MS-MS Experiments Research Articles

Exploring methandienone metabolites generated via homogenized camel liver: Advancements for anti-doping applications through High Resolution-Liquid Chromatography Mass Spectrometry analysis.

Anabolic steroids, also known as anabolic-androgenic steroids (AAS), encompass steroidal androgens such as testosterone, as well as synthetic counterparts with similar structures and effects. The misuse of AAS has increased over the years, leading to ethical and welfare concerns in sports. The World Anti-Doping Agency (WADA) and the International Federation for Equestrian Sports (FEI) have banned AAS in relevant sports. Methandienone is one of the most identified anabolic androgenic steroids in sports drug testing, Therefore, reliable detection methods are crucial for effective doping control and maintaining the integrity of the sports. This study explores the use of homogenized camel liver for detecting methandienone metabolites in camels. The biotransformation pathways of methandienone in homogenized camel liver tissues are analyzed using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) to identify and characterize the phase I and phase II metabolites. Chromatographic separation was achieved using a Thermo-Hypersil C18 column. The study has identified 11 methandienone metabolites (M1-M11), this includes 10 phase I and one phase II metabolite. A glucuronic acid conjugate of methandienone was observed in this study, but no sulfonic acid conjugations were found. The metabolites and their possible chemical structures, along with their fragmentation patterns are confirmed using MSMS (MS2) experiments in data-independent acquisition (DIA) mode. These findings serve as a vital tool for the rapid detection of methandienone, combating its illicit use in camel racing. Comprehensive screenings covering both the parent drug and its metabolites are recommended to improve detection accuracy and ensure regulatory compliance in sports doping. Future research should explore methandienone's metabolite profile in administered camel samples.

Sequence-Defined Tertiary Amine-Based Oligomer Employing a Scalable, Support-Free, and Protection/Deprotection-Free Iterative Strategy.

Sequence-defined oligomers (SDOs) with their unique monomeric sequence and customizable nature are attracting the attention of researchers globally. The structural and functional diversity attainable in SDOs makes this platform promising, albeit with challenges in the synthesis. Herein, we report the design and synthesis of a novel class of SDO by incorporating tertiary amines into the backbone from commercially available inexpensive materials. Tertiary amines were selected due to their various material and biomedical applications. Even though the synthesis and purification of amine compounds are challenging, their various significant applications, such as pharmaceuticals, catalysts, surfactants, corrosion inhibitors, dye intermediates, polymer additives, rubber accelerators, gas treating agents, agriculture, and analytical chemistry, make them fascinating. The synthetic strategy that is designed here is extremely efficient and economical for the scalable synthesis of the SDO and is support-free, protection-deprotection chemistry-free, and catalyst/template-free. Most importantly, no extra design and synthesis of the monomer is required here. The key reactions employed for the SDO synthesis are (i) transformation of the hydroxy group to a halide and (ii) substitution of the halide by the secondary amine units. Including the purifying processes, the multigram synthesis of 4-mer was completed in 12-14 h. The synthetic strategy was established by synthesizing two different sequences of SDOs. The SDOs are characterized by 1H NMR and LC-MS. The tandem MS (MS/MS) experiment was conducted in order to validate the sequences over the SDO chain. Furthermore, the SDO platform was advanced in two ways: (i) by increasing the chain length via attaching a linker, which provides a rapid method for increasing the tertiary amine over the SDO chain, and (ii) postsynthetic modification of SDO with other functional groups, including guanidine for biological importance and a well-known fluorophore dansyl group for material significance.

Carbon dots surface chemistry drives fluorescent properties: New tools to distinguish isobaric peptides

The possibility to design rational carbon dots surface functionalization for specific analytical and bioanalytical applications is hindered by the lack of a full knowledge of the surface chemical features driving fluorescent properties.In this model study, we have synthesized four different peptides, three of which are isobaric and not distinguishable by common MSMS experiments. After having characterized the peptides conformations by CD analyses, we have covalently bonded all four peptides to carbon dots by using different experimental procedures, which produce different functional groups on the carbon dots surface. The peptide orientations obtained on the differently functionalized surface of the nanoparticles were different and produced different fluorescent responses. The reported results indicate the possibility to design amino and carboxyl enriched surface carbon dots to answer specific chemical requirements, paving the way for the use of these nanoparticles as a versatile and useful new chemical and biochemical tool.

Towards Advances in Molecular Understanding of Boric Acid Biocatalyzed Ring-Opening (Co)Polymerization of δ-Valerolactone in the Presence of Ethylene Glycol as an Initiator.

Following our previous studies on the molecular level structure of (co)oligoesters obtained via anionic homo- and co-polymerization of novel β-substituted β-lactones, prepared by the atmospheric pressure carbonylation reaction of respective epoxides, the boric acid biocatalyzed ring-opening (co)polymerization of δ-valerolactone has been studied. As a co-monomer the 6-methy-ε-caprolactone, prepared by the one-pot oxidation of respective alcohol, and ethylene glycol as polymerization initiator were used. The obtained copolymers were characterized by 1H-NMR, GPC and ESI-MS, respectively in order to confirm their chemical structures and identity. Subsequently, tandem mass spectrometry (MS-MS studies) via collision-induced dissociation were utilized to characterize the fragmentation pattern. ESI-MS and NMR analyses confirmed the formation of random linear copolymer chains composed of different polyester repeat units. MS-MS experiments showed that fragmentation proceeds via ester bound cleavage along the (co)polyester chains. The innovative aspect of this contribution is related to the elaboration of the telechelic (co)polymers end-capped with hydroxyl end groups and well-defined molecular architectures, which could facilitate the development of new flexible macromolecular systems for potential biomedical applications.

Escherichia coli and Sf9 Contaminant Databases to Increase Efficiency of Tandem Mass Spectrometry Peptide Identification in Structural Mass Spectrometry Experiments.

Filtering of nonspecifically binding contaminant proteins from affinity purification mass spectrometry (AP-MS) data is a well-established strategy to improve statistical confidence in identified proteins. The CRAPome (contaminant repository for affinity purification) describes the contaminating background content present in many purification strategies. However, full contaminant lists for nickel-nitrilotriacetic acid (NiNTA) and glutathione S-transferase (GST) affinity matrices are lacking. Similarly, no Spodoptera frugiperda (Sf9) contaminants are available, and only the FLAG-purified contaminants are described for Escherichia coli. For MS experiments that use recombinant protein, such as structural mass spectrometry experiments (hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking, and radical foot-printing), failing to include these contaminants in the search database during the initial tandem MS (MS/MS) identification stage can result in complications in peptide identification. We have created contaminant FASTA databases for Sf9 and E. coli NiNTA or GST purification strategies and show that the use of these databases can effectively improve HDX-MS protein coverage, fragment count, and confidence in peptide identification. This approach provides a robust strategy toward the design of contaminant databases for any purification approach that will expand the complexity of systems able to be interrogated by HDX-MS.

Identification of opioids in surface and wastewaters by LC/QTOF-MS using retrospective data analysis

Opioids, both as prescription drugs and abuse substances, have been a hot topic and a focus of discussion in the media for the last few years. Although the literature published shows the occurrence of opioids and some of their metabolites in the aquatic environment, there are scarce data in the application of high resolution mass spectrometry (HRMS) for the analysis of these compounds in the environment. The use of HRMS allows increasing the number of opioids that can be studied as well as the detection of unknown opioids, their metabolites and potential transformation products. In this work, a retrospective analysis for the identification of opioids and their metabolites using a curated database was applied to surface water and wastewater samples taken in the state of Minnesota (U.S.) in 2009, which were previously analyzed by liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) for antidepressants. The database comprised >200 opioids including natural opiates (e.g. morphine and codeine), their semi-synthetic derivatives (e.g. heroin, hydromorphone, hydrocodone, oxycodone, oxymorphone, meperidine and buprenorphine), fully synthetic opioids (e.g. fentanyl, methadone, tramadol, dextromethorphan and propoxyphene), as well as some of their metabolites (e.g. 6-monoacetylcodeine, dextrorphan, EDDP, normorphine and O-desmethyltramadol). Moreover, additional MS-MS experiments were performed to confirm their identification, as well as to recognize fragmentation patterns and diagnostic ions for several opioids. These data provide a better understanding of the historical occurrence of opioids and their metabolites in surface waters impacted by wastewater sources. The concentrations of individual opioids in surface water and wastewater effluent varied from 8.8 (EDDP) to 1640 (tramadol) ngL−1 and from 12 (dihydrocodeine) to 1288 (tramadol) ngL−1, respectively. The opioids with higher overall frequency detections were tramadol, dextromethorphan and its metabolite, dextrorphan.

Oligomers in polyethylene furanoate - identification and quantification approach via LC-UV LC-MS response ratio

ABSTRACTPolyethylene furanoate polymer is intended to be used as a food contact material. A PEF polymer sample was investigated for its oligomer composition by solvent extraction and using HRLC-MS. The 20 oligomers found were divided into four groups: group I contains cyclic oligomers consisting of furandicarboxylic acid and monoethylene glycol units, group II comprises cyclic oligomers consisting of furandicarboxylic acid, monoethylene glycol units and one diethylene glycol unit, group III are cyclic oligomers were two monoethylene glycol units are substituted by diethylene glycol units and group IV are linear oligomers consisting of furandicarboxylic acid and monoethylene glycol units. Oligomers of group I account for around 87% of the total oligomer content, group II oligomers 12% and group III oligomers 1%. The contribution of group IV oligomers is very small: less than 0.05%. MS-MS experiments showed similar fragmentation patterns for all oligomers. The results of this study demonstrate that oligomers are abundant in the PEF material and are potential migrants to foods that are in contact with the polymer. Oligomers of group I and group II have the same absorption maxima in UV detection which was used to develop a quantification approach for these oligomers using dimethyl 2,5-furandicarboxylate as external standard.

Lipid changes within the epidermis of living skin equivalents observed across a time-course by MALDI-MS imaging and profiling.

BackgroundMass spectrometry imaging (MSI) is a powerful tool for the study of intact tissue sections. Here, its application to the study of the distribution of lipids in sections of reconstructed living skin equivalents during their development and maturation is described.MethodsLiving skin equivalent (LSE) samples were obtained at 14 days development, re-suspended in maintenance medium and incubated for 24 h after delivery. The medium was then changed, the LSE re-incubated and samples taken at 4, 6 and 24 h time points. Mass spectra and mass spectral images were recorded from 12 μm sections of the LSE taken at each time point for comparison using matrix assisted laser desorption ionisation mass spectrometry.ResultsA large number of lipid species were identified in the LSE via accurate mass-measurement MS and MSMS experiments carried out directly on the tissue sections. MS images acquired at a spatial resolution of 50 μm × 50 μm showed the distribution of identified lipids within the developing LSE and changes in their distribution with time. In particular development of an epidermal layer was observable as a compaction of the distribution of phosphatidylcholine species.ConclusionsMSI can be used to study changes in lipid composition in LSE. Determination of the changes in lipid distribution during the maturation of the LSE will assist in the identification of treatment responses in future investigations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12944-015-0089-z) contains supplementary material, which is available to authorized users.

Negative chemical ionization gas chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry and automated accurate mass data processing for determination of pesticides in fruit and vegetables.

Gas chromatography coupled to high resolution hybrid quadrupole time-of-flight mass spectrometry (GC-QTOF MS), operating in negative chemical ionization (NCI) mode and combining full scan with MSMS experiments using accurate mass analysis, has been explored for the automated determination of pesticide residues in fruit and vegetables. Seventy compounds were included in this approach where 50% of them are not approved by the EU legislation. A global 76% of the analytes could be identified at 1μgkg(-1). Recovery studies were developed at three concentration levels (1, 5, and 10μgkg(-1)). Seventy-seven percent of the detected pesticides at the lowest level yielded recoveries within the 70%-120% range, whereas 94% could be quantified at 5μgkg(-1), and the 100% were determined at 10μgkg(-1). Good repeatability, expressed as relative standard deviation (RSD <20%), was obtained for all compounds. The main drawback of the method was the limited dynamic range that was observed for some analytes that can be overcome either diluting the sample or lowering the injection volume. A home-made database was developed and applied to an automatic accurate mass data processing. Measured mass accuracies of the generated ions were mainly less than 5ppm for at least one diagnostic ion. When only one ion was obtained in the single-stage NCI-MS, a representative product ion from MSMS experiments was used as identification criterion. A total of 30 real samples were analyzed and 67% of the samples were positive for 12 different pesticides in the range 1.0-1321.3μgkg(-1).

Identification of Prometon, Deisopropylprometon, and Hydroxyprometon in Groundwater by High Resolution Liquid Chromatography/Mass Spectrometry

Prometon, a major soil sterilant, and its main transformation products, deisopropylprometon (N2-isopropyl-6-methoxy-1,3,5-triazine-2,4-diamine) and hydroxyprometon (4,6-bis(isopropylamino)-1,3,5-triazin-2-ol), were identified as the dominant triazine herbicides in groundwater samples from 51 locations in Colorado, USA, over a two-year time period. They were concentrated from water by solid phase extraction and detected using an ultrahigh pressure, liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC/QTOF-MS). The transformation products, deisopropylprometon and hydroxyprometon, were confirmed using MS-MS experiments. An original strategy was applied to form the degradation standards for deisopropylprometon and hydroxyprometon, which consisted of photo-degradation of prometon followed by MS-MS analysis. The concentration of prometon ranged from the detection limit of 3ng·L-1 to 87ng·L-1, hydroxyprometon ranged up to 50ng·L-1, and deisopropylprometon up to 100ng·L-1, with a frequency of detection of 80%, which was greater than the other triazines detected in the groundwater samples. A new ratio is proposed for prometon degradation called the “deisopropylprometon to prometon ratio” or the DIP ratio, as an indicator of prometon residence time in groundwater. Furthermore, these data suggest that prometon is more of an issue for groundwater contamination in urban areas rather than agricultural areas.

Analysis of Biomolecules by Atmospheric Pressure Visible-Wavelength MALDI-Ion Trap-MS in Transmission Geometry

We report the development of a new AP visible-wavelength MALDI-ion trap-MS instrument with significantly improved performance over our previously reported system (Int. J. Mass Spectrom. 315, 66-73 (2012)). A Nd:YAG pulsed laser emitting light at 532nm was used to desorb and ionize oligosaccharides and peptides in transmission geometry through a glass slide. Limits of detection (LODs) achieved in MS mode correspond to picomole quantities of oligosaccharides and femtomole quantities of peptides. Tandem MS (MS/MS) experiments enabled identification of enzymatically digested proteins and oligosaccharides by comparison of MS/MS spectra with data found in protein and glycan databases. Moreover, the softness of ionization, LODs, and fragmentation spectra of biomolecules by AP visible-wavelength MALDI-MS were compared to those obtained by AP UV MALDI-MS using a Nd:YAG laser emitting light at 355nm. AP visible-wavelength MALDI appears to be a softer ionization technique then AP UV MALDI for the analysis of sulfated peptides, while visible-wavelength MALDI-MS, MS/MS, and MS/MS/MS spectra of other biomolecules analyzed were mostly similar to those obtained by AP UV MALDI-MS. Therefore, the methodology presented will be useful for MS and MS(n) analyses of biomolecules at atmospheric pressure. Additionally, the AP visible-wavelength MALDI developed can be readily used for soft ionization of analytes on various mass spectrometers.

Simultaneous Screening and Identifying Four Categories of Particular Flavonoids in the Leaves of Murraya exotica L. by HPLC-DAD-ESI-MS-MS

Polymethoxylated flavonoids (PMFs), the particular flavonoid subclass in which all or almost all hydroxyls are capped by methylation, have high oral bioavailability and various activities. A sensitive high-performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS-MS) method was established to screen and identify the PMFs in the leaves of Murraya exotica. Eight PMF standards, including two polymethoxylated flavones, two polymethoxylated flavanones, two polymethoxylated chalcones and two PMF glycosides, were first to be analyzed in positive mode by collision-induced dissociation MS-MS. On the basis of the ESI-MS, characterizations were deduced, and in the results of the extracted ion chromatogram MS-MS experiment, 26 PMFs, including 18 flavones, five flavanones or chalcones and three PMF glycosides, were screened out from the complex extract of the leaves of M. exotica. Among them, 24 PMFs were hydroxylated polymethoxyflavonoids, whereas the rest were all permethoxylated PMFs. This was the first systematic report on the presence of PMFs in the leaves of M. exotica. The results indicated that the established analytical method could be adopted as a rapid, effective technique for the structural characterization of PMFs from the complex extracts of traditional Chinese medicines.

Unusual N-glycan Structures Required for Trafficking Toxoplasma gondii GAP50 to the Inner Membrane Complex Regulate Host Cell Entry Through Parasite Motility

Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.

Identification of biotransformation products of macrolide and fluoroquinolone antimicrobials in membrane bioreactor treatment by ultrahigh-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was applied for the identification of transformation products (TPs) of fluoroquinolone (norfloxacin and ciprofloxacin) and macrolide (azithromycin, erythromycin, and roxitromycin) antimicrobials in wastewater effluents from a Zenon hollow-fiber membrane bioreactor (MBR). The detected TPs were thoroughly characterized using the accurate mass feature for the determination of the tentative molecular formulae and MS-MS experiments for the structural elucidation of unknowns. Several novel TPs, which have not been previously reported in the literature, were identified. The TPs of azithromycin and roxithromycin, identified in MBR effluent, were conjugate compounds, which were formed by phosphorylation of desosamine moiety. Transformation of fluoroquinolones yielded two types of products: conjugates, formed by succinylation of the piperazine ring, and smaller metabolites, formed by an oxidative break-up of piperazine moiety to form the 7-[(2-carboxymethyl)amino] group. A semi-quantitative assessment of these TPs suggested that they might have contributed significantly to the overall balance of antimicrobial residues in MBR effluents and thus to the overall removal efficiency. Determination of TPs during a period of 2 months indicated a conspicuous dynamics, which warrants further research to identify microorganisms involved and treatment conditions leading to their formation.

Identification of a new antimicrobial lysine-rich cyclolipopeptide family from Xenorhabdus nematophila

Entomopathogenic bacteria of the genus Xenorhabdus are known to be symbiotically associated with soil dwelling nematodes of the Steinernematidae family. These bacteria are transported by their nematode hosts into the hemocoel of the insect larvae, where they proliferate and produce insecticidal proteins, inhibitors of the insect immune system and antimicrobial molecules. In this study, we describe the discovery of a new family (PAX) of five antimicrobial compounds produced by fermentation of the Xenorhabdus nematophila F1 strain and purified by cation exchange chromatography and reversed phase chromatography. The chemical structure of PAX 3, a lysine-rich cyclolipopetide, was obtained from the analysis of homo and heteronuclear 2D NMR and confirmed by MS-MS experiments. The five members of the PAX family showed significant activity against plants and human fungal pathogens and moderate activity against few bacteria and yeast. No cytotoxicity was observed on CHO or insect cells.

An electrodynamic ion funnel interface for greater sensitivity and higher throughput with linear ion trap mass spectrometers

An electrospray ionization interface incorporating an electrodynamic ion funnel has been designed and implemented on a linear ion trap mass spectrometer (Thermo Electron, LTQ). We found ion transmission to be greatly improved by replacing the standard capillary–skimmer interface with the capillary–ion funnel interface. An infusion study using a serial dilution of a reserpine solution showed that ion injection (accumulation) times to fill the ion trap at a given automatic gain control (AGC) target value were reduced by ∼90% which resulted in an ∼10-fold increase in peak intensities. In liquid chromatography tandem MS (LC–MS/MS) experiments performed using a global protein digest sample from the bacterium, Shewanella oneidensis, more peptides and proteins were identified when the ion funnel interface was used in place of the standard interface. This improvement was most pronounced at lower sample concentrations, where extended ion accumulation times are required, resulting in an ∼2-fold increase in the number of protein identifications. Implementation of the ion funnel interface on a LTQ Fourier transform (FT) mass spectrometer showed a ∼25–50% reduction in spectrum acquisition time. The duty cycle improvement in this case was due to the ion accumulation event contributing a larger portion to the total spectrum acquisition time.

ESI Mass Spectrometry of Non-Polar Fullerene Derivatives: Evaluations for Qualitative and Quantitative Analysis and Elucidation of Structural Information from the ESI MS-MS experiments

not Available.

Design of a sheathless capillary eletrophoresis-mass spectrometry probe for operation with a Z-spray ionization source.

The construction of a sheathless interface for capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS), for operation with a Z-Spray source on a Micromass Quattro-LC triple quadrupole mass spectrometer is described. Designing the interface involved machining a probe compatible with the setup already in place on the mass spectrometer, i.e., MegaFlow-Z ESI. The probe was made of Lexan with the same dimensions as the ESI probe supplied with the instrument. The electrical connection at the electrospray end of the CE capillary was made possible by gold-coating (sheathless CE-ESI-MS). The probe design as well as the electrical and power supply requirements are described in detail. Experiments were performed using this interface, and CE separations of mixtures containing pmole and sub-pmole amounts of peptides were monitored by on-line MS. For a standard peptide mixture (10(-4) M), separation efficiency was typically characterized by N > 10(4) theoretical plates with S/N > 400. Using the same experimental setup, it was also possible to conduct on-line CE-ESI-tandem MS (MS/MS) experiments on the same peptide mixture, and to determine the sequence of the peptides. MS/MS scan functions for different precursor ions were used either alternately or sequentially and the results from both methods were compared. The possibility of peptide mass mapping was explored, and CE-ESI-MS results were obtained for the digestion products of equine myoglobin. Separation efficiencies and S/N values were similar to those obtained for standard peptides. A complete map of the digestion products was obtained.

Separation and identification of ceramides in the human stratum corneum by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and electrospray multiple-stage mass spectrometry profiling

A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum coracum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramdies than that achievable by thin-layer chromatography.

Determination of Diarrheic Shellfish Toxins in Mussels b Microliquid Chromatography-Tandem Mass Spectrometry

A fast, sensitive, and specific procedure for determining toxins that cause diarrheic shellfish poisoning (DSP) using microliquid chromatography coupled with tandem mass spectrometry (micro-LC-MS-MS) is reported. The lipophylic polyether acidic toxins okadaic acid (OA), its isomer dinophysistoxin-2 (DTX-2), the 35-methylokadaic acid dinophysistoxin-1 (DTX-1), and the novel toxin dinophysistoxin-1B (DTX-2B; recently isolated from Irish mussels) were extracted from shellfish tissues with acetone and chromatographed by isocratic elution at 10 microL/min with CH3 CN-H2O, 80 + 20 (v/v), containing 0.1% trifluoroacetic acid, through a C18 reversed-phase column (1.0 mm id). The chromatograph is coupled via an ion spray interface to an atmospheric pressure ionization source. Collision-induced-dissociation (CID) ion mass spectra of the protonated molecule, [M + H]+, at m/z 805 for OA, DTX-2, and DTX-2B and at m/z 819 for DTX-1, were obtained in MS-MS experiments to identify 2 diagnostic fragment ions for each analyte that could be used for selected-reaction-monitoring (SRM) micro-LC-MS-MS analysis. The CID spectrum of DTX-2B confirmed it to be a new OA isomer, like DTX-2. Standard curves obtained by SRM micro-LC-MS-MS were linear (r2 > or = 0.9992) over the range 0.05-1.00 micrograms/mL (i.e., 0.10-2.00 micrograms toxin/g hepatopancreas), and a detection limit of 15 pg/injection was obtained for each DSP toxin. Average recoveries ranged from 95 to 101%, and coefficients of variation ranged from 1.8 to 3.4%. This novel SRM micro-LC-MS-MS method was used to confirm acidic DSP toxins in Irish and Italian toxic mussels. It offers a high degree of specificity because analyte confirmation is based on retention time, molecular weight, structural information obtained from the presence of 2 diagnostic fragments for each analyte, and ion ratios. OA was found in both Irish (< or = 0.7 micrograms/g hepatopancreas) and Italian (< or = 1.5 micrograms/g hepatopancreas) mussels. DTX-1 was found only in Italian mussels (< or = 0.3 micrograms/g hepatopancreas). DTX-2 (< or = 6.1 micrograms/g hepatopancreas) and DTX-2B (< or = 0.08 micrograms/hepatopancreas) were unique to Irish shellfish.