Novel IAC-LC-ESI-MS(2) analytical set-up for ochratoxin A determination in pork.

A reliable exposure assessment of mycotoxin contamination relies on their unambiguous identification and accurate quantification. With such purpose, a new analytical methodology was developed for evaluation of ochratoxin A (OTA) contamination in pork. Briefly OTA extraction from minced muscle samples involved the use of acidified methanol, ultrasound treatment and centrifugation, followed by an immunoaffinity column (IAC) clean-up step before mass spectrometric detection (precursor-to-fragment transitions: m/z 404→m/z 358 and m/z 404→m/z 386) in positive ESI mode using SRM scanning. The method combines green chemistry principles (e.g. absence of highly toxic solvents commonly used in this matrix) with a validation according to the guidelines laid down by the 2002/657/EC European Decision parameters: recoveries varied between 98.5% and 100.6%, limits of detection (LOD) and quantification (LOQ) were estimated at 0.06 and 0.19 µg/kg, whereas decision limit (CCα) and detection capability (CCß) were 0.01 and 0.50 µg/kg, respectively. The proposed analytical set-up was successfully applied to twenty pork samples commercially acquired.

Evaluation of enniatins A, A1, B, B1 and beauvericin in Portuguese cereal-based foods.

Sixty-one samples of Portuguese cereal-based foods were analysed for the occurrence of emerging mycotoxins called enniatins (A, A1, B and B1) and beauvericin. Samples were extracted with a mixture of acetonitrile/water (85/15, v/v) using an Ultra-Turrax homogeniser, and mycotoxins were detected with liquid chromatography coupled to a mass spectrometer. This method was validated and adequate values of recovery (70-103%) and relative standard deviation (<15%) were obtained. Signal suppression/enhancement was studied and matrix-matched calibration used to minimise this effect, but no additional clean-up step was necessary. The mass spectrometer was operated in selected reaction monitoring (SRM) mode and with selected transitions for each compound to quantify and to qualify them. Fifty-nine per cent of samples were contaminated. The percentages of enniatins were 53%, 49%, 44% and 16% for A1, B, B1, and A, respectively, and for beauvericin it was 1.6%. For the total samples, the mean contamination was 30, 24, 15, 2.1 and 0.1 ng g⁻¹ for enniatins A1, B, B1 and A, and beauvericin, respectively. The wheat-based samples showed higher levels and greater prevalence than any other cereals monitored. These results were used to estimate the daily intake of ENs from wheat-based cereal by the Portuguese population. At the same time, the usefulness of this method in the analysis of other important mycotoxins (aflatoxin B1, ochratoxin A, deoxynivalenol, T-2 toxin, fumonisin B1 and zearalenone) was evaluated.

Determinants of ochratoxin A exposure--a one year follow-up study of urine levels.

Dietary exposure to the ochratoxin A (OTA) occurring in Portugal is characterized by a high frequency of contamination of the consumed foodstuffs, although at low levels. The exposure bears significance for the total food consumed, and not for a particular one. Biomonitoring studies are thus fundamental in simplifying the evaluation of exposure, with no need to examine the entire range of consumed foodstuffs. Biomonitoring studies further allow the identification of host factors as predictors of OTA exposure in epidemiologic studies, the results of which are merited for targeting intervention strategies by public health authorities and advising official regulatory decisions. Using a longitudinal approach, this study examined factors related to OTA exposure in the adult population over a one-year period. Anthropometric measures, season of the year and region were the selected factors correlated with OTA exposure biomarker. Urine samples from 95 inhabitants from six Portuguese main geographical areas were assayed through spectrofluorimetric detection. Exposure to OTA proved to markedly increase in winter, and gender differences were observed only in summer, which might be related to different dietary patterns not only between seasons, but also between genders. The same rationale may also serve the observed statistically significant differences between some regions. No other strong association upon the remaining determinants under testing was observed. These observations reinforce the need for OTA exposure evaluation, possibly specifically targeting the staple foods or dietary habits that sustain potential predictors or determinants of exposure.

Ochratoxin A survey in Portuguese wine by LC-FD with direct injection.

Wine and grape juices were identified as one of the most important sources of ochratoxin A (OTA), a mycotoxin with diverse toxic effects that naturally appears in food and foodstuffs all over the world. The aim of this study was to assess the OTA levels in Portuguese wines through the application of a simple and accurate method based on liquid chromatography (LC) with direct injection, followed by fluorescence detection (FD). Randomly selected wine samples were used to evaluate the performance of direct injection as efficient, fast, inexpensive and safe sample preparation method. The proposed method was successfully validated. The limit of quantification (LOQ) was 1.0 microg/L and OTA recoveries from wine samples, spiked at the three fortification levels, were higher than 85.4%, with RSDs lower than 9.6% for both red and white wines. The presence of OTA was confirmed by methyl ester derivatization followed by LC analysis. Data on OTA levels were obtained for 60 Portuguese red and white wine samples. OTA was found in 12 samples, nine (26%) red wine samples and three (12%) white wine samples. Only one red wine sample and one white wine sample presented a contamination level above the LOQ, with 1.23 and 2.4 microg/L, respectively. It should be pointed out that this white wine sample exceeded the EC maximum permitted level of 2.0 microg/L. The safe dose established as 120 ng/kg body weight/week was not exceeded by the weekly intake estimated for the samples contaminated above the LOQ.

Mycotoxin food and feed regulation and the specific case of ochratoxin A: a review of the worldwide status.

Mycotoxins are naturally occurring contaminants whose presence in food- and feedstuffs cannot be completely avoided. The safety and economic impact arising from the commercialization of mycotoxin-contaminated food and feed supply chain is considerable and acknowledged. To protect consumers from mycotoxin-derived health risks, many countries have adopted regulations or guidelines to limit exposure. Similar to regulation for other mycotoxins, the regulatory status of ochratoxin A (OTA) lacks consensus. Although this was one of the first fungal toxins to be subjected to compliance control due to its toxic properties, the conflict between the prime objective of consumer health safeguard and the economic interests of producers and traders remains. One of the key challenges facing policymakers is to balance these conflicting demands and reach consensual regulatory actions or limits. The noteworthy transboundary implications are recognized, both in the case of absence as the unsubstantiated tightening of regulatory limits. This paper scrutinizes the rationales and implications of mycotoxin regulation, with special attention devoted to OTA. In view of the ongoing debate concerning this complex issue, a review of the arguments and suggested strategies proposed by different parties is also made. The specific case of OTA regulation in food and feed is updated and analysed at a European Union and global level.

Influencing factors on bread-derived exposure to ochratoxin A: type, origin and composition.

The nearly ubiquitous consumption of cereals all over the world renders them an important position in international nutrition, but concurrently allocates exposure to possible contained contaminants. Mycotoxins are natural food contaminants, difficult to predict, evade, and reduce, so it is important to establish the real contribution of each contaminated food product, with the aim to evaluate mycotoxin exposure. This was the key objective of this survey and analysis for ochratoxin A content on 274 samples of commercialized bread in the Portuguese market, during the winter 2007. Different bread products were analyzed through an HPLC-FD method, including traditional types, novel segments, and different grain based bread products. A wide-ranging low level contamination was observed in all regions and types of bread products analyzed, especially in the Porto and Coimbra regions, and in the maize and whole-grain or fiber-enriched bread. Nevertheless, the exposure through contaminated wheat bread continues to be the most significant, given its high consumption and dominance in relation to the other types of bread.

Fluoroquinolone antibiotics determination in piggeries environmental waters.

To trace the contamination and evaluate the environmental impact of piggeries, the occurrence of norfloxacin (NOR), ciprofloxacin (CIPRO), enrofloxacin (ENRO) and sarafloxacin (SARA) residues were evaluated in water for swine consumption, piggeries' wastewaters, drainage waters and a river receiving piggery effluents. Water samples were acidified before being percolated through Oasis HLB cartridges or through a tandem system consisting of strong anion-exchange and Oasis HLB cartridges. A liquid chromatography with fluorescence detection (LC-FD) method developed in previous studies, based on the use of a monolithic column, was successfully applied. NOR, CIPRO, ENRO and SARA had good linear responses, with mean regression coefficients higher than 0.9989. The limits of quantification (LOQ) for waters for swine consumption, and a river receiving piggery effluents, were 0.025 ug L(-1) for NOR, CIPRO and ENRO and 0.05 ug L(-1) for SARA. For wastewaters and drainage waters, the LOQs were 0.05 ug L(-1) for CIPRO and ENRO and 0.1 ug L(-1) for SARA. Recoveries obtained for spiked samples ranged from 70.8 to 89.1%, and precision data were under 12.7%. The method was successfully applied to 18 environmental water samples, collected from piggeries in Alentejo, Portugal, and the first Portuguese data are presented. ENRO was detected in a total of 8 samples, at concentrations ranging between 0.31 microg L(-1) and 63 microg L(-1). CIPRO, its degradation product, and SARA were present in 2 samples, in the ranges 0.48-0.83 microg L(-1) and 1.8-14 microg L(-1), respectively. No fluoroquinolones were detected in the waters for swine consumption or in the river water samples.

Determination of fluoroquinolone residues in poultry muscle in Portugal.

A total of 98 poultry samples, including chicken and turkey muscle, were analysed, using a sensitive and reliable analytical method based on liquid chromatography (LC) with spectrofluorimetric detection, for simultaneous determination of four fluoroquinolone (FQ) antibiotics, namely enrofloxacin (ENRO), ciprofloxacin (CIPRO), norfloxacin (NOR), and sarafloxacin (SARA). The method involved extraction with 0.15 mol L(-1) HCl and clean-up by solid-phase extraction using Oasis HLB cartridges. Chromatographic separation was carried out on a C(18) TSK gel column, in isocratic mode, with 0.025 mol L(-1) H(3)PO(4) solution, adjusted to pH 3.0 with tetrabutylammonium hydroxide-methanol (78:22) as mobile phase. Good linearity over the investigated concentration range was observed, with mean values of correlation coefficients higher than 0.9989 for all the analytes studied. The limits of quantification (LOQ), expressed as the lowest fortification level with acceptable precision were 15 microg kg(-1) for ENRO, CIPRO, and NOR, and 30 microg kg(-1) for SARA; these values are in compliance with requirements for monitoring of maximum residues levels (MRLs). Overall recoveries from spiked samples ranged from 80% to 92% with relative standard deviations (RSD) lower than 6.1%. Of the chicken and turkey samples analysed, 44.2% and 37.8%, respectively, were contaminated. The levels found in the analysed poultry samples, collected from markets of Oporto and Coimbra, located in the north and central zones of Portugal, respectively, were lower than 114.2 and 87.6 microg kg(-1) in chicken and turkey muscle samples, respectively. One positive chicken sample was contaminated with ENRO at levels higher than the MRL.

Photo-induced fluorescence of magnesium derivatives of tetracycline antibiotics in wastewater samples.

An analytical strategy, for the determination of tetracyclines (TCs), based on a HPLC system coupled with a photo-reactor followed by post-column derivatization was developed. Higher fluorescence emission after coupling the resulting photo-fragments with magnesium ions was observed for the determination of minocycline (MC), epitetracycline (ETC), tetracycline (TC) and doxycycline (DC). The manifold included a HPLC system with a photo-reactor (PTFE tubing helically coiled around a low-pressure mercury lamp), a mixing T-piece and a fluorescence detector. The derivatization reagent was delivered at 0.5 mL min(-1) by a pump. After HPLC separation using a gradient system with a mobile phase containing oxalic acid 0.02 M and acetonitrile, TCs were irradiated for 60 s, and the resulting photo-fragments were mixed with the post-column derivatization reagent, and the magnesium derivatives of TCs were detected by fluorimetry (lambda(exc) 386 nm, lambda(em) 500 nm). The results obtained showed a significant increase of sensitivity due to photodegration of TCs, 45.4%, 37.6% and 25.3% for MC, TC and ETC respectively. For DC an increase of only 1.5% was observed. The developed method was successfully applied to TCs determination in hospital and municipal wastewater samples using solid phase extraction with Oasis HLB cartridges. The LOQs were 0.25, 0.15, 01 and 0.25 microg L(-1) for TC, ETC, MC and DC, respectively. The recovery values oscillated between 107.1% and 92.4% for fortification of 2.5 microg L(-1) of each antibiotic.

Tetracycline antibiotics in hospital and municipal wastewaters: a pilot study in Portugal.

This study investigated the occurrence of tetracyclines (TCs), namely minocycline (MIN), TC, and its epimer epitetracycline (ETC), and doxycycline (DC), in four hospital wastewater effluents and its fate in municipal wastewater treatment plants (WWTPs), in Coimbra, Portugal. Analytical determination was carried out by solid-phase extraction followed by liquid chromatography with fluorescence detection. A gradient system with a mobile phase containing oxalic acid 0.02 M and acetonitrile was used. After postcolumn derivatization with magnesium reagent, TCs were detected at lambda(exc) 386 nm and lambda(em) 500 nm. The proposed method allowed good sensitivity, accuracy, and precision. LOQs were 0.5 microg l(-1) for ETC and TC and 15 and 5 microg l(-1) for MIN and DC, respectively. The recovery values ranged between 66.4% and 117.1%, and intraday and interday repeatability was lower than 6.8%. The method was successfully used to determine the presence of the above-mentioned TCs in 24 wastewater composite samples obtained from hospital effluents and from influent and effluent of the WWTP located in Coimbra, Portugal. MIN and TC were found in 41.7% of the samples; ETC and DC were found in 25% and 8.3% of the samples, respectively. The levels found ranged from 6 to 531.7 microg l(-1) in hospital effluents, while its concentrations in WWTP ranged from 95.8 to 915.3 microg l(-1). A seasonal influence in the concentrations found has also been observed, the levels found in samples collected during spring being higher than those observed in samples collected during autumn; however, these are only preliminary results. The WWTP removal rate ranged between 89.5% and 100%.

A review on ochratoxin A occurrence and effects of processing of cereal and cereal derived food products.

Ochratoxin A (OTA) continues to grab global attention and concern for the hazard and impact that embody for both human and animals, based on its toxicity and occurrence. Despite OTA has been described in a myriad of foodstuffs, cereal and its derivatives remain the major contributors to OTA exposure. For that reason, a critical review on OTA occurrence reported by recent studies worldwide focusing on unprocessed and processed cereal foodstuffs is made in this work. Special attention is drawn to the major cereal derived products, namely flour, bread, breakfast cereals, baby/infant foods and the inherently involved technological food processing methods and its influence on the redistribution and chemical modification of OTA. The paper further examines the factors that influence the OTA content of cereal and its derived products, explicitly the different ecological niches of the ochratoxigenic mycobiota -Aspergillus spp. and Penicillium verrucosum, the agricultural practice involved, harvest procedures and storage conditions, the type of grain, and the nature and extent of technological processing as well as the ultimate stages of analytical quality level of the sampling and analysis of the suspected ingredients or foods.

Monitoring of ochratoxin A exposure of the Portuguese population through a nationwide urine survey--Winter 2007.

Ochratoxin A (OTA) is a mycotoxin produced by a variety of fungi, such as Penicillium verrucosum and Aspergillium spp., which has been found to have a wide number of potentially deadly toxic effects, and can enter the human organism through a variety of means. It then finds its way into the bloodstream and, after a lengthy process, is eventually excreted through the urine. It can thus be detected in its original form not only in blood samples but also in this biological medium. As such, and in an attempt to evaluate the exposure of the Portuguese population to this mycotoxin, morning urine samples were collected during the Winter of 2007, from each of five geographically distinct Portuguese locations--Bragança, Porto, Coimbra, Alentejo, and Algarve--and subjected to extraction by immunoaffinity columns and to OTA quantification through liquid chromatography coupled with fluorescence detection. Prevalent incidence was higher than 95% with Coimbra being the exception (incidence of 73.3%). In nearly all locations, the OTA content of most samples was found to be above the limit of quantification (LOQ) of 0.008 ng/ml. Indeed, excluding Coimbra, with an OTA content level of 0.014 ng/ml, all regions featured content values over 0.021 ng/ml.

Ochratoxin A exposure assessment of the inhabitants of Lisbon during winter 2007/2008 through bread and urine analysis.

A survey on the occurrence of ochratoxin A (OTA) in 41 bread samples was carried out in the Portuguese capital, Lisbon. Maize (5) and wheat bread (36) and 43 representative urine samples from the Lisbon region were assayed for OTA levels using immunoaffinity column cleanup (IAC) and HPLC with fluorimetric detection (LC­FD). The percentage of OTA-positive samples was slightly higher for maize bread (80%) than wheat bread (70.8%), although, due to its higher consumption, the latter contributes more to OTA exposure, featuring a higher estimated daily intake (EDI). In the urine samples analyzed, both female and male residents displayed similarly high levels of OTA frequency and average contamination. In summary, OTA is a food contaminant of concern and may constitute a hazard for public health through consumption of cereal-based products.

Ochratoxin A in the morning and afternoon portions of urine from Coimbra and Valencian populations.

The widespread contamination of foodstuffs and beverages by mycotoxins, such as ochratoxin A (OTA), has made the monitoring of human contamination levels essential. By using a sensitive, accurate and speedy method that combines extraction with 5% NaHCO(3), immunoaffinity column clean-up and HPLC with fluorescence detection, the human exposure to OTA through urine analysis can be monitored. This method is less invasive than blood monitoring and has the potential to be a good marker of human exposure. The limit of quantification of the method was 0.007 ng/mL of urine, with recoveries of OTA, from urine samples spiked at levels between 0.02 and 0.1 ng/mL, higher than 91% with RSD lower than 15.5%. This study evaluated OTA contamination levels in human urine sample fractions, collected in the morning and afternoon, in two populations, one from Coimbra city, in Portugal, and another from the Valencian community, in Spain. In the Coimbra population, 60 samples from 30 healthy individuals were analyzed, levels of OTA in 13 morning samples and 14 afternoon samples having been detected, with concentrations ranging from 0.011 to 0.208 and 0.008 to 0.11 ng/mL respectively. In the Valencia population, 62 samples from 31 healthy individuals were analyzed, with OTA being detected in 25 morning samples and 26 afternoon samples. The concentrations varied between 0.007 and 0.124 ng/mL in the morning samples, and 0.008 and 0.089 ng/mL in the afternoon samples. Significant differences were found between the morning levels of OTA from both populations (P=0.033). For afternoon samples, significant differences were not found, P value=0.163.

Determination of fluoroquinolone antibiotics in hospital and municipal wastewaters in Coimbra by liquid chromatography with a monolithic column and fluorescence detection.

The main goal of this work was determination of residues of the antibiotics ofloxacin (OFLO), norfloxacin (NOR), ciprofloxacin (CIPRO), and enrofloxacin (ENRO) in wastewater samples. The samples, after acidification to pH 4.5 and addition of EDTA, were extracted on an anion-exchange cartridge in tandem with an Oasis HLB cartridge. The LC-FD method, developed in previous studies, was based on application of a monolithic C(18) column. The limit of quantification (LOQ) of the method was 250 ng L(-1) for OFLO, 25 ng L(-1) for NOR and CIPRO, and 50 ng L(-1) for ENRO. Mean recovery ranged between 75 and 121% for OFLO, NOR, CIPRO, and ENRO. A total of 14 wastewater samples were analyzed; these were collected from four hospitals and from influent and effluent from a wastewater-treatment plant in Coimbra, Portugal, during spring and autumn. CIPRO was present in all the samples, NOR was detected second most often, followed by OFLO. ENRO was found at concentrations under the LOQ in five hospital samples, and the highest level was found in influent from the WWTP.

Estimated intake of the sweeteners, acesulfame-K and aspartame, from soft drinks, soft drinks based on mineral waters and nectars for a group of Portuguese teenage students.

In a survey of levels of acesulfame-K and aspartame in soft drinks and in light nectars, the intake of these intense sweeteners was estimated for a group of teenage students. Acesulfame-K was detected in 72% of the soft drinks, with a mean concentration of 72 mg l(-1) and aspartame was found in 92% of the samples with a mean concentration of 89 mg l(-1). When data on the content of these sweeteners in soft drinks were analysed according to flavour, cola drinks had the highest mean levels for both sweeteners with 98 and 103 mg l(-1) for acesulfame-K and aspartame, respectively. For soft drinks based on mineral water, aspartame was found in 62% of the samples, with a mean concentration of 82 mg l(-1) and acesulfame-K was found in 77%, with a mean level of 48 mg l(-1). All samples of nectars contained acesulfame-K, with a mean concentration of 128 mg l(-1) and aspartame was detected in 80% of the samples with a mean concentration of 73 mg l(-1). A frequency questionnaire, designed to identify adolescents having high consumption of these drinks, was completed by a randomly selected sample of teenagers (n = 65) living in the city of Coimbra, in 2007. The estimated daily intakes (EDI) of acesulfame-K and aspartame for the average consumer were below the acceptable daily intakes (ADIs). For acesulfame-K, the EDI was 0.7 mg kg(-1) bw day(-1) for soft drinks, 0.2 mg kg(-1) bw day(-1) for soft drinks based on mineral waters, and 0.5 mg kg(-1) bw day(-1) for nectars, representing 8.0%, 2.2%, and 5.8% of the ADI, respectively. A similar situation was observed for aspartame. In this way, the EDI for soft drinks was 1.1 mg kg(-1) day(-1), representing only 2.9% of the ADI. In respect of nectars, the EDI was 0.2 mg kg(-1) bw day(-1), representing 0.5% of the ADI. Soft drinks based on mineral waters showed the lowest EDI values of 0.3 mg kg(-1) bw day(-1), accounting for 0.7% of the ADI.

Levels of ochratoxin A in serum from urban and rural Portuguese populations and estimation of exposure degree.

Urban and rural population exposure to ochratoxin A (OTA) in central zone of Portugal was investigated in three places: Coimbra, Verride and Ereira. The analytical method proposed for the determination of ochratoxin A involved extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column (IAC), high performance liquid chromatography (HPLC) with spectrofluorimetric detection (FD) for separation and identification of ochratoxin A, and confirmation with HPLC-FD after OTA methylation in serum. The limit of quantification of the proposed method was 0.1 microg/L for serum and 0.05 microg/L for blood. OTA recoveries in serum ranged from 70.3% to 115.3% for levels at 0.25 microg/L and 0.5 microg/L, respectively, with a within-day RSD between 8.0% and 16.2%. Ochratoxin A serum levels were evaluated in an hundred and four donors from Coimbra city, Verride, and Ereira. The study revealed a frequency of detection of 100%. The ratio of ochratoxin A level in serum to whole blood was 2.0+/-0.7. The overall concentrations range from 0.25 to 2.49 microg/L, 0.14 to 1.91 microg/L, and 0.19 to 0.96 microg/L, for samples of Verride, Ereira, and Coimbra, respectively. The mean concentration and standard deviation were 0.78+/-0.53 microg/L, 0.44+/-0.31 microg/L, and 0.42+/-0.18 microg/L for the same samples. A significant difference was found in Verride population (P-value=0.000). Levels of OTA are clearly higher in males from rural areas than in females. For all samples, a significant difference was found in Verride male population (P-value=0.014).

Occurrence of fumonisins B1 and B2 in broa, typical Portuguese maize bread.

Fumonisin B(1) (FB(1)) and fumonisin B(2) (FB(2)) are mycotoxins mainly produced by Fusarium verticillioides, and Fusarium proliferatum, fungi species most commonly isolated from maize. The natural occurrence of FB(1) and FB(2) in broa, typical Portuguese maize bread, was evaluated in 30 samples. Twenty five were found positive with levels ranging from 142 to 550 microg kg(-1). The limit established by the European regulations was exceeded by 27% of the samples. The tolerable daily intake for fumonisin B(1), and B(2), alone or in combination, for all of the analysed samples, was lower than 2 microg kg(-1) body weight per day established by the European Commission.

Occurrence of fumonisins B1 and B2 in Portuguese maize and maize-based foods intended for human consumption.

Fumonisin B(1) (FB(1)) and fumonisin B(2) (FB(2)) are mycotoxins mainly produced by Fusarium verticillioides and Fusarium proliferatum, which are field pathogens of maize. A survey was conducted on the incidences of FB(1) and FB(2) in both maize and derived products purchased in Portugal. The analytical method involved extraction with methanol-water, clean-up by immunoaffinity column and derivatization with naphthalene-2,3-dicarboxaldehyde. Determination was carried out by high-performance liquid chromatography (HPLC) with spectrofluorimetric detection, with liquid chromatography/mass spectrometry (LC/MS) confirmation. The presence of FB(1) and FB(2) was determined in 67 samples of maize and maize-based foods, such as flour, semolina, starch, sweet maize, cornflakes and other breakfast cereals, and snacks collected in 2005. FBs were found in 15 samples at concentrations ranging from 113 to 2026 microg kg(-1). Two of the samples showed higher contamination levels than the limits established by the European Commission Regulation. None of the samples contained levels of fumonisins that would lead to an exposure exceeding the tolerable daily intake (TDI).

Ochratoxin A in nephropathic patients from two cities of central zone in Portugal.

Ochratoxin A (OTA) produced by Aspergillus and Penicillium genera contaminates several foods. OTA is nephrotoxic to all animal species studied so far, and most likely to humans, who show the longest half-life for elimination of this toxin among all examined species. OTA has other toxic effects such as teratogenicity, immunotoxicity, genotoxicity, and is also mutagenic and carcinogenic, all of which lead to life-threatening pathologies through several molecular pathways. A sensitive, specific and rapid method applying high performance liquid chromatography coupled to a spectrofluorimeter for the determination of ochratoxin A in human serum was validated. Serum samples were extracted with chloroform-orthophosphoric acid, and cleaned-up through immunoaffinity column (IAC). The separation and identification was performed by HPLC coupled to a spectrofluorimeter, and, after OTA methylation, the confirmation was achieved. Chromatographic separation of the analyte was performed on a reverse phase column with a mobile phase of water:acetonitrile:glacial acetic acid (49.5:49.5:1.0). Linearity was established between the range of 1 and 10 ng/ml. Under the optimized conditions, the recoveries were higher than 83.0% for all fortification levels. The intra-day precision oscillated between 8.0 and 5.0% at levels of 0.25 and 0.5 microg/l, while the inter-day precision was in the range of 10.7-16.0%. The limit of quantification of the method was 0.05 microg/l. The method is appropriate for quantitative determination of OTA in human serum and has been successfully applied to the analysis of OTA in haemodialysis patients from two principal cities of Portugal, in order to evaluate its exposure degree. Levels of OTA in Coimbra were higher than in Aveiro, 0.50 microg/l versus 0.49 microg/l. In respect to gender, levels of OTA were higher in males from Aveiro than in females, 0.52 microg/l versus 0.44 microg/l, and in Coimbra were similar, 0.50 microg/l versus 0.51 microg/l. However, in none of the cases, significant statistical differences were found.