Direct infusion electrospray ionization–ion mobility–mass spectrometry for comparative profiling of fatty acids based on stable isotope labeling

A rapid method for fatty acids (FAs) comparative profiling based on carboxyl-specific stable isotope labeling (SIL) and direct infusion electrospray ionization–ion mobility–mass spectrometry (ESI–IM–MS) is established. The design of the method takes advantage of the three-dimensional characteristics of IM–MS including drift time, m/z and ion intensity, for comparison of d0-/d6-2,4-dimethoxy-6-piperazin-1-yl pyrimidine (DMPP)-labeled FAs. In particular, without chromatographic separation, the method allowed direct FAs profiling in complex samples due to the advantageous priority of DMPP in signal enhancement as well as the extra resolution that IM–MS offered. Additionally, the d0-/d6-DMPP-labeled FAs showed expected features, including very similar drift times, 6 Da mass deviations, specific reporter ions, similar MS responses, and adherence to the drift time rule regarding the influence of carbon chain length and unsaturation on relative drift times. Therefore, the introduction of isotope analogs minimized the matrix effect and variations in quantification and ensured accurate identification of non-targeted FAs by those typical features. Peak intensity ratios between d0-/d6-DMPP-labeled ions were subsequently used in relative quantification for the detected FAs. The established strategy has been applied successfully in the rapid profiling of trace free FAs between normal and cancerous human thyroid tissues. Sixteen free FAs were found with the increased level with a statistically significant difference (p < 0.05) compared to the normal tissue samples. The integrated SIL technique and ESI–IM–MS are expected to serve as an alternative tool for high-throughput analysis of FAs in complex samples.     

Polymer architectures via mass spectrometry and hyphenated techniques: A review

This review covers the application of mass spectrometry (MS) and its hyphenated techniques to synthetic polymers of varying architectural complexities. The synthetic polymers are discussed as according to their architectural complexity from linear homopolymers and copolymers to stars, dendrimers, cyclic copolymers and other polymers. MS and tandem MS (MS/MS) has been extensively used for the analysis of synthetic polymers. However, the increase in structural or architectural complexity can result in analytical challenges that MS or MS/MS cannot overcome alone. Hyphenation to MS with different chromatographic techniques (2D × LC, SEC, HPLC etc.), utilization of other ionization methods (APCI, DESI etc.) and various mass analyzers (FT-ICR, quadrupole, time-of-flight, ion trap etc.) are applied to overcome these challenges and achieve more detailed structural characterizations of complex polymeric systems. In addition, computational methods (software: MassChrom2D, COCONUT, 2D maps etc.) have also reached polymer science to facilitate and accelerate data interpretation. Developments in technology and the comprehension of different polymer classes with diverse architectures have significantly improved, which allow for smart polymer designs to be examined and advanced. We present specific examples covering diverse analytical aspects as well as forthcoming prospects in polymer science.     

Flavonoids in Leguminosae: analysis of extracts of T. pratense L., T. dubium L., T. repens L., and L. corniculatus L. leaves using liquid chromatography with UV, mass spectrometric and fluorescence detection

Reversed-phase LC on C-18 bonded silica with a methanol–ammonium formate gradient was used to determine the main flavonoids in leaves of four species of the Leguminosae family. The detection modes were diode-array UV absorbance, fluorescence, and (tandem) mass spectrometry. LC–UV was used for a general screening, sub-classification, and the calculation of total flavonoid contents. LC–FLU was included to identify isoflavones on the basis of their native fluorescence. Most structural information regarding aglycons, sugar moieties, and acidic groups was derived from LC–MS in both the full-scan and extracted-ion mode, using negative-ion atmospheric pressure chemical ionization. MS/MS did not provide much additional information, because the same fragments were observed as in full-scan MS.In T. pratense and T. repens, the main constituents were flavonoid glucoside–(di)malonates, while T. dubium and L. corniculatus mainly contained flavonoid (di)glycosides. Satellite sets comprising an aglycon, the glucoside and glucoside–malonates or –acetates, were abundantly present only in T. pratense. Generally speaking, the main aglycons and sugars in the four plant species are surprisingly different. In addition, while the results for T. pratense are similar to those reported in the literature, there is little agreement in the case of the other species. Finally, total flavonoid contents ranged from 50–65 mg/g for L. corniculatus and T. dubium, to 15 mg/g for T. pratense and only 1 mg/g for T. repens.     

Characterization of polysorbate 85, a nonionic surfactant,by liquid chromatography vs. ion mobility separation coupled with tandem mass spectrometry

Liquid chromatography (LC) and ion mobility (IM) separation have been coupled with mass spectrometry (MS) and tandem mass spectrometry (MS²) to characterize a commercially important nonionic surfactant, polysorbate 85. The constituents of this amphiphilic blend contained a sorbitan or isosorbide core that was chain extended with poly(ethylene oxide) (PEO) and partially esterified at the PEO termini with oleic acid or, to a lesser extent, other fatty acids. Using interactive LC in reverse-phase mode, the oligomers of the surfactant were separated according to their hydrophobicity/hydrophilicity balance. On the other hand, IM spectrometry dispersed the surfactant oligomers by their charge and collision cross section (i.e. size/shape). With either separation method, an increased number of fatty ester groups and/or lack of the polar sorbitan (or isosorbide) core led to higher retention/drift times, enabling the separation of isobaric species or species with superimposed isotope patterns, so that their ester content could be conclusively identified by MS². LC–MS and IM–MS permitted the detection of several byproducts besides the major PEO-sorbitan oleate oligomers. LC–MS provides the separation resolution needed for quantitative determination of the degree of esterification. IM–MS, which minimizes analysis time and solvent use, is ideally suitable for a fast, qualitative survey of samples differing in their minor constituents or impurities.     

Application of isotope-dilution laser ablation ICP–MS for direct determination of Pu concentrations in soils at pg g<Superscript>−1</Superscript> levels

The methods available for determination of environmental contamination by plutonium at ultra-trace levels require labor-consuming sample preparation including matrix removal and plutonium extraction in both nuclear spectroscopy and mass spectrometry. In this work, laser-ablation inductively coupled plasma mass spectrometry (LA–ICP–MS) was applied for direct analysis of Pu in soil and sediment samples. Application of a LINA-Spark-Atomizer system (a modified laser ablation system providing high ablation rates) coupled with a sector-field ICP–MS resulted in detection limits as low as 3×10^–13 g g^–1 for Pu isotopes in soil samples containing uranium at a concentration of a few g g^–1. The isotope dilution (ID) technique was used for quantification, which compensated for matrix effects in LA–ICP–MS. Interferences by UH⁺ and PbO₂⁺ ions and by the peak tail of ²³⁸U⁺ ions were reduced or separated by use of dry plasma conditions and a mass resolution of 4000, respectively. No other effects affecting measurement accuracy, except sample inhomogeneity, were revealed. Comparison of results obtained for three contaminated soil samples by use of -spectrometry, ICP–MS with sample decomposition, and LA–ICP–IDMS showed, in general, satisfactory agreement of the different methods. The specific activity of ^239+240Pu (9.8±3.0 mBq g^–1) calculated from LA–ICP–IDMS analysis of SRM NIST 4357 coincided well with the certified value of 10.4±0.2 mBq g^–1. However, the precision of LA–ICP–MS for determination of plutonium in inhomogeneous samples, i.e. if "hot" particles are present, is limited. As far as we are aware this paper reports the lowest detection limits and element concentrations yet measured in direct LA–ICP–MS analysis of environmental samples.Sergei F. Boulyga is on leave from The Radiation Physics and Chemistry Problems Institute, 220109 Sosny, Minsk, Belarus.     

Characterisation of selected drugs with nitrogen-containing saturated ring structures by use of electrospray ionisation with ion-trap mass spectrometry

The electrospray ionisation–ion-trap mass spectrometry (ESI–MS ⁿ ) of selected drugs with nitrogen-containing saturated ring structures has been investigated. Sequential product-ion fragmentation experiments (MS ⁿ ) have been performed to elucidate degradation pathways for the [M+H]⁺ ions and their predominant fragment ions. These MS ⁿ experiments result in characteristic fragmentations in which functional groups are generally cleaved from the ring systems as neutral molecules such as H₂O, amines, alkenes, esters, carboxylic acids, etc. When such a nitrogen-containing drug molecule also contains a functional group, such as an ester, that on liberation as a neutral molecule has a significantly lower –H _f° value than that of the corresponding amine then the former is preferentially liberated. Furthermore, when an aromatic entity is present in these drug molecules together with the nitrogen-containing saturated ring structure fragmentation of the latter ring occurs with the former, predictably, being resistant to fragmentation. The structures of fragment ions proposed for ESI–MS ⁿ can be supported by electrospray ionisation–quadrupole time-of-flight mass spectrometry (ESI–QTOFMS). The data presented in this paper therefore provide useful information on the structure of these heterocyclic compounds which could be used to characterise unknown drug compounds isolated from natural sources, for example.     

Advances in analytical toxicology: the current role of liquid chromatography–mass spectrometry in drug quantification in blood and oral fluid

This paper reviews procedures for quantification of drugs in the biosamples blood, plasma, serum, or oral fluid (saliva, etc.) using liquid chromatography coupled with single-stage or tandem mass spectrometry (LC–MS, LC–MS–MS). Such procedures are important prerequisites for competent toxicological judgment and consultation in clinical and forensic toxicology. They cover blood (plasma, serum) analysis of amphetamines and related designer drugs, anesthetics, anticonvulsants, benzodiazepines, opioids, serotonergic drugs, tricyclic antidepressants, neuroleptics, antihistamines, beta-blockers, muscle relaxants, and sulfonylurea-type antidiabetics, and oral fluid analysis of amphetamines and related designer drugs, cocaine, benzoylecgonine, codeine, morphine, enantiomers of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), the nicotine metabolites cotinine and hydroxycotinine, and finally risperidone and its metabolite 9-hydroxyrisperidone. Basic information on the procedures is given in two tables and an example of quantification is illustrated in two figures. The pros and cons of such LC–MS procedures including sample work-up and ion suppression effects are critically discussed.     

Spectrophotometric determination of fluoride in aqueous solution using 2-hydroxy-1-(2-hydroxy-4-sulfo-1-naphthyl azo)-3-naphthoic acid

Fluoride (5–70 μg) in a final volume of 100 ml was determined. The analysis was based on the colored reaction of Al-HHSNNA and the bleaching effect of fluoride on it. The method is simple and rapid, and the accuracy is satisfactory when applied to different types of water. The procedure compared favorably with the conventional colorimetric method (D. F. Boltz and J. A. Howell (Eds.), Colorimetric Determination of Nonmetals, 2nd ed., pp. 10, 128, 1978). It is worth noting that the introduction of alizarinfluorine blue in 1958 and the pF electrode in 1966—both of fundamental importance—cannot be ignored in the introduction of the present method for fluoride as these still hold.     

Forensic discrimination of photocopy and printer toners. III. Multivariate statistics applied to scanning electron microscopy and pyrolysis gas chromatography/mass spectrometry

Copy toner samples were analyzed using scanning electron microscopy with X-ray dispersive analysis (SEM–EDX) and pyrolysis gas chromatography/mass spectrometry (Py–GC/MS). Principal component and cluster analysis of SEM data for 166 copy toner samples established 13 statistically different subgroups, with the presence or absence of a ferrite base being a major division. When toners were compared for which both SEM and reflection–absorption infrared spectral data were available, 41% of the samples could be assigned to specific manufacturers. Py–GC/MS on poly(styrene:acrylate)-based toners produced eight peaks relevant to toner differentiation. One third of the toners clustered in a small group that contained five statistically different subgroups. Of the 57 toners for which both Py–GC/MS and SEM data were available, 31 could be differentiated using the combined analytical results. The synergy of the complementary information provided by Py–GC/MS and SEM narrows matching possibilities for forensic investigations involving copied or laser printed documents.This is publication number 03-04 of the Laboratory Division of the Federal Bureau of Investigation. Names of commercial manufacturers are provided for identification only, and inclusion does not imply endorsement by the Federal Bureau of Investigation.     

Rapid Determination of Salicylic Acid in Plant Materials by Gas Chromatography–Mass Spectrometry

Summary Salicylic acid (SA) is a signaling compound in plants such as tobacco, cucumber, and tomato which can induce systemic acquired resistance. In the work discussed in this paper a simple, rapid, and sensitive method was developed for determination of salicylic acid in plant tissues by gas chromatography–mass spectrometry (GC–MS). SA from tomato leaves extracted with 9:1 (v/v) methanol–chloroform was derivatized by use of bis(trimethylsilyl)trifluoroacetamide (BSTFA) under the optimum reaction conditions (120 °C, 60 min). Quantitative analysis by GC–MS was performed in selected ion monitoring (SIM) mode using an internal standard. Procedures for sample preparation and reaction conditions were optimized. Analysis was completed within 2 h. A sensitivity of 10 ng g^–1 fresh weight and a relative standard deviation less than 5.0% for SA in tomato leaves were achieved. The method could be used for investigation of SA in plant tissues to monitor fast responses of plant defense.     

Development of tandem mass spectrometry: one perspective

The historical roots of tandem mass spectrometry or MS/MS (an invaluable tool for determining molecular structure and mixture analysis) are reviewed from the perspective of an active participant. The driving force for creating the first (and only) five-sector tandem mass spectrometer in the period 1962–1965 was to establish mechanisms of low energy ion–molecule reactions unequivocally and to evaluate the dependence of these reactions on ion kinetic energy. The invention and deployment of several other specialized instruments in the 1970s (drift cell and tandem ion cyclotron resonance spectrometers, hybrid tandems, and triple quadrupole instruments) also had fundamental studies of ion reactions as the research objective. The focus on low energy ion chemistry has evolved into collisional activation, the basis of analytical applications of MS/MS, as a natural evolution of scientific interest. The first such studies utilizing a crossed-beam hybrid MS/MS addressed single collision gas phase activation and has recently been extended to surface collision activation. The important parallel development of these methods and the metamorphosis of MS/MS from a suite of research tools into analytical practice using commercial instrumentation are discussed briefly.     

Determination of toxic and non-toxic arsenic species in urine by microwave assisted mineralization and hydride generation atomic absorption spectrometry

Flow injection — microwave oven — hydride generation — atomic absorption spectroscopy (FI-MO-HG-AAS) has been optimized for the determination of the total and toxic arsenic in urine with and without persulfate, respectively. With microwave oven assisted digestion of urine with 5% (w/v) K₂S₂O₈ and 5% (w/v) NaOH all arsenicals completely can be converted to arsenate, which is determined by HG-AAS to give the total concentration of the six species present in urine. The detection limits of 4–6 g l^–1, the relative standard deviation of 3–7% and the high sample throughput make the methods suitable for rapid routine on-line determination. Application of the proposed procedures to the analysis of urine from people on a diet rich in seafood revealed a significant increase in total urinary arsenic due to the rapid excretion of organoarsenicals. Efficient decomposition and quantitative recovery of all arsenic species in spiked urine is achieved by using 5% K₂S₂O₈ in 5% NaOH at 4.6 ml min^–1, microwave power of 700 W and a 1.5 m coil.     

Use of High-Performance Liquid Chromatography–UV and Gas Chromatography–Mass Spectrometry for Determination of the Imidacloprid Content of Honeybees, Pollen, Paper Filters, Grass, and Flowers

Imidacloprid is a new insecticide with a wide range of action. Because honeybees are very sensitive to this substance, two techniques (HPLC–UV and GC–MS) which enable its detection in several matrices of both animal and vegetable origin were used to monitor its possible presence in cultivated land. In the first method quantification of imidacloprid in honeybees was achieved by use of the external standard method; the detection limit was 50 mg kg^–1, the linear range 0.05–1 mg mL^–1, recovery 60–83%, and the imprecision (coefficient of variation) 8.6% for repeatability and 11.8% for reproducibility. Recovery from pollen was 71–98% in the range 0.05–0.5 mg kg^–1. The repeatability was 9.2–13.9%. Imidacloprid can often be found in the environment, not as a simple molecule but as a group of degradation products. The GC–MS method could be used to quantify all these species as oxidation products and to determine the initial quantity of imidacloprid by use of a conversion factor. The liquid chromatographic analysis could be used to detect, in a standard solution, 10 ng mL^–1 derivatized 6-chloronicotinic acid. The linearity was good (R=0.999) over a wide concentration range (10 g mL^–1–10 ng mL^–1). Several samples with different matrices (filter paper placed on an pneumatic corn seed drill, grass, flowers, honeybees, etc.) obtained during the sowing period for imidacloprid-treated corn were analyzed. The quantification limit (LOQ) was 0.005 mg kg^–1 for grass and flowers, 0.002 mg kg^–1 for honeybees, and 0.024 mg kg^–1 for paper filters.     

Determination of pyrimethanil and kresoxim-methyl in soils by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A method using headspace solid-phase microextraction (HS-SPME) then gas chromatography–mass spectrometry with selected ion monitoring (GC–MS, SIM) has been developed for determination of trace amounts of the fungicides pyrimethanil and kresoxim-methyl in soil and humic materials. Both fungicides were extracted on to a fused-silica fibre coated with 85 m polyacrylate (PA). Response-surface methodology was used to optimise the experimental conditions. For soil samples the linear dynamic range of application was 0.004–1.000 g g^–1 for pyrimethanil and 0.013–1.000 g g^–1 for kresoxim-methyl. The detection limits were 0.001 g g^–1 and 0.004 g g^–1 for pyrimethanil and kresoxim-methyl, respectively. HP-SPME–GC–MS analysis was highly reproducible—relative standard deviations (RSD) were between 6.7 and 12.2%. The method was validated by analysis of spiked matrix samples and used to investigate the presence of pyrimethanil and kresoxim-methyl above the detection limits in soil and humic materials.     

Phenolic-iodine Redox Products: Mass Spectrometry,Thermal and Other Physico-chemical Methods of Analyses

Three interesting new compounds formed as a result of phenols-iodine redox reactions were investigated by mass spectral fragmentation (MS) and thermal analyses (TA) as well as some other physicochemical methods as microanalysis and infra-red spectroscopy to elucidate their structures. The characterization of the compounds was satisfactorily achieved by using the above analytical tools and their proposed general formulae, were found to be C₂₄H₁₅O₈I (PC-IO ₃ ^– ), C₂₄H₁₄O₁₂ I₂ (PG-IO ₃ ^– ) and C₁₂H₈O₆I₂ (PG-IO ₄ ^– ).The fragmentation pathways of PC-IO ₃ ^– , PG-IO ₃ ^– and PG-IO ₄ ^– have been examined using electron ionization (EI) mass spectrometry in comparison with thermal analyses (TG and DTA). Both decomposition modes were investigated, and the fragmentation pathways were suggested. The combined application of mass spectrometry and thermogravimetry (MS and TG) in the analysis of the products allowed the characterization of the fragmentation pathway in MS.The major pathway in both techniques of PC-IO ₃ ^– is due to the loss of CHO followed by CH₃I+2H₂O. It is due to the loss of 2H₂O followed by the loss of 2CH₃I for PG-IO ₃ ^– . While for PG-IO ₃ ^– it is related to the loss of 2H₂O followed by loss of 2CH₃I molecule stepwise. Different stabilities for initial products and some fragments are discussed.This revised version was published online in November 2005 with corrections to the Cover Date.     

A pyrolysis-GC–MS investigation of poly(vinyl phenyl ketone)

The thermal decomposition process and pyrolysis products of poly(vinyl phenyl ketone) (PVPK) were investigated by thermogravimetric analysis (TGA) and on-line pyrolysis-gas chromatography–mass spectrometry (Py-GC–MS). TGA showed a largest weight loss rate around 380 °C. Py-GC–MS was used for the qualitative analysis of the pyrolysis products at 350, 500, 600, 700 and 850 °C. The major volatile thermal decomposition product was found to be 1-phenyl-2-propenone, which dominated all other volatile species especially under the least severe pyrolysis conditions (<600 °C). At higher temperatures a much wider range of pyrolysis products was obtained. The results have been interpreted assuming that primary random chain scission reactions occur followed by typical unzipping mainly producing monomer units; detachment of the side-group occurs only under more severe pyrolysis conditions. Py-GC–MS showed to be effective in PVPK detection in ink and paint formulations.     

Medium-size polarized basis sets for high-level-correlated calculations of molecular electric properties

Summary The first-order polarized basis sets for the use in high-level-correlated investigations of molecular electric properties have been generated for Pb, Bi, Po, and At. The performance of the standard [10.17.14.5/13.11.8.2] and extended [20.17.14.9/13.11.8.4] basis sets has been examined in nonrelativistic and quasirelativistic calculations for atoms and simple closed-shell hydrides. The relativistic contributions to electric dipole properties of those systems have been evaluated by using the recently developed quasirelativistic scheme. The predicted dipole polarizability of At is in good agreement with the results of other relativistic calculations. The calculated quasirelativistic dipole moments of BiH₃ (–0.499 a.u.), PoH₂ (–0.207 a.u.), and AtH (+0.036 a.u.) involve a significant relativistic contribution which amounts to —0.230 a.u., –0.177 a.u., and –0.097 a.u., respectively. The basis set details append this paper. They are also available as a part of the basis set library of the MOLCAS system.     

Anion exchangers functionalized by chelating reagents (AnChel) for preconcentration of trace elements: Capabilities and limitations

Summary Conventional anion exchangers (e.g., Adsorbex SAX, Amberlite IRA 410, Dowex 1X8, Lewatit MP 5080, TEAE cellulose) functionalized by means of sulfonated metal reagents (e.g., Arsenazo III, Eriochrome Red B, 8-hydroxyquinoline-5-sulfonic acid, Tiron and others) were investigated as collectors (AnChel) for analytical preconcentration of trace elements. In particular, the stability of AnChel strongly depending on competing anions, on the anion exchanger and on the metal reagent chosen, was characterized. Accordingly, for appropriate combinations of AnChel (e.g., Dowex 1X8/Arsenazo III) reagent distribution coefficients K_d in the range 10⁴ to 10⁵ (ml/g) could be attained on polystyrene-based anion exchangers even in concentrated salt solutions (e.g., 4 mol/l NaCl), but not on hydrophilic exchangers (e.g., SAX, TEAE cellulose). In general, the stability of AnChel against competing anions followed the order Cl^–>SO ₄ ^2– >NO ₃ ^– >ClO ₄ ^– . Reagent loadings of about 1 mmol/g (e.g., 8-hydroxyquinoline S) on the anion exchangers were obtainable. Trace metals precomplexed with the reagents cited could be separated (>90%) within 5 min and remobilized by acid (e.g. 2 mol/l HNO₃) within some 10 s (batch procedure). Using small columns filled with anion exchanger (e.g., 0.25 g Dowex 1X8) fast trace-matrix separations were carried out with 8-hydroxyquinoline S (Cu, Fe, In, Mn, Pb, Zn) in MgCl₂ solutions and with Arsenazo III (U, Th) in AlCl₃ solution, respectively. For subsequent trace determinations the flame-AAS (injection technique) was applied, except for Th and U [quantified by total reflection X-ray fluorescence (TXRF)].     

Ion-specific swelling behavior of uncharged poly(acrylic acid) gel

The ion-specific swelling behavior of poly(acrylic acid) (PAA) gel prepared by -ray irradiation was investigated as a function of salt concentration in the presence of 0.01 M HCl. The anion specificity for the swelling ratio was similar to that for many kinds of hydrogels, i.e., Cl^–<Br^–<NO₃ ^–<I^–, while the cation specificity proved to be rather unusual, i.e., Mg²⁺<Ca²⁺<Li⁺<Na⁺<K⁺<Cs⁺. In order to find any differences in the hydration of uncharged PAA from that of other polymers having typical polar groups, the hydrogen-bonding hydrations on the relevant polar groups were compared for small molecule analogues with an ab initio molecular orbital calculation. According to the results, the marked deswelling of PAA gel in the presence of strongly hydrated cations was ascribed to the unfavorable hydration to the acidic proton of PAA due to the reduced availability of water oxygen as well as to the destabilization of hydrophobic hydration developing around the uncharged PAA.     

Improved high-performance liquid chromatographic method for the determination of domoic acid and analogues in shellfish: effect of pH

Domoic acid (DA) is a naturally-occurring amino acid that causes a form of human intoxication called amnesic shellfish poisoning (ASP) following the consumption of shellfish. A rapid and sensitive HPLC-UV method has been developed for analysis of DA and analogues in shellfish without the need for SPE clean-up. Isocratic chromatographic separation of DA and its isomers from shellfish matrix interferences and from the prevalent amino acid, tryptophan, was achieved by careful control of the mobile phase pH. The optimised pH was found to be 2.5 when using a Luna(2) C₁₈ column. Sample extraction was verified with control extracts from shellfish spiked at 5.0 and 10.0 g/g of DA and with certified reference material. The average extraction efficiency was 98.5%. The calibration, based on mussel tissue spiked with DA standard, was linear in the range 0.05–5.0 g/ml (r=0.9999) and the detection limit (signal:noise 3:1) was better than 25 ng/ml. The DA assay achieved good precision; %RSD=1.63 (intra-day, n=6) and %RSD=3.7 (inter-day, n=8). This method was successfully applied to a variety of shellfish species, allowing the rapid screening of a large number of samples per day (20–30), without the need for SPE clean-up. Quantitative data were obtained for shellfish samples containing domoic acid in the concentration range 0.25–330 g/g. Using the same chromatographic conditions, LC-MS³ was used to determine DA and its isomers, isodomoic acid D and epi-domoic acid, in scallop tissues.