Urine biomonitoring of glyphosate in children: Exposure and risk assessment.

The use of glyphosate has been increasing over the years, making it one of the most consumed herbicides in the world. Although children are considered a vulnerable population, only four previous published studies determined glyphosate in the urine of non-occupationally exposed children. The paucity of epidemiological data and biomonitoring surveys are considered major gaps, that hinder the implementation of science driven policies in the protection of public health. The aim of the present study was to determine glyphosate in the urine of 41 Portuguese children (2-13 years old) and identify potential determinants of exposure. Glyphosate was detected in 95.1% of the samples (1.77 ± 0.86 µg/L), up to a maximum value of 4.35 µg/L. Glyphosate concentrations were higher in the urine of children aged 7-9 years, living near agricultural areas (<1 km), with a higher percentage of consumption of home-produced foods, and whose parents applied herbicides in the backyard. Risk assessment revealed an exposure representing 1-5.58% of the established Acceptable Daily Intake (ADI) of glyphosate (0.5 mg/kg bw/day). The results should be further analyzed considering the age of the participants, for which no adjusted ADI exists. This was the first published report of glyphosate exposure in the urine of Portuguese children.

Development and Validation of QuEChERS Followed by UHPLC-ToF-MS Method for Determination of Multi-Mycotoxins in Pistachio Nuts.

Pistachios are one of the types of tree nut fruits with the highest mycotoxin contamination, especially of aflatoxins, worldwide. This study developed a Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method that was followed by Ultra-High Performance Liquid Chromatography combined with Time-of-Flight Mass Spectrometry (UHPLC-ToF-MS) for the determination of mycotoxins in pistachios. Different approaches to dispersive solid phase extraction as a clean-up method for high lipid matrices were evaluated. For this, classic sorbents such as C18 (octadecyl-modified silica) and PSA (primary secondary amine), and new classes of sorbents, namely EMR-Lipid (enhanced matrix removal-lipid) and Z-Sep (modified silica gel with zirconium oxide), were used. The QuEChERS method, followed by Z-Sep d-SPE clean-up, provided the best analytical performance for aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), zearalenone (ZEA), toxin T2 (T2) and toxin HT-2 (HT2) in pistachios. The method was validated in terms of linearity, sensitivity, repeatability, interday precision and recovery; it achieved good results according to criteria imposed by Commission Regulation (EC) no. 401/2006. The method was applied to real samples and the results show that pistachios that are available in Portuguese markets are safe from mycotoxins that are of concern to human health.

Human Biomonitoring of Selected Hazardous Compounds in Portugal: Part II-Lessons Learned on Mycotoxins.

Human biomonitoring (HBM) data provide information on total exposure regardless of the route and sources of exposure. HBM studies have been applied to quantify human exposure to contaminants and environmental/occupational pollutants by means of determining the parent compounds, their metabolites, or even their reaction products in biological matrices. HBM studies performed among the Portuguese population are dispersed and limited. Thus, to overcome this knowledge gap, this work reviews the published Portuguese HBM information concerning mycotoxins detected in the urine, serum, milk, hair, and nails of different groups of the Portuguese population. This integrative approach to the available HBM data allows us to analyze the main determinants and patterns of exposure of the Portuguese population to the selected hazardous compounds, as well as to assess the potential health risks. We also aimed to identify the main difficulties and challenges of HBM through the analysis of the enrolled studies. Ultimately, this study aims to support national and European policies in promoting human health by summarizing the most important outcomes and lessons learned through the HBM studies carried out in Portugal.

Human Biomonitoring of Selected Hazardous Compounds in Portugal: Part I-Lessons Learned on Polycyclic Aromatic Hydrocarbons, Metals, Metalloids, and Pesticides.

Human biomonitoring (HBM) data provide information on total exposure regardless of the route and sources of exposure. HBM studies have been applied to quantify human exposure to contaminants and environmental/occupational pollutants by determining the parent compounds, their metabolites or even their reaction products in biological matrices. HBM studies performed among the Portuguese population are disperse and limited. To overcome this knowledge gap, this review gathers, for the first time, the published Portuguese HBM information concerning polycyclic aromatic hydrocarbons (PAHs), metals, metalloids, and pesticides concentrations detected in the urine, serum, milk, hair, and nails of different groups of the Portuguese population. This integrative insight of available HBM data allows the analysis of the main determinants and patterns of exposure of the Portuguese population to these selected hazardous compounds, as well as assessment of the potential health risks. Identification of the main difficulties and challenges of HBM through analysis of the enrolled studies was also an aim. Ultimately, this study aimed to support national and European policies promoting human health and summarizes the most important outcomes and lessons learned through the HBM studies carried out in Portugal.

Selected Pharmaceuticals in Different Aquatic Compartments: Part II-Toxicity and Environmental Risk Assessment.

Potential risks associated with releases of human pharmaceuticals into the environment have become an increasingly important issue in environmental health. This concern has been driven by the widespread detection of pharmaceuticals in all aquatic compartments. Therefore, 22 pharmaceuticals, 6 metabolites and transformation products, belonging to 7 therapeutic groups, were selected to perform a review on their toxicity and environmental risk assessment (ERA) in different aquatic compartments, important issues to tackle the water framework directive (WFD). The toxicity data collected reported, with the exception of anxiolytics, at least one toxicity value for concentrations below 1 µg L-1. The results obtained for the ERA revealed risk quotients (RQs) higher than 1 in all the aquatic bodies and for the three trophic levels, algae, invertebrates and fish, posing ecotoxicological pressure in all of these compartments. The therapeutic groups with higher RQs were hormones, antiepileptics, anti-inflammatories and antibiotics. Unsurprisingly, RQs values were highest in wastewaters, however, less contaminated water bodies such as groundwaters still presented maximum values up to 91,150 regarding 17α-ethinylestradiol in fish. Overall, these results present an important input for setting prioritizing measures and sustainable strategies, minimizing their impact in the aquatic environment.

Selected Pharmaceuticals in Different Aquatic Compartments: Part I-Source, Fate and Occurrence.

Potential risks associated with releases of human pharmaceuticals into the environment have become an increasingly important issue in environmental health. This concern has been driven by the widespread detection of pharmaceuticals in all aquatic compartments. Therefore, 22 pharmaceuticals, 6 metabolites and transformation products, belonging to 7 therapeutic groups, were selected to perform a systematic review on their source, fate and occurrence in different aquatic compartments, important issues to tackle the Water Framework Directive (WFD). The results obtained evidence that concentrations of pharmaceuticals are present, in decreasing order, in wastewater influents (WWIs), wastewater effluents (WWEs) and surface waters, with values up to 14 mg L-1 for ibuprofen in WWIs. The therapeutic groups which presented higher detection frequencies and concentrations were anti-inflammatories, antiepileptics, antibiotics and lipid regulators. These results present a broad and specialized background, enabling a complete overview on the occurrence of pharmaceuticals in the aquatic compartments.

Reviewing the Analytical Methodologies to Determine the Occurrence of Citrinin and its Major Metabolite, Dihydrocitrinone, in Human Biological Fluids.

Until now, the available data regarding citrinin (CIT) levels in food and the consumption of contaminated foods are insufficient to allow a reliable estimate of intake. Therefore, biomonitoring configuring analysis of parent compound and/or metabolites in biological fluids, such as urine or blood, is being increasingly applied in the assessment of human exposure to CIT and its metabolite, dihydrocitrinone (DH-CIT). Most studies report urinary levels lower for the parent compound when compared with DH-CIT. A high variability either in the mean levels or in the inter-individual ratios of CIT/DH-CIT between the reported studies has been found. Levels of DH-CIT in urine were reported as being comprised between three to seventeen times higher than the parent mycotoxin. In order to comply with this objective, sensitive analytical methodologies for determining biomarkers of exposure are required. Recent development of powerful analytical techniques, namely liquid chromatography coupled to mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography (UHPLC-MS/MS) have facilitated biomonitoring studies, mainly in urine samples. In the present work, evidence on human exposure to CIT through its occurrence and its metabolite, in biological fluids, urine and blood/plasma, in different countries, is reviewed. The analytical methodologies usually employed to evaluate trace quantities of these two molecules, are also presented. In this sense, relevant data on sampling (size and pre-treatment), extraction, cleanup and detection and quantification techniques and respective chromatographic conditions, as well as the analytical performance, are evidenced.

Environmental impact of pharmaceuticals from Portuguese wastewaters: geographical and seasonal occurrence, removal and risk assessment.

The occurrence, fate, geographical and seasonal influence and environmental risk assessment of eleven of the most consumed pharmaceuticals in Portugal were studied in wastewater treatment plants (WWTPs) influents and (WWI) and effluents (WWE). WWI and WWE samples, from two sampling campaigns (spring and summer), in 2013, were evaluated in 15 different WWTPs across the country, by solid phase extraction (SPE) and liquid chromatography coupled with tandem mass detection (LC-MS-MS). Lipid regulators were the most frequently found in WWI and WWE (184.1 and 22.3mg/day/1000 inhab., respectively), followed by anti-inflammatories (1339.4 and 15.0mg/day/1000 inhab., respectively), and antibiotics (330.7 and 68.6 mg/day/1000 inhab., respectively). Anxiolytics were the least detected with 3.3 and 3.4 mg/day/1000 inhab. in WWI and WWE, respectively. The mass loads, both in WWI and WWE, were higher in summer than those found during the spring season, being remarkable the high values registered in a region where population triplicates in this time of the year. The mean removal efficiency achieved was of 94.5%, nonetheless, between the different therapeutic groups, as well as within each group, important variations in removal were observed, going from not eliminated to 100%. In the summer higher efficiencies were observed regarding lipid regulators and antibiotics. Furthermore, an important outcome was the evaluation, by means of risk quotients (RQs), of the potential ecotoxicological risk posed by the selected pharmaceuticals to different aquatic organisms, exposed to the effluents studied. Ciprofloxacin, bezafibrate, gemfibrozil, simvastatin and diclofenac showed RQs higher than one, being expected that these pharmaceuticals might pose a threat to the three trophic levels (algae, daphnids and fish) evaluated. These results highlight the importance of these monitoring studies, as required by the Directive 2013/39/EU, in order to minimize their aquatic environmental contamination and support future prioritization measures.

Fluoroquinolones and Tetracycline Antibiotics in a Portuguese Aquaculture System and Aquatic Surroundings: Occurrence and Environmental Impact.

The growth of aquaculture over the past few years is widely recognized as one of the main sources of antibiotics, mainly fluoroquinolones (FQ) and tetracyclines (TC), in the aquatic environment, consequently, increasing the risk of the emergence of antibiotic bacterial resistance and promoting the spread of resistant genes. This study aimed to (1) develop and validate a multiresidue method for determination and quantification of ciprofloxacin (CIP), difloxacin (DIFL), enrofloxacin (ENR), norfloxacin (NOR), sarafloxacin (SARA), and oxytetracycline (OXY) in aquaculture waters and surrounding water bodies and (2) provide the first Portuguese data to utilize in assessment of risk of adverse effects. In addition, the potential environmental impact posed by these antibiotics to aquatic organisms, belonging to different trophic levels, when exposed to the studied aquaculture waters was also assessed. The analytical strategy comprised of solid-phase extraction (SPE) through Oasis HLB cartridges, and detection and quantification by liquid chromatography with tandem mass spectrometry (LC/MS(n)). Method detection limits (MDL) and method quantification limits (MQL) were in the range of 0.7-3 ng/L and 2.4-10 ng/L, respectively. Recoveries varied between 57.4 and 122.8%. The method was applied to 31 water samples collected from an aquaculture and surrounding water bodies located in north of Portugal. Residues of all antibiotics, except SARA and DIFL, were detected at concentrations ranging from 3 to 75.1 ng/L. Norfloxacin was the antibiotic present at highest frequency and concentration. Regarding the environmental impact assessment (EIA), a risk quotient higher than 1 was observed for NOR.

Virulence and plasmidic resistance determinants of Escherichia coli isolated from municipal and hospital wastewater treatment plants.

Escherichia coli is simultaneously an indicator of water contamination and a human pathogen. This study aimed to characterize the virulence and resistance of E. coli from municipal and hospital wastewater treatment plants (WWTPs) in central Portugal. From a total of 193 isolates showing reduced susceptibility to cefotaxime and/or nalidixic acid, 20 E. coli with genetically distinct fingerprint profiles were selected and characterized. Resistance to antimicrobials was determined using the disc diffusion method. Extended spectrum ß-lactamase and plasmid-mediated quinolone resistance genes, phylogroups, pathogenicity islands (PAIs) and virulence genes were screened by polymerase chain reaction (PCR). CTX-M producers were typed by multilocus sequence typing. Resistance to beta-lactams was associated with the presence of bla(TEM), bla(SHV), bla(CTX-M-15) and bla(CTX-M-32). Plasmid-mediated quinolone resistance was associated with qnrA, qnrS and aac(6')-Ib-cr. Aminoglycoside resistance and multidrug-resistant phenotypes were also detected. PAI IV(536), PAI II(CFT073), PAI II(536) and PAI I(CFT073), and uropathogenic genes iutA, papAH and sfa/foc were detected. With regard to the clinical ST131 clone, it carried bla(CTX-M-15), blaTEM-type, qnrS and aac(6')-lb-cr; IncF and IncP plasmids, and virulence factors PAI IV(536), PAI I(CFT073), PAI II(CFT073), iutA, sfa/foc and papAH were identified in the effluent of a hospital plant. WWTPs contribute to the dissemination of virulent and resistant bacteria in water ecosystems, constituting an environmental and public health risk.

Evaluation of storage and evaporation in the removal efficiency of D-norgestrel and progesterone in human urine.

Pharmaceuticals are emerging contaminants and it must be noted that approximately 70 % of them are excreted via urine. Therefore, urine usage implies the risk of transfer of pharmaceutical residues to agricultural fields and environment contamination. Thus, this study aimed on the development and validation of a LC-MS/MS method for D-norgestrel (D-NOR) and progesterone (PRO) determination in human urine, as well as the evaluation of the removal efficiency of two methods (storage and evaporation), and the effects of acidification with sulfuric acid. The storage process was evaluated for 6 weeks, while the evaporation was assessed at three different temperatures (50, 75, and 100 °C). All experiments were done with normal urine (pH = 6.0) and acidified urine (pH = 2.0, with sulfuric acid). The results of validation showed good method efficiency. In the second week of storage, higher hormone degradation was observed. In the evaporation method, both D-NOR and PRO were almost completely degraded when the volume was reduced to the lowermost level. Results also indicate that acidification did not affect degradation. Overall, the results showed that combination of two methods can be employed for more efficient hormone removal in urine.

Non-Steroidal Anti-Inflammatory Drugs in the Aquatic Environment and Bivalves: The State of the Art.

In recent years, contaminants of emerging concern have been reported in several environmental matrices due to advances in analytical methodologies. These anthropogenic micropollutants are detected at residual levels, representing an ecotoxicological threat to aquatic ecosystems. In particular, the pharmacotherapeutic group of non-steroidal anti-inflammatories (NSAIDs) is one of the most prescribed and used, as well as one of the most frequently detected in the aquatic environment. Bivalves have several benefits as a foodstuff, and also as an environment bioindicator species. Therefore, they are regarded as an ideal tool to assess this issue from both ecotoxicological and food safety perspectives. Thus, the control of these residues in bivalves is extremely important to safeguard environmental health, also ensuring food safety and public health. This paper aims to review NSAIDs in bivalves, observing their consumption, physicochemical characteristics, and mechanisms of action; their environmental occurrence in the aquatic environment and aquatic biota; and their effects on the ecosystem and the existent legal framework. A review of the analytical methodologies for the determination of NSAIDs in bivalves is also presented.

Development and validation of an analytical method based on QuEChERS followed by UHPLC-ToF-MS for the determination of tropane alkaloids in buckwheat (Fagopyrum esculentum L.) and buckwheat products.

A method was developed for the determination of tropane alkaloids (TAs), including atropine, scopolamine, anisodamine and homatropine in buckwheat and related products. This work presents an optimised methodology based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction procedure followed by ultra-high performance liquid chromatography combined with time-of-flight mass spectrometry for the determination of TAs (atropine, scopolamine, anisodamine and homatropine) in buckwheat samples. The analytical methodology was successfully validated, demonstrating good linearity, low limit of quantification, repeatability (RSDr < 15%), inter-day precision (RSDR < 19%) and recovery (74-113%). Finally, 13 commercial samples of buckwheat were analysed and the results demonstrated that they were in compliance with the current European regulations regarding TAs.

The potential of citrus by-products in the development of functional food and active packaging.

Food by-product valorization has become an important research area for promoting the sustainability of the food chain. Citrus fruits are among the most widely cultivated fruit crops worldwide. Citrus by-products, including pomace, seeds, and peels (flavedo and albedo), are produced in large amounts each year. Those by-products have an important economic value due to the high content on bioactive compounds, namely phenolic compounds and carotenoids, and are considered a valuable bio-resource for potential applications in the food industry. However, green extraction techniques are required to ensure their sustainability. This chapter addresses the main components of citrus by-products and their recent applications in food products and active food packaging, towards a circular economy. In addition, the concern regarding citrus by-products contamination (e.g. with pesticides residues and mycotoxins) is also discussed.

Analysis of Antibiotics in Bivalves by Ultra-High Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry.

The presence of pharmaceuticals in aquatic ecosystems mostly originates from wastewater treatment plants (WWTPs) and such a situation can be responsible for significant negative impacts on natural ecosystems, such as estuarine and coastal areas. Bioaccumulation of pharmaceuticals, namely antibiotics, in exposed organisms is known to have remarkable effects on different trophic levels of non-target organisms such as algae, invertebrates and vertebrates, including the emergence of bacterial resistance. Bivalves are a highly appreciated seafood product, as they are fed by filtering water, and can bioconcentrate chemicals, being ideal for biomonitoring environmental health hazards in coastal and estuarine ecosystems. To use this sentinel species, an analytical strategy was developed to be used in accessing antibiotics, from human and veterinary medicine, and evaluate their occurrence as emerging pollutants in aquatic environments. The optimized analytical method was fully validated according to the European requirements defined by the Commission Implementing Regulation 2021/808. The validation comprised the following parameters: specificity, selectivity, precision, recovery, ruggedness, linearity, and the decision limit CCα, as well as the limit of detection (LoD) and limit of quantification (LoQ). The method was validated for 43 antibiotics to allow their quantification in both contexts, environmental biomonitoring and food safety.

The Bright and Dark Sides of Herbal Infusions: Assessment of Antioxidant Capacity and Determination of Tropane Alkaloids.

Herbal infusions are highly popular beverages consumed daily due to their health benefits and antioxidant properties. However, the presence of plant toxins, such as tropane alkaloids, constitutes a recent health concern for herbal infusions. This work presents an optimized and validated methodology based on the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction procedure followed by Ultra-High Performance Liquid Chromatography combined with Time-of-Flight Mass Spectrometry (UHPLC-ToF-MS) for the determination of tropane alkaloids (atropine, scopolamine, anisodamine, and homatropine) in herbal infusions, in accordance with criteria established by Commission Recommendation EU No. 2015/976. One of the seventeen samples was contaminated with atropine, exceeding the current European regulation regarding tropane alkaloids. In addition, this study evaluated the antioxidant capacity of common herbal infusions available on Portuguese markets, indicating the high antioxidant capacity of yerba mate (Ilex paraguariensis), lemon balm (Melissa officinalis), and peppermint (Mentha x piperita).

Occurrence of zearalenone in dairy farms - A study on the determinants of exposure and risk assessment.

The Azorean dairy industry is based on a semi-intensive model. Due to the few storage facilities and the large climatic fluctuations that characterize the Azores, the complete feed mixture (total mixed ration - TMR) is susceptible to the proliferation of a variety of fungi and mycotoxin contamination. Thus, chronic ingestion of these xenobiotics may lead to increased susceptibility to disease, loss of reproductive performance and, in the case of dairy cattle, decreased productivity and quality of milk produced. Since it is impossible to eliminate completely the presence of mycotoxins, it is essential to ensure the implementation of strategies to reduce their concentration in products intended for food and feed, as well as to monitor and control the levels present in food. This study aimed to evaluate the occurrence zearalenone (ZEA) in feed (TMR) in four dairy cattle farms on the Azorean Island of São Miguel and relate it to the occurrence of this mycotoxin in the milk produced, by associating several production and health indicators translated by the monthly milk contrast. To this end, the monthly milk contrast data, the determination of ZEA in the ingested feed and the (individual) milk samples from the four farms included in the study were used. Eighty-three (98.8%) of the milk samples under study had detectable ZEA levels (1.56 ± 1.36 µg/L), higher than reported in previous similar studies. Although ZEA concentration was not significantly associated with any production indicator analyzed (days in milk, age at calving, milk yield, protein content, butyrate content, somatic cell concentration and urea), it was found that the production regime and type of feed management is a major factor in the exposure of animals to high mycotoxin contents. Further studies are recommended to ensure continued monitoring and reduction of the risk associated with exposure of animals and humans to ZEA.

Risk Assessment of Nine Coccidiostats in Commercial and Home-Raised Eggs.

The poultry industry, in order to prevent and control coccidiosis caused by Eimeria spp., widely uses coccidiostats as feed additives. The main objective of this study was to evaluate the presence of nine coccidiostats in 62 egg samples by UHPLC-MS/MS. Overall, detection frequency and average concentration were 90.3% (56/62) and 106.3 µg kg-1, respectively. Only diclazuril and nicarbazin were detected. Diclazuril, only found in home-raised eggs, showed an overall detection frequency of 8.1% (5/62), with average and maximum concentrations of 0.46 ± 1.90 µg kg-1 and 13.6 µg kg-1, respectively. Nicarbazin presented an overall higher frequency, 88.7% (55/62), with levels up to 744.8 µg kg-1. Additionally, four samples (6.5%) presented both nicarbazin and diclazuril. Home-raised egg samples (n = 28) showed a detection frequency of 89.3%, with nicarbazin found in more samples (85.7% vs. 17.9%) and at higher levels (266.3 ± 169.4 µg kg-1 vs. 0.91 ± 2.78 µg kg-1) when compared to diclazuril. In supermarket samples (n = 34), only nicarbazin was detected in 31 samples (91.1%), with an average of 167.6 ± 62.2 µg kg-1. Considering the average contamination scenario, consumers' health should not be adversely affected by egg consumption. In every scenario considered, children were the most vulnerable population group.

Mycotoxins in Rice Correlate with Other Contaminants? A Pilot Study of the Portuguese Scenario and Human Risk Assessment.

Rice is the second most important cereal crop and is vital for the diet of billions of people. However, its consumption can increase human exposure to chemical contaminants, namely mycotoxins and metalloids. Our goal was to evaluate the occurrence and human exposure of aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEN), and inorganic arsenic (InAs) in 36 rice samples produced and commercialized in Portugal and evaluate their correlation. The analysis of mycotoxins involved ELISA, with limits of detection (LODs) of 0.8, 1 and 1.75 µg kg-1 for OTA, AFB1, and ZEN, respectively. InAs analysis was carried out by inductively coupled plasma mass spectrometry (ICP-MS; LOD = 3.3 µg kg-1). No sample showed contamination by OTA. AFB1 was present in 2 (4.8%) samples (1.96 and 2.20 µg kg-1), doubling the European maximum permitted level (MPL). Concerning ZEN, 88.89% of the rice samples presented levels above the LOD up to 14.25 µg kg-1 (average of 2.75 µg kg-1). Regarding InAs, every sample presented concentration values above the LOD up to 100.0 µg kg-1 (average of 35.3 µg kg-1), although none surpassed the MPL (200 µg kg-1). No correlation was observed between mycotoxins and InAs contamination. As for human exposure, only AFB1 surpassed the provisional maximum tolerable daily intake. Children were recognized as the most susceptible group.

Comparison of three solid-phase extraction processes in quantification of ciprofloxacin and enrofloxacin in pork meat.

Due to strong implications for food safety, control of fluoroquinolones residues in swine meat should be undertaken to verify compliance of the contamination levels with the maximum residue limits recently updated by Commission Regulation (EU) No. 37/2010 of 22 December 2009. Solid-phase extraction is widely used in antibiotic analysis in food of animal origin. In this study, the results of a comparative study using different types of solid-phase extraction columns, HLB, MCX, and MAX, for ciprofloxacin and enrofloxacin analysis, in pork meat, are presented. In addition, diverse sample treatments for defatting, precipitate proteins, eliminate cations, and increase the ionic strength, were used to obtain the most suitable method of analysis. Only the MCX's use followed by liquid chromatography with fluorescence detection resulted in chromatograms that allow the quantification at maximum residue limits. The validation method, in terms of CCα and CCß, recovery and precision determination, was according to the EU Decision 2002/657/EC. This procedure was used in the analysis of 50 samples of pork meat of Portuguese origin. Only two samples presented residues of enrofloxacin at 30 and 42 µg/kg, values under the legal maximum residue limit.