Liquid chromatography/electrospray ionization tandem mass spectrometry analysis of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)

A quantitative liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed for the analysis of the explosive, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). In negative ionization mode, HMX forms an acetate adduct ion [M + CH(3)COO](-), m/z 355, in the presence of a small amount of acetic acid in the mobile phase. The ESI collision-induced dissociation (CID) spectrum of m/z 355 was acquired and the transitions m/z 355 --> 147 and m/z 355 --> 174 were chosen for the determination of HMX in samples. Using this quantification technique, the method detection limit was 1.57 microg/L and good linearity was achieved in the range 5-500 microg/L. This method will help to unambiguously analyze environmentally relevant concentrations of HMX.     

An alternative sampling algorithm for use in liquid chromatography/tandem mass spectrometry experiments

In liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses of complex peptide mixtures, dynamic exclusion functions are used to minimize repeat selections of identical precursors for collision induced dissociation (CID). We describe a new algorithm for the dynamic exclusion of m/z values during LC/MS/MS. Full-scan based peak exclusion (Fulspec) uses a simplified model of chromatographic peak formation to detect and exclude contaminants present throughout the run or that lead to broad peaks. Therefore, instead of excluding peptides from fragment analysis according to a rigidly predefined time window, the chromatographic properties of the detected analytes are used. The algorithm was tested on two datasets derived from previously published experiments. Fulspec achieves a distribution of CID spectra with minimal tailing on the retention time axis, without resorting to rigid exclusion of m/z values. The procedure further excludes intensities with a bias towards low-quality CID spectra. This combination frees up valuable analytical capacity. The underlying intensity vs. quality analyses challenge the assumption that abundant precursors automatically give the best identifications. Further validation of the algorithm will require its incorporation by equipment manufacturers into the instrument control programs.     

Characterisation of a series of acetals/ketals of bis(2-nitrophenyl) ethanediol and bis(4,5-dimethoxy-2-nitrophenyl) ethanediol under APCI mass spectrometric conditions

A series acetals/ketals of aldehydes and ketones formed by the reaction of two photolabile protecting groups, bis(2-nitrophenyl) ethanediol and bis(4,5-dimethoxy-2-nitrophenyl) ethanediol (I and II, respectively), were analysed under EI, LSIMS, ESI and APCI conditions to obtain molecular weights as well as structural information. The EI and LSIMS techniques failed to give molecular weight information. The positive ESI yielded [M + H](+) ions only for I; however, with added Na(+) both I and II formed [M + Na](+) adducts. But upon decomposition, the [M + Na](+) ions yielded Na(+) ion as the only product ion. Similarly, under negative ion ESI conditions both I and II gave molecular weight information by forming adduct ions with halide anions (F(-), Cl(-), Br(-) and I(-)); however, they did not give structural information as they resulted in only the halide anion as the abundant fragment ion upon dissociation. All the compounds formed abundant M(-*) ions under negative ion APCI conditions, and their MS/MS spectra showed characteristic fragment ions; hence the acetals/ketals of I and II could be successfully characterized under negative ion APCI conditions.     

Solid-phase microextraction liquid chromatography/tandem mass spectrometry for the analysis of chlorophenols in environmental samples

Liquid chromatography with atmospheric pressure chemical ionisation mass spectrometry (LC/APCI-MS), using negative ion detection in a triple quadrupole instrument, was used for the determination of chlorophenols (CPs) in environmental samples. In-source collision-induced dissociation (CID) was compared with MS/MS fragmentation. In general, less fragmentation was observed in MS/MS as compared with in-source CID, with the latter providing more intense fragment ions due to chemical ionisation. Under MS/MS conditions [M - H - HCl](-) was the main fragment ion observed for all compounds except for pentachlorophenol, which showed no fragmentation. For multiple reaction monitoring (MRM) acquisition mode, the transition from [M - H](-) to [M - H - HCl](-) was selected, leading to detection limits down to 0.3 ng injected. Direct and headspace-solid-phase microextraction (HS-SPME) were used as preconcentration procedures for the analysis of CPs in wood and in industrially contaminated soils. CPs were quantified by standard addition, which led to good reproducibility (RSD between 4 and 11%) in both SIM and MRM modes, and detection limits down to ng/g. The combination of MS/MS and in-source CID allowed confirmation of the presence of CPs in environmental samples.     

Investigation of the electrochemical oxidation products of zotepine and their fragmentation using on-line electrochemistry/electrospray ionization mass spectrometry

When zotepine, an antipsychotic drug, was electrochemically oxidized using electrospray ionization mass spectrometry (ESI-MS) coupled with a microflow electrolytic cell, [M + 16 + H]+ (m/z 348), [M-H]+ (m/z 330) and [M-14 + H]+ (m/z 318) were observed as electrochemical oxidation product ions (M represents the zotepine molecule). Although a major fragment ion that was derived from the dimethyl aminoethyl moiety was observed only at m/z 72 in the collision-induced dissociation (CID) spectrum of zotepine, new fragments such as m/z 315 and 286 ions could be generated in the CID spectrum by combining electrochemical oxidation and CID. Since these fragments were relatively specific with high ion strength, it was thought that they would be useful for developing a sensitive LC-MS/MS assay. The S-oxide and N-demethylated products were detected by electrolysis assuring that a portion of P450 metabolites of zotepine could be mimicked by the electrochemistry/electrospray ionization mass spectrometry (EC/ESI-MS) system.     

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.     

The application of "double isolation" in Fourier transform ion cyclotron resonance sustained off-resonance irradiation collisionally-induced dissociation tandem mass spectrometry to remove labile isobaric impurities

This study reports the application of "double isolation" in sustained off-resonance irradiation collisionally-induced dissociation tandem mass spectrometry (SORI-CID-MS/MS) to remove radio- frequency (RF) fragment ions of very close mass isobaric ions (0.02 m/z apart). Analyses were performed with a fraction of a biological extract isolated from a macroalgae containing the mycosporine-like amino acid asterina-330. Direct isolation of the precursor ion by narrowing the isolation window proved ineffective as it impinged upon the required ion thus substantially reducing its intensity. By increasing the correlated sweep time, ejection efficiency of the isolation was improved, but caused the unwanted side-effect of RF fragmentation of labile ions. Finally, by skipping the ion activation step and performing a second isolation (in the MS(3) module) the RF fragments from the first isolation were removed leaving a very pure isolation of the required precursor ion and allowed a very clean CID fragmentation. We demonstrated that the m/z 272.1351 ion is derived from the loss of NH(3) from m/z 289.1620 isobaric impurity and is not related to asterina-330. This application represents a powerful tool to remove unwanted ions in the MS/MS spectrum that result from fragmentation of isobaric ions.     

Liquid chromatographic/electrospray ionization tandem mass spectrometric study of the phenolic composition of cocoa (Theobroma cacao)

Liquid chromatography coupled with ionspray mass spectrometry in the tandem mode (LC/MS/MS) with negative ion detection was used for the identification of a variety of phenolic compounds in a cocoa sample. Gradient elution with water and acetonitrile, both containing 0.1% HCOOH, was used. Standard solutions of 31 phenolic compounds, including benzoic and cinnamic acids and flavonoid compounds, were studied in the negative ion mode using MS/MS product ion scans. At low collisional activation, the deprotonated molecule [M - H](-) was observed for all the compounds studied. For cinnamic and benzoic acids, losses of CO(2) or formation of [M - CH(3)](-*) in the case of methoxylated compounds were observed. However, for flavonol and flavone glycosides, the spectra present both the deprotonated molecule [M - H](-) of the glycoside and the ion corresponding to the deprotonated aglycone [A - H](-). The latter ion is formed by loss of the rhamnose, glucose, galactose or arabinose residue from the glycosides. Different fragmentation patterns were observed in MS/MS experiments for flavone-C-glycosides which showed fragmentation in the sugar part. Fragmentation of aglycones provided characteristic ions for each family of flavonoids. The optimum LC/MS/MS conditions were applied to the characterization of a cocoa sample that had been subjected to an extraction/clean-up procedure which involved chromatography on Sephadex LH20 and thin-layer chromatographic monitoring. In addition to compounds described in the literature, such as epicatechin and catechin, quercetin, isoquercitrin (quercetin-3-O-glucoside) and quercetin-3-O-arabinose, other compounds were identified for the first time in cocoa samples, such as hyperoside (quercetin-3-O-galactoside), naringenin, luteolin, apigenin and some O-glucosides and C-glucosides of these compounds.     

Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry

Three samples of albumin derived from human plasma (pharmaceutical grade, HSA) obtained from different commercial sources were investigated for their micro-heterogeneities by means of electrospray ionization (ESI) ion trap mass spectrometry (ITMS). The study covered MS analyses of the intact proteins as well as on the tryptic peptide level. The intact protein samples were analyzed without any separation step except for simple desalting. With these samples we observed in the positive ion ESI mass spectra that the multiply charged ion signals of HSA consisted of a number of fully or partly resolved peaks with relative intensities depending on the analyzed sample. The non-modified form of HSA was detected in the three HSA preparations at m/z values of 66448 +/- 3.6, 66450 +/- 0.6 and 66451 +/- 3.2 ([MH]+), respectively. The value calculated from the amino acid sequence was 66439. The second compound present with high intensity (in two cases the base peak in the deconvoluted mass spectrum) is interpreted as a modified HSA, and the molecular mass increase in relation to the unmodified HAS was between 116 and 118 Da (m/z of 66 564, 66 567 and 66 569), suggesting the presence of a covalently bound cysteine residue. A further peak in the deconvoluted ESI spectra was found in all three samples with rather low signal/noise ratio at m/z 66 619, 66 621 and 66 613, respectively, which may correspond to a non-enzymatic glycation described in the literature. The verification of the proposed covalent HSA modifications was subsequently done on the peptide level using high-performance liquid chromatography (HPLC)/ESI-MS and HPLC/ESI-MS/MS including low-energy collision-induced dissociation (CID). Prior to the tryptic digestion, the HSA samples were alkylated without a prior reduction step. Following this procedure we detected peptides of the sequence T21-41 that included the Cys-34 residue in both forms: cysteinylated (m/z 639.15 [M+4H]4+) as well as vinylpyridine-alkylated (m/z 635.69 [M+4H]4+, which means in its previously native free SH form). In the next step on-line LC/ESI low-energy CID MS/MS experiments were performed to verify these two proposed structures. By means of MS/MS analysis of the mentioned ions the described modification (cysteinylation) at the Cys-34 residue could be proven. This abundant modification of HSA in pharmaceutical-grade preparations could be unambiguously identified as cysteinylation at the Cys-34 residue. On the other hand, the proposed non-enzymatic glycation was not detectable on the peptide level in the on-line HPLC/ESI-MS mode, maybe due to the low concentration in the three samples under investigation.     

Complexes of dibromo(ethylenediamine)-palladium(II) observed from aqueous solutions by electrospray mass spectrometry

The dimeric complex of dibromo(ethylenediamine)palladium(II) observed in water was investigated using electrospray mass spectrometry. One microM aqueous solutions of Pd(en)Br2 yielded a variety of previously unreported species. The most abundant ion observed was attributable to the Pd(en)Br2.Pd(en)Br+ dimeric complex at m/z 568.7 (most abundant stable isotopes). The characteristics of the oligomeric complexes were examined using collision-induced dissociation (CID) up to MS6. The most common loss mechanism observed was loss of HBr leaving an unsaturated Pd(II) center. Fragmentation of the ethylenediamine ligand was also observed during CID experiments. Loss of Pd was only observed as the final step in the CID process when other loss mechanisms had been exhausted. A number of calculations were carried out at the B3-LYP/SBKJC[d] level of theory in an attempt to elucidate the structure of the [2M-Br]+ dimer.     

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of anatoxins in cyanobacteria and drinking water

Anatoxin-a (AN) and homoanatoxin-a (HMAN) are potent neurotoxins produced by a number of cyanobacterial species. A new, sensitive liquid chromatography/multiple tandem mass spectrometry (LC/MS(n)) method has been developed for the determination of these neurotoxins. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer in positive ion mode. The [M+H](+) ions at m/z 166 (anatoxin-a) and m/z 180 (homoanatoxin-a) were used as the precursor ions for multiple MS experiments. MS(2)bond;MS(4) spectra displayed major fragment ions at m/z 149 (AN), 163 (HMAN), assigned to [Mbond;NH(3)+H](+); m/z 131 (AN), 145 (HMAN), assigned to [Mbond;NH(3)bond;H(2)O+H](+), and m/z 91 [C(7)H(7)](+). Although the chromatographic separation of these neurotoxins is problematic, reversed-phase LC, using a C(18) Luna column, proved successful. Calibration data for anatoxin-a using spiked water samples (10 mL) in LC/MS(n) modes were: LC/MS (25-1000 microg/L), r(2) = 0.998; LC/MS(2) (5-1000(microg/L), r(2) = 0.9993; LC/MS(3) (2.5-1000 microg/L), r(2) = 0.9997. Reproducibility data (% RSD, N = 3) for each LC/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L. The detection limit (S/N = 3) for AN was better than 0.03 ng (on-column) for LC/MS(3) which corresponded to 0.6 microg/L.     

Electrospray ionisation mass spectral studies on hydrolysed products of sulfur mustards

Off-site detection of the hydrolysed products of sulfur mustards in aqueous samples is an important task in the verification of Chemical Weapons Convention (CWC)-related chemicals. The hydrolysed products of sulfur mustards are studied under positive and negative electrospray ionisation (ESI) conditions using an additive with a view to detecting them at trace levels. In the presence of cations (Li(+), Na(+), K(+) and NH(4) (+)), the positive ion ESI mass spectra of all the compounds include the corresponding cationised species; however, only the [M+NH(4)](+) ions form [M+H](+) ions upon decomposition. The tandem mass (MS/MS) spectra of [M+H](+) ions from all the hydrolysed products of the sulfur mustard homologues were distinct and allowed these compounds to be characterised unambiguously. Similarly, the negative ion ESI mass spectra of all the compounds show prominent adducts with added anions (F(-), Cl(-), Br(-), and I(-)), but the [M-H](-) ion can only be generated by decomposition of an [M+F](-) ion. The MS/MS spectra of the [M-H](-) ions from all the compounds result in a common product ion at m/z 77. A precursor ion scan of m/z 77 is shown to be useful in the rapid screening of these compounds in aqueous samples at trace levels, even in the presence of complex masking agents, without the use of time-consuming sample preparation and chromatography steps. An MS/MS method developed to measure the detection limits of the hydrolysed products of sulfur mustards found these to be in the range of 10-500 ppb.     

An unexpected ion-molecule adduct in negative-ion collision-induced decomposition ion-trap mass spectra of halogenated benzoic acids

The ion observed at m/z 145 when product ion spectra of iodobenzoate anions are recorded using ion-trap mass spectrometers corresponds to the adduct ion [I(H(2)O)](-). The elements of water required for the formation of this adduct do not originate from the precursor ion but from traces of moisture present in the helium buffer gas. A collision-induced decomposition (CID) spectrum recorded from the [M-H](-) ion (m/z 251) derived from 3-iodo[2,4,5,6-(2)H(4)]benzoic acid also showed an ion at m/z 145. This observation confirmed that the m/z 145 is not a product ion resulting from a direct neutral loss from the carboxylate anion. (79)Bromobenzoate anions produce similar results showing an ion at m/z 97 for [(79)Br(H(2)O)](-). The ion-molecule reaction observed here is unique to ion-trap mass spectrometers since a corresponding ion was not observed under our experimental conditions in spectra recorded with in-space tandem mass spectrometers such as triple quadrupole or quadrupole time-of-flight instruments.     

Unusual collision-induced dissociation of fluorated and non-fluorated alpha-nitrotoluene analogs in a gas chromatograph triple-stage quadrupole mass spectrometer under electron-capturing negative-ion chemical ionization conditions

Unusual collision-induced dissociation (CID) of perfluorated and non-perfluorated alpha-nitrotoluene analogs in a gas chromatograph triple-stage quadrupole (TSQ) mass spectrometer (GC-QqQ-MS) under electron-capturing negative-ion chemical ionization conditions is reported. CID of [M - 1]- of alpha-nitro-2,3,4,5,6-pentafluorotoluene (C6F5CH2-NO2) and alpha-nitro-2,5-difluorotoluene (C6H3F2CH2-NO2) produced an intense ion with m/z 66. By using 15N- or 18O-labelled C6F5CH2-NO2 analogs, we found that this anion has the formula C3NO. By contrast, CID of [M - 1]- of alpha-nitrotoluene (C6H5CH2-NO2) and alpha-nitro-3,5-difluorotoluene (C6H3F2CH2-NO2) produced an anion with m/z 86 with the formula C3H4NO2. The expected CID of the C-N-bond of all alpha-nitrotoluene analogs to form the nitrite anion (NO2-, m/z 46) did not occur. We propose mechanisms for the formation of the anions C3NO and C3H4NO2 in the collision chamber of the TSQ mass spectrometer. The most likely structures for the anion C3NO are :C=C=C=N--O and N triple bond C-C triple bond C--O-. The unique CID behavior of C6F5CH2--NO2 can be utilized to unequivocally identify and accurately quantify nitrite in biological fluids by GC-tandem MS.     

Analysis of fungal cyclopentenone derivatives from Hygrophorus spp. by liquid chromatography/electrospray-tandem mass spectrometry

Fruitbodies of the genus Hygrophorus (Basidiomycetes) contain a series of anti-biologically active compounds. These substances named hygrophorones possess a cyclopentenone skeleton. LC/ESI-MS/MS presents a valuable tool for the identification of such compounds. The mass spectral behaviour of typical selected members of this group under positive and negative ion electrospray conditions is discussed. Using the ESI collision-induced dissociation (CID) mass spectra of the [M + H]+ and [M - H]- ions, respectively, the compounds can be classified with respect to the substitution pattern at the cyclopentenone ring and the type of oxygenation at C-6 (hydroxy/acetoxy or oxo function) of the side chain. The elemental composition of the fragment ions was determined by ESI-QqTOF measurements. Thus, in case of the negative ion CID mass spectra an unusual loss of CO2 from the deprotonated molecular ions could be observed.     

Structural determination of hexadecanoic lysophosphatidylcholine regioisomers by fast atom bombardment tandem mass spectrometry

The structural determination of sn-1 and sn-2 hexadecanoic lysophosphatidylcholine (LPC) regioisomers was carried out using fast atom bombardment tandem mass spectrometry (FAB-MS/MS). The collision-induced dissociation (CID) of protonated and sodiated molecules produced diverse product ions due mainly to charge remote fragmentations. Based on the information obtained from the CID spectra of protonated and sodiated molecules, sn-1 and sn-2 hexadecanoic LPC isomers could be discriminated. Especially, the abundance ratio of the diagnostic ion pair [m/z 224/226] in the CID spectra of [M + H](+) ions was shown to be greatly different. Moreover, the CID-MS/MS spectra of sodium-adducted molecules for hexadecanoic LPC isomers showed characteristic product ions such as [M + Na - 103](+), [M + Na - 85](+), and [M + Na - 59](+), by which their regio-specificity can be differentiated.     

Comparison of negative and positive ion electrospray tandem mass spectrometry for the liquid chromatography tandem mass spectrometry analysis of oxidized deoxynucleosides

Oxidized deoxynucleosides are widely used as biomarkers for DNA oxidation and oxidative stress assessment. Although gas chromatography mass spectrometry is widely used for the measurement of multiple DNA lesions, this approach requires complex sample preparation contributing to possible artifactual oxidation. To address these issues, a high performance liquid chromatography (HPLC)-tandem mass spectrometric (LC-MS/MS) method was developed to measure 8-hydroxy-2'-deoxyguanosine (8-OH-dG), 8-hydroxy-2'-deoxyadenosine (8-OH-dA), 2-hydroxy-2'-deoxyadenosine (2-OH-dA), thymidine glycol (TG), and 5-hydroxy-methyl-2'-deoxyuridine (HMDU) in DNA samples with fast sample preparation. In order to selectively monitor the product ions of these precursors with optimum sensitivity for use during quantitative LC-MS/MS analysis, unique and abundant fragment ions had to be identified during MS/MS with collision-induced dissociation (CID). Positive and negative ion electrospray tandem mass spectra with CID were compared for the analysis of these five oxidized deoxynucleosides. The most abundant fragment ions were usually formed by cleavage of the glycosidic bond in both positive and negative ion modes. However, in the negative ion electrospray tandem mass spectra of 8-OH-dG, 2-OH-dA, and 8-OH-dA, cleavage of two bonds within the sugar ring produced abundant S1 type ions with loss of a neutral molecule weighing 90 u, [M - H - 90]-. The signal-to-noise ratio was similar for negative and positive ion electrospray MS/MS except in the case of thymidine glycol where the signal-to-noise was 100 times greater in negative ionization mode. Therefore, negative ion electrospray tandem mass spectrometry with CID would be preferred to positive ion mode for the analysis of sets of oxidized deoxynucleosides that include thymidine glycol. Investigation of the fragmentation pathways indicated some new general rules for the fragmentation of negatively charged oxidized nucleosides. When purine nucleosides contain a hydroxyl group in the C8 position, an S1 type product ion will dominate the product ions due to a six-membered ring hydrogen transfer process. Finally, a new type of fragment ion formed by elimination of a neutral molecule weighing 48 (CO2H4) from the sugar moiety was observed for all three oxidized purine nucleosides.     

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.     

Nano-electrospray ionization time-of-flight mass spectrometry of gangliosides from human brain tissue

A general approach for the detection and structural elucidation of brain ganglioside species GM1, GD1 and GT1 by nano-electrospray ionization quadrupole time-of-flight (nanoESI-QTOF) mass spectrometry (MS), using combined data from MS and MS/MS analysis of isolated native ganglioside fractions in negative ion mode and their permethylated counterparts in the positive ion mode is presented. This approach was designed to detect and sequence gangliosides present in preparatively isolated ganglioside fractions from pathological brain samples available in only very limited amounts. In these fractions mixtures of homologue and isobaric structures are present, depending on the ceramide composition and the position of the sialic acid attachment site. The interpretation of data for the entire sequence, derived from A, B, C and Y ions by nanoESI-QTOFMS/MS in the negative ion mode of native fractions, can be compromised by ions arising from double and triple internal cleavages. To distinguish between isobaric carbohydrate structures in gangliosides, such as monosialogangliosides GM1a and GM1b, disialogangliosides GD1a, GD1b and GD1c or trisialogangliosides GT1b, GT1c and GT1d, the samples were analysed after permethylation in the positive ion nanoESI-QTOFMS/MS mode, providing set of data, which allows a clear distinction for assignment of outer and inner fragment ions according to their m/z values. The fragmentation patterns from native gangliosides obtained by low-energy collision induced dissociation (CID) by nanoESI-QTOF show common behaviour and follow inherent rules. The combined set of data from the negative and positive ion mode low-energy CID can serve for the detection of structural isomers in mixtures, and to trace new, not previously detected, components.     

Differentiation of Boc- alpha,beta- and beta,alpha-peptides and a pair of diastereomeric beta,alpha-dipeptides by positive and negative ion electrospray tandem mass spectrometry (ESI-MS/MS)

Positive and negative ion electrospray ionization (ESI) tandem mass spectral study of a new series of hybrid peptides, viz, BocN-alpha,beta-peptides and BocN-beta,alpha-peptides, synthesized from C-linked carbo-beta3-amino acids [Caa (S)] and L-Ala has been carried out. The alpha,beta-peptides have been differentiated from beta,alpha-peptides by the collision-induced dissociation (CID) of [M + H]+ and [M - H]- ions in positive and negative ion ESI-MS respectively. The fragment ion [M + H - C(CH3)3 + H]+ formed from [M + H]+ ions by the loss of 2-methyl-prop-2-ene in alpha,beta-peptides with L-Ala at the N-terminus is insignificant or totally absent for beta,alpha-peptides which have the Caa (S) at N-terminus. The fragment ion [M - H-C(CH3)3OH - HNCO]- formed from [M - H]- of beta,alpha-peptide acids is totally absent for alpha,beta-peptide acids. This has been attributed to the absence of the beta-methylene group in alpha,beta-peptides, and the participation of the beta-methylene group in the loss of HNCO in beta,alpha-peptide acids is confirmed by the deuteration experiments. The CID of [M + H-Boc + H]+ ions of these peptides also produce characteristic fragmentation. In the CID spectra of alpha,beta-peptides, the b(n)+ ions and the resulting y(n)+ ions occur at a mass difference of 243 and 71 Da corresponding to the successive losses of Caa and L-Ala, whereas a mass difference of 71 and 243 Da is observed for beta,alpha-peptides. In contrast to the CID of protonated peptides, the CID of [M - H]- ions of the alpha,beta- and beta,alpha-peptide acids do not give b(n)- ions and show abundant z(n) (-) ions. Further, a pair of diastereomeric dipeptide esters and acids have been distinguished by the CID of [M + H]+ ions. The loss of 2-methyl-prop-2-ene is more pronounced for Boc-NH-Caa(R)-D-Ala-OCH3 (21) and Boc-NH-Caa(R)-D-Ala-OH (23) with Caa (R) at the N-terminus, whereas it is totally absent for Boc-NH-Caa (S)-D-Ala-OCH3 (22) and Boc-NH-Caa(S)-D-Ala-OH (24) peptides, which have Caa (S) at the N-terminus. Thus, on the basis of our previous and present studies, we propose that the CID of [M + H]+ ions provides a simple and useful method for distinguishing the configuration of Caa (S or R) at the N-terminus of BocN-carbo beta,alpha- and beta,beta-dipeptides.