Intact glucosinolate analysis in plant extracts by programmed cone voltage electrospray LC/MS: performance and comparison with LC/MS/MS methods

We present a comprehensive, sensitive, and highly specific negative ion electrospray LC/MS method for identifying all structural classes of glucosinolates in crude plant extracts. The technique is based on the observation of simultaneous maxima in the abundances of the m/z 96 and 97 ions, generated by programmed cone voltage fragmentation, in the mass chromatogram. The abundance ratios lie in the range 1:2-1:4 ([m/z 96]/[m/z 97]). Examination of the corresponding full-scan mass spectra allows individual glucosinolates of all structural classes to be identified rapidly and with confidence. The use of linearly programmed cone voltage fragmentation enhances characteristic fragment ions without compromising the abundance of the analytically important [M - H]- ion and its associated (and analytically useful) sulfur isotope peaks. Detection limits are in the low nanogram range for full-scan, programmed cone voltage spectra. Comparison of the technique with LC/MS/MS methods (product ion, precursor ion, and constant neutral loss scans) has shown that the sensitivity and selectivity of the programmed cone voltage method is superior. Data obtained on a variety of plant extracts confirmed that the methodology was robust and reliable.     

Characterisation of glucosinolates using electrospray ion trap and electrospray quadrupole time-of-flight mass spectrometry

Twelve naturally occurring glucosinolates displaying alkenyl, hydroxylated, methylsulphinyl, aromatic and indole side chains were investigated by both negative and positive ion electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). In order to resolve the MS/MS spectra obtained from the anion and cation molecular ions of glucosinolates, the different fragments were investigated by MSn experiments using an ion trap spectrometer. The MS3 spectra obtained permitted possible fragmentation schemes to be proposed. These were supported by accurate mass measurements of some characteristic diagnostic ions with the help of a quadrupole time-of-flight instrument. The negative ion ESI-MS/MS behaviour of the different glucosinolates investigated in this study confirmed previously described patterns and revealed new interesting structural informative fragments. Some are common to all the glucosinolates and others are highly specific for a type of variable side chain. The positive ion ESI-MS/MS fragments obtained from the [MNa+Na]+ or [MK+K]+ molecular ions did not provide complementary specific diagnostic ions. Nevertheless, when compared with the negative ion mode, the daughter ions appeared more homogenous and with a better relative abundance for all of the 12 compounds studied. Moreover, the positive ion mode appeared to be more efficient than the negative mode for the study of methoxylated glucosinolates and should be useful to detect the glucosinolates present as organic salts in crude plant extracts.     

Positive and negative electrospray ionisation tandem mass spectrometry as a tool for structural characterisation of acid released oligosaccharides from olive pulp glucuronoxylans

Xylo-oligosaccharides with degrees of polymerisation 5-13, formed by partial acid hydrolysis from an extract representative of olive pulp glucuronoxylans (GX), were analysed by electrospray ionisation mass spectrometry (ESI-MS), both in positive and negative modes. The positive spectrum showed the presence of xylo-oligosaccharides in the mass range between m/z 500 and 1500 corresponding to singly [M+Na](+) charged ions of neutral (Xyl(7-9)) and acidic xylo-oligosaccharides (Xyl(5-9)MeGlcA), and doubly [M+2Na](2+) charged ions of Xyl(9-13) and Xyl(7-11)MeGlcA. Ammonium adducts [M+NH(4)](+) were also observed for Xyl(5-9)MeGlcA. The negative spectra showed the contribution of ions in the mass range between m/z 600 and 1400, ascribed to the deprotonated molecules [M-H](-) of Xyl(3-9)MeGlcA. Tandem mass spectrometry (MS/MS) of the major ions observed in the MS spectra was performed. The MS/MS spectra of the [M+Na](+) adducts showed the loss of MeGlcA residues as the major fragmentation pathway and glycosidic fragment ions of Xyl(n) and Xyl(n)MeGlcA structures. The MS/MS spectra of the [M+NH(4)](+) adducts suggests the occurrence of isomers of Xyl(5-9)MeGlcA oligosaccharides with the MeGlcA residue at the reducing end and at the non-reducing end of the molecules, although other structural isomers can also occur. Both glycosidic bond and cross-ring cleavages in the MS/MS spectra of the [M-H](-) ion suggest the occurrence of Xyl(3-9)MeGlcA with the substituting group at the reducing end position of the xylose backbone, as the main fragmentation ions. The results obtained by ESI-MS/MS, both in positive and negative modes, of Xyl(7-13)- and Xyl(5-11)MeGlcA, allow to identify fragmentation patterns of the structural isomers with MeGlcA linked to the terminal xylosyl residues of the oligosaccharides. The occurrence of these higher molecular weight oligosaccharides with a low substitution pattern allows to infer a scatter and random distribution of MeGlcA along the xylan backbone of olive pulp.     

Positional identification of fluorine in methyl per-O-acetyl-x-deoxy-x-fluoro-alpha-D-hexopyranosides by electron impact and chemical ionisation mass spectrometry.

Fully acetylated methyl x-deoxy-x-fluoro-alpha-D-glucopyranosides have been studied using electron impact and ammonia chemical ionisation mass spectrometry. Mass analysed metastable ion kinetic energy spectroscopy (MIKE), collisional activation (CID), and accelerated voltage scanning have been used to evaluate complete fragmentation schemes. Characteristic differences in the fragmentation of positional isomers were noted on analysis of the spectra, and these make it possible to determine the location of fluorine in the molecules studied. Collisionally activated fragmentation of [M-OCH3]+ ions, produced by electron impact, provides an alternative method for localisation of the fluorine atoms. To the contrary, MIKE and CID spectra of [M + NH4]+ cluster ions produced by chemical ionisation did not afford such structural information.     

Analysis of N-glycans from recombinant immunoglobulin G by on-line reversed-phase high-performance liquid chromatography/mass spectrometry

An on-line reversed-phase (RP) high-performance liquid chromatography/mass spectrometry (MS) method has been developed for profiling and characterizing N-glycans from recombinant immunoglobulin G antibodies. In this method, released N-glycans are derivatized at their reducing end with 2-aminobenzamide (2AB) and separated on a RP column with on-line fluorescence and MS detection. The method achieves good resolution of all major glycans and segregates glycan types (high-mannose, hybrid, and complex) to different regions of the chromatogram, thus allowing accurate quantification of N-glycans from the fluorescent signal alone. Moreover, the mobile phase used allows high quality on-line MS detection. The 2AB-labeled N-glycans demonstrate good ionization efficiency in electrospray and generate primarily doubly charged [M+2H](2+) ions. The mass and structural information can be readily obtained from the on-line MS and tandem MS data. As little as 70 fmol glycan species can be detected and identified.     

Immunocytochemical and molecular data guide peptide identification by mass spectrometry: orcokinin and orcomyotropin-related peptides in the stomatogastric nervous system of several crustacean species.

In order to identify new orcokinin and orcomyotropin-related peptides in crustaceans, molecular and immunocytochemical data were combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the crayfish Procambarus clarkii, four orcokinins and an orcomyotropin-related peptide are present on the precursor. Because these peptides are highly conserved, we assumed that other species have an identical number of peptides. To identify the peptides, immunocytochemistry was used to localize the regions of the stomatogastric nervous system in which orcokinins are predominantly present. One of the regions predominantly containing orcokinins was a previously undescribed olive-shaped neuropil region within the commissural ganglia of the lobsters Homarus americanus and Homarus gammarus. MALDI-TOF MS on these regions identified peptide masses that always occur together with the known orcokinins. Seven peptide ions occurred together in the peptide massspectra of the lobsters. Mass spectrometric fragmentation by MALDI-MS post-source decay (PSD) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI Q-TOF MS) collision-induced dissociation (CID) were used in the identification of six of these masses, either as orcokinins or as orcomyotropin-related peptides and revealed three hitherto unknown peptide variants, two of which are [His13]-orcokinin ([M+H]+ = 1540.8 Da) and an orcomyotropin-related peptide FDAFTTGFGHN ([M+H]+ = 1213.5 Da). The mass of the third previously unknown orcokinin variant corresponded to that of an identified orcokinin, but PSD fragmentation did not support the suggested amino acid sequence. CID analysis allowed partial de novo sequencing of this peptide. In the crab Cancer pagurus, five orcokinins and an orcomyotropin-related peptide were unambigously identified, including the previously unknown peptide variant [Ser9-Val13]-orcokinin ([M+H]+ = 1532.8 Da).     

Characterization of betaines using electrospray MS/MS

Betaines are an important class of naturally occurring compounds that function as compatible solutes or osmoprotectants. Because of the permanent positive charge on the quaternary ammonium moiety, mass spectrometric analysis has been approached by desorption methods, including fast atom bombardment and plasma desorption mass spectrometry. Here we show that electrospray ionization MS gives comparable results to plasma desorption MS for a range of authentic betaine standards and betaines purified from plant extracts by ion exchange chromatography. A distinct advantage of electrospray ionization MS over plasma desorption MS is the capability of obtaining product ion spectra via MS/MS of selected parent ions, and hence structural information to discriminate between ions of identical mass.     

Selective analysis of phenolic compounds in propolis by HPLC-MS/MS

Phenolic compounds (flavonoids and phenolic acid derivatives) are major active constituents of the resinous fraction of propolis, and also represent its allergenic principles. We have developed a chromatography electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) method to characterise the polyphenolic fraction of propolis rapidly and quali-quantitatively. With precursor ion scanning, selective detection of caffeic esters was easily achieved, confirming the identification of prenyl caffeate, benzyl caffeate and phenylethyl caffeate by comparison with synthetic standards. The ionisation and fragmentation behaviour of the major propolis flavonoids was rationalised and applied to selected real samples. Taken together, the results of this study show that the introduction of precursor ion analysis leads to a significant improvement in the characterisation of the phenolic fraction of propolis, paving the way to the establishment of a better quality control for this important natural remedy. Copyright (c) 2007 John Wiley & Sons, Ltd.     

Mass spectrometric analysis of catechol-histidine adducts from insect cuticle

Adducts of catechols and histidine, which are produced by reactions of 1,2-quinones and p-quinone methides with histidyl residues in proteins incorporated into the insect exoskeleton, were characterized using electrospray ionization mass spectrometry (ESMS), tandem electrospray mass spectrometry (ESMS-MS, collision-induced dissociation), and ion trap mass spectrometry (ITMS). Compounds examined included adducts obtained from acid hydrolysates of Manduca sexta (tobacco hornworm) pupal cuticle exuviae and products obtained from model reactions under defined conditions. The ESMS and ITMS spectra of 6-(N-3')-histidyldopamine [6-(N-3')-His-DA, pi isomer] isolated from M. sexta cuticle were dominated by a [M + H]+ ion at m/z 308, rather than the expected m/z 307. High-resolution fast atom bombardment MS yielded an empirical formula of C14H18N3O5, which was consistent with this compound being 6-(N-1')-histidyl-2-(3, 4-dihydroxyphenyl)ethanol [6-(N-1')-His-DOPET] instead of a DA adduct. Similar results were obtained when histidyl-catechol compounds linked at C-7 of the catechol were examined; the (N-1') isomer was confirmed as a DA adduct, and the (N-3') isomer identified as an (N-1')-DOPET derivative. Direct MS analysis of unfractionated cuticle hydrolysate revealed intense parent and product ions characteristic of 6- and 7-linked adducts of histidine and DOPET. Mass spectrometric analysis of model adducts synthesized by electrochemical oxidative coupling of N-acetyldopamine (NADA) quinone and N-acetylhistidine (NAcH) identified the point of attachment in the two isomers. A prominent product ion corresponding to loss of CO2 from [M + H]+ of 2-NAcH-NADA confirmed this as being the (N-3') isomer. Loss of (H2O + CO) from 6-NAcH-NADA suggested that this adduct was the (N-1') isomer. The results support the hypothesis that insect cuticle sclerotization involves the formation of C-N cross-links between histidine residues in cuticular proteins, and both ring and side-chain carbons of three catechols: NADA, N-beta-alanyldopamine, and DOPET.     

Structural analysis of choline phospholipids by fast atom bombardment mass spectrometry and tandem mass spectrometry

The structures of intact choline phospholipids were determined by positive and negative ion mode fast atom bombardment mass spectrometry, tandem mass spectrometry, and B2/E and B/E constant linked scan mass spectrometry. The molecular weight of the choline lipid could be clearly determined by the appearance of [M + H]+ or [M + Na]+ in the positive ion mode and triplet ions, e.g., [M - 15]-, [M - 60]-, and [M - 86]-, in the negative ion mode. The structures of the triplet ions were assigned to [M - CH3]-, [M - HN(CH3)3]-, and [M - CH2 = CHN(CH3)3]-, respectively, by the MS/MS of each triplet ion, and the origin of the triplet ions was found as the matrix-ion adduct to the target molecule by using the B2/E linked scan technique. The polar group could be identified by the existence of ions indicating glycerophosphocholine and its cleavage products and by the presence of the triplet ions in the negative ion mode. Positional determination of the distribution of constituent fatty acyl groups was carried out by comparing the intensity of deacylated ions from positions 1 and 2 in the positive ion mode and of the ions produced by MS/MS of the triplet ions. From the mass number of the [RCOO]- ion which appeared in the negative ion mode, the molecular weight and degree of unsaturation of the fatty acyl group were determined. The position of double bond(s) in the acyl group was determined from the MS/MS of the [RCOO]- ion.     

Analysis of derivatised steroids by matrix-assisted laser desorption/ionisation and post-source decay mass spectrometry

Neutral steroids are difficult to analyse using desorption ionisation methods coupled with mass spectrometry (MS). However, steroids with an unhindered ketone group can readily be derivatised with the Girard P (GP) reagent to give GP hydrazones. Steroid GP hydrazones contain a quaternary nitrogen atom and are readily desorbed in the matrix-assisted laser desorption/ionisation (MALDI) process, giving an improvement in sensitivity of two orders of magnitude. Steroids without a ketone group, but with a 3beta-hydroxy-Delta5 function, can be readily converted to 3-oxo-Delta4 steroids and subsequently derivatised to GP hydrazones for MALDI analysis. In addition to giving strong [M]+ ions upon MALDI, steroid GP hydrazones give informative post-source decay (PSD) spectra. By using the accurate mass of the precursor-ion measured by MALDI-MS, in combination with the structural information encoded in its PSD spectrum, steroid structures can readily be determined.     

Gas chromatography/mass spectrometry of bacterial amines

Bacterial amines were examined by gas chromatography/mass spectrometry. Under electron impact all trifluoroacetamides exhibited peaks at m/z 69 due to [CF3]+. Many trifluoroacetamides also showed peaks at m/z 97 corresponding to the [COCF3]+ ion fragment. The spectra of n-alkyl and aralkyl trifluoroacetamides were consistent with the spectra and their interpretations in the earlier literature. Molecular ions were of low abundance for all alkyl trifluoroacetamides having alkyl chains longer than two carbon atoms. Chemical ionization gave molecular weight information in all cases. Most peaks observed were molecular addition products, e.g. [M + H]+ and [M + NH4]+. Application of chemical ionization mass spectrometry to analysis of bacterial amines revealed the production of beta-phenylethylamine, n-decylamine, 1,4-diaminobutane and 1,5-diaminopentane by Clostridium histolyticum; whereas both Clostridium bifermentans and Clostridium oedematiens produced beta-phenylethylamine. The latter organism also produced a peak with a retention time similar to that of an authentic amylamine derivative.     

Isolation and characterisation of procyanidins from Rumex obtusifolius

An acetone:water (7:3) extract obtained from the leaves of Rumex obtusifolius was fractionated into procyanidin oligomer and polymer fractions using a linear gradient and a simple step method on Sephadex LH-20. The chemical characteristics of the procyanidin fractions were studied by 13C-NMR spectroscopy, acid-catalysed degradation in the presence of benzyl mercaptan, matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) MS and electrospray ionisation (ESI) MS. The 13C-NMR showed that the polymer fraction consisted predominantly of procyanidin polymers, some with galloyl groups attached. The thiolysis reaction products indicated a mean degree of polymerisation (DP) of 4.3 for the step method, and a range of 2.3-8.2 mean DP for the gradient fractionation, with epicatechin as the most abundant flavan-3-ol extension unit, while the terminal units consisted of equal proportions of catechin, epicatechin and epicatechin gallate. Singly charged ions observed in MALDI-TOF/MS showed a range of oligomeric procyanidins and their polygalloyl derivatives. These species (in the range DP 2-7) were also observed by ESI/MS but the spectra were more complex due to overlapping multiply charged ions. Isolation of oligomers from the Sephadex LH-20 fraction by chromatography on polyamide and C18 yielded B1, B2, B3 and B7 dimers, an A-type trimer and a B2 3,3'-O-digallate.     

Fragmentation pattern of underivatised xylo-oligosaccharides and their alditol derivatives by electrospray tandem mass spectrometry

Maltopentaose and olive pulp xylo-oligosaccharides and the correspondent alditol derivatives were analysed by ESI-MS and ESI-MS/MS. The ESI-MS spectrum of maltopentaose and maltopentaose alditols showed [M+Na]⁺and [M+H]⁺ ions. ESI-MS spectrum of xylo-oligosaccharides and their alditols showed [M+Na]⁺of neutral (Xyl_3–6) and acidic (Xyl_2–3MeGlcA and Xyl_2–3GlcA) xylo-oligosaccharides. The ESI-MS/MS spectra of maltopentaose and underivatised xylo-oligosaccharides presented fragments of glycosidic cleavages attributed to B/Z and C/Y ions. On the other hand, MS/MS spectra of the correspondent alditols showed glycosidic cleavages unambiguously identified as B-type and Y-type ions. Y-type fragment ions showed higher abundance in the MS/MS spectra of the alditol derivatives when compared to the non-reduced samples. The study of the oligoxylosyl alditols fragmentation permits to distinguish fragmentation pathways that occur both from the reducing end and from the non-reducing end of the xylan chain, allowing to obtain more information about the localization of the acidic substituent along the glucuronoxylan backbone.     

Taxoids profiling of suspension Taxus chinensis var. mairei cells in response to shear stress

To develop an optimal bioprocess for paclitaxel (Taxols) supply, taxoid biosynthetic pathway regulation must be better understood. The main taxoid metabolites (paclitaxel, baccatin III, taxol C, etc.) in Taxus cell culture showed great difference under shear stress. However, the regulating mechanism of taxoids metabolism under shear stress remained elusive. Here an efficient metabolic profiling approach combined with multivariate analysis was employed to profile taxoids changes of Taxus cells under laminar shear stress. A total of 21 taxoids were identified and quantified by ultra-performance liquid chromatography coupled with Q-TOF mass spectrometry. The result showed the contents of paclitaxel and baccatin III were reduced by shear stress, indicating the inhibitory effect of shear stress on paclitaxel biosynthesis. The levels of other taxoids uninvolved in paclitaxel biosynthesis were decreased except several metabolites. Further analysis of mapping measured taxoids concentrations onto paclitaxel biosynthesis pathway illustrating proposed intermediates and “off-pathway” metabolites revealed shear stress might disrupt the appropriate cyclization process of geranylgeranyl-pyrophosphate, aggravate the inappropriate order of hydroxylations and acylations, and not be good for functional group oxetane formation. These findings revealed the possible mechanism for shear stress limiting paclitaxel production and might have important biotechnological applications to increase the yields of paclitaxel and relevant precursors.     

Identification of the major metabolites of resveratrol in rat urine by HPLC-MS/MS

To identify the major metabolites of resveratrol in rat, rat urine samples were pretreated by using solid-phase extraction technique (SPE) with polyamide cartridges. And a LC-MS/MS method with electrospray ionisation (ESI), negative ion mode and collision induced dissociation (CID), was used to elucidate the structures of the major metabolites of resveratrol. According to the results of our experiment, we found that the main metabolites of resveratrol were resveratrol monoglucuronide (M1), dihydroresveratrol monosulfate (M2), resveratrol monosulfate (M3) and dihydroresveratrol (M4).     

Analysis of sesterterpenoids from Aspergillus terreus using ESI‐QTOF and ESI‐IT

Introduction – Biosynthesis of terretonin was studied due to the interesting skeleton of this series of sesterterpenoids. Very recently, López‐Gresa reported two new sesterterpenoids (terretonins E and F) which are inhibitors of the mammalian mitochondrial respiratory chain. Mass spectrometry (MS), especially tandem mass spectrometry, has been one of the most important physicochemical methods for the identification of trace natural products due to it rapidity, sensitivity and low levels of sample consumption. The potential application prospect and unique skeleton prompted us to study structural characterisation using MS. Objective – To obtain sufficient information for rapid structural elucidation of this class of compounds using MS. Methodology – The elemental composition of the product ions was confirmed by low‐energy ESI‐CID‐QTOF‐MS/MS analyses. The fragmentation pathways were postulated on the basis of ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ spectra. Common features and major differences between ESI‐QTOF‐MS/MS and IT‐MSⁿ spectra were compared. For ESI‐QTOF‐MS/MS/MS experiments, capillary exit voltage was raised to induce in‐source dissociation. Ammonium acetate or acetic acid were added into solutions to improve the intensity of [M + H]⁺. The collision energy was optimised to achieve sufficient fragmentation. Some fragmentation pathways were unambiguously proposed by the variety of abundance of fragment ions at different collision energies even without MSⁿ spectra. Results – Fragmentation pathways of five representative sesterterpenoids were elucidated using ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ in both positive‐ and negative‐ion mode. The key group of characterising fragmentation profiles was ring B, and these fragmentation patterns are helpful to identify different types of sestertepenoids. Conclusion – Complementary information obtained from fragmentation experiments of [M + H]⁺ (or [M + NH₄]⁺) and [M ? H]^? precursor ions is especially valuable for rapid identification of this kind of sesterterpenoid.     

Spectroscopic characterization of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

The properties of the [4Fe-4S] cluster in glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis have been investigated using low temperature magnetic circular dichroism, electron paramagnetic resonance (EPR), and resonance Raman spectroscopies. The Raman spectra of the native enzyme in the Fe-S stretching region show a [4Fe-4S]2+ cluster that is structurally very similar to those in simple redox proteins. Photochemical reduction mediated by 5-deazaflavin with oxalate as the electron donor resulted in [4Fe-4S]+ clusters with a mixture of ground state spin multiplicities. Magnetic circular dichroism and EPR studies of samples ranging in concentration from 0.15 to 0.4 mM concur in finding S = 3/2 [4Fe-4S]+ clusters with predominantly axial and positive zero field splitting as the dominant species. The EPR studies also revealed minor contributions from S = 1/2 [4Fe-4S]+ centers and an S = 5/2 species. The latter becomes the dominant component in more concentrated samples (approximately 2 mM), and arguments are presented in favor of assignment to S = 5/2 [4Fe-4S]+ clusters rather than adventitiously bound high spin Fe(III) ions. The concentration-dependent spin state heterogeneity of the [4Fe-4S]+ cluster in glutamine phosphoribosylpyrophosphate amidotransferase is discussed in light of the magnetic and electronic properties of the [4Fe-4S]+ centers in other enzymes and proteins.     

Development and validation of an LC/MS/MS assay for mycophenolic acid in human peripheral blood mononuclear cells

The aim was to develop a LC/MS/MS method able to quantify mycophenolic acid (MPA) in the peripheral blood mononuclear cells (PBMCs) of transplanted patients. PBMCs were isolated from blood by a density gradient separation. The chromatographic separation was carried out on a Zorbax Stable Bond CN, 150 mmx2.1 mm, and MS/MS detection was performed after positive electrospray ionisation of the protonated parent ion. The calibration range was from 0.25 to 100 ng/sample. Extraction from the cells and ionisation recoveries reached 73.5 and 37.9%, respectively. Inaccuracy was always <10% with CVs<15%. MPA was stable at room temperature in the autosampler over 48 h and at -20 degrees C over 1.5 months. Application to clinical samples taken from patients treated with mycophenolate mofetil indicated that the method is suitable for measuring intracellular MPA.