ABSTRACT
A novel extraction and clean-up method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish samples. Raw shellfish material was extracted with an acidic acetonitrile/water (80:20, v/v) solution, whilst being homogenised. During the homogenisation the sample extraction solution was cooled with ice water. Subsequently, the extract was frozen at -20 degrees C for at least 4h. During freezing, two layers were formed, only the lower predominantly aqueous layer was used for the determination. The final extract solution was cleaned-up using a combination of Oasis HLB and Carbograph activated carbon SPE columns. The developed extraction and clean-up methods combined with gradient elution liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS) has resulted in a method which can determine the analogs GTX 1-5, C1-2, DcGTX 2-3, DcSTX, Neo, STX in a single analysis with an overall detection limit of 313 microg STXdiHCL-eq./kg shellfish meat. The use of the developed extraction method with post-column high performance liquid chromatography (HPLC) with fluorescence detection (FLD) method provided an overall limit of detection of 89 microg STXdiHCL-eq./kg shellfish meat for the same toxins. Both post-column HPLC-FLD and LC-MS/MS was used to investigate the Norwegian PSP toxin profile. It was found that the PSP toxins could be detected in shellfish samples from the Norwegian coastline for 10 months of the year, from March till December. The toxin profile consisted mainly of the carbamate toxins, GTX 1-4, Neo and STX, in terms of both concentrations and contribution to the overall toxicity. In addition, several of the n-sulfo-carbamoyl toxins were either detected in the samples at relatively low concentrations or their presence in the samples were indicated but could not be confirmed by the post-column HPLC-FLD and LC-MS/MS analyses.
Subject(s)
Food Contamination/analysis , Marine Toxins/chemistry , Shellfish/analysis , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid , Environmental Monitoring , Fluorescence , Norway , Seasons , Solid Phase ExtractionABSTRACT
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).
Subject(s)
Chromatography, Liquid/methods , Food Analysis/methods , Mass Spectrometry/methods , Toxins, Biological/analysis , Animals , Biological Assay , Ethers, Cyclic/analysis , Furans/analysis , Furans/metabolism , Heterocyclic Compounds, 3-Ring/analysis , Hydrocarbons, Cyclic/analysis , Hydrolysis , Imines/analysis , Kainic Acid/analogs & derivatives , Kainic Acid/analysis , Macrolides , Marine Toxins/analysis , Methanol/chemistry , Mice , Mollusca , Mollusk Venoms , Okadaic Acid/analysis , Oxocins/analysis , Pyrans/analysis , Pyrans/metabolism , Reproducibility of Results , Sensitivity and Specificity , Shellfish , Spiro Compounds/analysis , Time FactorsABSTRACT
195 patients with headache were examined by general practitioners and neurologists, and their diagnoses were compared. The general practitioners diagnosed migraine in 116 cases, the neurologists in 144 cases, and in 104 cases their diagnoses agreed. Thus, migraine can be difficult to diagnose, and this study indicates underdiagnosis of this common condition by general practitioners. This may result in overuse of analgesics and alternative therapies, and too little prescription of specific migraine medication. Greater familiarity with the diagnostic criteria of the International Headache Society should make it easier to arrive at a correct migraine diagnosis.
Subject(s)
Migraine Disorders/diagnosis , Adolescent , Adult , Aged , Clinical Competence , Family Practice , Female , Humans , Male , Middle Aged , Migraine Disorders/drug therapy , NorwayABSTRACT
195 patients with headache were examined by general practitioners and neurologists, and their diagnoses were compared. The general practitioners diagnosed migraine in 116 cases, the neurologists in 144 cases, and in 104 cases their diagnoses agreed. Thus, migraine can be difficult to diagnose, and this study indicates underdiagnosis of this common condition by general practitioners. This may result in overuse of analgesics and alternative therapies, and too little prescription of specific migraine medication. Greater familiarity with the diagnostic criteria of the International Headache Society should make it easier to arrive at a correct migraine diagnosis.
Subject(s)
Family Practice , Migraine Disorders/diagnosis , Neurology , Adolescent , Adult , Aged , Diagnostic Errors , Female , Humans , Male , Middle AgedABSTRACT
Clinical, Doppler and angiographic findings are described in 53 consecutive patients who presented with amaurosis fugax (AF) in a total of 57 eyes; 4 had non-simultaneous attacks in both eyes. Atherosclerotic lesions were detected on Doppler or angiographic (conventional arteriography and/or intravenous digital subtraction angiography) examination in 36 (63%) of the relevant precerebral internal carotid arteries (ICA) in 34 patients. Nineteen (53%) of these lesions caused a diameter reduction of more than 75%. Patient age was the most important factor in predicting the presence of relevant carotid occlusive disease, all 36 lesions being found in patients over 50 years of age. Two unusual cases of AF are described; in one AF was caused by stenosis of the ipsilateral ophthalmic artery, and in another by occlusion of the brachiocephalic artery with a steal syndrome from the right common carotid artery (CCA) to the right subclavian artery.
