Detection of cylindrospermopsin and its decomposition products in raw and cooked fish (Oreochromis niloticus) by analytical pyrolysis (Py-GC/MS)

The presence of the toxin cylindrospermopsin is increasingly frequent in samples from different ecosystems and it is a serious problem both at environmental level and for animal and human health. To be able to prevent CYN exposure risk, it is important to have suitable analytical methods, but also quick and economical ones. Analytical pyrolysis coupled to GC/MS (Py-GC/MS) represents an important alternative for the rapid detection, characterization or “fingerprinting” of different materials. However, it has been less studied with cyanotoxins up to date. The present work aims to investigate: 1) the suitability of Py-GC/MS for detection of CYN and its decomposition products in raw and cooked fish samples before consumption and 2) the influence of the different cooking methods on the presence of different CYN degradation products detected by Py-GC/MS. For first time, these results present that Py-GC/MS could be a rapid and economical alternative for the detection and monitoring of CYN and its degradation products (DP. m/z 290.1, 169.1 and 336.2) in raw or cooked fish. Moreover, the changes induced in CYN and DP by cooking could be amenable and detected by Py-GC/MS.Ministerio de Economía y Competitividad AGL2015-64558-R, CGL2016-78937-

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

Detección de cilindrospermopsina a través de sus productos de descomposición en músculo de pescado crudo y cocinado: utilidad de la pirólisis analítica (Py-GC/MS)

Póster con 7 figuras, 1 tabla presentado en el 2º Congreso Iberoamericano y 6º Ibérico de Cianotoxinas 3-5 junio 2019 MurciaDebido a la capacidad que tiene la citotoxina cilindrospermopsina (CYN) de acumularse en diversos organismos acuáticos, como los peces, es importante disponer de métodos analíticos adecuados que permitan su detección en muestras de pescado contaminado con la toxina. El objetivo de este trabajo fue poner a punto un método de pirólisis analítica (Py-GC/MS) con el que detectar CYN y algunos de sus fragmentos de descomposición en músculo de escado crudo y cocinado. Músculos de tilapia (Oreochromis niloticus) (4g; n=5) contaminados con 50 ng CYN/g peso seco fueron cocinados 2 min por distintas técnicas (microondas, asado, hervido o vapor). Se tomó como control positivo músculo con CYN sin cocinar y como control negativo músculo sin CYN y no cocinado. Todas las muestras fueron congeladas (-80°C) y posteriormente liofilizadas hasta análisis. Las muestras se pirolizaron durante 1 minuto a 350 °C en ausencia de oxígeno en un pirolizador de doble disparo acoplado a un sistema de cromatografía de gases con detector selectivo de masas. La Py-GC/MS directa permitió la detección de CYN (PM 416) en músculo de pescado contaminado a un tiempo de retención de 24,2 min, así como de 3 posibles fragmentos de descomposición característicos de la toxina con PM 290 (15,9 min), 169 (22,4 min) y 336 (25,25 min). Se observaron además variaciones en la abundancia relativa de cada fragmento según el tipo de cocinado, siendo característico el aumento del fragmento con PM 336 tras el cocinado en microondas. El fragmento con PM 290 se observó cuando el pescado fue cocinado por técnicas que no implican agua. De manera general, las técnicas de cocinado que conllevan el empleo de agua (especialmente el hervido), mostraron una abundancia relativa menor tanto de CYN, como de los distintos fragmentos, lo que sugiere la pérdida de la toxina a través del agua del cocinado.Ministerio de Economía y Competitividad (AGL2015-64558-R y CGL2016-78937-R, MINECO/FEDER, UE)

Pirólisis analítica (Py-GC/MS) para la determinación de cilindrospermopsina en músculo de pescado cocinado

Póster presentado en el XXIII Congreso Español de Toxicología y VII Iberoamericano. 26- 28 Junio 2019La cianotoxina cilindrospermopsina (CYN) es una sustancia hepatotóxica cada vez más frecuente a nivel mundial que puede acumularse en una gran variedad de animales acuáticos y transmitirse fácilmente lo largo de la cadena alimentaria. Por ello, es importante disponer de métodos analíticos adecuados que permitan su detección en muestras de pescado contaminado, tanto crudo como ya cocinado. El objetivo de este trabajo es optimizar un método basado en pirólisis analítica para su aplicación en la detección de CYN y sus productos de descomposición en carne de pescado cruda y cocinada. Para ello, utilizamos músculo de tilapia (Oreochromis niloticus) en filetes (4 g, n=5) a los que se les inyectó CYN pura (50 ng CYN/g peso seco) y se cocinaron durante 2 min mediante diferentes técnicas como el asado, microondas, hervido o vapor. Se mantuvo un grupo sin cocinar como control positivo y otro sin CYN y sin cocinar como control negativo. Todas las muestras se congelaron (-80°C) y se liofilizaron hasta ser analizadas. La Py-GC/MS se llevó a cabo en un pirolizador de doble disparo acoplado a un sistema de cromatografía de gases. Las muestras de pescado liofilizado (1,8-2,9 mg) se situaron en un crisol de pirólisis y se sometieron a 350 °C durante 1 min en ausencia de oxígeno. En las condiciones cromatográficas utilizadas la Py-GC/MS directa permitió la detección de la CYN (PM 416,1) en músculo de pescado contaminado a un tiempo de retención de 24,24 min, así como de 3 fragmentos de descomposición característicos con PM 336,2; 169,1 y 290,1 a 25,25 min, 22,45 min y 15,92 min, respectivamente. Además, se observa una variación en la abundancia relativa de los fragmentos en función del tipo de cocinado. De manera general, las técnicas de cocinado que conllevaban el uso de agua (especialmente el hervido), mostraron una abundancia relativa menor tanto de CYN, como de los distintos fragmentos, lo que parece indicar una pérdida de la toxina a través del agua del cocinado.Ministerio de Economía y Competitividad (AGL2015-64558-R y CGL2016-78937-R, MINECO/FEDER, UE).N

Analytical Pyrolysis of fish (Oreochromis niloticus) muscle. Effect of different cooking method

Póster (P-FA-34 ) presentado en la XVIII Reunión de la Sociedad Española de Cromatografía y Técnicas Afines (SECyTA 2018), Granada, del 2 al 4 de Octubre de 2018.In this communication a detailed analytical pyrolysis of tilapia fish (Oreochromis niloticus) muscle is described. The fish, supplied by Valenciana de Acuicultura (fish hatchery of Valencia, Spain), were acclimatized in the laboratory and for 15 days in two aquariums (8 individuals/aquarium) with 96 L of tap-water at a constant temperature (21 ± 2°C). Fish were fed daily (0.3 g/day) with commercial fish food only (Dibaq S.L., Segovia, Spain). After acclimation, were dissected and each muscle sample was cut into approximately 4 g portions. Fish muscle samples were cooked for 2 min by boiling, steaming, microwaving and broiling. Briefly, for boiling and steaming, the fish muscle was introduced into the pot or onto the food steamer, respectively, with cool water, heated to boiling (100°C) and continued to boil for 2 min. A conventional household microwave oven (Samsung M17-13, 300W, 2450 MHz) was used for microwaving, and samples were broiled in Teflon pans for both sides of the fillet. A non-cooked fish muscle fillet was used as control group. The assays were always carried out by quintuplicate (n=5). All samples were kept at -80°C until freeze dry (Cryodos 80, Telstar, Tarrasa, Spain). The Py-GC/MS was performed using a double-shot pyrolyzer (Frontier Laboratories, model 2020i, Fukushima, Japan) attached to a GC system (Agilent Technologies, Palo Alto, CA,. USA, model 6890N). The muscle samples (2 mg freeze-dry tissue) were placed in crucible deactivated steel pyrolysis capsules and introduced into a preheated micro-furnace at (350 °C) for 1 min. The volatile pyrolysates were then directly injected into the GC/MS for analysis. The gas chromatograph was equipped with a low polar-fused silica (5%-phenyl-methylpolysiloxane) capillary column (Agilent J&W HP-5ms Ultra Inert, of 30 m × 250 μm × 0.25 μm film thickness. The oven temperature was held at 50 °C for 1 min and then increased to 100 °C at 30 °C min-1, from 100 °C to 300 °C at 10 °C min-1, and stabilized at 300 °C for 10 min, with a total analysis time of 32 min. The carrier gas was He at a controlled flow of 1 mL min-1. The detector consisted of a mass selective detector (Agilent Technologies, Palo Alto, CA. USA, model 5973N) and mass spectra were acquired at 70 eV ionizing energy. Compound assignment was achieved by single-ion monitoring (SIM) for the major homologous series and by comparison with published data reported in the literature or stored in digital NIST 14 (Maryland, USA) and Wiley 7 (Weinheim, Germany) libraries. In a first analytical step, a detailed pyrolysis fingerprint of raw fish muscle tissue is produced and the effect of the pyrolysis temperature from 150 to 550 ºC in 100 ºC increments is studied in both, i) applying each temperature to a different sample or ii) sequentially (multi-shot) applying each temperature to the same sample. In a second phase, after stablishing an optimum pyrolysis temperature of 350 ºC for 1 minute, the effect of the different cooking methods (boil, steam,microwave and broil cooking) in the fish muscle pyrolyzates was studied.Projects CGL2016-78937-R and AGL2015-64558-R co-financed by FEDER Funds. Desiré Monis and Alba Carmona for technical assistance.N

Detection of cylindrospermopsin and its decomposition products in raw and cooked fish (Oreochromis niloticus) by analytical pyrolysis (Py-GC/MS)

10 páginas.- 8 figuras.- 3 tablas.- referencias.- Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125469The presence of the toxin cylindrospermopsin is increasingly frequent in samples from different ecosystems and it is a serious problem both at environmental level and for animal and human health. To be able to prevent CYN exposure risk, it is important to have suitable analytical methods, but also quick and economical ones. Analytical pyrolysis coupled to GC/MS (Py-GC/MS) represents an important alternative for the rapid detection, characterization or “fingerprinting” of different materials. However, it has been less studied with cyanotoxins up to date. The present work aims to investigate: 1) the suitability of Py-GC/MS for detection of CYN and its decomposition products in raw and cooked fish samples before consumption and 2) the influence of the different cooking methods on the presence of different CYN degradation products detected by Py-GC/MS. For first time, these results present that Py-GC/MS could be a rapid and economical alternative for the detection and monitoring of CYN and its degradation products (DP. m/z 290.1, 169.1 and 336.2) in raw or cooked fish. Moreover, the changes induced in CYN and DP by cooking could be amenable and detected by Py-GC/MS.The authors wish to thank Ministerio de Economía y Competitividad, Spain (MINECO) Projects AGL2015-64558-R and CGL2016-78937-R, co-funded with FEDER, UE fundsPeer reviewe

Pyrolytic behaviour of microcystins and microcystin-spiked algal blooms

7 pages, 5 figures, 3 tables.This work describes the pyrolytic behaviour of microcystin (MC) standards using both conventional analytical pyrolysis (Py-GC–MS) and thermochemolysis with tetramethylammonium hydroxide (TMAH). In both cases, the pyrolytic patterns of the MCs at low pyrolysis temperature were structure-dependant, which confirms that pyrolytic techniques are suitable to readily distinguish between different types of MCs. However, the different types of amino acids conforming the structure of the MCs were distinguished only after TMAH/thermochemolysis. In order to examine the suitability of such techniques as a potential tool in the analysis of MC in cyanobacterial algae blooms, pyrolytic experiments using cyanobacterial cultures spiked with the microcystin LR (MC-LR) were also performed. The results suggest that pyrolytic techniques could be used for the rapid and safe detection of toxins in cyanobacterial blooms. Although promissory, further work directed to technique optimization would be necessary before this methodology is used as a routine detection method.Peer reviewe

Detection of carvacrol in biological tissues by analytical pyrolysis

Abstracts of the 52nd Congress of the European Societies of Toxicology (EUROTOX) Fibes Congress Center Seville, Spain, 04th-07th September 2016Oregano essential oil is being included in new packaging materials due to its bioactive properties. Carvacrol, main compound of this essential oil, with antioxidant and antimicrobial properties, is intended to be used in food packaging. According to the recommendation of the EFSA for the genotoxic assessment of substances in food, the in vivo negative results are acceptable when direct or indirect evidence supportive of exposure of the target tissues have been demonstrated. Previous studies reported negative results for comet assay in stomach and liver of rats orally exposed to carvacrol (81, 256 and 810 mg/kg bw). Hence, in this work, direct pyrolysis–gas chromatography–mass spectrometry (Py–GC/MS) analysis was performed using a double-shot pyrolyzer attached to a GC/MS system to ensure the exposure of such tissues to carvacrol. Results showed the presence of carvacrol in stomach and liver in all of the concentrations assayed. Moreover, pyrolysis technique showed clear and distinct dose–response relationships depending on the tissue evaluated. However, the ingestion of concentrations over 256 mg/kg bw in the liver, exhibited no-linear relation. This does not relate to the ratios predicted by the linear model found in the stomach. In this regard, the differences observed in those curves may be related to carvacrol metabolism in rats and the possible occurrence of saturation mechanisms limiting an excess of carvacrol metabolization/presence in liver. Therefore, our results indicate that Py–GC/MS is a valuable tool to evaluate the exposure of biological tissues to carvacrol avoiding sample pre-treatmenEuropean Soc Toxicol u projects AGL2012-38357-C02-0 y CGL2012-38655-C04-01, co-financed by FEDERN

Detection of the monoterpene carvacrol in mammal tissues by analytical pyrolysis (Py‐GC/MS)

Póster presentado en el la XVI Reunión Científica de la Sociedad Española de Cromatografía y Técnicas Afines (SECyTA2016) P‐NP‐1Eds: González-Pérez, José Antonio.-- Almendros Martín, Gonzalo.-- González-Vila, Francisco Javier.-- Rosa Arranz, José M. de laResponding to consumer demands on minimal processing and preservative‐free products, the use of essential oils (EOs) to extend shelf life of foods is on the spot in the food industry [1]. Carvacrol, main compound of Oregano EO, is registered as a flavouring in Europe; however, its use for other applications, such as active food packaging, may require higher concentrations and there is an increasing concern regarding exposure. Because of this, the European Food Safety Authority (EFSA) requires additional genotoxic studies data of substances which could be incorporated into food packaging like carvacrol. Here a detailed analytical pyrolysis (Py‐GC/MS) study is conducted as a complement to in vivo genotoxicity studies. Analytical pyrolysis was the technique chosen to search for carvacrol directly in viscera and to confirm that the compound effectively reached target tissues from orally exposed (0, 81, 256 or 810 mg carvacrol/kg bw, calculated according to carvacrol Maximum Tolerated Dose (MTD)) young adult male Wistar rats strain RjHan:WI*. Doses were prepared in corn oil at a final volume of 1 mL and during the treatment period, clinical signs, body weight, and food and water consumption were recorded daily. Rat stomach and liver composite samples were selected for pyrolysis and preserved at ‐80ºC until lyophilisation (Testal Cryodos, Madrid). Direct pyrolysis was performed in a double‐shot pyrolyzer (Frontier Lab 2020i) attached to a GC/MS system (Agilent 6890N + 973MSD). Detailed chromatographic conditions can be found in [2]. In short, lyophilized tissue (stomach and liver) were thoroughly homogenized and samples introduced (0.5 mg) into a preheated micro‐furnace at 500 ºC for 1 min and evolved gases transferred to the GC/MS for analysis. Compounds assignment was via single‐ion monitoring and by comparison with published and stored (NIST and Wiley libraries) data. In a previous study, it was determined that pyrolysis of carvacrol does not to produce major effects on its chemical structure and therefore was considered an adequate technique to detect the presence of the monoterpene in animal tissues. The analytical pyrolysis of target tissues of control rats was negative to any sign of carvacrol even when searching for the specific mass fragments (m/z 135 and 150). However, carvacrol was clearly detected in the tissues of rats treated at all doses. Furthermore, when normalizing the chromatograms to a common peak, a clear dose response was obtained. A conspicuous difference was found in the dose‐response curves between stomach and liver; whereas a direct lineal correlation could be drawn from the former, the response for the latter was best fit to a quadratic equation model. In this regard, the differences observed in those curves may be related to carvacrol metabolism in rats and the possible occurrence of saturation mechanisms limiting an excess of carvacrol metabolization/presence in liver. als received humane care.[1] M. Llana‐Ruiz‐Cabello, S. Pichardo, A. Baños, C. Nuñez, J.M. Bermúdez, E. Guillamón, S. Aucejo, A.M. Cameán, Food Sci. Technol‐LEB 64 (2015) 1354‐1361 [2] M. Llana‐Ruíz‐Cabello, S. Pichardo, N.T. Jiménez‐Morillo, J.M. Bermúdez, S. Aucejo. F.J. González‐Vila, A.M. Cameán, J.A. González‐Pérez (2016). J. Sci. Food Agr. 96 (2016) 3207‐3212 (*) The Ethics Committee on Animal Experimentation of the University of Sevilla approved the in vivo experiments. Moreover, in compliance with the Directive 2010/63/EU for the protection of animals used for scientific purposes all animN

Pirolisis analítica como método de detección de carvacrol en tejidos biológicos.

El aceite esencial de orégano (Origanum vulgare L. virens) (OEO) es uno de los extractos naturales más interesantes para la industria alimentaria por sus propiedades antioxidantes y antimicrobianas. El carvacrol, su compuesto mayoritario, es uno de los compuestos fenolicos que le confiere estas propiedades al aceite. Para evaluar la seguridad de estas sustancias, la Autoridad Europea de Seguridad Alimentaria (EFSA) exige una serie de ensayos tanto in vitro como in vivo que estudien la capacidad genotóxica de las mismas. Para poder evaluar in vivo la genotoxicidad de carvacrol mediante la combinación del test de micronúcleos (MN) y del ensayo cometa, es necesario confirmar la exposición de los tejidos a esta sustancia. En este sentido, en el presente trabajo se evaluó mediante técnicas de pirólisis analítica (Py-CG/MS) la presencia de carvacrol en estómago e hígado de ratas Wistar expuestas por vía oral a distintas dosis de carvacrol (81, 256, 810 mg/ kg). Los resultados muestran por primera vez la presencia del compuesto en todos los órganos analizados. Por ello, demostramos que la técnica de pirólisis analítica es útil para obtener información sobre la exposición de los tejidos biológicos al agente activo.Los autores agradecen al Ministerio de Ciencia e Innovación (AGL2012-38357-C02-01 cofinanciado con fondos FEDER y CGL2012-38655-C04-01) y a la Junta de Andalucía (AGR- 7252) la financiación de este proyecto. M Llana-Ruiz-Cabello agradece a la Junta de Andalucía por su beca predoctoral y NT Jiménez-Morillo por su beca FPI (BES-2013-062573).Peer Reviewe