Differentiation of diasteromeric α-sulfanyl-β-amino acid derivatives by electrospray ionization tandem mass spectrometry

A set of diastereomeric α-sulfanyl-β-amino acid derivatives, which are important building blocks for pharmaceuticals with potent biological activity, are studied by electrospray ionization tandem mass spectrometry. The collision induced dissociation (CID) spectra of [M+H](+), [M+NH(4)](+), [M+Na](+) and [M+Li](+) of the diastereomers were studied, among them the CID of [M+Na](+) and [M+Li](+) showed consistent differences in the relative abundance of characteristic ions that enabled distinction of the anti isomers from syn isomers. The decomposition pathways for the diagnostic ions were arrived at based on high-resolution mass spectrometry data, multiple mass spectrometry data, deuterium labeling experiments and the mass shift in accordance with the substituents located at different places. Loss of (R(1)-C(6)H(4)-CH=NH) and (Cat-NH-SO(2)R(2)) from [M+Cat](+), where Cat=Na and Li, and the product ions as a results of McLafferty rearrangement involving either >S=O or >C=O group were found to be diagnostic. The McLafferty rearrangement product ions involving >S=O group were more abundant in syn isomers while those involving >C=O group were more abundant in anti isomer. The selectivity observed in the decomposition of [M+Li](+) ions was found to be similar to that of [M+Na](+) ions, but in few cases the differences are marginal in the decomposition [M+Li](+) ions.     

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography–electrospray tandem mass spectrometry

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra ^® MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r ²?=?0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.     

Comparison of the isomeric α-amino acyl adenylates and amino acid phosphoramidates of adenosine by electrospray ionization tandem mass spectrometry

The isomeric α-amino acyl adenylates and amino acid phosphoramidates of adenosine were synthesized and analyzed in detail by electrospray ionization tandem mass spectrometry (ESI-MS(n)). In ESI-MS/MS of α-amino acyl adenylates, the novel rearrangement ion [cAMP-H](-) observed as the most intense signal was formed through the pentacoordinate phosphorus intermediate with a six-membered ring by nucleophilic attack of the 3'-hydroxyl group on the phosphorus atom. In contrast, for the amino acid phosphoramidate of adenosine, the phosphorus atom could be attacked not only by the carboxylic group to form the cyclic aminoacyl phosphoramidates (CAPAs), but also by the nitrogen atom on the nucleobase leading to intramolecular phosphoryl group migration. It was found that the sodium ion having multidentate binding ability played an essential role in this characteristic rearrangement. The proposed mechanisms were supported by the MS/MS study, deuterium-labeled experiments, high-resolution tandem mass spectrometry and moderate calculations at the B3LYP/6-31G* level. The characteristic fragmentation patterns of α-amino acyl phosphates and amino acid phosphoramidates allows identification of stereoisomers when either the phosphorylation is at the N-terminus or C-terminus of amino acids.     

Detection and quantification of plasma amyloid-β by selected reaction monitoring mass spectrometry

Amyloid-β (Aβ) in human plasma was detected and quantified by an antibody-free method, selected reaction monitoring mass spectrometry (SRM-MS) in the current study. Due to its low abundance, SRM-based quantification in 10 μL plasma was a challenge. Prior to SRM analysis, human plasma proteins as a whole were digested by trypsin and high pH reversed-phase liquid chromatography (RPLC) was used to fractionate the tryptic digests and to collect peptides, Aβ_1–5, Aβ_6–16, Aβ_17–28 and Aβ_29–40(42) of either Aβ_1–40 or Aβ_1–42. Among those peptides, Aβ_17–28 was selected as a surrogate to measure the total Aβ level. Human plasma samples obtained from triplicate sample preparations were analyzed, obtaining 4.20 ng mL⁻¹ with a CV of 25.3%. Triplicate measurements for each sample preparation showed CV of <5%. Limit of quantification was obtained as 132 pM, which corresponded to 570 pg mL⁻¹ of Aβ_1–40. Until now, most quantitative measurements of Aβ in plasma or cerebrospinal fluid have required antibody-based immunoassays. Since quantification of Aβ by immunoassays is highly dependent on the extent of epitope exposure due to aggregation or plasma protein binding, it is difficult to accurately measure the actual concentration of Aβ in plasma. Our diagnostic method based on SRM using a surrogate peptide of Aβ is promising in that actual amounts of total Aβ can be measured regardless of the conformational status of the biomarker.     

Differentiation of 2′-O- and 3′-O-methylated Ribonucleosides by tandem mass spectrometry

Recent studies revealed that the 3'-terminal nucleotides in plant microRNAs were methylated on the ribose at the 2' or 3' hydroxyl groups. Here we examined the fragmentation of the electrospray-produced [M + H]+ and [M - H]- ions of 2'- and 3'-O-methylated ribonucleosides. It turned out that the predominant fragmentation pathway for the [M + H]+ ions of ribose-methylated nucleosides was the neutral loss of the methylated ribose, which made it impossible to distinguish 2'-O-methylation from 3'-O-methylation by positive-ion MS/MS. However, characteristic fragment ions, resulting from the cleavage through the ribose rings, were produced for the [M - H]- ions of each pair of ribose-methylated nucleosides. In this respect, the neutral loss of a 90-Da fragment (C3H6O3) was observed for 2'-O-methylated cytidine, guanosine and adenosine, but not for their 3'-O-methylated counterparts. On the other hand, the neutral loss of a 60-Da fragment (C2H4O2) was found for 3'-O-methyluridine, but not for 2'-O-methyluridine.     

Simultaneous quantification of multiple classes of phenolic compounds in blood plasma by liquid chromatography–electrospray tandem mass spectrometry

A method using high performance liquid chromatography–electrospray tandem mass spectrometry (LC–ESI-MS/MS) in positive ion mode was developed for the simultaneous analysis of 30 phenolic compounds, including four estrogens, bisphenol A (BPA), 10 hydroxylated polybrominated dephenyl ethers (OH-PBDEs) and 15 bromophenols (BRPs), in blood plasma. In the present method, derivatization with dansyl chloride was employed, and all the derivertized target compounds were well resolved on a 100 mm Xbridge C18 column with acetonitrile and 0.1% formic acid as the mobile phases. Purification procedures, such as liquid–liquid extraction and silica-gel chromatography, were applied to reduce matrix effects in the sample extract and remove excess derivatizing reagents, thus permitting selective and sensitive detection of the target phenolic compounds. The limit of quantification for all analytes, with a signal-to-noise ratio of ∼10, was 2–30 pg/g (plasma weight) except for 6-OH-BDE-137 (30 pg/g) and 3-BRP (60 pg/g). The method was validated for recoveries (68–100%), accuracy (84–110%) and precision (3.7–11%) using charcoal-stripped bovine blood plasma spiked with all target compounds (500 and 5000 pg/mL). Finally, the method was applied to analyze six blood plasma samples from frogs and cormorants, where two natural estrogens, one BPA, one OH-PBDE and four BRPs were detected. The greatest total concentrations of estrogens coincided with the least total concentrations of other phenolic compounds for both species. The proposed method based on derivatization followed by LC–MS/MS provides a novel method to simultaneously monitor multiple groups of phenolic compounds in blood plasma.     

Reactions of aminoguanidine with α-dicarbonyl compounds studied by electrospray ionization mass spectrometry

Aminoguanidine possesses extensive pharmacological properties. This drug is recognized as a powerful α-dicarbonyl scavenger. In order to better elucidate the reactivity of aminoguanidine with α-dicarbonyls, aminoguanidine was reacted with several aldehydic and diketonic α-dicarbonyls. Electrospray ionization mass spectrometry is a suitable technique to study chemical and biochemical processes, and was selected for the purpose. In aminoguanidine reactions, triazines were detected and, other compounds that have never been reported before were identified. Triazine precursor forms were detected, namely tetrahydrotriazines and singly dehydrated tetrahydrotriazines. Moreover, species with bicyclic ring structures, and dehydrated forms, were also identified in aminoguanidine reactions. These species appear to result from tetrahydrotriazines and triazines reactions with one dicarbonyl molecule. Experiments revealed that these bicyclic species, in particular the ones resulting from triazines reactivity, could exist in solution, since they were both identified in the reactions of aminoguanidine and of a selected triazine with the dicarbonyls studied. The results obtained, regarding aminoguanidine/triazines reactivities, appear to support the capability of triazines to condensate and form polycyclic ring structures, and also to support literature mechanistic data for dihydroimidazotriazines formation via dihydroxyimidazolidine-triazines. The data obtained in this study may prove to be valuable to complement solution information, concerning the reactivity of amines with α-dicarbonyls, in particular.     

Determination of emerging and priority industrial pollutants in surface water and wastewater by liquid chromatography–negative electrospray ionization tandem mass spectrometry

A method for the simultaneous determination of six perfluoroalkyl compounds (perfluorooctanesulfonic acid (PFOS) and five perfluoroalkyl carboxylic acids), five phenolic compounds (nonylphenol (NP), bisphenol A (BPA), and methyl-, ethyl- and propylparabens), and the brominated flame retardant hexabromocyclododecane (HBCDD) in surface water and effluent wastewater has been developed. The selected pollutants include eight of the industrial pollutants (PFOS and derivatives, NP, and HBCDD) that could be regulated in surface water according to an European Union Directive proposal and four compounds of great concern because their estrogenicity (BPA and parabens). The method is based on solid-phase extraction and determination by high-performance liquid chromatography–triple quadrupole mass spectrometry in negative electrospray ionization mode. Method quantitation limits of NP, PFOS and derivatives, and HBCDD allow its application for routinely control of surface water according to the EU proposal of directive.

Determination of thyroxine and 3,5,3′-triiodothyronine by sub- and super-equivalence isotope dilution analysis

Trace amounts of thyroxine in model samples (160.0 ng.ml^–1 and 20.0 ng.ml^–1), and thyroxine and 3,5,3-triiodothyronine in blood plasma were determined by sub- superequivalence isotope dilution analysis and radio-immunoassay technique. Hormones were labelled by¹²⁵I. The separation of antibody-bound hormone from free hormone was performed by ultracentrifugation. The results show higher accuracy of the sub- super-equivalence isotope dilution method over that of radioimunoassay.     

Determination of pharmaceuticals from different therapeutic classes in wastewaters by liquid chromatography–electrospray ionization–tandem mass spectrometry

A sensitive analytical method has been developed and validated for simultaneous determination of pharmaceuticals from different therapeutic classes, i.e. five sulfonamide (SA) and trimethoprim antimicrobials and the anti-inflammatory drug diclofenac, in effluent wastewaters at trace levels. Effluent samples from treatment of wastewater were enriched by solid-phase extraction (SPE) using the Waters Oasis HLB cartridge. The analytes were identified and quantified by reversed-phase liquid chromatography-tandem mass spectrometry operated in the selected reaction monitoring (SRM) mode, using positive electrospray ionization. The pharmaceuticals were, consequently, quantified both by use of isotopically labelled internal standards and by standard addition methods to address the issue of matrix effects related to signal suppression by co-eluting compounds. Average recoveries from fortified samples were usually >70%, with relative standard deviations below 20%. Method detection limits in wastewater matrices were between 7.0 and 10 ng L(-1). Identification points (IPs) were used for unequivocal identification of target analytes in real samples. Diclofenac, trimethoprim, and sulfamethoxazole were mainly detected, in the concentration range 10 to 400 ng L(-1), in effluent samples collected from four different sewage-treatment plants in Greece.     

Quantification of clomiphene metabolite isomers in human plasma by rapid-resolution liquid chromatography–electrospray ionization–tandem mass spectrometry

Since the 1960s, clomiphene citrate is used for ovulation induction. Since nonresponse to clomiphene therapy is still not well understood, interindividual variability of clomiphene metabolism has been considered to be a plausible explanation. Therefore, a comprehensive, rapid, sensitive, and specific analytical method for the quantification of (E)- and (Z)-isomers of clomiphene and their putative N-desethyl, N,N-didesethyl, 4-hydroxy, and 4-hydroxy-N-desethyl metabolites, and the N-oxides in human plasma has been newly developed, using HPLC-tandem mass spectrometry and stable isotope-labeled internal standards. All standards other than the parent drug were synthesized in our laboratory. Following protein precipitation analytes were separated on a ZORBAX Eclipse plus C18 1.8 μm column with a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile and detected on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Lower limit of quantification for metabolites ranged from 0.06 ng/mL for clomiphene-N-oxides to 0.3 ng/mL for (E)-N-desethylclomiphene. The assay was validated according to FDA guidelines. Plasma levels of clomiphene and its metabolites were measured in two women after single-dose treatment with clomiphene.     

The mechanism of Tröger's base formation probed by electrospray ionization mass spectrometry

Using direct infusion electrospray ionization mass and tandem mass spectrometric experiments [ESI-MS(/MS)], we have performed on-line monitoring of some reactions used to form Tr?ger's bases. Key intermediates, either as cationic species or as protonated forms of neutral species, have been intercepted and characterized. The role of urotropine as the methylene source in these reactions has also been accessed. Reaction pathways shown by ESI-MS(/MS) have been probed by gas-phase ion/molecule reactions, and an expanded mechanism for Tr?ger's base formation based on the mass spectrometric data has been elaborated.     

Analysis of ancient Greco–Roman cosmetic materials using laser desorption ionization and electrospray ionization mass spectrometry

Microsamples of pink cosmetic powders from the Greco–Roman period were analyzed using two complementary analytical approaches for identification of the colouring agents (lake pigments originally manufactured from madder plants with an inert binder, usually a metallic salt) present in the samples. The first technique was a methanolic acidic extraction of the archaeological samples with an additional ethyl acetate extraction of the anthraquinone-type colouring agents which were identified using high performance liquid chromatography coupled to electrospray ionization with high resolution mass spectrometry (LC–ESI–HRMS), and the second was direct analysis of a microsample by laser desorption ionization–mass spectrometry (LDI–MS). The latter technique is well suited when the quantity of samples is very low. This soft ionization technique enables the detection of very small quantities of compounds using the combination of positive and negative-ion modes. It was also successfully applied for the direct analysis of some laboratory-made reference compounds. However, the presence of lead in one of these ancient samples induced a spectral suppression phenomenon. In this case and conditional on a sufficient quantity of available sample, the former method is better adapted for the characterization of these anthraquinone-type molecules. This study also confirmed that purpurin, munjistin, and pseudopurpurin are the principal colouring agents present in these ancient cosmetic powders constituted from madder plants. Presented at the Annual French National Symposium on Mass Spectrometry, Electrophoresis and Proteomics, 20–23 September 2007 in Pau, France.     

Characterization of rat liver microsomal and hepatocytal metabolites of brevetoxins by liquid chromatography–electrospray tandem mass spectrometry

Brevetoxins are natural neurotoxins that are produced by “red tide” algae. This class of compounds can cause neurotoxic shellfish poisoning and other health problems. Brevetoxin-2 is the most abundant among the nine brevetoxins that have been characterized, whereas brevetoxin-1 is the most toxic. In this study, brevetoxin-1 and brevetoxin-2 were incubated with rat liver hepatocytes and rat liver microsomes, respectively. After clean-up steps were taken to remove the proteins, samples were analyzed by liquid chromatography (LC) coupled with electrospray mass spectrometry (LC-MS). After incubation of brevetoxin-1, two metabolites were found: brevetoxin-1-M1 (molecular weight = 900 Da), and brevetoxin-1-M2 (molecular weight = 884 Da). The increase in molecular weight combined with evidence from tandem mass spectrometry showing an increased tendency for loss of water molecules, along with considerations of established precedents for chemical transformations led to the conclusion that brevetoxin-1-M1 was formed by converting one double bond in the E or F ring of brevetoxin-1 into a diol. The second metabolite (brevetoxin-1-M2) is proposed to be a hydrolysis product of brevetoxin-1 involving opening of the lactone ring with the addition of a water molecule. The incubation study of the other starting compound, brevetoxin-2, found two metabolites in the LC-ES-MS selected ion chromatogram. Brevetoxin-2-M1 (molecular weight = 912 Da) gave a large [M−H]⁻ peak at m/z 911, and its product ion mass spectrum allowed the deduction that this metabolite was the hydrolysis product of brevetoxin-2 involving conversion of the lactone to a carboxylic acid and an alcohol. The second metabolite (brevetoxin-2-M2, molecular weight = 896 Da) was deduced to have the same structure as that of brevetoxin-3 based on identical chromatographic retention times and similar mass spectra as those obtained for a brevetoxin-3 standard.     

Characterization of Nα-Fmoc-protected ureidopeptides by electrospray ionization tandem mass spectrometry (ESI-MS/MS): differentiation of positional isomers

Four pairs of positional isomers of ureidopeptides, FmocNH‐CH(R₁)‐φ(NH‐CO‐NH)‐CH(R₂)‐OY and FmocNH‐CH(R₂)‐φ(NH‐CO‐NH)‐CH(R₁)‐OY (Fmoc = [(9‐fluorenyl methyl)oxy]carbonyl; R₁ = H, alkyl; R₂ = alkyl, H and Y = CH₃/H), have been characterized and differentiated by both positive and negative ion electrospray ionization (ESI) ion‐trap tandem mass spectrometry (MS/MS). The major fragmentation noticed in MS/MS of all these compounds is due to ? N? CH(R)? N? bond cleavage to form the characteristic N‐ and C‐terminus fragment ions. The protonated ureidopeptide acids derived from glycine at the N‐terminus form protonated (9H‐fluoren‐9‐yl)methyl carbamate ion at m/z 240 which is absent for the corresponding esters. Another interesting fragmentation noticed in ureidopeptides derived from glycine at the N‐terminus is an unusual loss of 61 units from an intermediate fragment ion FmocNH = CH₂⁺ (m/z 252). A mechanism involving an ion‐neutral complex and a direct loss of NH₃ and CO₂ is proposed for this process. Whereas ureidopeptides derived from alanine, leucine and phenylalanine at the N‐terminus eliminate CO₂ followed by corresponding imine to form (9H‐fluoren‐9‐yl)methyl cation (C₁₄H₁₁⁺) from FmocNH = CHR⁺. In addition, characteristic immonium ions are also observed. The deprotonated ureidopeptide acids dissociate differently from the protonated ureidopeptides. The [M ? H]^? ions of ureidopeptide acids undergo a McLafferty‐type rearrangement followed by the loss of CO₂ to form an abundant [M ? H ? Fmoc + H]^? which is absent for protonated ureidopeptides. Thus, the present study provides information on mass spectral characterization of ureidopeptides and distinguishes the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparison of electrospray ionization and atmospheric pressure chemical ionization for multi-residue analysis of biocides,UV-filters and benzothiazoles in aqueous matrices and activated sludge by liquid chromatography–tandem mass spectrometry

This paper describes the development of a multi-residue method for the determination of 36 emerging organic pollutants (26 biocides, 5 UV-filters and 5 benzothiazoles) in raw and treated wastewater, activated sludge and surface water using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The target analytes were enriched from water samples adjusted to pH 6 by solid-phase extraction (SPE) on Oasis HLB 200 mg cartridges and eluted with a mixture of methanol and acetone (60/40, v/v). Extraction of freeze-dried sludge samples was accomplished by pressurized liquid extraction (PLE) using a mixture of methanol and water (50/50, v/v) as extraction solvent followed by SPE. LC–tandem MS detection was compared using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in positive and negative ionization mode. ESI exhibited strong ion suppression for most target analytes, while APCI was generally less susceptible to ion suppression but partially leading to ion enhancement of up to a factor of 10. In general, matrix effects could be compensated using stable isotope-labeled surrogate standards, indicated by relative recoveries ranging from 70% to 130%. In wastewater, activated sludge and surface water up to 33 analytes were detected. Maximum concentrations up to 5.1 and 3.9 μg L⁻¹ were found in raw wastewater for the water-soluble UV-filters benzophenone-4 (BZP-4) and phenylbenz-imidazole sulfonic acid (PBSA), respectively. For the first time, the anti-dandruff climbazole was detected in raw wastewater and in activated sludge with concentrations as high as 1.4 μg L⁻¹ and 1.2 μg g TSS⁻¹, respectively. Activated sludge is obviously a sink for four benzothiazoles and two isothiazolones, as concentrations were detected in activated sludge between 120 ng g TSS⁻¹ (2-n-octyl-4-isothiazolin-3-one, OIT) to 330 ng g TSS⁻¹ (benzothiazole-2-sulfonic acid, BTSA).     

Investigation of noncovalent complexes between β-cyclodextrin and polyamide acids containing N-methylpyrrole and N-methylimidazole by electrospray ionization mass spectrometry

Electrospray ionization (ESI) mass spectrometry was utilized to investigate noncovalent complexes between beta-cyclodextrin (beta-CD) and five novel polyamide acids containing N-methylpyrrole and N-methylimidazole. The 1:1 binding mode was specified by examining the binding stoichiometry from ESI mass spectra. It found that polyamide acids with beta-CD have binding affinities in the order: ImImImbetaCOOH > ImPyImbetaCOOH > ImPyPybetaCOOH > PyPyPybetaCOOH > NO(2)PyPyPybetaCOOH. The method gives, simultaneously, the binding constants between beta-CD and polyamide acids based on a novel linear equation.     

Ionization and transmission efficiency in an electrospray ionization—mass spectrometry interface

The ionization and transmission efficiencies of an electrospray ionization (ESI) interface were investigated to advance the understanding of how these factors affect mass spectrometry (MS) sensitivity. In addition, the effects of the ES emitter distance to the inlet, solution flow rate, and inlet temperature were characterized. Quantitative measurements of ES current loss throughout the ESI interface were accomplished by electrically isolating the front surface of the interface from the inner wall of the heated inlet capillary, enabling losses on the two surfaces to be distinguished. In addition, the ES current lost to the front surface of the ESI interface was spatially profiled with a linear array of 340-microm-diameter electrodes placed adjacent to the inlet capillary entrance. Current transmitted as gas-phase ions was differentiated from charged droplets and solvent clusters by measuring sensitivity with a single quadrupole mass spectrometer. The study revealed a large sampling efficiency into the inlet capillary (>90% at an emitter distance of 1 mm), a global rather than a local gas dynamic effect on the shape of the ES plume resulting from the gas flow conductance limit of the inlet capillary, a large (>80%) loss of analyte ions after transmission through the inlet arising from incomplete desolvation at a solution flow rate of 1.0 microL/min, and a decrease in analyte ions peak intensity at lower temperatures, despite a large increase in ES current transmission efficiency.