Hamstring strain injury prevention: current beliefs and practices of practitioners working in Major League Baseball.

CONTEXT: Hamstring strain injuries (HSIs) are the most frequently sustained injury in Major League Baseball (MLB). However, the beliefs and practices of MLB practitioners regarding HSI risk factors and prevention strategies in baseball athletes, have not been documented. OBJECTIVE: To document the current beliefs and practices of MLB practitioners in relation to HSI prevention. DESIGN: cross-sectional study. SETTING: Major League Baseball via an online survey. PARTICIPANTS: Athletic trainers, physical therapists and strength and conditioning coaches employed in MLB during the 2021 season. DATA COLLECTION AND ANALYSIS: An online survey was conducted with participants completing the survey once. Questions pertained to risk factor identification, the use and perceived effectiveness of preventative strategies, and barriers to implementation. Descriptive statistics were calculated for each question. RESULTS: 91 responses were received featuring respondents from 28 of 30 MLB organizations. The perceived most important intrinsic risk factor for first-time HSI was tolerance to high-speed running and previous HSI for recurrent injury. The perceived most important extrinsic risk factor for both first-time and recurrent HSI was internal communication between staff.The perceived most effective prevention strategies were managing overall workload, exposure to high-speed running, and periodization. The most used prevention strategies were core/lumbopelvic strengthening, resistance training and workload management.Approximately half (53%) of respondents reported barriers to effective implementation of HSI prevention strategies, including player and coach buy-in, compliance, training time constraints, and in-season scheduling/reduced recovery time. CONCLUSIONS: This was the first survey to investigate MLB practitioner beliefs and practices regarding HSI prevention. Responses from practitioners regarding their beliefs about risk factors and appropriate prevention strategies were varied, and discrepancies existed between the perceived most effective strategies and those most frequently employed.

Determination of pyrethrin and pyrethroid residues in animal fat using liquid chromatography coupled to tandem mass spectrometry.

A method was developed for the confirmatory and quantitative analysis of one pyrethrin and 18 pyrethroid residues in animal fat. Fat was extracted was collected from adipose tissue melted in an oven at 65 °C for 2 h. Fat samples (1 g) were dispersed with deactivated Florisil® sorbent and extracted with MeCN. Sample extracts were purified by cold temperature precipitation at -30 °C for 4 h and further purified using dispersive solid-phase extraction (d-SPE) clean-up in tubes containing 500 mg of Z-SEP+ and 125 mg of PSA bonded silica. Purified samples were analysed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) detection. Chromatographic separation was carried out on a Acquity C8 BEH column, using a binary gradient separation comprising of mobile phase A, 5 mM ammonium formate in water:MeOH (80:20, v/v,) and mobile phase B, 5 mM ammonium formate in MeOH. The mass spectrometer was operated in the positive electrospray ionisation mode (ESI(+)). Validation was performed following the 2002/657/EC guidelines. Trueness ranged between 84% and 143% and precision ranged between 3.9% and 29%. The developed method is particularly advantageous because the sample preparation procedure does not require complex sample extraction equipment and uses less solvent compared to other sample preparation protocols.

Development and validation of a quantitative confirmatory method for 30 ß-lactam antibiotics in bovine muscle using liquid chromatography coupled to tandem mass spectrometry.

A method was developed for the confirmatory and quantitative analysis of 30 ß-lactam antibiotic residues in bovine muscle. The method includes 12 penicillins (amoxicillin, ampicillin, cloxacillin, dicloxacillin, mecillinam, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, ticarcillin), 12 cephalosporins (cefacetrile, cefadroxil, cephalexin, cefalonium, cefazolin, cefoperazone, cefotaxime, cefquinome, cefuroxime, desacetyl cephapirin, desfuroylceftiofur cysteine disulfide, desfuroylceftiofur dimer), five carbapenems (biapenem, doripenem, ertapenem, imipenem, meropenem) and faropenem. Samples were extracted using a simple solvent extraction with acetonitrile:water (80:20, v/v) and C18 dispersive solid-phase extraction (d-SPE) clean-up, followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) detection. Chromatography was performed on a reversed phase CSH C18 column, using a binary gradient separation comprising of 0.01% formic acid and 0.2mM ammonium acetate in water (mobile phase A) and 0.01% formic acid in acetonitrile (mobile phase B). The mass spectrometer was operated in the positive electrospray ionisation mode (ESI(+)). Validation was performed following the 2002/657/EC guidelines. Trueness ranged between 69% and 143% and precision ranged between 2.0% and 29.9% under within-laboratory reproducibility conditions. The developed method uses minimal sample preparation and 30 test samples can be analysed by a single analyst in a single day. To the best of our knowledge, this is the first method for carbapenems in foodstuff that does not require derivatisation.

Rate of avascular necrosis and time to surgery in proximal humerus fractures.

PURPOSE: Avascular necrosis (AVN) of the humeral head is a devastating complication of proximal humeral fracture (PHF) that often results in long-term morbidity for the patient. Rates of AVN depend on the number of fracture fragments and are highly variable. The literature suggests that timely stable and anatomic reduction may decrease the rate at which AVN develops after PHF. To our knowledge, there is no literature published investigating a temporal relationship between the timing of PHF fixation and rates of AVN. METHODS: Operative records of one orthopedic trauma surgeon were used to identify patients that underwent open reduction internal fixation for PHF at our institution between 2007 and 2012. Radiographs at presentation were reviewed and used to classify the fractures into two, three or four parts. Date and time of the initial radiograph were recorded as were the date and time of available intra-operative fluoroscopic images. The time from presentation radiograph to operative fixation was calculated (hours). Available follow-up plain films were then reviewed and evaluated for the presence or absence of humeral head AVN. RESULTS: Time to surgery (less than or greater than 72 h) and patient age did not correlate with development of AVN after PHF (p > 0.26). Notably, the number of fracture fragments did influence the rate of AVN identified in patients with PHF (p = 0.002). CONCLUSION: Early operative intervention does not appear to decrease the rate of development of avascular necrosis after PHF.

Metabolomic analysis of human plasma reveals that arginine is depleted in knee osteoarthritis patients.

OBJECTIVE: To identify novel biomarker(s) for knee osteoarthritis (OA) using a metabolomics approach. METHOD: We utilized a two-stage case-control study design. Plasma samples were collected from knee OA patients and healthy controls after 8-h fasting and metabolically profiled using a targeted metabolomics assay kit. Linear regression was used to identify novel metabolic markers for OA. Receiver operating characteristic (ROC) analysis was used to examine diagnostic values. Gene expression analysis was performed on human cartilage to explore the potential mechanism for the novel OA marker(s). RESULTS: Sixty-four knee OA patients and 45 controls were included in the discovery stage and 72 knee OA patients and 76 age and sex matched controls were included in the validation stage. We identified and confirmed six metabolites that were significantly associated with knee OA, of which arginine was the most significant metabolite (P < 3.5 × 10(-13)) with knee OA patients having on average 69 µM lower than that in controls. ROC analysis showed that arginine had the greatest diagnostic value with area under the curve (AUC) of 0.984. The optimal cutoff of arginine concentration was 57 µM with 98.3% sensitivity and 89% specificity. The depletion of arginine in OA patients was most likely due to the over activity of arginine to ornithine pathway, leading to imbalance between cartilage repair and degradation. CONCLUSION: Arginine is significantly depleted in refractory knee OA patients. Further studies within a longitudinal setting are required to examine whether arginine can predict early OA changes.

A post-antibiotic era looms: can plant natural product research fill the void?

Antimicrobial resistance is increasing among certain pathogenic bacteria to the extent that treatment efficacy is no longer always assured. According to the CDC, as few as six new antibiotics have been released for use over the past 30 years. Resistance has already been observed to each of these. Eleven plant natural products have been approved for therapeutic use during the same period--none of them being antimicrobial agents. We have learned through experience that some microorganisms will inevitably overcome antibiotic treatment in certain situations, and then spread. It is clear that the rate of new antimicrobial development is insufficient to meet our current and future needs, which should be addressed in order to guarantee the effective future of antimicrobial chemotherapy. However, in recent years there has been an increase in the number of peer-reviewed reports of antimicrobial efficacy among plant-derived secondary metabolites. A limitation with these reports is the wide range of modified in vitro methods used to determine antimicrobial efficacy of these products, showing an absence of the type of standardisation that is the norm when testing the efficacy of single- or combined-agent conventional antimicrobials in the laboratory, thereby making inter-study comparison difficult. Overall, despite the large diversity in preparation and testing strategies used currently for natural product plant-derived antimicrobials, our investigations suggest that the field shows promise in the provision of novel antimicrobial agents, even as exemplified by our selected example, Inula helenium (Elecampane).

Influence of extraction technique on the anti-oxidative potential of hawthorn (Crataegus monogyna) extracts in bovine muscle homogenates.

Six extracts were prepared from hawthorn (Crataegus monogyna) leaves and flowers (HLF) and berries (HB) using solid-liquid [traditional (T) (HLFT, HBT), sonicated (S) (HLFS, HBS)] and supercritical fluid (C) extraction (HLFC, HBC) techniques. The antioxidant activities of HLF and HB extracts were characterised using in vitro antioxidant assays (TPC, DPPH, FRAP) and in 25% bovine muscle (longissimus lumborum) homogenates (lipid oxidation (TBARS), oxymyoglobin (% of total myoglobin)) after 24h storage at 4°C. Hawthorn extracts exhibited varying degrees of antioxidant potency. In vitro and muscle homogenate (TBARS) antioxidant activity followed the order: HLFS>HLFT and HBT>HBS. In supercritical fluid extracts, HLFC>HBC (in vitro antioxidant activity) and HLFC≈HBC (TBARS). All extracts (except HBS) reduced oxymyoglobin oxidation. The HLFS extract had the highest antioxidant activity in all test systems. Supercritical fluid extraction (SFE) exhibited potential as a technique for the manufacture of functional ingredients (antioxidants) from hawthorn for use in muscle foods.

Investigation of the migration of triclabendazole residues to milk products manufactured from bovine milk, and stability therein, following lactating cow treatment.

Triclabendazole (TCB) is a flukicide used in the treatment of liver fluke in cattle; however, its use is currently prohibited in lactating dairy cows. In this study, following administration of 10% Fasinex (triclabendazole, Novartis Animal Health UK Ltd., Camberley, UK) the milk of 6 animals was used to manufacture dairy products, to ascertain if TCB residues in milk migrate into dairy products. The detection limit of the ultra-high-performance liquid chromatography-tandem mass spectrometry method used was 0.67 µg/kg. The highest concentrations of TCB residue measured, within the individual cow milk yield, was 1,529 ± 244 µg/kg (n=6), on d 2 posttreatment. Days 2 and 23 posttreatment represented high and low residue concentrations, respectively. At each of these 2 time points, the milk was pooled into 2 independent aliquots and refrigerated. Milk products, including cheese, butter, and skim milk powder were manufactured using pasteurized and unpasteurized milk from each aliquot. The results for high residue milks demonstrated that TCB residues concentrated in the cheese by a factor of 5 (5,372 vs. 918 µg/kg for cheese vs. milk) compared with the starting milk. Residue concentrations are the sum of TCB and its metabolites, expressed as keto-TCB. Residues were concentrated in the butter by a factor of 9 (9,177 vs. 1,082 µg/kg for butter vs. milk) compared with the starting milk. For milk, which was separated to skim milk and cream fractions, the residues were concentrated in the cream. Once skim milk powder was manufactured from the skim milk fraction, the residue in powder was concentrated 15-fold compared with the starting skim milk (7,252 vs. 423 µg/kg for powder vs. skim milk), despite the high temperature (185 °C) required during powder manufacture. For products manufactured from milk with low residue concentrations at d 23 posttreatment, TCB residues were detected in butter, cheese, and skim milk powder, even though there was no detectable residue in the milk used to manufacture these products. Triclabendazole residues were concentrated in some milk products (despite manufacturing treatments), exceeding residue levels in the starting milk and, depending on the storage conditions, may be relatively stable over time.

Investigation of the persistence of triclabendazole residues in bovine milk following lactating-cow and dry-cow treatments.

Triclabendazole is a flukicide used in the treatment of liver fluke in cattle. However, its use in the treatment of liver fluke is prohibited in dairy cows. In this work, two independent studies were designed to investigate the persistence of triclabendazole residues in milk following the administration of 10% Fasinex® as dry-cow and lactating-cow treatments. In the dry-cow study, 36 in-calf dairy cows were treated with a commercial product, 10% Fasinex(®), at drying-off and three triclabendazole residues (triclabendazole, triclabendazole sulphoxide and triclabendazole sulphone) were monitored in the milk following calving, approximately 60 days post-treatment. No residues were measurable in the milk of the 36 cows tested - the LOQ of the method was 1.00 µg kg(-1). In the lactating-cow study, the persistence of four triclabendazole residues was investigated in the milk of six dairy cows. The highest levels of triclabendazole, triclabendazole sulphoxide, triclabendazole sulphone and keto-triclabendazole residues measured in individual milk samples were 244, 525, 1710 and 16 µg kg(-1), respectively. Residues of triclabendazole, triclabendazole sulphoxide, triclabendazole sulphone and keto-triclabendazole were detectable in milk for up to 5.5, 15.5, 20 and 5 days post-treatment, respectively. Triclabendazole sulphone was found to be the most important residue, accounting for >87% of marker residues at ≥3.5 days following drug administration. These results indicate that following treatment at drying-off, triclabendazole residues in milk post-calving are well below the current MRL. Therefore, triclabendazole is a suitable flukicide for use during the dry period.

Investigation of the persistence of rafoxanide residues in bovine milk and fate during processing.

Rafoxanide is an effective treatment for the control of fluke infections in animals, but it is currently not permitted for treating animals whose milk is intended for human consumption. In this study, the persistence of rafoxanide residues in milk, and their migration to dairy products, was investigated following the treatment of six lactating dairy cows with Curafluke 10% oral drench. The highest concentration of rafoxanide residues detected in the individual cows milk ranged from 249 to 627 µg kg(-1) and occurred at 2-3 days post-treatment. At 2 and 23 days post-treatment (representing high and low residue concentrations) the milk was pooled into two independent aliquots, each containing the full day's milk produced by three cows. Milk products were made from pasteurised and unpasteurised milk. Pasteurisation appeared to have little impact on the stability of the residues. Rafoxanide concentrated sixfold in the cheese (week 0) compared to the starting milk (2070 vs. 349 µg kg(-1)) but was four times lower in whey (75 µg kg(-1)). Rafoxanide residues were up to 14 times higher in butter (week 0) than in the starting milk (5468 vs. 376 µg kg(-1)). Residues were found to further concentrate in butter and cheese at longer storage and ripening times, respectively. Skim-milk powder was manufactured from skim milk, and residues were 10-fold higher than in the starting skim milk (5468 vs. 376 µg kg(-1)) despite the 185°C temperature required for the process. Rafoxanide residues were stable in this skim-milk powder when stored at ambient temperature for at least 1 year. Results showed that detectable rafoxanide residues were excreted in milk for 47 days, and concentrated in the fat-based products. The analytical ranges of the UHPLC-MS/MS method used were 1.0-200 µg kg(-1) (milk and whey) and 10-2000 µg kg(-1) (other dairy products).

First evidence of azaspiracids (AZAs): A family of lipophilic polyether marine toxins in scallops (Argopecten purpuratus) and mussels (Mytilus chilensis) collected in two regions of Chile.

Azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 following the consumption by consumers of intoxicated shellfish (Mytilus edulis). These azaspiracids have now been identified in mussels (Mytilus chilensis) and scallops (Argopecten purpuratus) from two Chilean locations. This is the first report of the occurrence of azaspiracid toxins in these species (Mytilus chilensis and Argopecten purpuratus) from Chile. The areas studied were Bahía Inglesa (III Region, 27 degrees SL) and Chiloé Archipelago, both important scallop and mussels farming areas. Separation of azaspiracid (AZA1), azaspiracid isomer (AZA6) and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3), was achieved using reversed-phase LC and toxins were identified using a turbo electrospray ionisation (ESI) source, to a triple quadrupole mass spectrometer. In mussels, AZA1 was the predominant toxin in mussel hepatopancreas with AZA2, AZA3 and AZA6 present in approximate equivalent amounts in the remaining tissues, 20-30% of the AZA1 level. AZA2 predominated in the scallop samples with the toxin almost entirely present in the hepatopancreas (digestive gland). AZA1 was only observed in some of the scallop samples and was present at 12-15% of the AZA2 levels. Whilst the levels of AZAs in Chilean samples are below the EU regulatory limit of 160mug/kg, it is significant that this toxin is present in Pacific Ocean species. Consequently measures should be taken by regulatory authorities to implement regular seafood monitoring to ensure safety of harvested product.

Impacts of microcystins on the feeding behaviour and energy balance of zebra mussels, Dreissena polymorpha: a bioenergetics approach.

Microcystins are produced by bloom-forming cyanobacteria and pose significant health and ecological problems. To investigate the impacts of these biotoxins on the physiology of the zebra mussels, Dreissena polymorpha, a series of short-term feeding experiments were conducted in the laboratory. We used five microalgal diets consisting of single-cell suspensions of the green algae, Chlorella vulgaris, the diatom, Asterionella formosa, the cryptophyte, Cryptomonas sp. and two strains of the toxic cyanobacterium, Microcystis aeruginosa (strains CCAP 1450/06 and CCAP 1450/10). A sixth diet was a mixture of the diatom and the CCAP 1450/10 cyanobacterial strain. The low-toxicity strain CCAP 1450/06 contained 7.4 microg l(-1) of the MC-LR variant while the very toxic strain CCAP 1450/10 contained 23.8 microg l(-1) of MC-LR and 82.9 microg l(-1) of MC-LF. A flow-through system was designed to measure the following feeding parameters: clearance, filtration, ingestion and absorption rates. Ultimately the scope for growth (SFG) was determined as a net energy balance. We observed that mussels cleared the cyanobacterial species containing MC-LF (mean+/-95% confidence interval) at a significant lower rate (498+/-82 ml h(-1) g(-1) for the single cell suspension and 663+/-100 ml h(-1) g(-1) for the mixture diet) than all of the non-toxic species and the cyanobacterium containing MC-LR (all above 1l h(-1) g(-1)). The same pattern was observed with all the feeding parameters, particularly absorption rates. Furthermore, MC-LF caused an acute irritant response manifested by the production of 'pseudodiarrhoea', unusually fluid pseudofaeces, rich in mucus and MC-LF-producing Microcystis cells, ejected through the pedal gape of the mussels. This overall response therefore demonstrates selective rejection of MC-LF-producing cyanobacteria by zebra mussels, enhancing the presence of the very toxic MC-LF-producing M. aeruginosa in mixed cyanobacterial blooms and in the benthos. Finally, we observed that the SFG (mean+/-95% confidence interval) of mussels feeding on M. aeruginosa containing MC-LF was significantly lower (34.0+/-18.8 J h(-1) g(-1) for the single cell suspension and 83.1+/-53.0 J h(-1) g(-1) for the mixture diet) than for mussels ingesting non-toxic diets, except for C. vulgaris (all above 200 J h(-1)g(-1)). This reveals a sublethal, stressful effect of microcystins (particularly MC-LF) on the feeding behaviour and energy balance of the zebra mussel.

Sick individuals and sick populations: 20 years later.

Twenty years after Geoffrey Rose published his classic paper, the central messages remain highly relevant to modern public health policy and practice. The individual and population approaches are fundamentally different but both are needed. Recent examples of powerful population approaches prove Rose's point that norms can change benefiting the most deprived. Individual approaches have also succeeded but their protection of the most deprived communities is limited. Consumerism in health and over-reliance on individual approaches risk widening health inequalities.

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 microg/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 microg/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 microg/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.

Azaspiracid poisoning, the food-borne illness associated with shellfish consumption.

Azaspiracid poisoning (AZP) is a recently discovered toxic syndrome that was identified following severe gastrointestinal illness from the consumption of contaminated mussels (Mytilus edulis). The implicated toxins, azaspiracids, are polyethers with unprecedented structural features. Studies toward total toxin synthesis revealed that the initial published structures were incorrect and they have now been revised. These toxins accumulate in bivalve molluscs that feed on toxic microalgae of the genus Protoperidinium, previously considered to be toxicologically benign. Although first identified in shellfish from Ireland, azaspiracid contamination of several types of bivalve shellfish species has now been confirmed throughout the western coastline of Europe. Toxicological studies have indicated that azaspiracids can induce widespread organ damage in mice and that they are probably more dangerous than previously known classes of shellfish toxins. The exclusive reliance on live animal bioassays to monitor azaspiracids in shellfish failed to prevent human intoxications. This was a consequence of poor sensitivity of the assay and the fact that azaspiracids are not exclusively found in the shellfish digestive glands used for toxin testing. The strict regulatory control of azaspiracids in shellfish now requires frequent testing of shellfish using highly specific and sensitive methods involving liquid chromatography-mass spectrometry.

Food safety implications of the distribution of azaspiracids in the tissue compartments of scallops (Pecten maximus).

Azaspiracids, a new class of shellfish toxins, have been implicated in several recent incidents of human intoxications following the consumption of mussels (Mytilus edulis). A study was undertaken to examine the distribution of azaspiracid poisoning (AZP) toxins in scallops (Pecten maximus) and individual shellfish were dissected into five tissue fractions for the determination of toxin composition. Separation of the predominant azaspiracids, AZA1-3, was achieved using reversed-phase liquid chromatography with detection by positive electrospray multiple tandem mass spectrometry. The AZP toxin composition was determined in the adductor muscle (meat), gonad (roe), hepatopancreas (digestive glands), mantle and gill of scallops. Substantial differences in the AZP toxin levels between tissue compartments were observed and toxins were concentrated predominantly, about 85%, in the hepatopancreas. There was also a significant variation in the total toxin levels between individual scallops from the same sample batch and the RSD was 60% (n = 9). Interestingly, although all three AZP toxins were present in phytoplankton and mussels, AZA3 was not detected in the scallop samples examined. It was concluded that to improve food safety, only the adductor muscle and gonad of scallops should be permitted for sale to the public.

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of yessotoxins in shellfish.

Yessotoxins are a group of large polyether toxins, produced by marine dinoflagellates, which cause widespread contamination of filter-feeding shellfish. A new, sensitive liquid chromatography-mass spectrometry (LC-MS) method has been developed for the determination of yessotoxin (YTX) and 45-hydroxy-yessotoxin (45-OHYTX), a major metabolite in shellfish. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap MS in negative mode. The molecular related ion species at m/z 1141 [M-2Na+H]- was used as the parent ion for multiple MS experiments. MS-MS and MS3 gave major fragment ions at m/z 1061 [1141-SO3H]- and m/z 945 [1061-C9H12O]-. Predominant ions, that are due to the fragmentation of the backbone structure of YTXs, were observed at the MS4 stage. Reversed-phase LC using a C16 amide column was preferable to C18 phases for the separation of YTX and 45-OHYTX. Optimum calibration and reproducibility data were obtained for YTX using LC-MS-MS; r 2=0.9960, RSD < or = 6.3% at 0.25 microg YTX/g (n=5). The detection limit (S/N=3) was 30 pg YTX on-column which corresponded to 3 ng/g shellfish tissue.

DSP toxin profile in the coastal waters of the central Adriatic Sea.

A monitoring program, carried out in 1996 and 1997, has confirmed that toxic compounds, other than the most frequently detected toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1), are involved in DSP phenomena in the Adriatic Sea. Toxicity was assessed by the mouse bioassay; the content and the nature of the toxic components were established through fluorometric HPLC analysis combined with mass spectrometry. A rare pectenotoxin-2 (PTX-2) derivative, 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2SA), was the exclusive contaminant of samples collected from the central Adriatic in 1996. Contrary to its marked oral toxicity, intraperitoneally 7-epi-PTX-2SA displayed no toxic effects, hampering its detection by the mouse bioassay. In 1997, its concentration and frequency of appearance were lower than in 1996, with concomitant occurrence of OA, DTX-2, and a new unidentified component related to the DSP toxic group of compounds. This is the first report on the occurrence of DTX-2 in Adriatic mussels. A survey of the phytoplankton community in the surrounding seawater has established the presence of Prorocentrum micans and several potentially toxic species from the Dinophysis genus. A case of unexplained toxicity, associated with the occurrence of Gonyaulax polyedra, suggested possible shellfish contamination with yessotoxin (YTX).

Azaspiracid shellfish poisoning: unusual toxin dynamics in shellfish and the increased risk of acute human intoxications.

A number of recent acute human intoxications in Europe from the consumption of Irish mussels have been attributed to the presence of a new class of toxins named azaspiracids. The study demonstrates that azaspiracids behave differently from other polyether toxins, and this accounts for most false-negative results in the mouse bioassay employed by regulatory agencies to detect azaspiracids. Typically, polyether toxins are concentrated in the digestive glands of shellfish, but this is not always the situation with azaspiracids. Liquid chromatography-mass spectrometry (LC-MS), especially multiple tandem MS methods, have been applied to demonstrate that azaspiracid (AZA1) and its methyl- and demethyl- analogues, AZA2 and AZA3 respectively, are distributed throughout shellfish tissues. Using conventional mouse bioassay protocols, only 0-40% of the total azaspiracid content of shellfish was used in the assay, which could directly account for false-negative results. It was also observed that the toxin profiles differed significantly in various mussel tissues with AZA1 as the predominant toxin in the digestive glands and AZA3 predominant in the remaining tissues.