. Corresponding diagnostic ions were only minor peaks in negative ion MS/MS spectra. It was shown that positive ion ESI MS/MS is the most promising method for further development of LC-MS methods for anabolic steroid glucuronides.     

Mobile phase variations in thermospray liquid chromatography-mass spectrometry of pesticides

The effect of four different mobile phase compositions with reversed-phase methanol-water (50:50) + 0.05 M ammonium acetate, methanol-water (50:50) + 0.05 M ammonium formate, acetonitrile-water (50:50) + 0.05 M ammonium acetate and acetonitrile-water (50:50) + 0.05 M ammonium formate were compared in filament-on thermospray liquid chromatography-mass spectrometry for the determination of carbamate and chlorotriazine pesticides. In the positive-ion mode, [M + H]+ and [M + NH4]+ were generally the base peaks for the chlorotriazines and the carbamates, respectively. Depending on the mobile phase used, other adduct ions obtained corresponded to [M + CH3CN + H]+, [M + CH3OH + NH4]+, [M + CH3COONH4 + NH4 - 2H2O]+, [M + CH3CN + NH4]+, [M + CH3COONH4 + H - H2O]+ and the dimer [2M + H]+. In the negative-ion mode, [M - H]- and adducts with the ionizing additive [M + CH3COO]- or [M + HCOO]- were obtained. Other ions for the carbamates carbaryl and oxamyl corresponded to [M - CONHCH3 + CH3COOH]- and [M - CON(CH3)2 + HCOO]-, respectively. The variation of mobile phase composition provides additional structural information in thermospray liquid chromatography-mass spectrometry with no appreciable loss of sensitivity. Applications are reported for the determination of carbamate and chlorotriazine pesticides at the ng/g level in spiked and real soil samples, respectively.     

Detection and characterization by mass spectrometry of radical adducts produced by linoleic acid oxidation

The formation of linoleic acid radical species under the oxidative conditions of the Fenton reaction (using hydrogen peroxide and Fe (II)) was monitored by FAB-MS and ES-MS using the spin trap 5,5-dimethyl-1-pyrrolidine-N-oxide, DMPO. Both the FAB and ES mass spectra were very similar and showed the presence of ions corresponding to carbon- and oxygen centered spin adducts (DMPO/L*, DMPO/LO*, and DMPO/LOO*). Cyclic structures, formed between the DMPO oxygen and the neighboring carbon of the fatty acid, were also observed. Electrospray tandem mass spectrometry of these ions was performed to confirm the proposed structure of these adducts. All MS/MS spectra showed an ion at m/z 114, correspondent to the [DMPO + H]+, and a fragment ion due to loss of DMPO (loss of 113 Da), confirming that they are DMPO adducts. ES-MS/MS spectra of alkoxyl radical adducts (DMPO/LO*) showed an additional ion at m/z 130 [DMPO - O + H]+, while ES MS/MS of peroxyl radical adducts (DMPO/LOO*) showed a fragment ion at m/z 146 [DMPO - OO + H]+, confirming both structures. Other fragment ions were observed, such as alkyl acylium radical ions, formed by cleavage of the alkyl chain after loss of water and the DMPO molecule. The identification of fragment ions observed in the MS/MS spectra of the different DMPO adducts suggests the occurrence of structural isomers containing the DMPO moiety both at C9 and C13. The use of ES tandem mass spectrometry, associated with spin trapping experiments, has been shown to be a valuable tool for the structural characterization of carbon and oxygen-centered spin adducts of lipid radicals.     

Collision-activated cleavage of a peptide/antibiotic disulfide linkage: possible evidence for intramolecular disulfide bond rearrangement upon collisional activation

Ceftiofur is an important veterinary beta-lactam antibiotic whose bioactive metabolite, desfuroylceftiofur, has a free thiol group. Desfuroylceftiofur (DFC) was reacted with two peptides, [Arg8]-vasopressin and reduced glutathione, both of which have cysteine residues to form disulfide-linked peptide/antibiotic complexes. The products of the reaction, [vasopressin + (DFC-H) + (DFC-H) + H]+, [(vasopressin+H) + (DFC-H) + H]+ and [(glutathione-H) + (DFC-H) + H]+, were analyzed using collision-activated dissociation (CAD) with a quadrupole ion trap tandem mass spectrometer. MS/MS of [vasopressin + (DFC-H) + (DFC-H) + H]+ resulted in facile dissociative loss of one and two covalently bound DFC moieties. Loss of one DFC resulted from either homolytic or heterolytic dissociation of the peptide/antibiotic disulfide bond with equal or unequal partitioning of the two sulfur atoms between the fragment ion and neutral loss. Hydrogen migration preceded heterolytic dissociation. Loss of two DFC moieties from [vasopressin + (DFC-H) + (DFC-H) + H]+ appears to result from collision-activated intramolecular disulfide bond rearrangement (IDBR) to produce cyclic [vasopressin + H]+ (at m/z 1084) as well as other cyclic fragment ions at m/z 1084 +/- 32 and +64. The cyclic structure of these ions could only be inferred as MS/MS may result in rearrangement to non-cyclic structures prior to dissociative loss. IDBR was also detected from MS(3) experiments of [vasopressin + (DFC-H) + (DFC-H) + H]+ fragment ions. MS/MS of [(glutathione-H) + (DFC-H) + H]+ resulted in cleavage of the peptide backbone with retention of the DFC moiety as well as heterolytic cleavage of the peptide/antibiotic disulfide bond to produce the fragment ion: [(DFC-2H) + H]+. These results demonstrate the facile dissociative loss by CAD of DFC moieties covalently attached to peptides through disulfide bonds. Published in 2004 by John Wiley & Sons, Ltd.     

Determination of acetaldehyde in tobacco smoke using N-methyl-4-hydrazino-7-nitrobenzofurazan and liquid chromatography/mass spectrometry

A liquid chromatography/mass spectrometry (LC/MS) method for the determination of carbonyl compounds based on derivatization with N-methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH) has been developed. Atmospheric pressure chemical ionization (APCI) in the positive mode proved the most versatile ionization technique for MNBD-hydrazones. APCI/MS spectra were recorded and the detection limits were determined for [M+H]+, 13C2 acetaldehyde MNBD-hydrazone has been synthesized and characterized. It is applied as internal standard for the quantification of acetaldehyde. Tobacco smoke has been investigated concerning its carbonyl content. Acetaldehyde was identified as main product and quantified by LC/MS using internal standardization. The result is in good agreement to quantification data obtained with UV/vis detection.     

Establishment of mass spectrometric fingerprints of novel synthetic cholesteryl neoglycolipids: The presence of a unique <Emphasis Type="Italic">C</Emphasis>-glycoside species during electrospray ionization and during collision-induced dissociation tandem mass spectrometry

In this study we evaluated the fragmentation pattern of 16 novel amphiphilic neoglycolipid cholesteryl derivatives that can be efficiently used to increase cationic liposomal stability and to enhance gene transfer ability. These neoglycolipids bear different sugar moieties, such as D-glucosamine, N-acetyl-D-glucosamine, N-trideuterioacetyl-D-glucosamine, N-acetyllactosamine, L-fucose, N-allyloxycarbonyl-D-glucosamine, and some of their per-O-acetylated derivatives. Regardless of the structure of the tested neoglycolipid, QqToF-MS analysis using electrospray ionization (ESI) source showed abundant protonated [M+H]+ species. We also identified by both QqToF-MS and low-energy collision tandem mass spectrometry (CID-MS/MS) of the [M+H]+ ion, the presence of specific common fingerprint fragment ions: [Cholestene]+, sugar [oxonium]+, [(Sugar-spacer-OH)+H]+, [oxonium-H2O]+, and [(Cholesterol-spacer-OH)+H]+. In addition, we observed a unique ion that could not be rationally explained by the expected fragmentation of these amphiphilic molecules. The structure of this ion was tentatively proposed with that of a C-glycoside species formed by a chemical reaction between the sugar portion and the cholesterol. MS/MS analysis of this unique [C-glycoside]+ confirmed the validity of the proposed structure of this ion. The presence of an amino group at position C-2 and free hydroxyl groups of the sugar motif is crucial for the formation of a "reactive" sugar oxonium ion that can form the [C-glycoside]+ species. In summary, we precisely established the fragmentation patterns of the tested series of neoglycolipid cholesteryl derivatives and authenticated their structure as well; moreover, we speculated on the formation of a C-glycoside with the ESI source under atmospheric pressure and in the collision cell during MS/MS analysis.     

Analysis of primary aromatic amines using precolumn derivatization by HPLC fluorescence detection and online MS identification

2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and 2-(9-acridone)-acetic acid (AAA), two novel precolumn fluorescent derivatization reagents, have been developed and compared for analysis of primary aromatic amines by high performance liquid chromatographic fluorescence detection coupled with online mass spectrometric identification. PPIA and AAA react rapidly and smoothly with the aromatic amines on the basis of a condensation reaction using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as dehydrating catalyst to form stable derivatives with emission wavelengths at 380 and 440 nm, respectively. Taking six primary aromatic amines (aniline, 2-methylaniline, 2-methoxyaniline, 4-methylaniline, 4-chloroaniline, and 4-bromoaniline) as testing compounds, derivatization conditions such as coupling reagent, basic catalyst, reaction temperature and time, reaction solvent, and fluorescent labeling reagent concentration have also been investigated. With the better PPIA method, chromatographic separation of derivatized aromatic amines exhibited a good baseline resolution on an RP column. At the same time, by online mass spectrometric identification with atmospheric pressure chemical ionization (APCI) source in positive ion mode, the PPIA-labeled derivatives were characterized by easy-to-interpret mass spectra due to the prominent protonated molecular ion m/z [M + H](+) and specific fragment ions (MS/MS) m/z 335 and 295. The linear range is 24.41 fmol-200.0 pmol with correlation coefficients in the range of 0.9996-0.9999, and detection limits of PPIA-labeled aromatic amines are 0.12-0.21 nmol/L (S/N = 3). Method repeatability, precision, and recovery were evaluated and the results were excellent for the efficient HPLC analysis. The most important argument, however, was the high sensitivity and ease-of-handling of the PPIA method. Preliminary experiments with wastewater samples collected from the waterspout of a paper mill and its nearby soil where pollution with aromatic amines may be expected show that the method is highly validated with little interference in the chromatogram.     

Influence of the labeling group on ionization and fragmentation of carbohydrates in mass spectrometry

The ionization and fragmentation behaviors of carbohydrate derivatives prepared by reaction with 2-aminobenzamide (AB), 1-phenyl-3-methyl-5-pyrazolone (PMP), and phenylhydrazine (PHN) were compared under identical mass spectrometric conditions. It has been shown that the intensities of signals in MS spectra depend on the kind of saccharides investigated and reducing end labels used. PMP sialyllactose, when ionized by ESI/MALDI, produced a mixture of [M + H]+, [M + Na]+, [M - H + 2Na]+ ions in the positive mode and [M - H]-, [M + Na - 2H]- ions in the negative mode. The AB and PHN derivatives formed abundant [M + H]+ and [M - H]- ions in ESI, and by matrix-assisted laser desorption/ionization (MALDI) produced abundant [M + Na]+ ions. PMP- and reduced AB-sialyllactose produced only Y-type fragment ions under both MS/MS sources. In the electrospray ionization (ESI)-MS/MS spectrum of PHN-sialyllactose, abundant ions corresponded to B, Z cleavages and in its MALDI-MS/MS spectrum, the abundant ions were consistent with Y glycosidic cleavages with the concurrence of B, C, and cross-ring fragment ions. In the MALDI-MS spectra of oligosaccharides acquired immediately after derivatization, it was possible to detect only PHN derivatives. After purification, spectra of all three types of derivatives showed high signal-to-noise ratios with the most abundant ions observed for AB reduced saccharides. [M + Na]+ ions were the dominant products and their fragmentation patterns were influenced by the type of the labeling and the kind of oligosaccharide considered. In the MALDI-PSD and -MS/MS spectra of AB-derivatized glycans, higher m/z fragment ions corresponded to B and Y cleavages and the loss of bisecting GlcNAc appeared as a weak signal or was not detected at all. Fragmentation patterns observed in the spectra of hybrid/complex PHN and PMP glycans were more comparable-higher m/z fragments corresponded to B and C glycosidic cleavages. For PHN glycans, the abundance of ions resulting from the loss of bisecting GlcNAc depended on the number of residues linked to the 6-positioned mannose. Also, PHN and PMP derivatives produced cross-ring cleavages with abundances higher than observed in the spectra of AB derivatized oligosaccharides. For high-mannose glycans, the most informative cleavages were provided by AB and PHN type of labeling. Here, PMP produced dominant Y-cleavages from the chitobiose while other ions produced weak signals.     

Discovering metabolites of post-harvest fungicides in citrus with liquid chromatography/time-of-flight mass spectrometry and ion trap tandem mass spectrometry

In this study, we benefit from the combination of liquid chromatography (LC)/time-of-flight (TOF) MS accurate mass measurements to generate elemental compositions of ions and LC/ion trap multiple MS (MSn) providing complementary structural information, which is useful for the elucidation of unknown organic compounds at trace levels in complex food extracts. We have applied this approach to investigate different citrus fruits extracts, and we have identified two post-harvest fungicides (imazalil and prochloraz), the main degradation product of imazalil ([M + H]+, m/z 257) and a non-previously reported prochloraz degradation product ([M + H]+, m/z 282). The database-mediated identification of the parent compounds was based on the generated elemental composition obtained from accurate mass measurements and additional qualitative information from the high resolution chlorine isotopic clusters of both the protonated molecules (imazalil, [M + H]+ 297.0556, <0.1 ppm error, 2-Cl; prochloraz, [M + H]+ 376.0381, 1.9 ppm error, 3-Cl) and their characteristic fragments ions (imazalil: m/z 255 and 159; prochloraz: m/z 308 and 266). The correlation between the structural information provided by ion trap MS/MS fragmentation pathways of the parent species and the TOF accurate mass elemental composition data of the degradation products were the key to elucidate the structures of the degradation products of both post-harvest fungicides. Finally, where standards were not available (prochloraz), further confirmation was obtained by synthesizing the proposed degradation product by acid hydrolysis of the parent standard and confirmation by LC/TOF-MS.     

Analysis of polycyclic aromatic hydrocarbons by liquid chromatography/tandem mass spectrometry using atmospheric pressure chemical ionization or electrospray ionization with tropylium post-column derivatization

Polycyclic aromatic hydrocarbons (PAHs) with four to six rings are potent carcinogens. This study analyzed ten of the sixteen US EPA priority PAHs using reversed-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) in selected reaction monitoring mode with two ionization sources: positive atmospheric pressure chemical ionization (APCI+) or positive elecrtrospray ionization (ESI+) with tropylium post-column derivatization. Several factors were investigated, including mobile phases, stationary phases of columns and chromatographic temperature, to determine how optimal separation and sensitivity might be achieved. Methanol used as an organic mobile phase provided better sensitivities for most PAHs than acetonitrile, although some PAHs co-eluted. Acidic buffers did not increase analyte signals. Use of Restek Pinnacle II PAH columns (250 x 4.6 mm or 250 x 2.1 mm, 5 microm) with water/acetonitrile gradient at 27 degrees C made possible a good separation of the ten analytes. [M]+. were the best precursor ions in both APCI and ESI, although fluoranthene could not be detected in ESI mode when tropylium post-column derivatization was performed. [M-28]+ and [M-52]+ were the major product ions of PAHs after collision-induced dissociation, a result of neutral losses of C(2)H(4) and (C(2)H(2))(2), respectively. Chromatographic separation for PAH isomers was crucial because the mass spectra were so similar that even MS/MS could not distinguish them from each other. The recoveries of sample preparations of PAHs spiked onto air-sampling filters ranged between 77.5 and 106% with relative standard deviations between 1.1 and 15.9%. This method was validated by analyzing NIST SRM 1649a (urban dust), producing results comparable with the certified PAH concentrations. The detection limits using APCI and ESI interfaces, defined as three times the noise levels, ranged between 0.23 and 0.83 ng and between 0.16 and 0.84 ng of on-column injection, respectively.     

Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

Gestrinone was studied by high performance liquid chromatography (HPLC) for screening and by gas chromatography/mass spectrometry (GC/MS) for confirmation. When the chromatograms of blank, spiked urine and dosed urine were compared by HPLC, two unknown metabolites were found and these were excreted as the conjugated forms. Metabolites 1 and 2 were tested by LC/MS and LC/MS/MS and both had parent ions at m/z 325. The fragment ion of metabolite 1 was at m/z 263 and ions for metabolite 2 were m/z 307 [MH - H(2)O](+), 289, 279 and 241. LC/MS/MS of m/z 263 as the parent ion of metabolite 1 gave fragment ions at m/z 245 and 217, which were assumed to be [263 - H(2)O](+) and [235 - H(2)O](+), respectively. The trimethylsilyl (TMS)-enol-TMS ether derivative of gestrinone displayed three peaks in its GC/MS chromatogram, formed by tautomerism.     

Determination of patulin in apple juice by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

An HPLC-MS-MS method with selected reaction monitoring (SRM) for the determination of patulin in apple juice samples is described. Mass spectrometric detection was accomplished following atmospheric pressure chemical ionization (APCI) in both positive and negative ion modes. Collision induced dissociation (CID) of the protonated molecular ion led initially to the loss of H2O (fragment m/z 137). At higher energies CO is lost from both the protonated parent molecule (fragment m/z 127) and the dehydrated molecular ion (fragment m/z 109). In contrast, CID of the deprotonated molecular ion led initially to the fragment at m/z 109 corresponding to the loss of either CO2 or acetaldehyde, followed at higher CID energy by the loss of H2O (fragment m/z 135) and CO (fragment m/z 125) from the deprotonated molecular ion. Detection in the negative ion mode proved superior and a linear response was observed over the injected range from 6 to 200 ng patulin. Apple juice samples spiked with patulin between 10 and 135 microg/l were analyzed following liquid-liquid extraction with ethyl acetate and clean up with sodium carbonate. Utilizing reversed-phase HPLC with acetonitrile-water (10:90) at 0.5 ml/min, levels down to 10 microg/l were readily quantified and a detection limit of 4 microg/l was attainable at a signal-to-noise (SIN) ratio of 4. The MS data for the spiked samples compared well to the UV data and when plotted against each other displayed a correlation coefficient (R) of 0.99.     

Novel phosphoryl derivatization method for peptide sequencing by electrospray ionization mass spectrometry

A one-step phosphoryl derivatization method has been used in a peptide sequencing procedure for electrospray ionization tandem mass spectrometry (ESI-MS/MS). The sodiated derivatized peptides exhibit very simple dissociation patterns, in which two kinds of fragment ions, [b(n) + OH + Na]+ and [a(n) + Na]+, are formed. Since the amino acid residues are lost sequentially from the C-terminus, peptide sequences can be identified easily. The fragmentation efficiency of peptides increased as a result of the phosphorylation, and also provided peaks of useful intensity at lower m/z. A peptide with lysine at the C-terminus was derivatized and analyzed by ESI-MS/MS. Similar mass spectra, from which the sequence could be read out, were obtained. This is a novel derivatization method yielding neutral derivatives that should be suitable for peptide sequencing by LC/ESI-MS/MS.     

Electrospray ionization tandem mass spectrometric studies to probe the interaction of Cu(II) with amoxicillin简

This study describes the application of electrospray ionization mass spectrometry(ESI-MS) to investigate copper ion interaction with amoxicillin. ESI mass spectra of Cu–amoxicillin complexes show complex ions at m/z 828, 792, 753, 731, 428, 388 and 366 corresponding to [63Cu+(2A-H)+2H2 O]+, [63Cu+(2A-H)]+, [2A+Na]+, [2A+H]+, [63Cu+(A-H)]+, [A+Na]+and [A+H]+(where A = amoxicillin). Based on the observed m/z values of Cu–amoxicillin complex ions, it is found that the Cu–amoxicillin ratios are 1:1 and 1:2, and the copper ions exhibited three feasible coordination numbers(2, 4 and 6) with amoxicillin complexes. The structures and coordination numbers of copper–amoxicillin complex ions were probed from their collisionally activated dissociation(CAD) spectra. Based on these results, it is confirmed that the copper ions could form stable tetrahedral and octahedral complexes with amoxicillin. This study validates the applicability of ESI-MS for probing copper–amoxicillin complex ions.     

Analysis of neutral oligosaccharides for structural characterization by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

We have acquired multi-stage mass spectra (MSn) of four branched N-glycans derived from human serum IgG by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF-MS) in order to demonstrate high sensitivity structural analysis. [M+H]+ and [M+Na]+ ions were detected in the positive mode. The detection limit of [M+Na]+ in MS/MS and MS3 measurements for structural analysis was found to be 100 fmol, better than that for [M+H]+. The [M+H]+ ions subsequently fragmented to produce predominantly a Y series of fragments, whereas [M+Na]+ ions fragmented to give a complex mixture of B and Y ions together with some cross-ring fragments. Three features of MALDI-QIT-CID fragmentation of [M+Na]+ were cleared by the analysis of MS/MS, MS3 and MS4 spectra: (1) the fragment ions resulting from the breaking of a bond are more easily generated than that from multi-bond dissociation; (2) the trimannosyl-chitobiose core is either hardly dissociated, easily ionized or it is easy to break a bond between N-acetylglucosamine and mannose; (3) the fragmentation by loss of only galactose from the non-reducing terminus is not observed. We could determine the existence ratios of candidates for each fragment ion in the MS/MS spectrum of [M+Na]+ by considering these features. These results indicate that MSn analysis of [M+Na]+ ions is more useful for the analysis of complicated oligosaccharide structures than MS/MS analysis of [M+H]+, owing to the higher sensitivity and enhanced structural information. Furthermore, two kinds of glycans, with differing branch structures, could be distinguished by comparing the relative fragment ion abundances in the MS3 spectrum of [M+Na]+. These analyses demonstrate that the MSn technology incorporated in MALDI-QIT-TOF-MS can facilitate the elucidation of structure of complex branched oligosaccharides.     

Tandem mass spectra of divalent metal ion adducts of glycosyl sulfides, sulfoxides and sulfones; distinction among stereoisomers

The tandem mass spectra of the divalent metal ion (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Ni2+, Co2+ and Zn2+) adducts of acetylated 1,2-trans-glycosyl sulfides, sulfoxides and sulfones were examined using low energy collision-induced dissociation on a Quattro II quadrupole tandem mass spectrometer. Abundant doubly charged ions, such as [3M + Met]2+ and [2M + Met]2+, were observed with alkaline earth metal chlorides. The other ions observed were [M + MetCl]+, [M + MetOAc]+, [M + MetO2SPh]+ and [2M + MetCl]+. The deprotonated metal adducts [M + Met-H]+ were seen only in the sulfones. The divalent metal ion adducts showed characteristic fragmentation pathways for the glycosyl sulfides, sulfoxides and sulfones, depending on the site of metal attachment. The doubly charged metal ion adducts dissociate to two singly charged ions, [M + MetOAc]+ and [M - OAc]+, in the sulfides and sulfoxides. In the sulfones, the adducts dissociate to [M + MetO2SPh]+ and [M - O2SPh]+. In contrast to the alkaline earth metals, which attach to the acetoxy functions, the transition metals attach to the sulfide and sulfoxide functions. The metal chloride adducts display characteristic fragmentation for the sulfides, sulfoxides and sulfones. The glucosyl, mannosyl and galactosyl sulfides, sulfoxides and sulfones could be differentiated on the basis of the stereochemically controlled MS/MS fragmentations of the metal chloride adducts.     

Study of phytochelatins and other related thiols as complexing biomolecules of As and Cd in wild type and genetically modified Brassica juncea plants

The accumulation of As and Cd in Brassica juncea plants and the formation of complexes of these elements with bioligands such as glutathione and/or phytochelatins (PCs) is studied. The genetic manipulation of these plants to induce higher As and Cd accumulation has been achieved by overexpressing the genes encoding for gamma-glutamyl cysteine synthetase (gamma-ECS) and glutathione synthetase (GS). These two enzymes are responsible for glutathione (GSH) formation in plants, which is the first step in the production of PCs. The biomass produced in both the wild type and the genetically modified plants, has been evaluated. Additionally, the total Cd and As concentration accumulated in the plant tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS) after extraction. Speciation studies on the extracts were conducted using size exclusion liquid chromatography (SEC) coupled online with ICP-MS to monitor As, Cd and S. For further purification of the As fractions, reversed phase high performance liquid chromatography (RP-HPLC) was used. Structural elucidation of the PCs and other thiols, as well as their complexes with As and Cd, was performed by electrospray-quadrupole-time-of-flight (ESI-Q-TOF). In both the Cd and As exposed plants it was possible to observe the presence of oxidized PC2 ([M + H]+, m/z 538), GS-PC2(-Glu) ([M + H]+, m/z 716) as well as reduced GSH ([M + H]+, m/z 308) and oxidized glutathione (GSSG) ([M + H]+, m/z 613). However, only the GS plants exhibited the presence of As(GS)3 complex ([M + H]+, m/z 994) that was further confirmed by MS/MS. This species is reported for the first time in B. juncea plant tissues.     

Determination and excretion study of gestrinone in human urine by high performance liquid chromatography and gas chromatography/mass spectrometry

Gestrinone was studied by HPLC for screening and by GC/MS for confirmation. Three unknown peaks were found by HPLC which are probably the metabolites of gestrinone, and conjugated gestrinone in dosed human urine. The metabolites and gestrinone were excreted as the conjugated forms. The total amounts of metabolite 1 and conjugated gestrinone, recovered after 48 h, were 0.20 and 0.32 mg, respectively. When metabolite 1 was tested by LC/MS and LC/MS/MS, the parent ion was m/z 327, [MH](+), and fragment ions were seen at m/z 309 [MH - H(2)O](+), 291 [MH - 2H(2)O](+), 283, 263 and 239. The TMS-enol-TMS ether derivative of gestrinone has three peaks in the GC/MS chromatogram formed by tautomerism. The reproducibility of the derivatization method was stable and recoveries were over 87% when spiked into blank urine.     

Electrospray ionization mass spectrometry monitoring of indigo carmine degradation by advanced oxidative processes

The degradation of the dye indigo carmine in aqueous solution induced by two oxidative processes (H(2)O(2)/iodide and O(3)) was investigated. The reactions were monitored by electrospray ionization mass spectrometry in the negative ion mode, ESI(-)-MS, and the intermediates and oxidation products characterized by ESI(-)-MS/MS. Both oxidative systems showed to be highly efficient in removing the color of the dye aqueous solutions. In the ESI(-)-MS of the indigo carmine solution treated with H(2)O(2) and H(2)O(2)/iodide, the presence of the ions of m/z 210 (indigo carmine in its anionic form, 1), 216, 226, 235, and 244 was noticeable. The anion of m/z 235 was proposed to be the unprecedented hydroperoxide intermediate 2 formed in solution via an electrophilic attack by hydroxyl and hydroperoxyl radicals of the exocyclic C=C bond of 1. This intermediate was suggested to be rapidly converted into the anionic forms of 2,3-dioxo-1H-indole-5-sulfonic acid (3, m/z 226), 2-amino-alpha-oxo-5-sulfo-benzeneacetic acid (4, m/z 244), and 2-amino-5-sulfo-benzoic acid (5, m/z 216). In the ESI(-)-MS of the indigo carmine solution treated with O(3), two main anions were detected: m/z 216 (5) and 244 (4). Both products were proposed to be produced via an unstable ozonide intermediate. Other anions in this ESI(-) mass spectrum were attributed to be [4 - H + Na](-) of m/z 266, [4 - H](2-) of m/z 121.5, and [5 - H](2-) of m/z 107.5. ESI-MS/MS data were consistent with the proposed structures for the anionic products 2-5.