液相色谱-串联质谱法同时测定贝类中大田软海绵酸、鳍藻毒素、蛤毒素和虾夷扇贝毒素

建立了同时测定贝类中大田软海绵酸(okadaic acid, OA)及其衍生物鳍藻毒素(dinophysistoxin-1, DTX-1)、蛤毒素(pectenotoxin-2, PTX-2)和虾夷扇贝毒素(yessotoxin, YTX)的液相色谱-串联质谱分析方法。样品经甲醇提取,固相萃取柱净化,C18色谱柱分离,经含甲酸和甲酸铵的乙腈-水溶液为流动相梯度洗脱,选择反应监测(SRM)模式检测,正、负离子切换扫描,基质标准校正,外标法定量。结果表明,OA、DTX-1和YTX的线性范围为2.0～200.0 μg/L,定量限(以信噪比(S/N)≥10计)为1.0 μg/kg; PTX-2的线性范围为1.0～100.0 μg/L,定量限为0.5 μg/kg;几种化合物的添加平均回收率为83.1%～105.7%,相对标准偏差(RSD)为3.16%～9.29%。成功应用本法对黄海灵山湾海域采集的贝类样品进行了分析,发现部分样品中含有大田软海绵酸、鳍藻毒素、蛤毒素和虾夷扇贝毒素。     

Liquid chromatography with mass spectrometry--detection of lipophilic shellfish toxins

A rapid multiple toxin method based on liquid chromatography with mass spectrometry (LC/MS) was developed for the detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1), DTX-2, yessotoxin (YTX), homoYTX, 45-hydroxy-YTX, 45-hydroxyhomo-YTX, pectenotoxin-1 (PTX-1), PTX-2, azaspiracid-1 (AZA-1), AZA-2, and AZA-3. Toxins were extracted from shellfish using methanol-water (80%, v/v) and were analyzed using a C8 reversed-phase column with a 5 mM ammonium acetate-acetonitrile mobile phase under gradient conditions. The method was validated for the quantitative detection of OA, YTX, PTX-2, and AZA-1 in 4 species (mussels, Mytilus edulis; cockles, Cerastoderma edule; oysters, Crassostrea gigas; king scallop, Pecten maximus) of shellfish obtained from United Kingdom (UK) waters. Matrix interferences in the determination of the toxins in these species were investigated. The validated linear range of the method was 13-250 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. Recovery and precision ranged between 72-120 and 1-22%, respectively, over a fortification range of 40-160 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. The limit of detection, reproducibility, and repeatability of analysis showed acceptable performance characteristics. A further LC/MS method using an alkaline hydrolysis step was assessed for the detection of OA, DTX-1, and DTX-2 in their esterified forms. In combination with the LC/MS multiple toxin method, this allows detection of all toxin groups described in Commission Decision 2002/225/EC.     

Comparison of different fluorimetric HPLC methods for analysis of acidic polyether toxins in marine phytoplankton

The human toxic syndrome, diarrhetic shellfish poisoning (DSP), is caused by polyether toxins that are present in bivalve molluscs but originate from some species of marine phytoplankton. During the last few years different HPLC methods with fluorescence detection (FLD) have been proposed for analysis of marine toxins, including polyether toxins, in shellfish and phytoplankton. Several derivatization reagents have been proposed in the literature, with the aim of converting the acidic DSP toxins into their corresponding fluorescent derivatives. In this work we report results obtained from HPLC–FLD analysis of extracts from phytoplankton, including Dinophysis spp., harvested off the south-west coast of Ireland. Three different reagents were used for fluorescent derivatization: 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (BrDMEQ), 9-chloromethylanthracene (CA), and in situ 9-anthracenyldiazomethane (ADAM). Derivatization was performed under conditions previously optimised. The DSP derivatives were cleaned using different SPE procedures then analysed by HPLC–FLD. In this study, the use of BrDMEQ, CA, and in situ ADAM was compared in terms of sensitivity and selectivity. Evaluation of HPLC methods for analysis of DSP toxin derivatives was also conducted; the presence of okadaic acid (OA), dinophysistoxin-2 (DTX-2), and pectenotoxin-2 seco acids (PTX1SAs) was detected in the sample extracts studied.     

Validation of a confirmatory method for lipophilic marine toxins in shellfish using UHPLC-HR-Orbitrap MS

Lipophilic marine toxins are produced by harmful microalgae and can accumulate in edible filter feeders such as shellfish, leading to an introduction of toxins into the human food chain, causing different poisoning effects. During the last years, analytical methods, based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), have been consolidated by interlaboratory validations. However, the main drawback of LC-MS/MS methods remains the limited number of compounds that can be analyzed in a single run. Due to the targeted nature of these methods, only known toxins, previously considered during method optimization, will be detected. Therefore in this study, a method based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS) was developed. Its quantitative performance was evaluated for confirmatory analysis of regulated lipophilic marine toxins in shellfish flesh according to Commission Decision 2002/657/EC. Okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX), and 13-desmethyl spirolide C (SPX-1) were quantified using matrix-matched calibration curves (MMS). For all compounds, the reproducibility ranged from 2.9 to 4.9 %, repeatability from 2.9 to 4.9 %, and recoveries from 82.9 to 113 % at the three different spiked levels. In addition, confirmatory identification of the compounds was effectively performed by the presence of a second diagnostic ion (¹³C). In conclusion, UHPLC-HR-Orbitrap MS permitted more accurate and faster detection of the target toxins than previously described LC-MS/MS methods. Furthermore, HRMS allows to retrospectively screen for many analogues and metabolites using its full-scan capabilities but also untargeted screening through the use of metabolomics software. Figure

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Electrospray tandem mass spectrometry of alendronate analogues: fingerprints for characterization of new potential prodrugs

1-hydroxymethylene-1,1-bisphosphonic acids (HMBPs) are important drugs for the treatment of a variety of bone diseases. Since these compounds have no chromophore, their detection is challenging and mass spectrometry (MS) appears to be an appropriate sensitive tool. Our work deals with the analysis by electrospray ionization tandem mass spectrometry (ESI-MSn) of the well-known nitrogen-containing HMBP alendronate and of three analogues, considered as potential prodrugs. These four molecules share a common structure with different protecting groups on the phosphonic acid and on the amine functions. We describe the dissociation mechanisms of nitrogen-containing HMBPs in positive ion mode and we compare, in negative ion mode, our results with literature data. In both modes, the dissociations are essentially losses of ROH, and of phosphorus-containing species (HPO2, ROP(OH)2 and ROPO(OH)2), where R=H, C6H5, or CH3OC6H5. These fingerprints will be of great value for differentiating alendronate from its potential prodrugs in complex biological mixtures.     

Mass spectrometry of polycyclic tetracarboxylic ('ARN') acids and tetramethyl esters

Polycyclic C(80) tetracarboxylic (so-called 'ARN') acids are found as calcium salts in deposits which form in certain oilfield pipelines and equipment. Characterisation of these acids is important for improving the prediction and hence avoidance or minimisation of oilfield deposition problems. Although several of the acids have been isolated and characterised (as regioisomeric mixtures) by nuclear magnetic resonance spectroscopy, mass spectrometric methods are likely to be much more useful for the routine analysis of oils and deposits containing the acids. A publication summarising the mass spectra of the purified acids and major derivatives might thus be a very useful source of reference for scientists and technologists studying these unusual compounds. We now report the characterisation of several of the purified acids and of the tetramethyl esters by electrospray ionisation mass spectrometry (ESI-MS) in both positive ion and negative ion modes, by multistage ESI-MS with a suggested rationalisation of the ions produced, by positive ion atmospheric solids analysis probe (ASAP) atmospheric pressure chemical ionisation (APCI), and by positive ion electron ionisation (EI)-MS. Tentative identifications of C(80) acyclic, mono-, bi- and tricylic tetraacids and the δ(13)C isotope values of a mixture of the semi-pure acids determined by MS are also reported for the first time.     

Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).     

Mass spectrometric analysis of the marine lipophilic biotoxins pectenotoxin-2 and okadaic acid by four different types of mass spectrometers

The performances of four different mass spectrometers [triple-quadrupole (TQ), time-of-flight (ToF), quadrupole ToF (Q-ToF) and ion trap (IT)] for the detection of the marine lipophilic toxins pectenotoxin-2 (PTX2) and okadaic acid (OA) were investigated. The spectral data obtained with the different mass spectrometric analyzers were used to propose fragmentation schemes for PTX2 in the positive electrospray mode and for OA in the negative electrospray mode. TQ data were used to obtain product ions, while ToF and Q-ToF-MS produced accurate mass data of the precursor ion and product ions, respectively. IT data provided a better understanding of the fragmentation pathways using MS(n) experiments. With respect to analytical performance, all four mass analyzers showed a good linearity (R(2) > 0.97) and repeatability (CV < 20%). Detection limits (LoDs) (S/N = 3) were the lowest on triple-quad MS: 12.2 and 2.9 pg on-column for PTX2 and OA, respectively.     

Fast atom bombardment mass spectrometry of salts of substituted methylene bisphosphonic acids and other analogues of pyrophosphoric acid

Fast atom bombardment mass spectra have been obtained of salts of analogues of inorganic pyrophosphoric acid, e.g. substituted methylene bisphonates, and positive ion fast atom bombardment mass spectrometry (FABMS) has proved to be more successful than negative ion FABMS for the analysis of these salts. Owing to the low molecular weight of these analogues, FAMBS of the free acids and the sodium salts does not always give results which are easy to interpret as interference by peaks from the matrix can occur. However, the potassium salts in a 60% glycerol matrix, when bombarded by argon atoms, give good FAB spectra which are relatively free of interfering peaks.     

Characterization of complex assemblages of organic acids in geological samples by negative electrospray ionization mass spectrometry using a double‐focusing magnetic sector field mass spectrometer

Four different geological sample types (a crude oil, a crude oil asphaltene, a reservoir core extract and a reservoir core asphaltene) have been characterized by negative ionization electrospray mass spectrometry at low and high mass resolution using a double‐focusing magnetic sector field mass spectrometer. The mass range, shape of the spectra and the signal distribution of the acidic constituents as well as the average molecular weights, the total ion abundance and signal intensity in the spectra were compared for the different sample types. Nominal mass classes have been evaluated and Kendrick mass plots were generated in order to identify homologous series. For the crude oil sample, accurate mass assignments were made by high‐resolution double‐focusing magnetic sector field mass spectrometry (DFMSFMS) and were compared with those obtained by negative ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). With both instrument types, compounds with the molecular composition C_nH_2n+zO₂, among which carboxylic acids predominated, were the main acidic compound class detectable in negative ESI mass spectra. Good agreement was achieved for the double bond class distribution and the carbon number distribution of the O₂ class. In addition, minor compound classes could be identified using FTICRMS. Copyright © 2010 John Wiley & Sons, Ltd.     

Confirmation of okadaic acid, dinophysistoxin-1 and dinophysistoxin-2 in shellfish as their anthrylmethyl derivatives using UV radiation

A rapid and simple method for confirmation of the diarrhetic shellfish poisons (DSP): okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) using fluorescence detection following derivatization with 9-chloromethylanthracene, has been established as an alternate to LC/MS. Exposure of the anthrylmethyl derivatives of OA, DTX-1 and DTX-2 to near UV light (300-400 nm) resulted in the loss of these compounds to below detection limits within 30 min, with a concurrent appearance of two additional compounds. Based on the mass spectral evidence, we propose that these newly formed compounds are the decarboxylation products of the derivatized diarrhetic shellfish poisons. UV radiation is, therefore, proposed as a rapid and simple confirmation technique for these DSP in mussel samples.     

Chemical ionization mass spectrometry of derivatives of L-dopa and L-tryptophan and their detection in tumour samples

Conditions have been established for the detection by chemical ionization mass spectrometry (CIMS) of compounds related to L -dopa (3-hydroxy-L -tyrosine) and L -tryptophan without derivatization. The least fragmentation of the parent ion occurs in positive ion CIMS when methylamine is used as reagent gas. The compounds can also be detected by negative ion CIMS using carbon tetrachloride as reagent gas. While the total ion current in the latter technique is lower than that obtained with positive ion CIMS, the background noise in the mass spectra of samples obtained from natural sources is greatly reduced. CIMS has been used to show the presence of aromatic amino acids in acid extracts of samples of two different classes of tumour without sample derivatization.     

Mass spectral studies on synthetic analogs of organophosphorus toxin occurring in dinoflagellate algae

A series of organophosphorus compounds related to PB-1 toxin [O,O-diphenyl N- cyclooctylphosphoramidate] occurring in dinoflagellate algae as fish toxin have been synthesized and subjected to mass spectral studies under electron ionization. The fragmentation pattern obtained for the compounds has been substantiated by performing tandem mass spectrometry experiments in product ion scan mode.     

Structural study of spirolide marine toxins by mass spectrometry

The spirolides are a family of marine biotoxins derived from the dinoflagellate Alexandrium ostenfeldii, recently isolated from contaminated shellfish and characterized. A crude phytoplankton extract has been extensively studied for mass spectrometric determination and characterization of several known spirolides and previously unreported compounds. The complex sample was initially analyzed by full-scan mass spectrometry in an ion-trap instrument, enabling identification of several components. Subsequent analysis by selected-ion monitoring in a triple-quadrupole instrument resulted in the confirmation of the identities of the compounds detected in the ion trap. Purification of the crude extract was performed using an automated mass-based fractionation system, yielding several fractions with different relative contributions of the spirolide components. Collision-induced dissociation (CID) in the triple-quadrupole instrument produced significant fragment ions for all identified species. Selective enrichment of some minor compounds in certain fractions enabled excellent CID spectra to be generated; this had previously been impossible, because of interferences from the major toxins present. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry was then performed for accurate determination of the masses of MH+ ions of all the species present in the sample. Additionally, infrared multiphoton dissociation in the FTICR instrument generated elemental formulae for product ions, including those formed in the previous collisional activation experiments. Collection of these results and the fragmentation scheme proposed for the main component of the extract, 13-desmethyl spirolide C, from part I of this study, enabled elucidation of the structures of some uncharacterized spirolides and some biogenetically related compounds present at previously unreported masses.     

Determination of domoic acid in seawater and phytoplankton by liquid chromatography-tandem mass spectrometry

Domoic acid (DA) is an algal neurotoxin produced by diatoms primarily of the genus Pseudo-nitzschia and is responsible for the human intoxication syndrome known as amnesic shellfish poisoning. A method has been developed to determine DA in seawater and phytoplankton matrices by liquid chromatography-tandem mass spectrometry for both quantitation and confirmation purposes. Sample extraction and clean-up was achieved on a C18 solid-phase extraction (SPE) cartridge. An acidic condition is critical for retaining hydrophilic DA on the cartridge. Direct injection of SPE eluate for analysis is recommended in order to avoid loss of DA by drying with heat prior to resuspension and injection. DA was quantified using the fragments produced from the protonated DA ion through multiple reaction monitoring (MRM). Recoveries exceeded 90% for all seawater samples spiked with DA and approximated 98% of toxin standard added to cultured phytoplankton material. Acceptable reproducibility (ca. 5% or less) was obtained for all intra-day and inter-day samples. The detection limit was 30 pg/ml level with a 20 microl injection volume, which demonstrated the value of this method for not only confirming DA production by minimally toxic phytoplankton species, but also for investigating the potentially important role of dissolved DA in marine food webs.     

Desorption electrospray ionization imaging mass spectrometry of lipids in rat spinal cord

Imaging mass spectrometry allows for the direct investigation of tissue samples to identify specific biological compounds and determine their spatial distributions. Desorption electrospray ionization (DESI) mass spectrometry has been used for the imaging and analysis of rat spinal cord cross sections. Glycerophospholipids and sphingolipids, as well as fatty acids, were detected in both the negative and positive ion modes and identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation and accurate mass measurements. Differences in the relative abundances of lipids and free fatty acids were present between white and gray matter areas in both the negative and positive ion modes. DESI-MS images of the corresponding ions allow the determination of their spatial distributions within a cross section of the rat spinal cord, by scanning the DESI probe across the entire sample surface. Glycerophospholipids and sphingolipids were mostly detected in the white matter, while the free fatty acids were present in the gray matter. These results show parallels with reported distributions of lipids in studies of rat brain. This suggests that the spatial intensity distribution reflects relative concentration differences of the lipid and fatty acid compounds in the spinal cord tissue. The “butterfly” shape of the gray matter in the spinal cord cross section was resolved in the corresponding ion images, indicating that a lateral resolution of better than 200 μm was achieved. The selected ion images of lipids are directly correlated with anatomic features on the spinal cord corresponding to the white and the gray matter.     

Acidity trends in alpha,beta-unsaturated sulfur, selenium, and tellurium derivatives: comparison with C-, Si-, Ge-, Sn-, N-, P-, As-, and Sb-containing analogues

The gas-phase acidity of CH3-CH2XH (X=S, Se, Te), CH2=CHXH (X=S, Se, Te) and PhXH (X=S, Se) compounds was measured by means of Fourier transform ion cyclotron resonance mass spectrometry. To analyze the role that unsaturation plays on the intrinsic acidity of these systems, a parallel theoretical study, in the framework of the G2 and the G2(MP2) theories, was carried out for all ethyl, ethenyl (vinyl), ethynyl, and phenyl O-, S-, Se-, and Te-containing derivatives. Unsaturated compounds are stronger acids than their saturated analogues, because of the strong pi-electron donor ability of the heteroatoms that contributes to a large stabilization of the unsaturated anions. Ethynyl derivatives are stronger acids than vinyl compounds, while phenyl derivatives have an intrinsic acidity intermediate between that of the corresponding vinyl and ethynyl analogues. The CH2=CHXH vinyl compounds (enol-like) behave systematically as slightly stronger acids than their CH3-C(H)X (keto-like) tautomers. Vinyl derivatives are stronger acids than ethyl compounds, because the anion stabilization attributable to unsaturation is greater than that undergone in the neutral compounds. Conversely, the enhanced acidity of the ethynyl derivatives with respect to the vinyl compounds is due to two concomitant effects, the stabilization of the anion and the destabilization of the neutral compound. The acidities of ethyl, vinyl, and ethynyl derivatives containing heteroatoms of Groups 14, 15, and 16 of the periodic table are closely related, and reflect the differences in electronegativity of the CH3CH2-, CH2=CH-, and CH[triple chemical bond]C- groups.     

Determination of traces of five antifouling agents in water by gas chromatography with positive/negative chemical ionisation and tandem mass spectrometric detection.

A highly selective and sensitive gas chromatography-mass spectrometry methodology has been developed for the determination of five antifouling compounds, currently licensed for use in marine antifouling paints. The procedure uses an ion trap mass spectrometer provided with an external ion source that allows the combined use, in the same analysis, of positive (PCI) and negative (NCI) chemical ionisation and tandem mass spectrometric fragmentation (MS-MS). Ionisation and fragmentation processes were optimised individually for each compound, thus, permitting maximum sensitivity and selectivity to be obtained. A complete validation study, including those aspects that affect both correct quantification and unequivocal confirmation, demonstrated the good performance of the proposed method. Detection limits obtained were lower than 0.005 microg l(-1), except for Irgarol 1051 (0.050 microg l(-1)). The method was applied to real seawater samples from different     

Detection, characterization and identification of crucifer phytoalexins using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry

We have analyzed 23 crucifer phytoalexins (e.g. brassinin, dioxibrassinin, cyclobrassinin, brassicanals A and C) by HPLC with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) using both negative and positive ion modes. Positive ion mode ESI-MS appeared more sensitive than negative ion mode ESI-MS in detecting this group of compounds. A new HPLC separation method, new LC-MS and LC-MS(2) data and proposed fragmentation pathways, LC retention times, and UV spectra for selected compounds are reported.     

Fragmentation study of hexanitrostilbene by ion trap multiple mass spectrometry and analysis by liquid chromatography/mass spectrometry

The fragmentation pathways of three explosive compounds with similar structures, hexanitrostilbene (HNS), cyclotrimethylene trinitramine (RDX), and 2,4,6-trinitrotoluene (TNT), have been investigated by multiple mass spectrometry (MSn, n = 1, 2, 3) with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources. The electron capture mechanism for these compounds in negative ion APCI and ESI mode differs from the usual negative ion mechanism, deprotonation or addition of other species. This was shown for HNS and TNT, which both gave a [M]- anion but not a [M-H]- ion in APCI, and the [M]- anion of HNS was observed in ESI. The quantitative analysis of HNS was performed by liquid chromatography (LC)/ESI-MS, and the results obtained by the internal standard (ISTD) method were compared with those from the external standard (ESTD) method, demonstrating that both quantitation approaches are useful, with good sensitivity, reproducibility and linearity, and ESTD is preferable in routine applications.