The detection of red pigment-concentrating hormone (RPCH) in crustacean eyestalk tissues using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry: [M + Na]+ ion formation in dried droplet tissue preparations

Red pigment-concentrating hormone (RPCH), an octapeptide found in crustaceans and insects with the sequence pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2, is an N- and C-terminally blocked uncharged peptide. These structural features are shared with many members of the larger adipokinetic hormone (AKH)/RPCH peptide family in insects. We have applied vacuum UV matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron mass spectrometry (FTMS) to the direct analysis of crustacean sinus gland tissues, using 2,5-dihydroxybenzoic acid (DHB) as the MALDI matrix, and have found that RPCH is detected in the cationized, [M + Na]+, form under conditions where other peptides in the direct tissue spectra are protonated without accompanying [M + Na]+ or [M + K]+ satellite peaks. The [M + H]+ ion for RPCH is not detected in tissue samples or for an RPCH standard, even when care is taken to eliminate metal ions. This behavior is not unprecedented; however, both direct tissue spectra and SORI-CID spectra provide no clues to suggest that the ionizing agent is a metal cation. In this communication, we characterize the MALDI-FTMS ionization and SORI-CID mass spectra of the [M + Na]+ and [M + K]+ ions from RPCH, and report on the detection of this neuropeptide in sinus gland tissues from the lobster Homarus americanus and the kelp crab Pugettia producta. We describe two strategies, an on-probe extraction procedure and a salt-doping approach, that can be applied to previously analyzed MALDI tissue samples to enhance and unmask sodiated peptides that may otherwise be mistaken for novel neuropeptides.     

Electrospray ionization mass spectrometric analysis of microcystins, cyclic heptapeptide hepatotoxins: modulation of charge states and [M+H]+ to [M+Na]+ ratio

Electrospray ionization mass spectrometry was used to develop a rapid, sensitive, and accurate method for determination and identification of hepatotoxic microcystins, cyanobacterial cyclic heptapeptides. To optimize the electrospray ionization conditions, factors affecting charge state distribution, such as amino acid components of sample, proton affinity of the additives, and additive concentration, were investigated in detail and a method for controlling charge states was developed to provide molecular-related ions for assignment of molecular weight and reasonably abundant precursor ions for MS/MS analysis. A procedure for identification of microcystins consisting of known amino acids was proposed: for microcystins giving abundant [M + 2H]2+ ions, the addition of nitrogen-containing bases to the aqueous sample solution is effective to obtain an increased intensity of [M + H]+ ions, whereas the addition of Lewis acids containing nitrogen can produce increased abundances of [M + 2H]2+ ions for microcystins giving weak [M + 2H]2+ ions. Microcystins possessing no arginine residue always give sodium adduct ions [M + Na]+ as the base peak, and these are difficult to fragment via low energy collision-induced dissociation to yield structurally informative products; the addition of oxalic acid increases [M + H]+ ion abundances, and these fragment readily.     

Improved protonation, collision-induced decomposition efficiency and structural assessment for 'red tide' brevetoxins employing nanoelectrospray mass spectrometry

Brevetoxins are a group of natural neurotoxins found in blooms of red tide algae. Previous electrospray mass spectrometry (ES-MS) studies show that all brevetoxins have high affinities for sodium ions, and they form abundant sodium adduct ions, [M + Na]+, in ES-MS, even when trace contamination is the only source of sodium ions. Attempts to obtain informative product ions from the collision-induced decomposition (CID) of [M + Na]+ brevetoxin precursor ions resulted only in uninformative sodium ion signals, even under elevated collision energies. In this study, a nano-ES-MS approach was developed wherein ammonium fluoride was used to form cationic [M + NH4]+ adducts of brevetoxin-2 and brevetoxin-3; a significant increase in the abundance of protonated brevetoxin molecules [M + H]+ also resulted, whereas the abundance of sodium adducts of brevetoxins [M + Na]+ was observed to decrease. Under CID, both [M + NH4]+ and [M + H]+ gave similar, abundant product ions and thus underwent the same types of fragmentation. This indicated that ammonium ions initially attached to brevetoxins forming [M + NH4]+ easily lose neutral ammonia in a first step in the gas phase, leaving protonated brevetoxin [M + H]+ to readily undergo further fragmentation under CID.     

Liquid chromatography/tandem mass spectrometric study and analysis of xanthone and secoiridoid glycoside composition of Swertia chirata, a potent antidiabetic

Swertia chirata is a bitter plant, used in the Indian system of medicine (Ayurveda) for various human ailments. The bioactive constituents include the xanthone and secoiridoid glycosides consisting of mangiferin, amarogentin, amaroswerin, sweroside and swertiamarin. Methanolic extracts of S. chirata possess constituents with antidiabetic activities, which was investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Preliminary HPLC analyses were performed on a reversed-phase C18 column using gradient elution. In the LC/ESI-MS spectra, predominant [M+H]+ and [M+Na]+ ions were observed in positive ion mode and provided molecular mass information. The five components of S. chirata were structurally correlated and confirmed based on the fragmentation characteristics and information available in the literature. The fragmentation behavior of [M+H]+/[M+Na]+ ions of these components were deduced from the collision-induced dissociation (CID) spectra obtained from the selective on-column information-dependant acquisition (IDA) approach. Xanthone-C-glycoside showed characteristic fragment ions due to fragmentation in the C-glycosidic unit while iridoid-O-glycosides showed characteristic fragment ions due to cleavage in the glycoside linkage and retro-Diels-Alder (RDA) cleavage within an iridoid aglycone. Furthermore, on the basis of this information, an analytical assay was developed and validated to determine relative concentrations of mangiferin, amarogentin, amaroswerin, sweroside and swertiamarin. The detection was carried out using multiple reaction monitoring (MRM) in positive ionization mode with a total analysis time of 3.5 min. The method was successfully applied to standardize four different batches of herbal preparation on the basis of relative concentration of five bioactive components.     

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.     

Electrospray mass spectrometry and fragmentation of N-linked carbohydrates derivatized at the reducing terminus

Derivatives were prepared from N-linked glycans by reductive amination from 2-aminobenzamide, 2-aminopyridine, 3-aminoquinoline, 2-aminoacridone, 4-amino-N-(2-diethylaminoethyl)benzamide, and the methyl, ethyl, and butyl esters of 4-aminobenzoic acid. Their electrospray and collision-induced dissociation (CID) fragmentation spectra were examined with a Q-TOF mass spectrometer. The strongest signals were obtained from the [M + Na]+ ions for all derivatives except sugars derivatized with 4-amino-N-(2-diethylaminoethyl)benzamide which gave very strong doubly charged [M + H + Na]2+ ions. The strongest [M + Na]+ ion signals were obtained from the butyl ester of 4-aminobenzoic acid and the weakest from 2-aminopyridine. The most informative spectra were recorded from the [M + Li]+ or [M + Na]+ ions. These spectra were dominated by ions produced by sequence-revealing glycosidic cleavages and "internal" fragments. Linkage-revealing cross-ring cleavage ions were reasonably abundant, particularly from high-mannose glycans. Although the nature of the derivative was found to have little effect upon the fragmentation pattern, 3-aminoquinoline derivatives gave marginally more abundant cross-ring fragments than the other derivatives. [M + H]+ ions formed only glycosidic fragments with few, if any, cross-ring cleavage ions. Doubly charged molecular ions gave less informative spectra; singly charged fragments were weak, and molecular ions containing hydrogen ([M + 2H]2+ and [M + H + Na]2+) fragmented as the [M + H]+ singly charged ions with no significant cross-ring cleavages.     

刺五加寡糖的电喷雾多级串联质谱研究

采用小柱层析法从刺五加中分离得到刺五加寡糖类系列化合物(刺五加二糖刺五加六糖).实验结果表明,在正离子模式下的ESI-MS谱中,此类化合物呈现出特征的加合离子峰簇[M+Na]⁺/[M+K]⁺或[M+H₂O+Na]⁺/[M+H₂O+K]⁺,可以确定其分子量;在负离子模式下的ESI-MS谱中,刺五加寡糖易形成[M-H]^-/[M+nH₂O-H]^-(n<3).还利用电喷雾多级串联质谱(ESI-MSⁿ)对刺五加三糖进行了系统的研究,推断出刺五加三糖的组成与结构.     

Structural determination of saponins extracted from starfish by fast atom bombardment collision-induced dissociation mass spectrometry.

Three saponins were extracted and isolated from starfish by reversed-phase high performance liquid chromatography (HPLC), and analyzed by fast atom bombardment mass spectrometry (FAB-MS). Their molecular weight information could be obtained by the presence of abundant [M+Na]+ ions and weak [M+H]+ ions in FAB-MS spectra. Moreover, high resolution mass measurements of their [M+Na]+ ions were performed at the resolution of 10000 to elucidate the element composition of extracted saponins. The collision-induced dissociation (CID) of sodium-adducted molecules [M+Na]+ yielded diverse product ions via dissociated processes. In the collision-induced dissociation (CID)-MS/MS analysis of [M+Na]+ ion, the sulfate-containing saponins produced characteristic ions such as SO4Na+, [NaHSO4+Na]+, [M+Na-sugar]+ and [M+Na-2sugar]+ ions, whereas the sulfate-free compound showed characteristic ions produced by cleavage of sugar moiety and side chain of aglycone. The fragmentation patterns could provide information on the linkage position of sugar groups in aglycone and sulfate groups.     

Fragmentation study of iridoid glucosides through positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry

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 mass spectral study of a series of six naturally occurring iridoids through in-source fragmentation of the protonated [M+H]+, deprotonated [M--H]- and sodiated [M+Na]+ ions. This led to the unambiguous determination of the molecular masses of the studied compounds and allowed CID spectra of the molecular ions to be obtained. Valuable structural information regarding the nature of both the glycoside and the aglycone moiety was thus obtained. Glycosidic cleavage and ring cleavages of both aglycone and sugar moieties were the major fragmentation pathways observed during CID, where the losses of small molecules, the cinnamoyl and the cinnamate parts were also observed. The formation of the ionized aglycones, sugars and their product ions was thus obtained giving information on their basic skeleton. The protonated, i.e. [M+H]+ and deprotonated [M--H]-, ions were found to fragment mainly by glycosidic cleavages. MS/MS spectra of the [M+Na]+ ions gave complementary information for the structural characterization of the studied compounds. Unlike the dissociation of protonated molecular ions, that of sodiated molecules also provided sodiated sugar fragments where the C0+ fragment corresponding to the glucose ion was obtained as base peak for all the studied compounds.     

Electrospray tandem mass spectrometry of alkali-cationized BocN-carbo-alpha,beta- and -beta,alpha-peptides: Differentiation of positional isomers

Dissociation pathways of a series of alkali-cationized hybrid peptides, viz., Boc-alpha,beta- and -beta,alpha-carbopeptides, synthesized from C-linked carbo-beta3-amino acids [Caa (S)] and alpha-alanine (L-Ala), have been investigated by electrospray ionization tandem mass spectrometry. The positional isomers (six pairs) of the cationized alpha,beta- and beta,alpha-peptides can be differentiated by the collision-induced dissociation (CID) spectra of their [M + Cat-Boc + H]+ ions which give characteristic series of alkali-cationized C- (x(n)+, y(n)+, z(n)+) and N-terminal (a(n)+, b(n)+, c(n)+) ions. Another noteworthy difference is cationized beta,alpha-peptides eliminate a molecule of ammonia whereas this pathway is absent for alpha,beta-peptides. This is useful for identifying the presence of a beta-amino acid at the N-terminus. The CID spectra of [M + Cat-Boc + H]+ ions of these peptide acids show abundant rearrangement [b(n) + 17 + Cat]+ (n = 1 to n-1) ions which is diagnostic for distinguishing between alpha- and beta-amino acid at the C-terminus. MS(n) experiments of [b(n) + Li-H]+ ions from these hybrid peptides showed the loss of CO and 72 u giving rise to [a(n) + Li-H]+ and cationized nitrile product ions which render support to earlier proposals that b(n)+ or [b(n) + Cat-H]+ ions have protonated or cationized oxazolinone structures, respectively.     

Evaluation of different quantitative approaches for the determination of noneasily ionizable molecules by different atmospheric pressure interfaces used in liquid chromatography tandem mass spectrometry: Abamectin as case of study

The liquid chromatography tandem mass spectroscopy residue determination of compounds without any acidic or basic centers such as abamectin has been investigated. Several approaches regarding the interface used and adduct formation have been compared. The low acidity of the hydroxyl groups only made deprotonation feasible using the atmospheric pressure chemical ionization (APCI) interface. To obtain sufficient sensitivity for residue analysis, the Ion Sabre APCI interface was necessary. However, the sensitivity attained was lower than for monitoring adducts in positive ion mode. Using electrospray ionization, different adducts with Na+, NH4+, and Li+ were tested and compared. The best results were obtained for the ammoniated adduct in electrospray ionization (ESI) because of its high sensitivity and the presence of several product ions with similar abundance. The highest sensitivity was reached using an in-source fragment as precursor ion, leading to a limit of detection (LOD) of 2 microg/L with low relative standard deviation. The relatively high abundance of other transitions allowed abamectin confirmation at concentrations close to the LOD (6 microg/L). Alkali ions were found to be a suitable alternative to determine and confirm abamectin at residue levels. The [M + Na]+ also presented various product ions with similar abundance, which allowed confirmation at LOD levels. However, this LOD was found to be almost four times higher than with [M + NH4]+ because of the poor sensitivity of the transitions obtained. Although the use of Li+ facilitated the fragmentation of the adduct [M + Li]+, with similar sensitivity to [M + NH4]+, this fragmentation preferentially generated only one product ion, which did not allow confirmation at concentration levels lower than 15 microg/L. The use of APCI for monitoring adducts was also feasible, but with less sensitivity. The sensitivity increased with the Ion Sabre APCI, although it was still five times lower than with ESI. Other adduct formers such as Co2+ and Ni2+ also were tested with unsatisfactory results.     

Tandem mass spectrometric analysis of distinct fragmentation patterns to [M+Na]/z and [M+H]/z of dendritic Al(III) and Zn(II) quinolates

The synthesis of two novel dendritic aluminum and zinc quinolates, which are soluble in common solvents, was monitored effectively by positive ion electrospray ionization mass spectrometry (ESI-MS). Through tandem mass spectrometric analysis of the complexes, distinct fragmentation pathways for sodium adduct molecular ion [M+Na]+ and protonated molecular ion [M+H]+ were observed.     

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.     

Collision-induced fragmentation of underivatized N-linked carbohydrates ionized by electrospray

The electrospray mass spectra and collision-induced fragmentation of neutral N-linked glycans obtained from glycoproteins were examined with a Q-TOF mass spectrometer. The glycans were ionized most effectively as adducts of alkali metals, with lithium providing the most abundant signal and caesium the least. Singly charged ions generally gave higher ion currents than doubly charged ions. Addition of formic acid could be used to produce [M + H]+ ions, but these ions were always accompanied by abundant cone-voltage fragments. The energy required for collision-induced fragmentation was found to increase in a linear manner as a function of mass with the [M + Na]+ ions requiring about four times as much energy as the [M + H]+ ions for complete fragmentation of the molecular ions. Fragmentation of the [M + H]+ ions gave predominantly B- and Y-type glycosidic fragments whereas the [M + Na]+ and [M + Li]+ ions produced a number of additional fragments including those derived from cross-ring cleavages. Little fragmentation was observed from the [M + K]+ and [M + Rb]+ ions and the only fragment to be observed from the [M + Cs]+ ion was Cs+. The [M + Na]+ and [M + Li]+ ions from all the N-linked glycans gave abundant fragments resulting from loss of the terminal GlcNAc moiety and prominent, though weaker, ions as the result of 0,2A and 2,4A cross-ring cleavages of this residue. Most other ions were the result of successive additional losses of residues from the non-reducing terminus. This pattern was particularly prominent with glycans containing several non-reducing GlcNAc residues where successive losses of 203 u were observed. Many of the ions in the low-mass range were products of several different fragmentation routes but still provided structural information. Possibly of most diagnostic importance was an ion formed by loss of 221 u (GlcNAc molecule) from an ion that had lost the 3-antenna and the chitobiose core. This latter ion, although coincident in mass with some other 'internal' fragments, often provided additional information on the composition of the antennae. Other ions defining antenna composition were weak cross-ring fragments produced from the core branching mannose residue. Glycans containing Gal-GlcNAc residues showed successive losses of this moiety, particularly from the B-type fragments resulting from loss of the reducing-terminal GlcNAc residue. The [M + Na]+ and [M + Li]+ ions from high-mannose and hybrid glycans gave a series of ions of composition (Man)nNa/Li+ where n = 1 to the total number of glycans in the molecule, allowing these sugars to be distinguished from the more highly processed complex glycans. Other ions in the spectra of the high-mannose glycans were diagnostic of chain branching but insufficient information was available to determine their mode of formation.     

Elucidation of O-Phosphoryl and N-Phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

Mass spectroscopic characteristics of phosphoryl amino acids were studied in detail by positive and negative electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). Besides N-diisopropyloxyphosphoryl amino acids (N-DIPP-AA), O-phospho- and O-diisopropyloxyphosphoryl amino acids (O-DIPP-AA) were studied and compared to N-DIPP-AA. The fragmentation pathways of [M H]^ ,[M Na]^ and [M-H]^- ions of phosphoryl amino acids were summarized. In addition to several similar patterns, each of them showed its characteristic fragmention.     

Structural characterization of flavonol di-O-glycosides from Farsetia aegyptia by electrospray ionization and collision-induced dissociation mass spectrometry

This study reports the application of mass spectrometric methods to characterize unknown flavonoids of the herb Farsetia aegyptia Turra (Crucifereae). High-performance liquid chromatography was performed in combination with UV-photodiode array detection (LC/UV-DAD) and electrospray ionization mass spectrometry (LC/ESI-MS) in both positive and negative ion modes. Collision-induced dissociation (CID) mass spectral data were obtained off-line by nanospray (nano-ESI) analysis, which provided a wealth of information and led to the structural proposal of the flavonol di-O-glycosides present in the herb extract. In addition to the mass spectral data, we also report NMR data for the major compound which allowed the completion of its structural elucidation. The Farsetia aegyptia Turra herb extract was found to contain three flavonol di-O-glycosides containing a monosaccharidic residue linked to the 3-O position and a disaccharidic residue linked to the 7-O position; the major compound was characterized as the new flavonoid, isorhamnetin 3-O-alpha-L-arabinoside 7-O-[beta-D-glucosyl-1 --> 2]-alpha(L)rhamnoside. Different types of CID spectra, i.e., low-energy [M+H]+, [M+Na]+ and [M--H]- spectra as well as high-energy [M+Na]+ spectra, were evaluated with respect to their utility to locate the O-linked saccharidic residues in flavonol di-O-glycosides and to determine the sequence in the disaccharidic part. In agreement with previously published data, the 3-O-glycosyl residue was more readily lost from the protonated molecule than the 7-O-glycosyl residue. The opposite behavior was noted for the fragmentation of the deprotonated and sodiated molecules. Radical ions were observed in the high-energy [M+Na]+ CID spectra which provided supporting information on the glycosylation positions.     

Electrospray ionization tandem mass spectrometry of 3-[4-bis-N,N-(2- chloroethyl)aminophenyl]acetates of estrane series

Collision-induced fragmentation of the [M + Na](+) and [M + H](+) ions generated from 3-[4-bis-N,N-(2-chloroethyl)aminophenyl]acetates in the estrane series under electrospray/ionization was studied. Some regularities in fragmentation pathways depending on the nature of functional groups were established. Formation of the [(M + Na) NaCl](+) ions along with [(M + Na) HCl](+) ions from the [M + Na](+) ions was explained using quantum chemical calculations for some simplified models.     

Reduction of nitriles to amines in positive ion electrospray ionization mass spectrometry

Some compounds readily form [M+46]+ adduct ions during positive ion electrospray ionization mass spectrometry ((+)ESI-MS) analysis. These [M+46]+ ions were characterized as [M+CH3CH2NH2+H]+ by accurate mass determination. Ethylamine involved in the adduct was proposed to be the reduction product of acetonitrile and this was confirmed using deuterated acetonitrile. Other nitrile-containing compounds tested, including isobutyronitrile and benzonitrile, also formed the adduct ions of the respective amine forms under (+)ESI-MS conditions. Hydrogen/deuterium exchange experiments demonstrated that the reductive hydrogen originated from water. Reduction of nitriles (R-CN) to their respective amines (R-CH2NH2) under (+)ESI-MS conditions expands the ability to identify nitrile-containing chemical unknowns.     

Characterization of limonin glucoside metabolites from human prostate cell culture medium using high-performance liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

The metabolism of limonin 17-beta-D-glucopyranoside (LG) by non-cancerous (RWPE-1) and cancerous (PC-3) human prostate epithelial cells was investigated using high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation and tandem mass spectrometry (MS/MS). During positive ion LC/ESI-MS, LG formed an abundant sodiated species ([M+Na]+) while the protonated molecule was barely observable. [M+Na]+ further fragmented into the less abundant [LARL+H]+ and a predominantly protonated aglycone molecule (limonin) due to in-source fragmentation. The major metabolite, limonin A-ring lactone (LARL), formed an abundant protonated molecule that was fragmented into a protonated molecule of limonin by loss of one molecule of water. In MS/MS by collisionally activated dissociation (CAD), LG produced the sodiated aglycone, [aglycone+Na]+, while LARL fragmented into [M+H]+ of limonin and fragment ions resulted by further loss of water, carbon monoxide and carbon dioxide, indicating the presence of oxygenated-ring structures. The limits of detection of LG were 0.4 and 20 fmol in selected-ion monitoring (SIM) and selected-reaction monitoring (SRM) detection, respectively.