Biomedical subjects

Michael A Quilliam

Publications and source records attributed to Michael A Quilliam.

17 recordsLinked to original sources

PubMed2006

Fit-for-purpose shellfish reference materials for internal and external quality control in the analysis of phycotoxins.

The need for reference materials for quality control of analysis of foodstuffs has been stressed frequently. This has been particularly true in the phycotoxins field, where there is a great shortage of both pure calibration standards and reference materials. Worldwide there are very few independent bodies that produce certified reference materials for phycotoxins, the main producers currently being the National Research Council Canada and the Japanese Food Research Laboratory. Limited availability of contaminated shellfish and algae, as well as the time and knowledge necessary for the production of adequate reference materials, continuously lead to limited editions of certified reference materials and even more limited production of in-house reference materials. The restricted availability of in-house quality control materials promotes the rapid use of the limited certified reference materials, which in turn hampers the production of the suite of materials required globally for complete protection of public health. This paper outlines the various options that analysts can pursue in the use of reference materials for internal and external quality control, with a view to optimising the efforts of both reference materials users and reference materials producers. For this purpose, the logical sequence is reviewed from the discovery of a new bioactive compound in shellfish, through initial method development up to regulation for food safety purposes including accepted reference methods. Subsequently, the requirements for and efforts typically spent in the production and characterisation of laboratory reference materials, certified reference materials and other test materials used in inter-laboratory studies or proficiency testing, in the area of marine biotoxins are evaluated. Particular emphasis is put on practical advice for the preparation of in-house reference materials. The intricate link between reference material characterisation and method performance is outlined to give guidance on the appropriate in-house method validation in the rapidly developing field of phycotoxins.

Animals↗

PubMed2006

Discovery of fatty acid ester metabolites of spirolide toxins in mussels from Norway using liquid chromatography/tandem mass spectrometry.

Cultured mussels sampled in the spring of 2002 and 2003 from Skjer, a location in the Sognefjord, Norway, tested positive in the mouse bioassay for lipophilic toxins. In a previous report, it was established that a number of spirolides, cyclic imine toxins produced by the phytoplankton Alexandrium ostenfeldii, were present in the mussels and were responsible for the observed toxicity. The main toxin proved to be a new compound named 20-methyl spirolide G. In subsequent studies, a delayed onset of spirolide-like symptoms in the mouse bioassay exceeding the usual time limit of 20 min was observed in some samples, with symptoms and death appearing as long as 45-50 min after injection. It is well known that shellfish can extensively metabolize other toxins, such as okadaic acid and the dinophysistoxins, to fatty acid acyl esters and it is also known that a delayed onset of toxic symptoms with such metabolites can occur. Analyses performed with liquid chromatography/tandem mass spectrometry (LC/MS/MS) have revealed a complex mixture of esters of 20-methyl spirolide G in the contaminated mussels. Precursor ion scanning has delineated the range of fatty acid esters involved, while product ion scanning has provided information on structure. Identity was also supported through reaction of 20-methyl spirolide G with palmitic anhydride, which produced a derivative with a retention time and spectrum identical with one putative metabolite, 17-O-palmitoyl-20-methyl spirolide G.

Acylation↗

PubMed2006

Spirolides isolated from Danish strains of the toxigenic dinoflagellate Alexandrium ostenfeldii.

Using LC/MS methodology, spirolides were detected in two clonal isolates of Alexandrium ostenfeldii isolated from Limfjorden, Denmark. Examination of the LC/MS profiles of extracts from these Danish cultures revealed the presence of two dominant peaks representing two previously unidentified spirolide components and one minor peak identified as the previously reported desmethyl spirolide C (1). Culturing of these clonal strains, LF 37 and LF 38, of A. ostenfeldii resulted in the accumulation of sufficient cell biomass to allow for the isolation and structure elucidation of two new spirolides, 13,19-didesmethylspirolide C (2) and spirolide G (3). While 2 was found to differ from 1 only in that it contained one less methyl group, 3 was the first spirolide to be isolated that contained a 5:6:6-trispiroketal ring system. The effect of this new feature on the toxicity of 3 relative to other spirolides is presently being pursued.

Animals↗

PubMed2006

Identification of pectenotoxin-11 as 34S-hydroxypectenotoxin-2, a new pectenotoxin analogue in the toxic dinoflagellate Dinophysis acuta from New Zealand.

A new pectenotoxin, which has been named pectenotoxin-11 (PTX11), was isolated from the dinoflagellate Dinophysis acuta collected from the west coast of New Zealand. The structure of PTX11 was determined as 34S-hydroxypectenotoxin-2 by tandem mass spectrometry and UV and NMR spectroscopy. PTX11 appears to be only the third pectenotoxin identified as a natural biosynthetic product from algae after pectenotoxin-2 and pectenotoxin-12. The LD50 of PTX11 determined by mouse intraperitoneal injection was 244 microg/kg. The LD(min) of PTX11 in these experiments was 250 microg/kg. No signs of toxicity were recorded in mice following an oral dose of PTX11 at 5000 microg/kg. No diarrhea was observed in any of the animals administered with the test substance by either route of administration. Unlike pectenotoxin-2 (PTX2), PTX11 was not readily hydrolyzed to its corresponding seco acid by enzymes from homogenized green-lipped mussel (Perna canaliculus) hepatopancreas.

Animals↗

PubMed2005

Hydrophilic interaction liquid chromatography--mass spectrometry for the analysis of paralytic shellfish poisoning (PSP) toxins.

Hydrophilic interaction liquid chromatography (HILIC) was examined for the separation of paralytic shellfish poisoning (PSP) toxins using the stationary phase TSK-gel Amide-80. The parameters tested included type of organic modifier and percentage in the mobile phase, buffer concentration, pH, flow rate and column temperature. Using mass spectrometric (MS) detection, the HILIC column allowed the determination of all the major PSP toxins in one 30 min analysis with a high degree of selectivity and sensitivity. The high percentage of organic modifier in the mobile phase and the omission of ion pairing reagents, both favored in HILIC, provided limits of detection (LOD) in the range 50-100 nM in selected ion monitoring (SIM) mode on a single quadrupole LC-MS system. LOD in selected reaction monitoring (SRM) mode on a sensitive triple quadrupole system were as low as 5-30 nM. Excellent linearity of response was observed.

Animals↗

PubMed2005

Quantitative 1H NMR with external standards: use in preparation of calibration solutions for algal toxins and other natural products.

We examine the use of external standards for quantitative measurement by 1H NMR of solution concentrations of natural products and other low molecular weight, hydrogen-containing compounds and show that precision and accuracy ca. 1% is obtainable with a commercial 11.7 T spectrometer when standards and analytes are contained in separate but identical sealed precision glass NMR tubes. Numerous factors contributing to the intensity of the NMR signals are evaluated. Precise measurements of 360 degrees pulse lengths for each sample provide direct corrections for variations in probe Q-factor that enable samples in different solvents to be compared, provided single-coil excitation and detection is used throughout. Samples need not be prepared in deuterated solvents if the 1H spectra of the solvents are simple enough for peak suppression by presaturation. The approach is particularly suitable for hazardous materials kept in sealed tubes and for the preparation of certified calibration solution reference materials for use with LC-MS and other techniques where deuterated solvents should be avoided.

Biological Products↗

PubMed2005

Hydrophilic interaction liquid chromatography/mass spectrometry for determination of domoic acid in Adriatic shellfish.

This paper describes a new method for sensitive, specific and direct determination of domoic acid (DA), the causative toxin of amnesic shellfish poisoning (ASP) syndrome, in shellfish. It is based on combination of hydrophilic interaction liquid chromatography with mass spectrometry (HILIC/MS). The high percentage of organic modifier in the mobile phase and the omission of ion-pairing reagents, both favoured in HILIC, result in enhanced detection limits with MS detection. The new method was set up either on an ionspray ion trap MS instrument operating in MS and MS/MS scanning acquisition modes, or on a turboionspray triple-quadrupole MS system operating in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) acquisition modes. Positive and negative ion experiments were performed. MRM experiments are recommended for screening contaminated shellfish tissue and for quantitative analyses due to highest sensitivity and selectivity. The minimum detection levels for the toxin in tissue were found to be 63 and 190 ng/g in positive and negative MRM experiments, respectively, which are well below the regulatory limit for DA in tissue (20 microg/g). Application to shellfish samples collected in the Adriatic Sea (Italy) in the period 2000-2004 demonstrated for the first time in Italy the presence of DA as a new toxin that has entered the Adriatic Mytilus galloprovincialis toxin profile.

Animals↗

PubMed2005

Isolation and identification of (44-R,S)-44,55-dihydroxyyessotoxin from Protoceratium reticulatum, and its occurrence in extracts of shellfish from New Zealand, Norway and Canada.

44,55-Dihydroxyyessotoxin (1) was isolated from extracts of Protoceratium reticulatum and identified by analysis of its one- and two-dimensional NMR and mass spectra. In addition, LC-MS methods revealed the presence of compounds tentatively identified as (44-R,S)-44,55-dihydroxy-41a-homoyessotoxin (2) and (44-R,S)-44,55-dihydroxy-9-methyl-41a-homoyessotoxin (3). LC-MS analyses indicate that 1 is a constituent of P. reticulatum in New Zealand and Norway, and it was present in three species of mussels from New Zealand, Norway, and Canada.

Animals↗

PubMed2005

Detection and identification of spirolides in norwegian shellfish and plankton.

Mussels sampled in the spring of 2002 and 2003 from Skjer, a location in Sognefjord, Norway, tested positive in the mouse bioassay for lipophilic toxins. The symptoms, which included cramps, jumping, and short survival times (as low as 4 min), were not characteristic of toxins previously observed in Norway. A survey of the algae present at the aquaculture sites showed that the toxicity correlated with blooms of Alexandrium ostenfeldii. Up to 2200 cells/L were found at the peak of one bloom. In Canadian waters, this alga is known to be a producer of the cyclic imine toxins, spirolides. Analysis of mussel extracts from Skjer in the spring of 2002 and 2003, using liquid chromatography tandem mass spectrometry, revealed the presence of several new spirolides. The same compounds were also found in algal samples dominated by A. ostenfeldii, which had been sampled from Skjer in February 2003. A large-scale extraction of mussel digestive glands and chromatographic fractionation of the extracts allowed the isolation and structure elucidation of the main spirolide, 20-methyl spirolide G, with a molecular weight of 705.5. This is the first confirmed occurrence of spirolides in mussels and plankton from Norway.

Acetals↗

PubMed2004

Analysis of cyanobacterial toxins by hydrophilic interaction liquid chromatography-mass spectrometry.

The combination of hydrophilic interaction liquid chromatography with electrospray mass spectrometry (HILIC-MS) has been investigated as a tool for the analysis of assorted toxins produced by cyanobacteria. Toxins examined included saxitoxin and its various analogues (1-18), anatoxin-a (ATX-a, 19), cylindrospermopsin (CYN, 20), deoxycylindrospermopsin (doCYN, 21), and microcystins-LR (22) and -RR (23). The saxitoxins could be unequivocally detected in one isocratic analysis using a TSK gel Amide-80 column eluted with 65% B, where eluent A is water and B is a 95% acetonitrile/water solution, both containing 2.0 mM ammonium formate and 3.6 mM formic acid. The analysis of ATX-a, CYN and doCYN required 75% B isocratic. Simultaneous determination of 1-21 was also possible by using gradient elution. HILIC proved to be suitable for the analysis of microcystins, but peak shape was not symmetric and it was concluded that these compounds are best analysed using existing reversed-phase methods. The HILIC-MS method was applied to the analysis of field and cultured samples of Anabaena circinalis and Cylindrospermopsis raciborskii. In general, the method proved quite robust with similar results obtained in two different laboratories using different instrumentation.

Bacterial Toxins↗

PubMed2004

Isolation of pectenotoxin-2 from Dinophysis acuta and its conversion to pectenotoxin-2 seco acid, and preliminary assessment of their acute toxicities.

We have developed a simple and effective method for isolating pectenotoxin-2 (PTX-2) from Dinophysis cells collected from a natural bloom. A two-step extraction procedure followed by two column chromatography steps produced PTX-2 in high purity suitable for use as an analytical standard and for toxicological studies. Incubation of purified PTX-2 with the supernatant from ultracentrifuged blue (Mytilus edulis) or Greenshell (Perna canaliculus) mussel hepatopancreas homogenate caused rapid conversion to pectenotoxin-2 seco acid (PTX-2 SA). Purification of PTX-2 SA was achieved by solvent extraction followed by column chromatography. PTX-2 and PTX-2 SA were fully characterized by LC-MS and NMR, and full (1)H and (13)C NMR assignments were obtained. Okadaic acid C(8)-diol ester was isolated during the purification of PTX-2, and its identity confirmed by NMR and LC-MS analyses. Pectenotoxin-2 seco acid methyl ester, identified by LC-MS, was also produced during the hydrolytic procedure due to the presence of methanol. PTX-2 was acutely toxic to mice by i.p. injection (LD(50)=219 microg/kg) but no effects were seen with PTX-2 SA at 5000 microg/kg. Neither PTX-2 nor PTX-2 SA was overtly toxic to mice by the oral route at doses up to 5000 microg/kg. No diarrhea was observed in mice dosed with either compound, suggesting that pectenotoxins do not belong in the diarrhetic shellfish poison group.

Administration, Oral↗

PubMed2004

Isolation of 41a-homoyessotoxin and the identification of 9-methyl-41a-homoyessotoxin and nor-ring A-yessotoxin from Protoceratium reticulatum.

41a-homoyessotoxin (1), a new analogue of yessotoxin (7), was isolated from extracts of Protoceratium reticulatum grown in culture. In addition, 9-methyl-41a-homoyessotoxin (2) and a nor-ring A-yessotoxin (3) were identified as the major components of a mixed fraction. The structural information was initially obtained from LC-UV and LC-MS3 chromatograms, with subsequent definitive structure determination by NMR, LC-MS/MS, and high-resolution MS. Full 1H and indirectly detected 13C NMR assignments for all but two carbon atoms were obtained from ca. 100 microg of 1. Full 1H NMR assignments for 2 and 3 and identification of three new heptanor-41-oxo-analogues of 3 (4-6) during LC-MS3 analysis of a fraction containing 1-3 and 7 are also reported.

Animals↗

PubMed2004

A novel pectenotoxin, PTX-12, in Dinophysis spp. and shellfish from Norway.

Two novel pectenotoxins (PTXs) were detected by LC-MS in solid phase extracts of net hauls taken at Flødevigen, Norway, in June 2002 that were dominated by Dinophysis acuminata and Dinophysis norvegica. The new compounds were isolated as minor components from a large collection of a Dinophysis acuta-dominated bloom obtained from Skjer, Sognefjorden, Norway, in October 2002. LC-MS and NMR analyses revealed that the new components, 36S-PTX-12 and 36R-PTX-12, occurred as a pair of equilibrating diastereoisomers differing from PTX-2 in that they contained an exocylic olefinic methylene rather than a methyl group at C-38. Analyses of shellfish extracts revealed that PTX-12 accumulated in Norwegian blue mussels (Mytilus edulis) and cockles (Cerastoderma edule), along with PTX-12 seco acids occurring as a complex mixture of diastereoisomers. LC-MS analysis of algal cells picked from the net haul from Flødevigen revealed that PTX-12 predominated in D. acuta and D. norvegica, whereas PTX-2 was the predominant pectenotoxin in D. acuminata. Preliminary observations indicate that the relative contents of PTX-2 and PTX-12 vary between sites and years in Norway, even within a single species of Dinophysis. Our data also suggest that heterotrophic dinoflagellates may accumulate toxins from their prey.

Animals↗

PubMed2003

The role of chromatography in the hunt for red tide toxins.

An overview is given of the different approaches that have been used to identify toxins responsible for seafood poisoning incidents, to investigate the origins of toxins, and to monitor seafood on a routine basis. It is shown that advancements in our knowledge of toxins and our ability to protect the public have often followed key developments in separation and analysis technologies. Specific examples of research in this field are presented to illustrate the significant role that chromatographic methods play. The presentation will be given in an order that reflects the typical sequence of investigations that follow a new toxin episode.

Animals↗

PubMed2003

Liquid chromatography-mass spectrometry of spiroketal stereoisomers of pectenotoxins and the analysis of novel pectenotoxin isomers in the toxic dinoflagellate Dinophysis acuta from New Zealand.

The acid-catalyzed inter-conversion of spiroketal isomers of pectenotoxins PTX1, PTX6 and PTX2 were studied by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS-MS). Using a C8-silica reversed-phase column and a mobile phase of aqueous acetonitrile containing 2 mM ammonium formate and 50 mM formic acid, the known spiroketal stereoisomers of PTX1 eluted in order of PTX1, PTX4 and PTX8, while those of PTX6 eluted in the order PTX6, PTX7 and PTX9. Acid treatment of PTX2 yielded two novel spiroketal stereoisomers, which have been named PTX2b and PTX2c. LC-MS-MS spectra obtained for the [M+NH4]- ions of PTX2, PTX2b and PTX2c were essentially identical. As an application of the LC-MS-MS methodology, a sample of the toxic dinoflagellate Dinophysis acuta collected from the coast of New Zealand was analyzed for pectenotoxins. PTX2 and a new pectenotoxin, which has been named PTX11, were detected as the most predominant compounds. Novel PTX2 and PTX11 isomers were also found in the D. acuta although the levels of these compounds were low.

Animals↗

PubMed2002

Detection of paralytic shellfish poisoning (PSP) toxins in shellfish tissue using MIST Alert, a new rapid test, in parallel with the regulatory AOAC mouse bioassay.

In parallel trials with the mouse bioassay, MIST Alert for Paralytic Shellfish Poisoning (PSP), a rapid diagnostic test for PSP, detected 100% of the toxic extracts in over 2100 regulatory samples. Toxic extracts contained at least 80 microg saxitoxin equivalents (STX equiv.) in 100 g of shellfish tissue, or more, as measured by the regulatory AOAC mouse bioassay. Only one potentially toxic sample, which contained 78 and 86 microg STX equiv./100 g shellfish tissue in two different mouse bioassays, was recorded as negative in one replicate of MIST Alert. All other toxic extracts among more than 2100 regulatory shellfish tissue samples were detected by MIST Alert for PSP. The MIST Alert for PSP also detected the majority of extracts containing PSP toxin greater than 32 microg STX equiv./100 g, which is the mouse bioassay detection limit. The MIST Alert for PSP gave a false positive result compared to the mouse bioassay at an average rate of about 14% over all sites, although some differences were seen between sites. Further analysis by high performance liquid chromatography (HPLC) of the (false positive) extracts showed that many contained PSP toxicity in the range of 20-40 microg STX equiv./100 g, below the level detectable by the mouse bioassay. The MIST Alert for PSP gave false positive results from extracts containing less than 20 microg STX equiv./100 g shellfish tissue only about 6% of the time. The PSP family of toxin analogues can occur in any combination in naturally contaminated shellfish tissue and the antibody mixture in the MIST Alert tests detect each of the different PSP toxin analogues with different efficacy. It is therefore impossible to provide an exact detection limit for the MIST Alert that would be applicable for all possible toxin profiles. Through the experience of comparison testing with the regulatory mouse bioassay in many parts of the world, with over 2100 different samples, the MIST Alert for PSP has proven its ability to detect all types of profiles of the PSP toxin analogues. The detection limit for MIST Alert for PSP was about 40 microg STX equiv./100 g for the 'average' profile of PSP toxin analogues. Since the detection limit depends on the toxin profile in the individual extract, it will also vary depending on the profile of analogues most commonly found at each geographic location. This was observed in our study. Over all sites in the trials, approximately 5% of samples below 40 microg STX equiv./100 g were positive, and 5% of samples between 40-80 microg STX equiv./100 g were negative. This is a reflection of the different analogue profiles found in naturally contaminated extracts, even after acid hydrolysis using the AOAC extraction method.

Animals↗

PubMedDate not supplied

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.

Animals↗