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.

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.

Carmines (E120) in coloured yoghurts: a case-study contribution for human risk assessment.

Carmines (E120) are used worldwide as natural food colouring agents of animal origin, with a widespread application, including yoghurts. Despite being considered safe for human health, carmines are known to cause allergic reactions. Our goal was to evaluate the presence of carmines in different yoghurts with a label declaration of E120, purchased in Portugal, and, for the first time, to assess the human risk. The analytical methodology, recommended by JECFA, was based on acid hydrolysis of the samples followed by spectrophotometric UV-Vis analysis at 494 nm. This methodology allowed for a limit of quantification (LOQ) of 39.0 mg/kg and recovery rates higher than 97.7%. All the samples had carmines at levels above the LOQ, ranging between 43.8 and 193.8 mg/kg, with an average of 125.2 ± 34.5 mg/kg. In total, 8 (26.7%) samples exceeded the European Union (EU) maximum permitted level (MPL) established for carmines in this foodstuff, 150 mg/kg. Solid yoghurts presented higher average levels, 137.2 mg/kg, when compared to liquid samples, 107.2 mg/kg, with a significant statistical difference (p= 0.0236) being observed. No significant statistical difference was observed between white and private labels, whose average levels were very similar, 125.4 vs 125 mg/kg, respectively. Although some samples were above the allowable values, the estimated daily intake (EDI), designed for the different scenarios of different yoghurt types, did not exceed the established ADI, 5 mg/kg bw/day. According to the obtained results, carmine ingestion through the consumption of yoghurt poses low risk to the Portuguese consumers. However, children were the most vulnerable population group with a calculated risk value of up to 10% considering the mean content scenario. These first findings point out the need to reinforce surveillance programmes and monitoring studies, contributing to an increased awareness regarding carmine exposure, however it must be emphasised that yoghurt has evident nutritional benefits depending on a healthful consumer choice.

Fumonisins determination in urine by LC-MS-MS.

The present work shows the optimization and validation results of an analytical methodology based on immunoaffinity clean-up (IAC) followed by liquid chromatography with tandem mass detection (LC-MS-MS) for the analysis of the mycotoxins fumonisin B(1) (FB(1)) and B(2) (FB(2)) in human urine samples, in order to evaluate fumonisins exposure in two Portuguese populations. As far as we know, immunoaffinity clean-up procedure was used, for the first time, in the analysis of fumonisins (FBs) in urine. Using this analytical methodology, the limit of quantification achieved was 10 ng mL(-1) for FB(1) and for FB(2). Recoveries were higher than 73.4% for fortification levels between 10 and 100 ng mL(-1) and intra-day and inter-day repeatability were lower than 8.6%. The natural occurrence of FB(1) and FB(2) in 68 human urine samples obtained from the central zone of Portugal was studied. None of the studied samples presented detectable levels of FB(1) and FB(2).

Citrinin in Foods and Supplements: A Review of Occurrence and Analytical Methodologies.

Citrinin (CIT) deserves attention due to its known toxic effects in mammalian species and its widespread occurrence in food commodities, often along with ochratoxin A, another nephrotoxic mycotoxin. Human exposure, a key element in assessing risk related to food contaminants, depends upon mycotoxin contamination levels in food and on food consumption. Commercial supplements, commonly designated as red rice, usually used in daily diets in Asiatic countries due to their medicinal properties, may pose a health problem as a result of high CIT levels. In addition to the worldwide occurrence of CIT in foods and supplements, a wide range of several analytical and detection techniques with high sensitivity, used for evaluation of CIT, are reviewed and discussed in this manuscript. This review addresses the scientific literature regarding the presence of CIT in foods of either vegetable or animal origin, as well as in supplements. On what concerns analytical methodologies, sample extraction methods, such as shaking extraction and ultrasonic assisted extraction (UAE), clean-up methods, such as liquid-liquid extraction (LLE), solid phase extraction (SPE) and Quick, Easy, Cheap, Effective, Rugged and Safe (QuECHERS), and detection and quantification methods, such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC), capillary electrophoresis (CE), biosensors, and ELISA, are also reviewed.

Ochratoxin A in Beers Marketed in Portugal: Occurrence and Human Risk Assessment.

Ochratoxin A (OTA) is produced by fungi present in several agricultural products with much relevance to food safety. Since this mycotoxin is widely found in cereals, beer has a potential contamination risk. Therefore, it was deemed essential to quantify, for the first time, the levels of OTA in beer, a cereal-based product that is marketed in Portugal, as well as to calculate the human estimated weekly intake (EWI) and risk assessment. A total of 85 samples were analyzed through immunoaffinity clean-up, followed by liquid chromatography-fluorescence detection (LC-FD). This analytical methodology allowed a limit of quantification (LOQ) of 0.43 µg/L. The results showed that 10.6% were contaminated at levels ranging between

Ochratoxin A and Portuguese children: Urine biomonitoring, intake estimation and risk assessment.

Ochratoxin A (OTA) is a mycotoxin detected worldwide. Urine biomonitoring is a more realistic and non-invasive way to assess exposure when compared to traditional methods based on food occurrence and consumption data. Few studies have investigated children's exposure to OTA, although it is a more susceptible population. Our main goal was the OTA biomonitoring in urine of Portuguese children to better characterize the health risk of this population. The validated analytical methodology was based on an immunoaffinity clean-up, followed by LC-FD. First morning samples were collected, between 2018 and 2019, from 85 healthy children, 41 boys and 44 girls, aging between 2 and 13 years old. Overall, from the 85 analysed samples 79 (92.94%) were found positive. The average OTA concentration level was of 0.020 ng/mL (29.41 ng/g of creatinine), with a maximum value of 0.052 ng/mL (114.45 ng/g of creatinine). No statistical differences were found between the OTA contamination levels, the anthropometric data and dietary habits considered. Depending on the excretion rate considered for the PDI calculation, the risk assessed for the average OTA contamination varied from 10% to 194%, indicating a potential concern. This is the first study regarding OTA exposure in Portuguese children, showing their clear exposure.

Artificial sweeteners in non-alcoholic beverages: Occurrence and exposure estimation of the Portuguese population.

Artificial sweeteners such as acesulfame-potassium (ACE-K). saccharin (SAC), and aspartame (ASP), play a vital role in the food industry. Despite apparent benefits, several studies report adverse reactions on consumers. EFSA is currently re-evaluating several individual sweeteners, including ASP, Ace-K and SAC that shall be completed by 31 December 2020. Our goal was to evaluate, by liquid chromatography with ultraviolet detection (LC-UV), their levels in non-alcoholic beverages from the Portuguese market (soft drinks, sport drinks, energy drinks, and nectars), and estimate the exposure of the Portuguese adolescent/adult population. ACE-K was found in 85.7% of the samples, in mean levels of 97.4 mg/L. ASP and SAC, detected in 37.5% and 33.9% of the samples, were found in averages of 161.5 and 55.0 mg/L, respectively. The maximum limits set for ASP were exceeded in one energy drink. SAC exceeded the maximum permitted level (MPL) level in 3 soft drinks based on tea extracts. In 4 samples SAC was found but not mentioned in the label, and in one of these samples exceeded the MPL. The estimated daily intake (EDI), calculated in mg per kilogram of body weight per day, of ACE-K, SAC and ASP was found to be below the acceptable daily intake (ADI), both for Portuguese adolescents and adult population. Traditional soft drinks accounted for the highest EDIs representing 1.8%, 2.3% and 0.6% for ACE-K, SAC and ASP, respectively, of the ADI. Considering the previous report in Portugal it is observed that the population is currently exposed to a greater amount of artificial sweeteners, which may indicate a trend for more artificial sweeteners' usage in order to reduce sugar intake.

Ochratoxin A in the Portuguese Wine Market, Occurrence and Risk Assessment.

Ochratoxin A (OTA) is mainly found in cereals and cereal-based foodstuffs, but also in wine. Being one of the most consumed alcoholic drinks in Portugal and one of the main sources of human exposure to OTA, wine monitoring and exposure studies are essential. The analytical methodology consisted of the direct injection of the filtered samples into the liquid chromatograph, equipped with fluorescent detection (LC-FLD). Linearity was adequate, both in mobile phase and in matrix-matched solutions, with R2 values higher than 0.997. The limits of detection were 0.08 and 0.39 µg/L for white and red wine, respectively and recoveries were above 91.9%. One hundred wine samples acquired on the Portuguese market were investigated. In 5 samples the OTA was detected, with the red wine presenting higher frequency of contamination. Regarding the risk to human health it was observed that the estimated weekly intake (EWI) is considerably lower than the established tolerable weekly intake (TWI).

The mycoestrogen zearalenone in Portuguese flowing waters and its potential environmental impact.

This study provides the first environmental risk assessment (ERA) for zearalenone (ZEN), the only known mycoestrogen, based on a broad-scale investigation on its occurrence in rivers and creeks from Portugal. Water sample filtration and immunoaffinity columns (IAC) clean-up followed by liquid chromatograph with tandem mass spectrometry (LC/MS n ) provided an analytical method with good analytical performance. ZEN levels were determined for seven Portuguese rivers and one creek, during two different seasons, in a total of 38 samples collected upstream wastewater treatment plants (WWTPs). Overall, 23.7% were contaminated with ZEN at levels ranging between 5.6 and 82.6 ng/L. The highest concentration was observed during spring, although no statistically significant differences were observed between spring and autumn sampling campaigns. The potential ecotoxicological risk from ZEN to different trophic levels of aquatic organisms was evaluated by means of risk quotients (RQs) calculation. Although all the RQs obtained were lower than 1, our results confirm that ZEN is a relatively frequent contaminant in flowing waters in Portugal and might contribute to the overall estrogenic activity in the environment.

Determination of tetracycline antibiotic residues in edible swine tissues by liquid chromatography with spectrofluorometric detection and confirmation by mass spectrometry.

A sensitive and specific method is described for the simultaneous determination of oxytetracycline, tetracycline (TC), and chlortetracycline residues in edible swine tissues, by combining liquid chromatography with spectrofluorometric and mass spectrometry detection. The procedure involved a preliminary extraction with EDTA-McIlvaine buffer acidified at pH 4.0, followed by solid-phase extraction cleanup using a polymeric sorbent. The liquid chromatography analysis was performed with spectrofluorometric detection after postcolumn derivatization with magnesium ions. The limits of quantification were 50 microg/kg for muscle and 100 microg/kg for kidney tissues. The recovery values were greater than 77.8% for muscle and 65.1% for kidney. The method has been successfully used for the quantification of tetracyclines in swine tissues samples. The selective liquid chromatography mass spectrometric analysis for confirmation of oxytetracycline in one positive swine muscle sample was made by atmospheric pressure chemical ionization (APCI). The APCI mass spectra of the TCs gave the protonated molecular ion and two typical fragment ions, required for their confirmation in single ion monitoring scan mode in animal tissues.

A critical evaluation of different parameters for estimating pharmaceutical exposure seeking an improved environmental risk assessment.

A critical evaluation of the European Medicines Agency (EMA) Guideline on Environmental Risk Assessment (ERA) was performed on 16 of Portugal's most consumed pharmaceuticals in wastewater effluents (WWEs), the main route for aquatic contamination. The predicted environmental concentrations (PECs) were formulated based on the Guideline, after incorporating several refinements. The best approach was selected by comparing the measured environmental concentrations (MECs) to the PECs in WWEs. Finally, risk was assessed by comparing PECs to predicted no-effect concentrations (PNECs). The results showed that the default value of the penetration factor (Fpen) used by the EMA (0.01) was surpassed and that national consumption and excretion data were the two most important parameters for PEC calculations. The risk quotient between PECs and PNECs was higher than 1 for 12 pharmaceuticals, indicating a risk to all three trophic levels of aquatic organisms (algae, daphnids and fish). To improve the current ERA framework, suggestions were made for incorporating consumption and excretion data, changing the default value of Fpen to 0.04 and adding a safety factor of 10. Moreover, this evaluation should be performed for pharmaceuticals already on the market, and future ERAs should incorporate a risk-benefit analysis, an important risk-management step.

SSRIs antidepressants in marine mussels from Atlantic coastal areas and human risk assessment.

Selective serotonin reuptake inhibitors (SSRIs) are among the most prescribed antidepressants nation and worldwide. Their presence in the aquatic environment is known, causing growing pressures in heavily populated coastal areas. For instance, disruption of invertebrate endocrine systems has been described. Since discrete seawater sampling may not be suitable to evaluate such environmental risk, we aimed to assess SSRIs and metabolites in wild Mytilus galloprovincialis, and assess risk to human health. A total of 1225 mussels were sampled during one year along the Portuguese Atlantic coast. Each freeze-dried pool of 25 specimens was analyzed, in duplicate, by solid phase extraction (SPE) and liquid chromatography with tandem mass spectrometry (LC-MSn). Approximately 70% of the samples were contaminated with at least one, and up to 4 from the 7 targeted compounds. The cumulative SSRIs and metabolites content in mussels ranged between 3.17 and 33.93ngg-1 dry weight, in a mean level of 14.68±9.69ngg-1. Norfluoxetine was the most recurring SSRI and the one with the highest mean concentration, 13.52ngg-1. Although some differences were found between different sampling periods and locations, those were not statistically significant (p>0.05) and no correlation was found between wet weight and mean contamination levels. Regarding risk to human health following consumption of these filter-feeders, when considering the average sample contamination of positive samples and the average Portuguese and European consumption, the ratio between the estimated daily intake (EDI) and the acceptable daily intake (ADI) ranged from 0.45 to 5.50%, and from 1.60 to 19.41%, respectively. Therefore, although the resultant risk is lower for the Portuguese consumer than for the average EU consumer, apparently, no appreciable risk to human health is expected.

Human pharmaceuticals in Portuguese rivers: The impact of water scarcity in the environmental risk.

Pharmaceuticals occurrence and environmental risk assessment were assessed in Portuguese surface waters, evaluating the impact of wastewater treatment plants (WWTPs) and river flow rates. Twenty three pharmaceuticals from 6 therapeutic groups, including metabolites and 1 transformation product, were analysed in 72 samples collected from 20 different sites, upstream and downstream the selected WWTPs, in two different seasons. Analysis was performed by solid phase extraction followed by liquid chromatography coupled to tandem mass spectroscopy. Pharmaceuticals were detected in 27.8% of the samples. Selective serotonin reuptake inhibitors (SSRIs), anti-inflammatories and antibiotics presented the highest detection frequencies (27.8, 23.6 and 23.6%, respectively) and average concentrations (37.9, 36.1 and 33.5ngL-1, respectively). When assessing the impact of WWTPs, an increase of 21.4% in the average concentrations was observed in the samples located downstream these facilities, when compared with the upstream samples. Increased detection frequencies and concentrations were observed at lower flow rates, both when comparing summer and winter campaigns and by evaluating the different rivers. Risk quotients (RQs) higher than one were found for two pharmaceuticals, concerning two trophic levels. However, since Iberian rivers are highly influenced by water scarcity, in drought periods, the flow rates in these rivers can decrease at least ten times from the lowest value observed in the sampling campaigns. In these conditions, RQs higher than 1 would be observed for 5 pharmaceuticals, additionally, all the detected pharmaceuticals (11) would present RQs higher than 0.1. These results emphasize that the river flow rate represents an important parameter influencing pharmaceuticals concentrations, highlighting the ecotoxicological pressure, especially due to water scarcity in drought periods. This should be a priority issue in the environmental policies for minimizing its impact in the aquatic environment.

Assessing environmental risk of pharmaceuticals in Portugal: An approach for the selection of the Portuguese monitoring stations in line with Directive 2013/39/EU.

In line with the Directive 2013/39/EU the most representative surface waters, regarding pharmaceuticals contamination, were selected based on a Portuguese nationwide monitoring exercise. To meet this purpose, and given that wastewater treatment plants (WWTPs) are regarded as the major point sources of pharmaceuticals environmental contamination, the occurrence, fate and environmental risk assessment (ERA) of eleven of the most consumed pharmaceuticals, belonging to several therapeutic classes were assessed in 15 WWTPs (influents (WWIs) and effluents (WWEs)), from five different regions during one year (4 sampling campaigns). Results showed that all samples were contaminated with at least 1, and up to 8 from the 11 targeted pharmaceuticals. The highest concentrations observed were 150 and 33 µg L(-1) for WWI and WWE, respectively. Regarding temporal and spacial influence, winter, Alentejo, Algarve and Center regions presented higher mass loads. The ERA posed by 7 of the selected pharmaceuticals presented a risk quotient higher than 1 to the three trophic levels. Our findings highlighted that the rivers Mondego, Tagus, Ave, Trancão, Fervença and Xarrama should be selected as surface water monitoring stations. This study gives a good overview on pharmaceuticals contamination in WWTPs and its impact on surface waters in Portugal. Thus, a more integrative approach to rank and prioritize pharmaceuticals, based on an integrated assessment of ERA and exposure of surface water, was provided to support the future selection of the 6 most representative monitoring stations in Portugal, as required by the above mentioned directive.

Uptake, accumulation and metabolization of the antidepressant fluoxetine by Mytilus galloprovincialis.

Fluoxetine, a selective serotonin re-uptake inhibitor (SSRI) antidepressant, is among the most prescribed pharmaceutical active substances worldwide. This study aimed to assess its accumulation and metabolization in the mussel Mytillus galloprovincialis, considered an excellent sentinel species for traditional and emerging pollutants. Mussels were collected from Ria Formosa Lagoon, Portugal, and exposed to a nominal concentration of fluoxetine (75 ng L(-1)) for 15 days. Approximately 1 g of whole mussel soft tissues was extracted with acetonitrile:formic acid, loaded into an Oasis MCX cartridge, and fluoxetine analysed by liquid chromatography with tandem mass spectrometry (LC-MSn). After 3 days of exposure, fluoxetine was accumulated in 70% of the samples, with a mean of 2.53 ng g(-1) dry weight (d.w.) and norfluoxetine was only detected in one sample (10%), at 3.06 ng g(-1) d.w. After 7 days of exposure, the accumulation of fluoxetine and norfluoxetine increased up to 80 and 50% respectively, and their mean accumulated levels in mussel tissues were up to 4.43 and 2.85 ng g(-1) d.w., respectively. By the end of the exposure period (15 days), both compounds were detected in 100% of the samples (mean of 9.31 and 11.65 ng g(-1) d.w., respectively). Statistical analysis revealed significant accumulation differences between the 3rd and 15th day of exposure for fluoxetine, and between the 3rd and 7th against the 15th day of exposure for norfluoxetine. These results suggest that the fluoxetine accumulated in mussel tissues is likely to be metabolised into norfluoxetine with the increase of the time of exposure, giving evidence that at these realistic environmental concentrations, toxic effects of fluoxetine in mussel tissues may occur.

Reviewing the serotonin reuptake inhibitors (SSRIs) footprint in the aquatic biota: uptake, bioaccumulation and ecotoxicology.

Selective serotonin re-uptake inhibitors (SSRIs) antidepressants are amongst the most prescribed pharmaceutical active substances throughout the world. Their presence, already described in different environmental compartments such as wastewaters, surface, ground and drinking waters, and sediments, and their remarkable effects on non-target organisms justify the growing concern about these emerging environmental pollutants. A comprehensive review of the literature data with focus on their footprint in the aquatic biota, namely their uptake, bioaccumulation and both acute and chronic ecotoxicology is presented. Long-term multigenerational exposure studies, at environmental relevant concentrations and in mixtures of related compounds, such as oestrogenic endocrine disruptors, continue to be sparse and are imperative to better know their environmental impact.

Determination of tetracycline antibiotics in salmon muscle by liquid chromatography using post-column derivatization with fluorescence detection.

A simple and accurate cleanup procedure using polymeric sorbent was developed for the determination of oxytetracycline (OTC) and tetracycline (TC) residues in salmon muscle. It was applied to the analysis of 20 salmon samples during a month period. The OTC and TC residues were extracted with ethylenediaminetetracetic acid (EDTA)-McIlvaine buffer acidified at pH 4.0 and cleaned up by solid-phase extraction with a polymeric sorbent. The advantages of the polymeric sorbent over the silica-based sorbent in the cleanup of salmon muscle samples are described. A liquid chromatographic method with post-column derivatization and fluorescence detection is proposed because of its sensitivity and specificity. The average recoveries of OTC and TC from muscle salmon tissue fortified at 50, 100, and 200 microg/kg levels, ranged from 83.9 to 93.4% with a coefficient of variation between 4.09 and 5.80%. The limit of quantitation for OTC and TC in salmon muscle was 50 microg/kg.

SPE-LC-FD determination of polycyclic aromatic hydrocarbon monohydroxy derivatives in cephalopods.

A new analytical methodology, based on liquid chromatography with fluorescence detection (LC-FD), after extraction, enzymatic hydrolysis, and solid-phase extraction (SPE) through Oasis HLB cartridges, was developed and validated for the simultaneous determination of three monohydroxy derivatives of polycyclic aromatic hydrocarbons (PAHs). The optimized analytical method is sensitive, accurate, and precise, with recoveries between 62 and 110% and limits of detection of 227, 9, and 45 ng/g for 1-hydroxynaphthalene, 2-hydroxyfluorene, and 1-hydroxypyrene, respectively. Their levels were estimated in different cephalopod matrices (edible tissues and hemolymph). The methodology was applied to samples of the major cephalopod species consumed worldwide. Of the 18 samples analyzed, 39% were contaminated with 1-hydroxynaphthalene, which was the only PAH metabolite detected. Its concentration ranged from 786 to 1145 ng/g. This highly sensitive and specific method allows the identification and quantitation of PAH metabolites in forthcoming food safety and environmental monitoring programs.