ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

Characterization of N‐methylated amino acids by GC‐MS after ethyl chloroformate derivatization

Methylation is an essential metabolic process in the biological systems, and it is significant for several biological reactions in living organisms. Methylated compounds are known to be involved in most of the bodily functions, and some of them serve as biomarkers. Theoretically, all α‐amino acids can be methylated, and it is possible to encounter them in most animal/plant samples. But the analytical data, especially the mass spectral data, are available only for a few of the methylated amino acids. Thus, it is essential to generate mass spectral data and to develop mass spectrometry methods for the identification of all possible methylated amino acids for future metabolomic studies. In this study, all N‐methyl and N,N‐dimethyl amino acids were synthesized by the methylation of α‐amino acids and characterized by a GC‐MS method. The methylated amino acids were derivatized with ethyl chloroformate and analyzed by GC‐MS under EI and methane/CI conditions. The EI mass spectra of ethyl chloroformate derivatives of N‐methyl ( 1–18 ) and N,N‐dimethyl amino acids ( 19–35 ) showed abundant [M‐COOC₂H₅]⁺ ions. The fragment ions due to loss of C₂H₄, CO₂, (CO₂ + C₂H₄) from [M‐COOC₂H₅]⁺ were of structure indicative for 1–18 . The EI spectra of 19–35 showed less number of fragment ions when compared with those of 1–18 . The side chain group (R) caused specific fragment ions characteristic to its structure. The methane/CI spectra of the studied compounds showed [M + H]⁺ ions to substantiate their molecular weights. The detected EI fragment ions were characteristic of the structure that made easy identification of the studied compounds, including isomeric/isobaric compounds. Fragmentation patterns of the studied compounds ( 1–35 ) were confirmed by high‐resolution mass spectra data and further substantiated by the data obtained from ¹³C₂‐labeled glycines and N‐ethoxycarbonyl methoxy esters. The method was applied to human plasma samples for the identification of amino acids and methylated amino acids. Copyright © 2016 John Wiley & Sons, Ltd.     

Low-mass ions produced from peptides by high-energy collision-induced dissociation in tandem mass spectrometry

High-energy collision-induced dissociation (CID) mass spectrometry provides a rapid and sensitive means for determining the primary sequence of peptides. The low-mass region (below mass 300) of a large number of tandem CID spectra of peptides has been analyzed. This mass region contains several types of informative fragment ions, including dipeptide ions, immonium ions, and other related ions. Useful low-mass ions are also present in negative-ion CID spectra. Immonium ions (general structure [H₂N=CH-R]⁺, where R is the amino acid side chain) and related ions characteristic of specific amino acid residues give information as to the presence or absence of these residues in the peptide being analyzed. Tables of observed immonium and reiated ions for the 20 standard amino acids and for a number of modified amino acids are presented. A database consisting of 228 high-energy CID spectra of peptides has been established, and the frequency of occurrence of various ions indicative of specific ammo acid residues has been determined. Two model computer-aided schemes for analysis of the ammo-acid content of unknown peptides have been developed and tested against the database.     

Tandem mass spectrometric analysis of novel peptide‐modified gemini surfactants used as gene delivery vectors

Diquaternary ammonium gemini surfactants have emerged as effective gene delivery vectors. A novel series of 11 peptide‐modified compounds was synthesized, showing promising results in delivering genetic materials. The purpose of this work is to elucidate the tandem mass spectrometric (MS/MS) dissociation behavior of these novel molecules establishing a generalized MS/MS fingerprint. Exact mass measurements were achieved using a hybrid quadrupole orthogonal time‐of‐flight mass spectrometer, and a multi‐stage MS/MS analysis was conducted using a triple quadrupole‐linear ion trap mass spectrometer. Both instruments were operated in the positive ionization mode and are equipped with electrospray ionization. Abundant triply charged [M+H]³⁺ species were observed in the single‐stage analysis of all the evaluated compounds with mass accuracies of less than 8 ppm in mass error. MS/MS analysis showed that the evaluated gemini surfactants exhibited peptide‐related dissociation characteristics because of the presence of amino acids within the compounds' spacer region. In particular, diagnostic product ions were originated from the neutral loss of ammonia from the amino acids' side chain resulting in the formation of pipecolic acid at the N‐terminus part of the gemini surfactants. In addition, a charge‐directed amide bond cleavage was initiated by the amino acids' side chain producing a protonated α‐amino‐ε‐caprolactam ion and its complimentary C‐terminus ion that contains quaternary amines. MS/MS and MS³ analysis revealed common fragmentation behavior among all tested compounds, resulting in the production of a universal MS/MS fragmentation pathway. Copyright © 2017 John Wiley & Sons, Ltd.     

L‐Valine assisted distinction between the stereo‐isomers of D‐hexoses by positive ion ESI tandem mass spectrometry

The binary mixtures of 7 hexoses and 20 amino acids were investigated by electrospray ionization ion trap mass spectrometry (ESI‐ITMS). The adduct ions of the amino acid and the hexose were detected for 12 amino acids but not for the other 8 amino acids which are basic acidic amino acids and amides. The ions of amino acid–hexose complexes were further investigated by tandem mass spectrometry (MS/MS), and some of them just split easily into two parts whereas the others gave rich fragmentation, such as the complex ions of isoleucine, phenylalanie, tyrosine, and valine. We found that hexoses could be complexed by two molecules of valine but only by one molecule of the other amino acids. Among seven kinds of valine–hexose complexes coordinated by potassium ion, the MS² spectra of the ion at m/z 453 yielded unambiguous differentiation. And the fragmentation ions are sensitive to the stereochemical differences at the carbon‐4 of hexoses in the complexes, as proved by the MS². Copyright © 2010 John Wiley & Sons, Ltd.     

Phosphorylated serine and threonine residues promote site‐specific fragmentation of singly charged,arginine‐containing peptide ions

In order to investigate gas‐phase fragmentation reactions of phosphorylated peptide ions, matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI‐TOF/TOF (TOF: time‐of‐flight) spectra of synthetic arginine‐containing phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C‐terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI‐MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI‐MS/MS spectra of doubly charged phosphopeptides containing two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C‐terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI‐TOF/TOF spectra of phosphopeptides displaying N‐terminal fragment ions, abundant b–H₃PO₄ ions resulting from the enhanced dissociation of the pSer/pThr–X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were found to be particularly predominant in spectra of phosphopeptides containing pSer/pThr–Pro bonds. A quantitative evaluation of a larger set of MALDI‐TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine‐containing peptides increase the signal intensities of the respective y ions by almost a factor of 3. A less pronounced cleavage‐enhancing effect was observed in some lysine‐containing phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.     

MALDI TOF/TOF tandem mass spectrometry as a new tool for amino acid analysis

This is the first report of an application of collisionally induced fragmentation of amino acids (AA) and their derivatives by MALDI TOF/TOF tandem mass spectrometry (MS). In this work, we collected the data on high-energy fragmentation reactions of a large group of protonated amino acids and their derivatives with the goal of determining which product ions are analyte specific and if yields of these fragment could be used for quantitative analysis. From 34 different amino acids (20 alpha-amino acids, beta-amino acids, homocysteine, GABA, and modified AA Met sulfone and sulfoxide, hydroxyproline, etc.) we observed that high yields of the target specific immonium ions and fragmentation patterns are most similar to EI or FAB CID on sector instruments. The major exceptions were two highly basic amino acids, Arg and Orn. It is noted that neither beta-, gamma-, nor delta-amino acids produce immonium ions. As might be predicted from high-energy CID work on peptides from the sectors and TOF/TOF, the presence of specific indicator ions in MALDI tandem MS allows distinguishing isomeric and isobaric amino acids. These indicator ions, in combination with careful control of data acquisition, ensure quantitative analysis of amino acids. We believe our data provide strong basis for the application of MALDI TOF/TOF MS/MS in qualitative and quantitative analysis of amino and organic acids, including application in clinical medicine.     

Influences of peptide side chains on the metal ion binding site in metal ion-cationized peptides: Participation of aromatic rings in metal chelation

Aromatic side chains on amino acids influence the fragmentations of cationic complexes of doubly charged metal ions and singly deprotonated peptides. The metal ion interacts with an aromatic side chain and binds to adjacent amide nitrogens. When fragmentation occurs, this bonding leads to the formation of abundant metal-containing a-type ions by reactions that occur at the sites of amino acids that contain the aromatic side chain. Furthermore, formation of metal-containing immonium ions of the amino acids that contain the aromatic side chain also are formed. The abundant a-type ions may be useful in interpretation strategies in which it is necessary to locate in a peptide the position of an amino acid that bears an aromatic side chain.     

Chiral discrimination of β‐3‐homo‐amino acids using the kinetic method

Chiral discrimination of seven enantiomeric pairs of β‐3‐homo‐amino acids was studied by using the kinetic method and trimeric metal‐bound complexes, with natural and unnatural α‐amino acids as chiral reference compounds and divalent metal ions (Cu²⁺ and Ni²⁺) as the center ions. The β‐3‐homo‐amino acids were selected for this study because, first of all, chiral discrimination of β‐amino acids has not been extensively studied by mass spectrometry. Moreover, these β‐3‐homo‐amino acids studied have different aromatic side chains. Thus, the emphasis was to study the effect of the side chain (electron density of the phenyl ring, as well as the difference between phenyl and benzyl side chains) for the chiral discrimination. The results showed that by the proper choice of a metal ion and a chiral reference compound, all seven enantiomeric pairs of β‐3‐homo‐amino acids could be differentiated. Moreover, it was noted that the β‐3‐homo‐amino acids with benzyl side chains provided higher enantioselectivity than the corresponding phenyl ones. However, increasing or decreasing the electron density of the aromatic ring by different substituents in both the phenyl and benzyl side chains had practically no role for chiral discrimination of β‐3‐homo‐amino acids studied. When copper was used as the central metal, the phenyl side chain containing reference molecules (S)‐2‐amino‐2‐phenylacetic acid (L ‐Phg) and (S)‐2‐amino‐2‐(4‐hydroxyphenyl)‐acetic acid (L ‐4′‐OHPhg) gave rise to an additional copper‐reduced dimeric fragment ion, [Cu^I(ref)(A)]⁺. The inclusion of this ion improved noticeably the enantioselectivity values obtained. Copyright © 2010 John Wiley & Sons, Ltd.     

Electrospray tandem mass spectrometry of new porphyrin amino acid conjugates

Porphyrin amino acid conjugates with one or two porphyrin units were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The ESI-MS spectra of all the porphyrins studied, obtained in positive ion mode, show the presence of the corresponding protonated molecule [M+H]+; ESI-MS spectra of diporphyrinyl compounds also show the doubly charged ions [M+2H]2+. The fragmentations of these ions induced by collision with argon were studied (ESI-MS/MS). ESI-MS/MS gives detailed structural information about the amino acids associated with the porphyrin. Cleavage of the bonds in the vicinity of the porphyrin moiety and those involving the side chain of amino acid residues gives structural information about this type of association. A fragmentation common to all derivatives corresponds to the cleavage of the phenyl-CO bond. The expected cleavage of the amide bond, that links the porphyrin to the amino acid moiety, is a minor fragmentation, which in some cases is even absent. The MS/MS spectra of the monoporphyrinyl derivatives show product ions characteristic of the amino acid linked to the porphyrin; the fragmentation also indicates when the amino acids has a terminal carboxylic group or a terminal ester group. The fragmentations of the diporphyrinyl compounds occur mainly by the cleavage of the spacer, leading, in the case of the doubly charged ions, to predominantly mono-charged ions, indicating a preferential location of the two protons in separated porphyrinic units.     

Chemical cross‐linking with NHS esters: a systematic study on amino acid reactivities

Structure elucidation of tertiary or quaternary protein structures by chemical cross‐linking and mass spectrometry (MS) has recently gained importance. To locate the cross‐linker modification, dedicated software is applied to analyze the mass or tandem mass spectra (MS/MS). Such software requires information on target amino acids to limit the data analysis time. The most commonly used homobifunctional N‐hydroxy succinimide (NHS) esters are often described as reactive exclusively towards primary amines, although side reactions with tyrosine and serine have been reported. Our goal was to systematically study the reactivity of NHS esters and derive some general rules for their attack of nucleophilic amino acid side chains in peptides. We therefore studied the cross‐linking reactions of synthesized and commercial model peptides with disuccinimidyl suberate (DSS). The first reaction site in all cases was expectedly the α‐NH₂‐group of the N‐terminus or the ε‐NH₂‐group of lysine. As soon as additional cross‐linkers were attached or loops were formed, other amino acids were also involved in the reaction. In addition to the primary amino groups, serine, threonine and tyrosine showed significant reactivity due to the effect of neighboring amino acids by intermediate or permanent Type‐1 cross‐link formation. The reactivity is highly dependent on the pH and on adjacent amino acids. Copyright © 2009 John Wiley & Sons, Ltd.     

Factors affecting immonium ion intensities in the high-energy collision-induced decomposition spectra of peptides

Each amino acid in a peptide has a characteristic immonium ion (H₂N⁺?CHR), the presence of which in a mass spectrum can indicate the presence of that amino acid. High-energy collision-induced decomposition studies on small peptide ions formed by fast atom bombardment showed the relative intensities of these immonium ions to be dependent on the relative positions of the amino acids in the peptide chain: C-terminal, N-terminal or in-chain. Evidence in favour of competition in the formation of immonium ions is presented.     

Investigation of several unique tandem mass spectrometric fragmentation patterns of NFDEIDR, an orcokinin analog, and its N-terminal dimethylated form

Orcokinins are a family of myotropic neuropeptides widely present in various decapod crustaceans and insect species. The majority of the orcokinins identified to date share a conserved sequence of NFDEIDR at their N-termini. Electrospray ionization quadrupole time-of-flight tandem mass spectrometric (ESI-QTOF-MS/MS) analysis of doubly charged orcokinin precursor ions reveals the presence of a y(n - 1) + 10 peak, which is more intense than that for the y(n - 1) ion. To elucidate the identity of this novel fragment ion and understand the mechanism underlying this fragmentation, we employed a combined approach involving the use of isotopic N-terminal dimethylation, methyl esterification, and isotope-encoded NFDEIDR. Comparison of the fragmentation patterns of these chemically modified orcokinin analogs allowed the determination of the structure of the y(n - 1) + 10 ion as y(n - 1) + CO--H2O. The yx + CO--H2O ions, along with the yx + CO and yx + CO--NH3 ions, are also present in the MS/MS spectra of NFDEIDR and several other peptides. Additionally, we report two other unusual fragmentation ions in the MS/MS spectra of N-terminal dimethyl NFDEIDR (2+), which yields the novel fragment ions of the y(n - 1) + 38 ion and the [M+2H-59]2+ ion. These two ion series involve the neutral loss of the asparagine side chain. The same sets of ions are also present in other peptides with dimethyl-modified asparagines at the N-terminus. The competition between the side-chain loss and loss of dimethylamine is described. The loss of the side chain of N-terminal dimethyl Asp1 is reported as well. We also report for the first time the neutral loss of ammonia from the N-terminal amino group of Asn1 and the loss of CO2 from the side chain of aspartic acid.     

Methylating peptides to prevent adduct ion formation also directs cleavage in collision-induced dissociation mass spectrometry

We investigated the effect of N-terminal amino group and carboxyl group methylation on peptide analysis by electrospray mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Permethylation of the N-terminal amino group and the carboxyl groups can reduce metal ion adducts but does not enhance sensitivity in electrospray as previously observed for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. N-terminal trimethylated peptides exhibit collision-induced dissociation (CID) tandem mass spectra that differ from their unmodified analogs; the results support the mobile proton hypothesis of peptide fragmentation. A permanent positive charge at the N-terminus leads to competition between permanent-charge directed processes and loss of the N-terminal trimethyl amino group. Carboxyl methylation has no effect on fragmentation behavior other than to shift the mass of fragments containing methylated carboxyl groups. Comparison of regular and tandem mass spectra of different methylated peptides allowed probing the location of incomplete methylation, the proton displaced by alkali metal ions and the purity of a mass-selected methylated peptide ion.     

Characterization of N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives using electrospray ionization multi‐stage mass spectrometry

N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro‐inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion‐trap multi‐stage mass spectrometry (MSⁿ). The MS² collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N‐Fmoc/Z‐amino acids, dipeptide and tripeptide acids show the [M + Na‐NH₂CHO]⁺ ion, arising from the loss of formamide, as the base peak. Differently, the MS² CID spectra of [M + Na]⁺ ion of all the N‐Boc derivatives yield the abundant [M + Na‐C₄H₈]⁺ and [M + Na‐Boc + H]⁺ ions because of the loss of isobutylene and CO₂ from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N‐protected dipeptidyl and tripeptidyl‐N′‐formamides is provided by MS² and subsequent MSⁿ experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M‐H]^?, [M + HCOO]^? and [M + Cl]^? ions, the presence of in‐source CID fragment ions deriving from the involvement of the N‐protecting group. Furthermore, MSⁿ spectra of [M + Cl]^? ion of N‐protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C‐terminal gem‐diamino residue. The present paper represents an initial attempt to study the ESI‐MS behavior of these important intermediates and lays the groundwork for structural‐based studies on more complex partially modified retro‐inverso peptides. Copyright © 2013 John Wiley & Sons, Ltd.     

Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (b_n-1 + H₂O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H₂O and NH₃) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a y_m-1 ion, i.e., to the loss of the N-terminal residue following the a₁-y_m–1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N- and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.     

Ion‐pairing high‐performance liquid chromatography‐mass spectrometry of impurities and reduction products of sulphonated azodyes

An HPLC separation method with triethylammonium acetate mobile phase additive developed for the analysis of impurities in polysulphonated azo dyes provides good separation selectivity and compatibility with electrospray ionisation (ESI) mass spectrometry. The negative‐ion ESI mass spectra containing only peaks of deprotonated molecules [M–H]^– for monosulphonic acids, [M–xH]^x–, and sodiated adducts [M–(x + y)H + yNa]^x– for polysulphonic acids allow easy molecular mass determination of unknown impurities. Based on the knowledge of the molecular masses and of the fragment ions in the MS/MS spectra, probable structures of trace impurities in commercial dye samples are proposed. To assist in the interpretation of the mass spectra of complex polysulphonated azodyes, additional information can be obtained after chemical reduction of azodyes to aromatic amines. The structures of the non‐sulphonated reduction products can be determined by reversed‐phase HPLC/MS with positive‐ion atmospheric pressure chemical ionisation and of the sulphonated products by ion‐pairing HPLC/MS with negative‐ion ESI.     

Polarization induced electrospray ionization mass spectrometry for the analysis of liquid,viscous and solid samples

In this study, a polarization‐induced electrospray ionization mass spectrometry (ESI‐MS) was developed. A micro‐sized sample droplet was deposited on a naturally available dielectric substrate such as a fruit or a stone, and then placed close to (~2 mm) the orifice of a mass spectrometer applied with a high voltage. Taylor cone was observed from the sample droplet, and a spray emitted from the cone apex was generated. The analyte ion signals derived from the droplet were obtained by the mass spectrometer. The ionization process is similar to that in ESI although no direct electric contact was applied on the sample site. The sample droplet polarized by the high electric field provided by the mass spectrometer initiated the ionization process. The dielectric sample loading substrate facilitated further the polarization process, resulting in the formation of Taylor cone. The mass spectral profiles obtained via this approach resembled those obtained using ESI‐MS. Multiply charged ions dominated the mass spectra of peptides and proteins, whereas singly charged ions dominated the mass spectra of small molecules such as amino acids and small organic molecules. In addition to liquid samples, this approach can be used for the analysis of solid and viscous samples. A small droplet containing suitable solvent (5–10 µl) was directly deposited on the surface of the solid (or viscous) sample, placed close the orifice of mass spectrometer applied with a high voltage. Taylor cone derived from the droplet was immediately formed followed by electrospray processes to generate gas‐phase ions for MS analysis. Analyte ions derived from the main ingredients of pharmaceutical tablets and viscous ointment can be extracted into the solvent droplet in situ and observed using a mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural determination of sildenafil and its analogues in dietary supplements by fast‐atom bombardment collision‐induced dissociation tandem mass spectrometry

Sildenafil and its analogues, which are used as illegal additives in several dietary supplements, were isolated by liquid‐liquid extraction and column chromatography and analyzed by fast‐atom bombardment mass spectrometry (FAB‐MS). Structures of sildenafil and its derivatives were elucidated by FAB‐tandem mass spectrometry (MS/MS) with exact mass measurement in the positive‐ion mode. To find structurally diagnostic ions for the sildenafil analogues, authentic sildenafil was preferentially analyzed by high‐energy collision‐induced dissociation (CID)‐MS/MS. The CID‐MS/MS spectra of [M+H]⁺ precursor ions resulted in the formation of numerous characteristic ions via a series of dissociative processes. The product ions formed by CID provided important information on the modification of the piperazine ring, the phenylsulfonyl group and the pyrazolopyrimidine moiety of sildenafil. By interpreting their MS/MS spectra, the chemical structures of sildenafil analogues isolated from dietary supplements could be elucidated and fragmentation patterns were proposed. To clearly identify the sidenafil derivatives in dietary supplements, some of the derivatives such as acetildenafil, homosildenafil and hydroxyhomosildenafil which are not commercially available were synthesized and compared with their MS/MS spectra. In addition, high‐resolution mass measurements were conducted to obtain the elemental compositions of sildenafil and its analogues. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of the major metabolites of quinocetone in swine urine using ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Quinocetone (QCT), 3‐methyl‐2‐cinnamoylquinoxaline‐1,4‐dioxide, is a quinoxaline‐N,N‐dioxide used in veterinary medicine as a feed additive. QCT is broadly used in China to promote animal growth, but few studies have been performed to reveal the metabolism of QCT in animals until now. In the present study, the metabolites of QCT in swine urine were investigated using ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight mass spectrometry (UPLC/ESI‐QTOF‐MS). Multiple scans of metabolites in MS and MS/MS modes and accurate mass measurements were performed simultaneously through data‐dependent acquisition. Most measured mass errors were less than ±5 mDa for both protonated molecules and product ions using external mass calibration. The structures of metabolites and their product ions were easily and reliably characterized based on the accurate MS² spectra and known structure of QCT. As expected, extensive metabolism was observed in swine urine. Thirty‐one metabolites were identified in swine urine, most of which were reported for the first time. The results reveal that the N‐O group reduction at position 1 and the hydroxylation reaction occurring at the methyl group, the side chain or on the benzene ring are the main metabolic pathways of quinocetone in swine urine. There was abundant production of 1‐desoxyquinocetone and hydroxylation metabolites of 1‐desoxyquinocetone. The proposed metabolic pathway of quinocetone in vivo can be expected to play a key role in food safety evaluations. Copyright © 2010 John Wiley & Sons, Ltd.