Mass spectrometry characterization of Escherichia coli K4 oligosaccharides from 2-mers to more than 20-mers

The separation and characterization of oligosaccharides obtained by hyaluronidase [E.C. 3.2.1.35] digestion of Escherichia coli K4 polysaccharide using online high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) are presented. Complete identification and structural information for oligosaccharides containing 2-24 monomers (from 2- to 24-mers) were obtained. In particular, smaller K4 species, from 2-mers to 4-mers, exhibited mainly [M-H](-1) anions, whereas the 6- to 8-mers existed predominantly at the charge state of -2. The K4 oligomers from 10-mers to 14-mers were mainly represented by [M-3H](-3) anions while species from 16- to 20-mers were characterized by a charge state of -4. K4 oligosaccharides from 22- to 24-mers existed as [M-4H](-4) and [M-5H](-5) anions and, for this latter species, ions having a charge state of -6 appeared. For smaller K4 species, in particular from 6-mers to 10-mers, ESI-MS revealed anions related to the loss of one monosaccharide unit from the oligomers due to apparent collisional activation and ion source fragmentation. However, no odd-numbered anions were produced for K4 2/4-mer species or for oligosaccharides greater than 12-mers, while for K4 species 8/10-mer, ESI-MS revealed odd-numbered anions generally in low relative abundance making the interpretation of the spectra easier. The ESI-MS spectra of oligosaccharides separated by online HPLC were applied to the evaluation of the K4 polymerization process, confirming that the addition of fructose units is not critical for chain elongation as variously fructosylated oligomer species were detected directly on the K4 carbohydrate backbone.     

Characterisation of polyacetylenes isolated from carrot (Daucus carota) extracts by negative ion tandem mass spectrometry

The potential use of negative electrospray ionisation mass spectrometry (ESI-MS) in the characterisation of the three polyacetylenes common in carrots (Daucus carota) has been assessed. The MS scans have demonstrated that the polyacetylenes undergo a modest degree of in-source decomposition in the negative ionisation mode while the positive ionisation mode has shown predominantly sodiated ions and no [M+H](+) ions. Tandem mass spectrometric (MS/MS) studies have shown that the polyacetylenes follow two distinct fragmentation pathways: one that involves cleavage of the C3-C4 bond and the other with cleavage of the C7-C8 bond. The cleavage of the C7-C8 bond generated product ions m/z 105.0 for falcarinol, m/z 105/107.0 for falcarindiol, m/z 147.0/149.1 for falcarindiol-3-acetate. In addition to these product ions, the transitions m/z 243.2 → 187.1 (falcarinol), m/z 259.2 → 203.1 (falcarindiol), m/z 301.2 → 255.2/203.1 (falcarindiol-3-acetate), mostly from the C3-C4 bond cleavage, can form the basis of multiple reaction monitoring (MRM)-quantitative methods which are poorly represented in the literature. The 'MS(3) ' experimental data confirmed a less pronounced homolytic cleavage site between the C11-C12 bond in the falcarinol-type polacetylenes. The optimised liquid chromatography (LC)/MS conditions have achieved a baseline chromatographic separation of the three polyacetylenes investigated within 40 min total run-time.     

A comparative study of the fragmentation of neutral lactooligosaccharides in negative-ion mode by UV-MALDI-TOF and UV-MALDI ion-trap/TOF mass spectrometry

Structure analyses of underivatized neutral lacto oligosaccharides are systematically performed by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI TOF MS) and UV-MALDI ion-trap time-of-flight mass spectrometry (ion-trap/TOF MS) acquired in negative-ion mode. Interestingly, their fragmentation significantly differ each other. In postsource decay (PSD) in UV-MALDI TOF MS, cross-ring cleavage at the reducing terminal predominates. On the other hand, glycosyl bond cleavage (C-type fragmentation) takes place preferentially in collision induced dissociation (CID) in UV-MALDI ion-trap/TOF MS. The cross-ring cleavage in PSD similar to that in in-source decay occurs via a prompt reaction path characteristic of the UV-MALDI process itself. The product ion spectra of UV-MALDI ion-trap/TOF MS are similar to the electrospray ionization (ESI) ion-trap or quadrupole/TOF CID product ion spectra. During ion-trap/TOF MS experiments, the deprotonated molecular ions survive for several tens of milliseconds after CID event because the high internal energy chlorinated precursor ions are cooled by collisional cooling in the ion trap. The results obtained suggest that the PSD from the chlorinated precursor ion in UV-MALDI TOF MS might proceed as a two-step reaction; in the first, a high internal energy deprotonated molecular ion is generated as a reaction intermediate during the flight in the drift tube, and in the second, the rapid decomposition from the deprotonated molecular ion takes place.     

Tandem MS can distinguish hyaluronic acid from N-acetylheparosan

Isobaric oligosaccharides enzymatically prepared from hyaluronic acid (HA) and N-acetylheparosan (NAH), were distinguished using tandem mass spectrometry. The only difference between the two series of oligosaccharides was the linkage pattern (in HA 1-->3 and in NAH 1-->4) between glucuronic acid and N-acetylglucosamine residues. Tandem mass spectrometry afforded spectra in which glycosidic cleavage fragment ions were observed for both HA and NAH oligosaccharides. Cross-ring cleavage ions 0,2An and 0,2An-h (n is even number) were observed only in GlcNAc residues of NAH oligosaccharides. One exception was an 0,2A2 ion fragment observed for the disaccharide from HA. These cross-ring cleavage fragment ions are useful to definitively distinguish HA and NAH oligosaccharides.     

Structural differentiation of uronosyl substitution patterns in acidic heteroxylans by electrospray tandem mass spectrometry

The structures of two oligomers of acidic xylo-oligosaccharides (XOS) of the same molecular weight (634 Da), Xyl(2)MeGlcAHex and Xyl(2)GlcA(2) were differentiated by electrospray tandem mass spectrometry (ESI-MS/MS). These oligomers were present in a mixture of XOS obtained by acid hydrolysis of heteroxylans extracted from Eucalyptus globulus wood (Xyl(2)MeGlcAHex) and Olea europaea olive fruit (Xyl(2)GlcA(2)). In the ESI-MS spectra of the XOS, ions at m/z 657 and 652 were observed and assigned to [M + Na](+) and [M + NH(4)](+), respectively. The ESI-MS/MS spectrum of [M + Na](+) ion of Xyl(2)MeGlcAHex showed the loss of Hex residue from the reducing end followed by the loss of MeGlcA moiety. Simultaneously, the loss of a Xyl residue from either the reducing or the non-reducing ends was detected. On the other hand, the fragmentation of Xyl(2)GlcA(2) occurs mainly by the loss of one and two GlcA residues or by the loss of the GlcAXyl moiety, due to the glycosidic bond cleavage between the two Xyl residues. Loss of one and two CO(2) molecules was only observed for this oligomer, where the GlcA are in vicinal Xyl residues. The ESI-MS/MS spectra of [M + NH(4)](+) of both oligomers showed the loss of NH(3), resulting in the protonated molecule, where the presence of ions assigned as protonated molecules of aldobiuronic acid residues, [MeGlcA - Xyl + H](+) and [GlcA - Xyl + H](+), are diagnostic ions of the presence of MeGlcA and GlcA moieties in XOS. Since these structures occur in small amounts in complex acidic XOS mixtures and are very difficult, if possible, to isolate, tandem mass spectrometry revealed to be a powerful tool for the characterization of these types of substitution patterns present in heteroxylans.     

Fragmentation of deprotonated N-benzoylpeptides: Formation of deprotonated oxazolones

The fragmentation reactions of deprotonated N-benzoyl peptides, specifically hippurylglycine, hippurylglyclyclycine, and hippurylphenylalanine (hippuryl = N-benzoylGly) have been studied using MS2 and MS3 experiments as well as deuterium labeling. A major fragment ion is observed at m/z 160 ([C9H6NO2]-) which, upon collisional activation, mainly eliminates CO2 indicating that the two oxygen atoms have become bonded to the same carbon. This observation is rationalized in terms of formation of deprotonated 2-phenyl-5-oxazolone. Various pathways to the deprotonated oxazolone have been elucidated through MS3 experiments. Fragmentation of deprotonated N-acetylalanylalanine gives a relatively weak signal at m/z 112 which, upon collisional activation, fragments, in part, by loss of CO2 leading to the conclusion that the m/z 112 ion is deprotonated 2,4-dimethyl-5-oxazolone.     

The role of mobile protons in negative ion CID of oligosaccharides

Carbohydrates of all classes consist of glycoform mixtures built on common core units. Determination of compositions and structures of such mixtures relies heavily on tandem mass spectrometric data. Analysis of native glycans is often necessary for samples available in very low quantities and for sulfated glycan classes. Negative tandem mass spectrometry (MS) provides useful product ion profiles for neutral oligosaccharides and is preferred for acidic classes. In previous work from this laboratory, site-specific influences of sialylation on product ion profiles in the negative mode were elucidated. The present results show how the interplay of two other acidic groups, uronic acids and sulfates, determines product ion patterns for chondroitin sulfate oligosaccharides. Unsulfated chondroitin oligosaccharides dissociate to form C-type ions almost exclusively. Chondroitin sulfate oligosaccharides produce abundant B- and Y-type ions from glycosidic bond cleavage with C- and Z-types in low abundances. These observations are explained in terms of competing proton transfer reactions that occur during the collisional heating process. Mechanisms for product ion formation are proposed based on tandem mass spectra and the abundances of product ions as a function of collision energy.     

Identification of hyaluronic acid oligosaccharides by direct coupling of capillary electrophoresis with electrospray ion trap mass spectrometry

A new method for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with bacterial hyaluronidase (HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae) using online capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS) is presented. A fused-silica capillary coated with polyacrylamide was used with a 40 mM ammonium acetate buffer at pH 9.0 and a separation voltage of +30 kV applied to the inlet. Separation was achieved for oligosaccharides containing 4-16 monomers. The migration behavior follows the chain length of the oligomers, regardless of charge state. However, no linear relationship was found for the relation between mobility and chain length. Using an ion trap mass analyzer, complementary structural information was obtained by MS/MS and MS(n) experiments.     

Structural analysis of drug-DNA adducts by tandem mass spectrometry

The utility of electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) for the characterisation of ligand-oligonucleotide adducts is demonstrated with adducts formed between the oligonucleotide 5'-CACGTG-3' and both a platinating agent, cis-diamminedichloroplatinum(II) (cisplatin), and an alkylating ligand, n-bromohexylphenanthridinium bromide (phenC6Br). We have demonstrated previously that negative ion MS/MS spectra of alkylated oligonucleotides show a highly specific fragmentation pathway that enables the site of binding of the ligand to be readily identified. In comparison, the positive ion ESI-MS/MS spectra reported here also show a single major fragmentation pathway, but the dominant ion is the protonated ligand-base adduct. MS/MS of this ion confirms the site on binding of the ligand to the guanine base. MS/MS spectra of cisplatin adducts show much less specific fragmentation than alkylated adducts, particularly in the negative ion mode. This suggests that the ESI-MS/MS spectra of ligand-DNA adducts are strongly influenced by the extent to which the ligand weakens the glycosidic bond in the residue to which it is bound. For platinating agents, which do not labilise the glycosidic bond, additional experiments involving MS/MS of source-generated product ions were required to enable isomeric adducts to be distinguished.     

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MS(n) (part 1: methodology)

Underivatized neutral oligosaccharides from human milk were analyzed by nano-electrospray ionization (ESI) using a quadrupole ion trap mass spectrometer (QIT-MS) in the negative-ion mode. Under these conditions neutral oligosaccharides are observed as deprotonated molecules [M-H]- with high intensity. CID-experiments of these species with the charge localized at the reducing end lead to C-type fragment ions forming a "new" reducing end. Fragmentations are accompanied by cross-ring cleavages that yield information about linkages of internal monosaccharides. Several isomeric compounds with distinct structural features, such as different glycosidic linkages, fucosylation and branching sites were investigated. The rules governing the fragmentation behavior of this class of oligosaccharides were elucidated and tested for a representative number of certain isomeric glycoforms using the MS/MS and MS(n) capabilities of the QIT. On the basis of the specific fragmentation behavior of deprotonated molecules, the position of fucoses and the linkage type (Gal beta-->3 GlcNAc or Gal beta1-->4 GlcNAc) could be determined and linear and branched could be differentiated. Rules could be established which can be applied in further investigations of these types of oligosaccharides even from heterogenous mixtures.     

Different behavior of dextrans in positive-ion and negative-ion mass spectrometry

A mass spectrometric (MS) comparative study of dextran samples using two different ionization techniques (matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI)) in both positive- and negative-ion modes is reported. The experiments were carried out with two polydisperse dextrans (1000 and 8800 Da) and isomaltotriose. In the positive-ion mode, the expected alkali metal ion adducts of dextrans were observed in both techniques. In contrast, the expected preferential formation of deprotonated molecules [M - H](-) was not confirmed in negative mode MALDI time-of-flight (TOF) MS, where the series of ions [M(x)- H +42](-) or [M(x+1)- H - 120](-), coming either from some addition or fragmentation, were observed. In both ionization techniques, these ions formed the main distributions of dextrans in the negative-ion mode. It seems that the negative molecular ions formed from the alpha1 --> 6 linkage of polyglucose oligomers easily decompose, and the product ions [M - H - 120](-) markedly dominate. The fragmentation experiments and especially the investigation of the fundamental role of the nozzle-skimmer potential in ESI-MS supported our explanation of the observed behavior because its higher values caused higher fragmentation. The experiments with isomaltotriose excluded any addition of 42 Da during the MS procedures, which is not distinguishable from the loss of 120 Da in the case of polydisperse dextrans. MALDI-TOFMS was found to be more sensitive for the detection of higher oligosaccharides and ESI-MS more useful for structural studies.     

Structural analysis of isomeric chondroitin sulfate oligosaccharides using regioselective 6-O-desulfation method and tandem mass spectrometry

A strategy based on a regioselective 6-O-desulfation reaction and negative ion electrospray ionization tandem mass spectrometry (ESI-MS(n)) was developed for the structural delineation of isomeric chondroitin sulfate oligosaccharides. Product ions resulting from the glycosidic cleavage provided information about the number of sulfate groups in each sugar residue. After the regioselective 6-O-desulfation reaction, the number of sulfate groups on each residue was obtained using a tandem mass spectrometry analysis of the reaction product. The sulfation pattern could be obtained based on the product ions of analytes before and after the desulfation reaction. The strategy was demonstrated using a series of tetrasaccharides prepared from shark cartilage chondroitin sulfate D. Among the 12 identified tetrasaccharides, six structures had not been reported before.     

Systematic identification of N-acetylheparosan oligosaccharides by tandem mass spectrometric fragmentation

Recently, a useful procedure for the preparation of both even- and odd-numbered series of N-acetylheparosan (NAH) oligosaccharides was established. The present report describes findings when these NAH oligosaccharides were subjected to comparative mass spectrometry (MS)/MS fragmentation analysis by matrix-assisted laser desorption/ionization (MALDI)-LIFT-time-of-flight (TOF)/TOF-MS/MS, and electrospray ionization (ESI) collision-induced dissociation (CID) MS/MS. The resultant fragment ions were systematically assigned to elucidate fragmentation characteristics. In the MALDI-LIFT-MS/MS experiments, all the NAH oligosaccharides underwent unique glycosidic cleavages that included B-Y ion cleavages (nomenclature system of Domon and Costello, Glycoconjugate J. 1988; 5: 397) at the C-1 side, and C-Z ion cleavages at the C-4 side, with respect to glucuronic acid (GlcA). In addition, (0,2)A and/or (0,2)X cross-ring cleavages were observed for relatively small oligosaccharides. The former observation clearly reflects the occurrence of a GlcA-N-acetylglucosamine (GlcNAc) alternating structure of NAH, while the latter feature implies the occurrence of the -beta-1-4-glucuronide linkage. Extensive glycosidic cleavages were also observed in the ESI-CID-MS/MS fragmentation, though cleavage specificity was less evident than in the case of MALDI-LIFT-TOF/TOF-MS/MS. The information obtained in this study should be valuable for understanding both biosynthetic and degradation processes of NAH and its derivatives including heparin and heparan sulfate, as well as artificially modified NAH oligosaccharides.     

Structural characterization of steroidal saponins by electrospray ionization and fast-atom bombardment tandem mass spectrometry

The structural characterization of four steroidal saponin compounds involving two and three sugar groups, namely spirostanol saponins and furostanol saponins, were investigated by positive ion fast-atom bombardment (FAB), electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) techniques. Important structural information was obtained from collision-induced dissociation (CID) and FAB-MS spectra with different liquid matrices. It was found that a characteristic fragmentation involving the loss of 144 Da arising from the cleavage of the E-ring was observed when there was no sugar chain at the C-26 position. When a glucoside group was substituted at the C-26 position, this C-26 sugar moiety was preferentially eliminated. All of these compounds produced a major product ion with a stable skeleton structure at m/z 255. The results of this paper can assist structural analysis of mixtures of steroidal saponins.     

Characterisation of nicotine and related compounds using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their detection by liquid chromatography/electrospray ionisation mass spectrometry

Electrospray ionisation ion trap mass spectrometry (ESI-MS(n)) has been used to study the fragmentation patterns of nicotine and nine of its related compounds. From this study certain characteristic fragmentations are apparent with generally the pyrrolidine or piperidine ring being subject to chemical modifications. The structures of the product ions proposed for the ESI-MS(n) study have been supported by results from electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). Compounds with pyrrolidine and piperidine rings that possess an unsubstituted N atom have been shown to lose NH(3) at the MS(2) stage. Those compounds with N-methyl groups lose CH(3)NH(2) at the MS(2) stage. The loss of NH(3) or CH(3)NH(2) leaves the corresponding rings opened and this is followed by ring closure at the pyridine-2 carbon atom. Mono-N-oxides fragment in a similar way but the di-N-oxide can also fragment by cleavage of the bond between the pyridine and pyrrolidine rings. Cotinine also can undergo cleavage of this bond between the rings.This data therefore provides useful information on how substituents and the nature of the non-pyridine ring can affect the fragmentation patterns of nicotine and its related compounds. This information can be used in the characterisation of these compounds by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) which results in the separation of nicotine and its related compounds with limits of detection (LODs) ranging from 15 to 105 ng/mL. The use of LC/ESI-MS to study nicotine-containing samples resulted in the simultaneous and unambiguous identification of seven of the compounds discussed in this paper: cotinine identified at retention time 12.5 min (with its [M+H](+) ion at m/z 177), nornicotine 16.0 min (m/z 149), anatabine 18.0 min (m/z 161), myosmine 18.5 min (m/z 147), anabasine 20.4 min (m/z 163), nicotine 22.2 min (m/z 163), and nicotyrine 31.4 min (m/z 159). For quality control of nicotine replacement therapy products, these nicotine impurities can be readily identified and determined at levels up to 0.3% for single impurities and up to 1.0% for total impurities.     

Application of positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry to a study of the fragmentation of 6-hydroxyluteolin 7-O-glucoside and 7-O-glucosyl-(1 --> 3)-glucoside

Mass spectrometric methodology based on the combined use of positive and negative electrospray ionization, collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) has been applied to the structural characterization of 6-hydroxyluteolin 7-O-glucoside and 7-O-glucosyl-(1 --> 3)-glucoside. In-source fragmentation of both glycosides at an increased potential yielded the protonated and deprotonated aglycone, allowing CID spectra to be obtained. The differentiation between quercetin and 6-hydroxyluteolin aglycones was achieved by product ion analysis of the protonated and deprotonated aglycone (m/z 303 and 301), that showed the characteristic product ions (1,3)A at m/z 151 and 153 for quercetin, and m/z 167 and 169 for 6-hydroxyluteolin, consistent with the trihydroxylated A-ring skeleton. In the negative ion mode both glycosides were shown to undergo collision-induced homolytic and heterolytic cleavages of the O-glycosidic bond producing the aglycone radical-anion [Y0-H]-* and Y0(-) product ions. At lower collision energy, various fragmentations involving the glucose moieties were observed with a relatively higher abundance for the monoglucoside compared to the diglucoside. In the latter case both the inner and the terminal glucose residues were involved in the fragmentations, giving useful information on the 1 --> 3 interglycosidic linkage. CID MS/MS analysis of the sodiated molecules gave complementary information for the structural characterization of the studied compounds. Fragmentation mechanisms are proposed for the observed product ions.     

几种4-氨基吡啶衍生物的多级质谱裂解途径

为获得高效的海洋生物毒素河豚毒素(TTX)解毒剂,合成了一系列4-氨基吡啶类衍生物N-二异丙基磷酰化氨基酸-N-4-氨基吡啶;研究了N-二异丙基磷酰化氨基酸-N-4-氨基吡啶的多级质谱(ESI-MS/MS)裂解方式;提出了碎片离子m/z=95的裂解途径,并推测了其重排机理.结果表明,该类化合物具有相同分子量的碎片离子c/c′,是由两种母离子a或b离子裂解得到的.通过在吡啶环上引入氯原子可证实该裂解途径;而碎片离子m/z=95源于离子重排.     

Gas-phase fragmentation study of novel synthetic 1,5-benzodiazepine derivatives using electrospray ionization tandem mass spectrometry

The fragmentation patterns of a series of three novel synthesized 3-hydroxy-4-phenyl-tetrahydro-1,5-benzodiazepin-2-ones (1-3), possessing the same backbone structure, were investigated using electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) techniques. A simple methodology, based on the use of ESI (positive ion mode) and by increasing the declustering potential in the atmospheric pressure/vacuum interface, collision-induced dissociation (CID), was used to enhance the formation of the fragment ions. In general, the novel synthetic 1,5-benzodiazepine derivatives afforded, in the gas phase, both protonated and sodiated molecules. This led to the confirmation of the molecular masses and chemical structures of the studied compounds. Exact accurate masses were measured using a high-resolution ESI-quadrupole orthogonal time-of-flight (QqToF)-MS/MS hybrid mass spectrometer instrument.The breakdown routes of the protonated molecules were rationalized by conducting low-energy collision CID-MS/MS analyses (product ion- and precursor ion scans) using a conventional quadrupole-hexapole-quadrupole (QhQ) tandem mass spectrometer. All the observed major fragmentations for the 1,5-benzodiazepines occurred in the saturated seven-membered ring containing the nitrogen atoms. These formed a multitude of product ions by different breakdown routes. All the major fragmentations involved cleavages of the N-1-C-2 and C-3-C-4 bonds. These occurred with concomitant eliminations of glyoxal, benzene and ethyl formate, forming the product ion at m/z 119, which was observed in all the studied compounds. In addition, an unique simultaneous CID-MS/MS fragmentation was noticed for the 1,5-benzodiazepines 1 and 3, which occurred by a pathway dictated by the substituent located on the N-1-position. It was evident that the aromatic ring portion of the 1,5-benzodiazepines was resistant to CID-MS/MS fragmentation. Re-confirmation of the various geneses of the product ions was achieved by conducting a series of precursor ion scans. ESI-MS and CID-MS/MS analyses have thus proven to be a specific and very sensitive method for the structural identification of these novel 1,5-benzodiazepine derivatives.     

Multiple-stage mass spectrometry analysis of bisphenol A diglycidyl ether, bisphenol F diglycidyl ether and their derivatives

The fragmentation of bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their derivatives was studied by electrospray ionization tandem mass spectrometry. Multiple-stage mass spectrometry and accurate mass measurements were combined to establish the fragmentation pathways. BADGEs and BFDGEs tend to form ammonium adducts under electrospray conditions which fragmented easily. The fragmentation of [M+NH(4)](+) for BADGEs started with the cleavage of the phenyl-alkyl bond, which was followed by the α-cleavage of the ether group to generate the characteristic product ions at m/z 135, [C(9)H(11)O](+), and m/z 107, [C(7)H(7)O](+). The fragmentation of the BFDGE isomer mixtures was studied by on-line reversed-phase liquid chromatography coupled to multiple-stage mass spectrometry (LC/MS(n)). Information obtained from product ion spectra for each BFDGE isomer and its comparison with the fragmentation pathway of BADGE allowed each isomer and the chromatographic elution order to be identified.