Liquid Chromatography-electrospray Ionization-ion Trap Mass Spectrometry Research Articles

Detection of the Neurotoxic Amino Acid β-N-methylamino-L-alanine in Axenic Cultures of Cyanobacterial Isolates by the Application of Underivatized Method of Analysis

There is a growing demand to establish reliable method for the detection of cyanobacterial toxins in order to bewell informed on the prevalence of cyanotoxins in our environments. This study employed the underivatizedmethod of analysis to detect the production of BMAA by the axenic cultures of cyanobacterial strains SynechocystisNPLB 2 and Nostoc MAC PCC 8009 using liquid chromatography-electrospray ionization - ion trap massspectrometry (LC/ESI-ITMS). Cyanobacterial cultures were grown in the laboratory for 12 weeks after whichBMAA was extracted using methanol and analysed by LC/ESI-ITMS. Based on retention time, mass-to-chargeratio, and ratio of the product ions, the underivatized method of analysis employed in this study showed that bothSynechocystis NPLB 2 and Nostoc MAC PCC 8009 strains produced BMAA. The results of this study suggests thatthe LC/ESI-ITMS is a promising method for the analysis of BMAA in cyanobacterial matrices.

Characterization of fortimicin aminoglycoside profiles produced from Micromonospora olivasterospora DSM 43868 by high-performance liquid chromatography-electrospray ionization-ion trap-mass spectrometry.

In this study, an efficient high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-ion trap-tandem mass spectrometry (MS/MS) was developed for the identification of the biosynthetic congeners involved in the aminocyclitol aminoglycosidic fortimicin pathway from Micromonospora olivasterospora fermentation. The usage of both acid extraction (pH ∼2.5) followed by an cationic-exchanging SPE cleanup and pentafluoropropionic acid mediated ion-pairing chromatography with ESI-ion trap-MS/MS detection was determined to be sufficiently practical to profile the fortimicin (FOR) congeners produced in a culture broth. The limit of the quantification for the fortimicin A (FOR-A) standard spiked in the culture broth was ∼1.6 ng mL(-1). The average recovery rate was 93.6%, and the intra- and inter-day precisions were <5% with accuracy in the range from 87.1 to 94.2%. Moreover, the epimeric mixtures including FOR-KH, FOR-KR, and FOR-B were separately resolved through a macrocyclic glycopeptide (teicoplanin)-bonded chiral column. As a result, ten natural FOR pseudodisaccharide analogs were identified and semi-quantified in descending order as follows: FOR-A, FOR-B, DCM, FOR-KH plus FOR-KR, FOR-KK1, FOR-AP, FOR-KL1, FOR-AO, and FOR-FU-10. This is the first report on both the simultaneous characterization of diverse structurally closely related FORs derived from bacterial fermentation using HPLC-ESI-ion trap-MS/MS analysis and the chromatographic separation of the three FOR epimers.

Simultaneous determination of four designer drugs and their major metabolites by liquid chromatography-mass spectrometry.

A sensitive liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) method was utilized for the simultaneous analysis of four designer drugs and their in vitro metabolites in rat liver microsome S9 fraction. Four designer drugs, including methcathinone (MC), 3,4-methylenedioxymethcathinone (MDMC), 3,4-methylenedioxy-pyrovalerone (MDPV) and 4'-methyl-α-pyrrolidinopropiophenone (MPPP), were individually incubated with rat liver microsome S9 fraction, and the incubation mixtures were pooled together and analyzed by LC-ESI-ITMS simultaneously. Besides four designer drugs, five of their main metabolites were identified via the analysis of protonated molecules and tandem mass spectrometry data. Meanwhile, the quantification analysis of four designer drugs in rat liver microsome S9 fraction was performed, the calibration curves showed good linearity in the range of 0.01-5.0μg/mL and the detection limits were below 0.03μg/mL with RSDs less than 5.9% and recovery ratios above 77.4%. The experimental results not only showed that these designer drugs could be easily metabolized in rat liver microsome, and also displayed the superiorities of the method including time and cost saving, high efficiency, sensitivity and selectivity. The studies in this study indicated that the approach could be applied in the determination of illicit drugs and their metabolites in medical, pharmaceutical and forensic investigations.

Determination and quantitative analysis of designer drugs in human urine by liquid chromatography-mass spectrometry

A robust liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) method was developed and utilized for the determination and quantitative analysis of four designer drugs in human urine.

A sensitive HPLC assay for quantitative determination of polyamidoamine dendrimers after pre-column derivatization using FITC

A pre-column derivatization method was developed to analyze polyamidoamine (PAMAM) dendrimers. The fluorescein isothiocyanate (FITC) derivation method improved the retention of PAMAM dendrimers on the reversed-phase column of high performance liquid chromatography (RP-HPLC). The derivation benefited the UV-Vis detection of PAMAM, although the fluorescence of FITC was quenched by PAMAM dendrimers. The primary amine-terminated PAMAM dendrimers, generation 3 (PAMAM-NH2 (G3)), were quantitatively determined by UV-Vis spectroscopy over the concentration range of 10–80 μg mL−1, with the calibration curve expressed as A = 131.6C (μg mL−1) − 295.6 (R2 = 0.9901) and the detection limit of 2.7 μg mL−1. The binding ratio of FITC to PAMAM-NH2 (G3) was determined as 8 : 1 by liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-IT-MS).

Development of a Method for the Quantification of Caseinate Traces in Italian Commercial White Wines Based on Liquid Chromatography–Electrospray Ionization–Ion Trap–Mass Spectrometry

A method using the combination of size exclusion-solid phase extraction and ultrafiltration, followed by tryptic digestion and analysis of the protein digest by liquid chromatography-electrospray ionization-3D ion trap-mass spectrometry (LC-ESI-3D IT-MS), was developed for the detection and quantification of caseinate traces potentially resulting from fining processes in white wines. In particular, several tryptic peptides generated from the main proteins constituting caseinate (β-, αS1-, and αS2-caseins) were used as markers of its presence in the wine matrices; among them, the β-casein peptide GPFPIIV was found to be the best marker for quantification purposes. Method linearity and sensitivity were assessed on a series of Italian commercial white wines, first checked for the absence of any peptide signal attributable to caseins introduced during their production and subsequently spiked with increasing concentrations of caseinate, to provide samples for matrix-matched calibrations. Limits of detection ranging between 0.09 and 0.29 mg/L (S/N = 3), according to the wine, were achieved using a 10 mL sample volume and the MS signal of GPFPIIV as the response related to the caseinate concentration. Such levels are comparable or even lower than the one (0.25 mg/L) recently adopted as a threshold by European Union legislation concerning the indication of milk- and egg-derived fining agents on wine labels, that is, the most restrictive one among those currently proposed in the world.

Simultaneous analysis of analgesics and their major metabolites by liquid chromatography-electrospray ion trap mass spectrometry

A high-throughput analysis method applying liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) for the simultaneous analysis of analgesics and their in vitro metabolites is described. Four analgesics, including tramadol, ketamine, pethidine and methadone, were individually incubated with rat liver microsome S9 fraction initially. The incubation samples were pooled together for analysis by LC-ESI-ITMS in positive ion and full-scan detection modes. The analgesics and their metabolites were identified from the detection of the protonated molecules. The metabolite detection was confirmed by tandem mass spectrometry (MSn) analysis. In addition, the metabolic stabilities of the analgesics were determined by comparing the peak intensities between the control and incubated samples. The obtained results indicated that the method could provide a significant increase in analytical throughput and can be applied in the detection of samples in forensic analysis and in clinically addicted cases.

Mass spectrometry‐based chemotaxonomic classification of Penicillium species (P. echinulatum, P. expansum, P. solitum, and P. oxalicum) and its correlation with antioxidant activity

In this study, 4 Penicillium species (17 strains) were classified on the basis of metabolite profile (chemotaxonomy) by using liquid chromatography-electrospray ionization ion trap-mass spectrometry (LC-ESI-MS), gas chromatography-ion trap-mass spectrometry (GC-IT-MS) and multivariate statistical analysis. The LC-ESI-MS-based dendrogram was similar to the internal transcribed spacer (ITS)-based dendrogram, in that Penicillium oxalicum was separated from the other 3 species. Moreover, vermiculidiol, meleagrin, oxaline, glandicolin A and B, and secalonic acid D were identified as metabolites that enable discrimination of Penicillium species by partial least squares discriminant analysis (PLS-DA). Evaluation of the species-specific metabolites produced by P. expansum, P. echinulatum, and P. solitum revealed that the 3 species differed from each other. On the other hand, GC-IT-MS-based dendrogram revealed that P. expansum was clearly classified separately from the other 3 species, and this result correlated with the antioxidant activity of the 4 species: P. expansum had a higher radical scavenging activity than the other 3 species. The metabolites produced in higher amounts in P. expansum were gluconic acid (12, 29, 33); andrastin A (16), B (15), and C (17); chaetoglobosin C (14), a class of sugar (31, 32); and salicylic acid (28). The results of this study demonstrated that metabolite-based chemotaxonomy could be used not only as a classification method but also as a tool for evaluation of species-specific activities.

Validation of an LC–ESI-MS/MS method for the quantitation of phosphodiesterase-5 inhibitors and their main metabolites in rat serum and brain tissue samples

This work proposes a liquid chromatography-electrospray ionization ion trap mass spectrometry (LC-ESI-ITMS) method, for the quantification of sildenafil (SDF), tadalafil (TDF) and vardenafil (VDF) and their metabolites N-desmethylSDF, O-desethylSDF and N-desethylVDF, preceded by a sample preparation step based on protein and phospholipid elimination. A C8 column (150 mm × 4.6 mm, 5 μm) with ammonium formate (20mM) and acetonitrile as the mobile phase components have been used. This method has been validated, obtaining limits of quantification ranged from 1 to 2.5 ng/mL and 2 to 5 ng/g in serum and brain tissue respectively, while limits of detection ranged from 0.3 to 0.9 ng/mL in serum and 0.6 to 1.9 ng/g in brain tissue. Assay recoveries for low level QC samples were higher than 83% and the matrix effect ranged between 91% and 108% in serum and between 98% and 107% in brain tissue. The method has been applied to the quantification of these compounds in the serum and brain tissue of rats treated intraperitoneally with 10 mg/kg of SDF, TDF or VDF.

Identification and quantitation of auxins in plants by liquid chromatography/electrospray ionization ion trap mass spectrometry

Auxin is an important phylohormone, which regulates specific physiological responses such as division, elongation and differentiation of cells. A new method using liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS) has been developed for identification and quantitation of four auxins. Under the optimum conditions, four auxins (indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and 1-naphthylacetic acid) were completely separated and quantitated within 7 min with a minimum detection limit of 8.0 ng mL(-1) with relative standard deviations lower than 5.0%. This method also has been applied to analysis of auxins in Chinese cabbage where, even with a complicated serious background perturbation due to the natural biological matrix, the mean recoveries ranged from 77.5% to 99.8%. Finally, we discuss the MS-relevant properties of the identified auxins in detail.

Identification and quantitation of pyrethroid pesticide residues in vegetables by solid‐phase extraction and liquid chromatography/electrospray ionization ion trap mass spectrometry

A quantitative method consisting of solid-phase extraction (SPE) followed by liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS) analysis was developed for the identification and quantitation of ten pyrethroid pesticides commonly used in vegetables. The best HPLC separation was achieved using a gradient program of methanol/water mixture. For the vegetable samples, an SPE procedure to clean up the matrices was carried out prior to LC/MS analysis. Under the optimum conditions, the limits of quantification of the pyrethroid pesticides (tetramethrin, allethrin, fenpropathrin, lambda-cyhalothrin, cypermethrin, deltamethrin, fenvalerate, bioresmethrin, permethrin and bifenthrin) ranged from 0.03 to 0.1 mg kg-1 with relative standard deviations<20%, and the mean recoveries ranged from 69.5 to 102.5%. The proposed method has been successfully applied to the determination of pyrethroids in six vegetables with satisfactory results.

Characterization of Moenomycin Antibiotic Complex by Multistage MALDI-IT/RTOF-MS and ESI-IT-MS

Flavomycin is a commercially available antimicrobial growth promoter and an authorized additive for feeding stuffs in the EU and in the USA. As most antibiotically active products biosynthesized by microorganisms, it contains not only a single active compound but is a complex mixture of structurally closely related substances. Multistage matrix-assisted laser desorption/ionization-ion trap/reflectron time-of-flight mass spectrometry (MALDI-IT/RTOF-MS) and liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-IT-MS) were utilized for a detailed analysis of the constituents of the Flavomycin complex based on low-energy collision induced dissociation (CID). An optimal sample preparation for negative ion vacuum MALDI-MS for this compound class was developed. The MALDI-IT/RTOF-MS2 and -MS3 analysis starting with the precursor [M - H]- ions of these interesting phosphoglycolipids, named moenomycins, yielded a large variety of product ions that facilitated the structural characterization of this class of compounds. Based on the derived CID fragmentation pathway of the five known major constituents, namely moenomycin A, moenomycin A12, moenomycin C4, moenomycin C3. and moenomycin C1, four not yet described moenomycin-type constituents could be characterized. They were assigned as 4F-demethyl-6E-O-de-beta-D-glucopyranosyl-moenomycin A, 6B-N-de(2-hydroxy-5-oxo-1-cyclopenten-1-yl)-moenomycin A, 6B-hydroxy-6B-de[N-(2-hydroxy-5-oxo-1-cyclopenten-1-yl)amino]-moenomycin A, and 6C-hydroxy-moenomycin A. In addition, a moenomycin A carrying an oxygen in the moenocinol-group was found, which is most probably a chemical degradation product. These new compounds were verified by LC-ESI-IT-MS.

Online microdialysis‐dynamic nanoelectrospray ionization‐mass spectrometry for monitoring neuropeptide secretion

Although mass spectrometric approaches offer a sensitive method for identifying cell-cell signaling peptides, the high salt-containing environment of extracellular solutions often complicates characterization of these microscale samples. Accordingly, we have developed a miniature hollow-fiber microdialysis device optimized for desalting small-volume neuronal samples online, with the device directly connected to a modified dynamic nanoelectrospray ionization assembly interfaced with an ion trap mass spectrometer. Improvements over existing designs include placement of a capillary insert within the microdialysis fiber to minimize volume, as well as the use of a microinjector that enables 1 microl sample injections. We present detailed evaluation of peptide recoveries within the microdialysis fiber by liquid chromatography-electrospray ionization-ion trap-mass spectrometry analysis of tissue homogenate in artificial seawater with and without microdialysis. Analyte recoveries after microdialysis ranged from 6 to 78% with higher recoveries of more hydrophilic peptides, while little correlation between mass and percentage recovery was observed in the range studied (2000 to 6000 Da). Recoveries of peptides were the lowest for the analytes with the highest initial mass spectrometry signal intensity. Finally, we illustrate the utility of this microdialysis device for desalting neuropeptides secreted from preparations of the peptidergic bag cell neurons of the marine mollusk, Aplysia californica. Without microdialysis, the high concentration of salts ( approximately 0.5 M) prevented detection of peptides, whereas following online microdialysis-dynamic nanoelectrospray mass spectrometry of stimulated releasate, three peptides (acidic peptide, acidic peptide 1-24 and delta-bag cell peptide) were detected.

Sequencing and Mass Profiling Highly Modified Conotoxins Using Global Reduction/Alkylation Followed by Mass Spectrometry

A novel high-throughput method for characterizing heavily modified peptides from cone snail venom is described. Unpurified cone snail duct venom, consisting primarily of multiply disulfide-bonded peptides, is reduced and alkylated using a global procedure in order to simultaneously reduce and derivatize dozens of disulfide-bonded peptides. Samples of Conus victoriae venom are analyzed by online liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-MS) with collisionally induced dissociation (CID). Comparison of the mass profiles of peptides and CID spectra before and after the global reduction and alkylation enables cysteine-containing conopeptides to be ascertained. In this case, over 40 conotoxins are characterized based on only two LC-ESI-MS experiments in terms of mass, number of disulfide-linked cysteine residues (and hence, potential toxin superfamilies), relative hydrophobicity, and other posttranslational modifications. Using this technique, over half of the amino acids (by mass) of several peptides are defined prior to any detailed sequencing studies. Further comparison of the mass data with previously published genetic information allows sequence verification of three novel peptides, termed vc5b, vc6b and vc6c, based on both LC-ESI-MS CID and nanoelectrospray ionization-ion trap-mass spectrometry (nanoESI-MS) experiments. This global method is ideally suited to the use of larger genetic databases in order to efficiently sequence peptides in Conus venoms and is also applicable to analysis of other disulfide-rich classes of peptides such as defensins, chemokines, and snake, spider, or other venoms.

Simultaneous determination of androstenediol 3-sulfate and dehydroepiandrosterone sulfate in human serum using isotope diluted liquid chromatography–electrospray ionization-mass spectrometry

A simple method for simultaneous determination of androstenediol 3-sulfate (Adiol-3S) and dehydroepiandrosterone sulfate (DHEA-S) in human serum using isotope diluted liquid chromatography–electrospray ionization-ion trap-mass spectrometry (LC–ESI-ion trap-MS) was developed. After addition of deuterated internal standards ([ 2 H 5 ]Adiol-3S and [ 2 H 4 ]DHEA-S), human serum (100 μl) was deproteinized with acetonitrile and then applied to a solid-phase extraction cartridge, Oasis HLB. The obtained steroid sulfates fraction was washed with hexane and then analyzed by LC–ESI-MS operated in the negative ion mode. The quantification ranges of Adiol-3S and DHEA-S were 10–400 ng/ml and 0.05–8 μg/ml, respectively. The method does not require the chemical or enzymatic hydrolysis of the conjugates and purification with high-performance liquid chromatography, and shows satisfactory reproducibility and accuracy. The concentrations of these sulfates in the sera of healthy male volunteers ( n=14) were 19.2–245.3 mg/ml (Adiol-3S) and 0.175–5.16 μg/ml (DHEA-S), and those of patients with prostate cancer ( n=19) were 15.3–182.7 ng/ml (Adiol-3S; four samples, not detectable) and 0.110–2.421 μg/ml (DHEA-S).

Complete sequencing of anti-vancomycin fab fragment by liquid chromatography–electrospray ion trap mass spectrometry with a combination of database searching and manual interpretation of the MS/MS spectra

Sequencing of anti-vancomycin monoclonal antibody (mAb) Fab region (48,000 Da) was carried out using liquid chromatography–electrospray ionization ion trap mass spectrometry (LC/ESI-MS). Comprehensive strategies were employed to ensure complete sequence coverage. The sequence information was obtained from the spectra of collision-induced dissociation (CID) (MS/MS) of the protonated proteolytic peptides resulting from multiple enzymatic digestions of reduced/ S-carboxymethylated (RCM) light chain and Fd fragment. Database searching of the spectra against the published immunoglobulin G (IgG) sequences allowed the identification of all the peptides in constant domains as well as partial sequences in variable domains. The rest of the sequences were deduced by manual interpretation of the peptide tandem mass spectrometry (MS/MS) spectra. The analysis showed that the N-terminus of the heavy chain was modified by the conversion of a glutamine residue to pyroglutamic acid.

Determination of 2-Amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and Its Metabolite 2-Hydroxyamino-PhIP by Liquid Chromatography/Electrospray Ionization–Ion Trap Mass Spectrometry

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic aromatic amine found in cooked meat. It is metabolically activated by the human cytochrome P450 enzymes to form the carcinogenic metabolite N2-OH-PhIP. PhIP has been found to induce tumors in rats and is a suspected human carcinogen. In the present work, we have developed and validated a liquid chromatography–electrospray ionization/ion trap mass spectrometry (LC-ESI/ITMS) method for the determination of PhIP and N2-OH-PhIP. PhIP was incubated with microsomes prepared from the human liver; the PhIP and N2-OH-PhIP formed were isolated from the biomatrices by solid-phase extraction using C18 cartridges, with recoveries greater than 86%. Subsequently, the products were separated on a microbore reversed-phase C18 liquid chromatograph coupled to an ESI-ITMS. The ESI interface and the ITMS were tuned for various parameters, and data acquisition was performed in selective ion monitoring mode. The detection limit of PhIP and N2-OH-PhIP was 1 and 10 pg, respectively. The method is highly sensitive and selective, has simple sample preparation protocols, and should be applicable to the study of the metabolic activation of PhIP in various human tissues.

Direct analysis of microcystins by microbore liquid chromatography electrospray ionization ion-trap tandem mass spectrometry

Microcystins are a group of structurally similar cyclic heptapeptide hepatotoxins and tumor promoters, produced by cyanobacteria. A microbore liquid chromatography electrospray ionization ion-trap mass spectrometry (LC-ESI-ITMS) method has been developed which is capable of separating and detecting trace amounts of microcystin variants in environmental samples. Extracted water sample was loaded onto a LC trapping column and, using a column switching technique, the compounds of interest were back-flushed onto a 1-mm LC column. Structural elucidation was achieved using ion-trap with tandem mass spectrometry in the data dependent scan mode. Collision-induced dissociation to MS 3 allowed tentative identification of these cyclic peptides. Full-scan LC-ESI-MS mass spectrum was obtained when 250 pg of the authentic compound was injected onto the HPLC column, which represents the detection limit for microcystin-LR. This study demonstrated that LC-ESI-ITMS is a reliable and sensitive technique for analysing trace levels of microcystins.