Liquid Chromatography With Fluorescence Detection Research Articles

Mycotoxin Occurrence in Milk and Durum Wheat Samples from Tunisia Using Dispersive Liquid-Liquid Microextraction and Liquid Chromatography with Fluorescence Detection.

Food and feed contamination with mycotoxins is a major public health concern. Humans and animals are exposed to these toxins by consuming contaminated products throughout their lives. In this study, a method based on dispersive liquid-liquid microextraction (DLLME), followed by liquid chromatography with fluorescence detection (LC-FLD), was validated for the determination of aflatoxins (AFs) M1, B1, B2, G1, G2, zearalenone (ZEN), and ochratoxin A (OTA). The method was applied to 150 raw cow milk samples and 90 market durum wheat samples from two Tunisian climatic regions: the littoral region (Mahdia) and the continental region (Béja). This work was carried out to obtain more surveillance data to support rapid initiatives to assure safe foods and protect consumer health and to estimate the daily exposure of the Tunisian population consuming those products. AFG2 and OTA were found in wheat with incidences of 54.4 and 11.1%, respectively. On the other side, milk samples were contaminated by AFG2, AFB1, and AFB2 with incidences of 8.7%, 2.0%, and 0.67%, respectively. Some of the samples showed OTA concentrations above the maximum limit allowed by the European Union, which represents a health risk for consumers in Tunisia, where no legislation exists about the maximum content of mycotoxins in food.

Determination of synthetic cannabinoids in oral fluids by liquid chromatography with fluorescence detection after solid-phase extraction

Synthetic cannabinoids are one of the most consumed new psychoactive substances, being absolutely necessary the development of analytical methodologies for the determination of these substances in biological fluids. In this study, a liquid chromatography with fluorescence detection (LC-FD) method has been developed for the analysis of 8 synthetic cannabinoids in oral fluids. The method has been validated in terms of linearity, precision and extraction recoveries, giving limits of detection as low as 0.7 µg L−1, and limits of quantification of 2.6 µg L−1. Different silica and polymeric commercial solid sorbents such as C18, Supel-Select HLB, EB2 ExtrabondⓇ and SampliQ-OPT were tested, concluding that Supel-Select HLB provided quantitative recoveries for the extraction of synthetic cannabinoids in oral fluids.•Analysis of synthetic cannabinoids in oral fluids.•Analytical procedure based on liquid chromatography with fluorescence detection.•Sample treatment based on solid phase extraction with HLB cartridges.

Application of Microgel as a Sorbent for Bisphenol Analysis in Liquid Food Samples

Bisphenols are well-known endocrine disruptors that can easily migrate from plastic and can containers to food. Due to the complicated matrix and ultra-low concentrations of bisphenols in food, samples require extensive preparation before instrumental analysis. In this paper, an environmental sensitive microgel was employed as a sorbent for the preconcentration of four bisphenols, bisphenol A (BPA), bisphenol B (BPB), bisphenol E (BPE) and bisphenol F (BPF), from liquid food samples. Liquid chromatography with fluorescence detection (LC-FLD) was used for the quantification of bisphenols. By applying microgel solid-phase extraction procedure, the limits of detections achieved in liquid food samples can be lowered to 0.9 µg·L−1 for BPF and BPA, 2.3 µg·L−1 for BPE and 2.9 µg·L−1 for BPB. Only 5 mg of microgel was sufficient to achieve good recoveries (70.5–109%) with precision (RSD 0.21–5.01%, n = 3) for different analyzed liquid food samples spiked at concentration levels of 50 µg·L−1. In five out of twelve of the analyzed samples (pineapple, mandarin, peach, mushroom and pickles), they were contaminated with BPA, and the determined concentration was in the range of 6.2–22 µg·L−1; however, these results are below the specific migration limit (SML) set for BPA (50 µg·kg−1).

Simultaneous determination of N-methyl carbamate residues in pork tissues based on ultrasound assisted QuEChERS-dSPE extraction followed by reversed phase LC-FLD analysis

The presence of pesticide residues in animal tissues is a source of public awareness and health concerns. Therefore, to guarantee appropriate food safety and to enforce regulations, efficient, cost-effective and sensitive analytical methods are needed. This study set out to develop and validate a reliable and high-throughput analytical approach for the simultaneous determination of N-methyl carbamates (NMCs) in different parts of pork tissues (kidney, liver and muscle). An innovative sample preparation strategy based on ultrasound assisted QuEChERS extraction (UAE-QuEChERS), instead of manual agitation as original QuEChERS, was evaluated. The NMC insecticides were extracted with acidified (1% acetic acid, v/v) acetonitrile (ACN) and determined by reversed-phase high performance liquid chromatography with fluorescence detection (LC-FLD) after automatic post column derivatization and impurities removal through dispersive solid phase extraction (d-SPE). The separation of NMCs was carried out in a C8 column using H2O:ACN (90:10, v/v) and H2O:ACN (20:80, v/v) as mobile phases. The method showed a good linearity (r2 ≥ 0.991), with recoveries generally between 68.6 and 119%. Method limits of detection (LOD) ranging from 0.1 to 2 μg/kg, and method limits of quantification (LOQ) from 0.4 to 6 μg/kg, are satisfactory in terms of the maximum residue limits (MRLs) for NMCs. Suitable intra- and inter-day variations (RSD <12%) and low matrix effects were observed with sample-to-sample variation, for all assayed pork tissues. The utility of the method for implementation in regulatory and food laboratories was demonstrated by its application to the analysis of NMC insecticides in commercial pork tissues. In all cases, NMC residues were not present at concentrations above the LOQ determined for each type of pork tissue.

Efficient Aflatoxin B1 Sequestration by Yeast Cell Wall Extract and Hydrated Sodium Calcium Aluminosilicate Evaluated Using a Multimodal In-Vitro and Ex-Vivo Methodology.

In this work, adsorption of the carcinogenic mycotoxin aflatoxin B1 (AFB1) by two sequestrants—a yeast cell wall-based adsorbent (YCW) and a hydrated sodium calcium aluminosilicate (HSCAS)—was studied across four laboratory models: (1) an in vitro model from a reference method was employed to quantify the sorption capabilities of both sequestrants under buffer conditions at two pH values using liquid chromatography with fluorescence detection (LC-FLD); (2) in a second in vitro model, the influence of the upper gastrointestinal environment on the mycotoxin sorption capacity of the same two sequestrants was studied using a chronic AFB1 level commonly encountered in the field (10 µg/L and in the presence of feed); (3) the third model used a novel ex vivo approach to measure the absorption of 3H-labelled AFB1 in the intestinal tissue and the ability of the sequestrants to offset this process; and (4) a second previously developed ex vivo model readapted to AFB1 was used to measure the transfer of 3H-labelled AFB1 through live intestinal tissue, and the influence of sequestrants on its bioavailability by means of an Ussing chamber system. Despite some sorption effects caused by the feed itself studied in the second model, both in vitro models established that the adsorption capacity of both YCW and HSCAS is promoted at a low acidic pH. Ex vivo Models 3 and 4 showed that the same tested material formed a protective barrier on the epithelial mucosa and that they significantly reduced the transfer of AFB1 through live intestinal tissue. The results indicate that, by reducing the transmembrane transfer rate and reducing over 60% of the concentration of free AFB1, both products are able to significantly limit the bioavailability of AFB1. Moreover, there were limited differences between YCW and HSCAS in their sorption capacities. The inclusion of YCW in the dietary ration could have a positive influence in reducing AFB1′s physiological bioavailability.

Application of Six Detection Methods for Analysis of Paralytic Shellfish Toxins in Shellfish from Four Regions within Latin America.

With the move away from use of mouse bioassay (MBA) to test bivalve mollusc shellfish for paralytic shellfish poisoning (PSP) toxins, countries around the world are having to adopt non-animal-based alternatives that fulfil ethical and legal requirements. Various assays have been developed which have been subjected to single-laboratory and multi-laboratory validation studies, gaining acceptance as official methods of analysis and approval for use in some countries as official control testing methods. The majority of validation studies conducted to date do not, however, incorporate shellfish species sourced from Latin America. Consequently, this study sought to investigate the performance of five alternative PSP testing methods together with the MBA, comparing the PSP toxin data generated both qualitatively and quantitatively. The methods included a receptor binding assay (RBA), two liquid chromatography with fluorescence detection (LC-FLD) methods including both pre-column and post-column oxidation, liquid chromatography with tandem mass spectrometry (LC-MS/MS) and a commercial lateral flow assay (LFA) from Scotia. A total of three hundred and forty-nine shellfish samples from Argentina, Mexico, Chile and Uruguay were assessed. For the majority of samples, qualitative results compared well between methods. Good statistical correlations were demonstrated between the majority of quantitative results, with a notably excellent correlation between the current EU reference method using pre-column oxidation LC-FLD and LC-MS/MS. The LFA showed great potential for qualitative determination of PSP toxins, although the findings of high numbers of false-positive results and two false negatives highlighted that some caution is still needed when interpreting results. This study demonstrated that effective replacement methods are available for countries that no longer wish to use the MBA, but highlighted the importance of comparing toxin data from the replacement method using local shellfish species of concern before implementing new methods in official control testing programs.

An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems

The human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most essential conjugative enzymes, is responsible for the metabolism and detoxification of bilirubin and other endogenous substances, as well as many different xenobiotic compounds. Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices. Herein, an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices, using liquid chromatography with fluorescence detection (LC-FD), has been developed and validated. The newly developed LC-FD based assay has been confirmed in terms of sensitivity, specificity, precision, quantitative linear range and stability. One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate, enabling reliable quantification of lower amounts of active enzyme than any other method. The precision test demonstrated that both intra- and inter-day variations for this assay were less than 5.5%. Furthermore, the newly developed assay has also been successfully used to screen and characterize the regulatory effects of small molecules on the expression level of UGT1A1 in living cells. Overall, an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems, providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases, interactions with xenobiotics, and characterization modulatory effects of small molecules on this conjugative enzyme.

QUANTIFICATION OF HYALURONIC ACID AND METHYLSULFONYLMETHANE IN DIETARY SUPPLEMENTS

Objective: Osteoarthritis can be treated by taking oral supplements containing compounds that can nourish bones and joints such as hyaluronic acid,methylsulfonylmethane (MSM), chondroitin, glucosamine, and collagen. This study aimed to develop and validate tests for analyzing two compounds,namely, hyaluronic acid and MSM, simultaneously and to determine both their levels in a mixed sample.Methods: Hyaluronic acid derivatization was carried out using fluorenylmethyloxycarbonyl chloride and then analyzed by liquid chromatographywith fluorescence detection, while MSM was analyzed using gas chromatography. After the development of optimal conditions for each separation,system suitability tests were developed and calibration curves used for tests of accuracy and precision as well as for level determination. Hyaluronicwas detected at an excitation wavelength of 255 nm and emission wavelength of 330 nm. The mobile phase used was acetonitrile-acetate pH 4.2 (1: 4)with a flow rate of 1.0 mL/min.Results: The developed method was linear (r=0.9983) in the range of 5–50 ppm and the limits of detection (LOD) and quantitation (LOQ) were 3.55and 11.84 ppm, respectively. The initial column temperature for MSM analysis was 110°C and the mobile phase used was nitrogen gas at a flow rate of0.8 mL/min. The method was linear (r=0.9998) in the range of 4000–15,000 ppm and the LOD and LOQ were 332.90 and 1109.67 ppm, respectively.Conclusion: A simulated sample containing both compounds was assessed to contained 98.63% hyaluronic acid and 99.35% MSM.

Isolation and Characterization of Cylindrospermopsis raciborskii Strains from Finished Drinking Water.

In the summer of 2015, an intense cyanobacterial bloom producing geosmin/2-methylisoborneol (MIB) occurred in the Roxo freshwater reservoir in Alentejo, Portugal. The drinking water supplied from the Roxo water treatment plant (WTP) exhibited an unpleasant odor/taste and a significant cyanobacteria density was detected in the finished water at the exit of the WTP. Cyanobacteria were not evaluated downstream of the WTP, namely, at the city reservoir. The aim of this work was to isolate and characterize viable cyanobacteria present in finished water (exit of the WTP and city reservoir) that withstand conventional water treatment. Treated water samples collected at both sites were inoculated in Z8 culture medium to provide the conditions for putative cyanobacterial growth. After 30 days, filamentous cyanobacteria were observed in cultures inoculated with samples from the exit point of the WTP. Viable trichomes were isolated and identified as Cylindrospermopsis raciborskii by morphometric and molecular analysis. None of the isolates were cylindrospermopsin/microcystin producers, as confirmed by ELISA and amplification of corresponding genes (PS/PKS and mcyA-cd/mcyAB/mcyB). ELISA results were positive for saxitoxin, but saxitoxin and derivatives were not detected by liquid chromatography with fluorescence detection (LC-FLD), nor were their related genes (sxtA/sxtA4/sxtB/sxtM/sxtPer/sxtI). To our knowledge, this is the first report on the establishment of cultures of C. raciborskii that resisted water treatment processes.

A Rapid and Sensitive HPLC-FLD Method for the Determination of Retinol and Vitamin E Isomers in Human Serum

Background: Retinol and vitamin E are fat-soluble vitamins crucial for human health, yet their isomers’ distributions in the human body are still known roughly. In order to figure out the physical condition and evaluate the nutritional status of an individual, it is imperative to analyze retinol and VE isomers in human serum. Objective: This work aims to establish a rapid and simple high-performance liquid chromatography with fluorescence detection for simultaneous determination of retinol and vitamin E isomers in human serum. Methods: Separation was accomplished on a common C18 column thermostated at 25 oC, using a simple isocratic elution program of methanol/acetonitrile (85:15, v/v) at a flow rate of 1.0 mL/min. Fluorescence detection was operated using excitation/emission wavelengths of 329 nm/472 nm for retinol and 294 nm/338 nm for VE isomers, respectively. Results: Rapid separation was achieved within 13 min. Linear ranges of the method were 0.020-50.0 µg/mL, with correlation coefficients greater than 0.999. Detection limits and the quantification limits were 0.001-0.004 µg/mL and 0.003- 0.013 µg/mL, respectively. Mean recoveries were 84.1%- 98.2%, with intra-day and inter-day relative standard deviations less than 12.3% and 13.6%, respectively. This method has been applied to the simultaneous determination of retinol and 8 VE isomers in human serum samples with satisfactory results. Conclusion: A rapid, simple and robust method was developed for routine analysis of retinol and eight vitamin E isomers in human serum, providing a useful tool for clinical diagnosis and nutritional evaluation.

Monitoring of bisphenols in canned tuna from Italian markets

Monitoring of food contamination from bisphenols is a necessary process for the consumers' risk assessment. A method for the quali-quantitative analysis of Bisphenol A (BPA), Bisphenol B (BPB), Bisphenol A Diglycidyl Ether (BADGE), and Bisphenol F Diglycidyl Ether (BFDGE), by liquid chromatography with fluorescence detection (LC-FD), was performed and validated for their determination in 33 samples of tuna fish, canned in either oil or aqueous medium. Samples were collected in Italian markets. Tuna and the correspondent preservation medium were analyzed separately. Detected levels of bisphenols ranged from 19.1 to 187.0 ng/g in tuna matrix and from 6.3 to 66.9 ng/mL in oil medium. No bisphenols were found in aqueous medium. At least one of the analytes was found in 83% of the tuna samples in oil medium, whereas tuna samples in aqueous medium showed BPA alone in 67% of samples. 21% of the oil medium samples resulted positive for at least one bisphenol. On the basis of measured concentrations and general daily ingestion rate of canned tuna fish, the probable daily intake of BPA for Italian population was calculated.

Analysis of Ergot Alkaloids.

The principles and application of established and newer methods for the quantitative and semi-quantitative determination of ergot alkaloids in food, feed, plant materials and animal tissues are reviewed. The techniques of sampling, extraction, clean-up, detection, quantification and validation are described. The major procedures for ergot alkaloid analysis comprise liquid chromatography with tandem mass spectrometry (LC-MS/MS) and liquid chromatography with fluorescence detection (LC-FLD). Other methods based on immunoassays are under development and variations of these and minor techniques are available for specific purposes.

Simultaneous determination of camptothecin and 10-hydroxycamptothecine in the Camptotheca acuminate, its medicinal preparation and in rat plasma by liquid chromatography with fluorescence detection.

Camptotheca acuminata Decne is an important medicinal plant that contains various cytotoxic alkaloids, such as camptothecine (CPT) and 10-hydroxycamptothecine (HCPT). A rapid and sensitive liquid chromatography with fluorescence detection (LC-FLD) method for the quantification of CPT and HCPT is described. The separation was carried out on a DL-Cl8 column (4.6 × 150 mm, 5 µm), with the mobile phase of acetonitrile-sodium dihydrogen phosphate buffer (10 mm) using an gradient elution at the flow rate of 0.6 mL/min. The LC-FLD method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the above components. The lower detection limits of CPT and HCPT were 0.4 and 0.1 ng/mL, respectively. The precision was <1.58% and the mean recovery of the analytes was 96.0-98.6%. The LC-FLD method was successfully applied to determine CPT and HCPT in real samples including C. acuminate, HCPT injection and rat plasma.

Paralytic toxin profile of the marine dinoflagellate Gymnodinium catenatum Graham from the Mexican Pacific as revealed by LC-MS/MS

The paralytic shellfish toxin (PST) profiles of Gymnodinium catenatum Graham have been reported for several strains from the Pacific coast of Mexico cultured under different laboratory conditions, as well as from natural populations. Up to 15 saxitoxin analogues occurred and the quantity of each toxin depended on the growth phase and culture conditions. Previous analysis of toxin profiles of G. catenatum isolated from Mexico have been based on post-column oxidation liquid chromatography with fluorescence detection (LC-FLD), a method prone to artefacts and non-specificity, leading to misinterpretation of toxin composition. We describe, for the first time, the complete toxin profile for several G. catenatum strains from diverse locations of the Pacific coast of Mexico. The new results confirmed previous reports on the dominance of the less potent sulfocarbamoyl toxins (C1/2); significant differences, however, in the composition (e.g., absence of saxitoxin, gonyautoxin 2/3 and neosaxitoxin) were revealed in our confirmatory analysis. The LC-MS/MS analyses also indicated at least seven putative benzoyl toxin analogues and provided support for their existence. This new toxin profile shows a high similarity (> 80%) to the profiles reported from several regions around the world, suggesting low genetic variability among global populations.

Paralytic Shellfish Toxins in the Marine GastropodsZidona dufresneiandAdelomelon beckiifrom Argentina: Toxicity and Toxin Profiles

ABSTRACT The marine waters around Argentina are periodically affected by a range of phytoplankton species that are known producers of paralytic shellfish poisoning (PSP) toxins. The toxic dinoflagellates Alexandrium tamarense and Gymnodinium catenatum have been associated with PSP outbreaks in bivalve molluscs such as mussels. These bivalves are used as a food source by a number of species of carnivorous gastropods, including 2 species of marine snails, Zidona dufresnei and Adelomelon beckii. There is, consequently, a significant risk to consumers of the snails, which can accumulate PSP toxins after predation on the bivalves. Forty-one snail samples of carnivorous snails harvested between 1986 and 2012, representing 2 species (Z. dufresnei and A. beckii), were analyzed for PSP by the reference mouse bioassay (MBA) method and an alternative liquid chromatography with fluorescence detection (LC-FLD) method based on AOAC 2005.06. Sample toxicities determined by the 2 methods correlated well, with no statisti...

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.

Paralytic shellfish toxins in the sea scallop Placopecten magellanicus on Georges Bank: Implications for an offshore roe-on and whole scallop fishery

To protect public health from the potential risk of paralytic shellfish poisoning (PSP) in sea scallops, Placopecten magellanicus, from the Atlantic offshore U.S. waters of Georges Bank, harvesting of roe-on or whole scallops is banned. Only adductor muscles may be sold if harvested from Georges Bank, Gulf of Maine, or the PSP closure areas as far west as 71° West Longitude. Given the limited toxicity data available for sea scallops from this region both prior to and subsequent to implementation of this management strategy, this study sought a more extensive spatial and temporal evaluation of sea scallop gonad and viscera toxicities that would inform management decisions related to the roe-on and whole scallop fishery. Both overall toxicity and toxin composition were measured for sea scallop gonads and viscera collected from 232 stations in 2007 and 23 in 2010. Overall toxicity was assessed using two screening methods: field-deployable Jellett Rapid Tests (JRT) and quantitative, laboratory-based receptor binding assays (in 2007). Additionally, a quantitative liquid chromatography with fluorescence detection (LC-FD) method was used to determine toxin composition and overall toxicity (in 2010). The at-sea qualitative JRT screening tool results, whereby a positive indicated the sample contained at least 40μg saxitoxin (STX) equivalents 100g−1, were often inconsistent with results obtained using the quantitative methods. Sea scallop viscera toxicity represented the majority of toxin load in the organism and was often in excess of the regulatory guidance level. Sea scallop gonads accounted for a small percentage of total toxicity, but at times reached unsafe levels. Toxin composition in both the gonads and viscera was dominated by STX and gonyautoxin 2/3, as has been reported in previous studies. No predictive indices of gonad toxicity were found. Results at this time do not support a roe-on or whole scallop fishery on Georges Bank. While access restrictions to whole scallops on Georges Bank are unlikely to change based on these results, it seems plausible that a management strategy requiring at-sea testing of scallop gonads for PSP may be necessary in order for a safe roe-on scallop fishery from this region to be feasible.

Immunoaffinity in-tube solid phase microextraction coupled with liquid chromatography with fluorescence detection for determination of interferon α in plasma samples

The present work describes a sensitive and specific automated immunoaffinity in-tube solid-phase microextraction coupled with liquid chromatography with fluorescence detection (LC-FD) method for the determination of interferon α2a in plasma samples for therapeutic drug monitoring. To this end, the intrinsic selectivity of the monoclonal antibodies has been combined with in-tube solid phase microextraction by immobilization of these antibodies into the fused silica capillary. The in-tube SPME variables, such as plasma sample volume, draw/eject volume, number of draw-eject cycles, as well as desorption procedure have been optimized, in order to improve the sensitivity of the proposed method. Analyses of interferon α2a in plasma samples were carried out within 12min (sample preparation and LC analyses). The response of the proposed method was linear over a dynamic range 0.006-3.0MIUmL(-1), with a correlation coefficient of 0.998. The inter-day precision of the method had a coefficient of variation lower than 6.2%. The proposed automated method has adequate analytical sensitivity and selectivity for the determination of interferon α2a in plasma samples for therapeutic drug monitoring. The proposed method was successfully applied to the plasmas samples analyses from patients in therapy with interferon α-2a, demonstrating a rare application of in-tube SPME for biopharmaceutical (protein) analyses.

Searching ingredients polluted by polycyclic aromatic hydrocarbons in feeds due to atmospheric or pyrolytic sources

The primary aim of the proposed work is to propose the potential sources of pollution by polycyclic aromatic hydrocarbons (PAHs) in feeds and ingredients. To reach this propose the development of a simple, fast, quantitative and economic method for determining PAHs using liquid-liquid extraction (LLE), clean-up and detection by liquid chromatography with fluorescence detection (LC-FD) in polluting feeds and ingredients was developed. The overall method quantification limits range from 0.020 to 4.0μg/kg and analyte recoveries are between 70% and 105% with relative standard deviations (RSD) lower than 20%.Molecular patterns of PAHs were used to study their distribution in the selected samples by cluster analysis, separating them in two groups: contaminated by atmospheric or pyrolytic sources. In order to find a relationship between the nutritional composition (protein, fibre, ash and fat content), and the hypothetical toxicity of selected feeds, a partial least squared (PLS) analysis was used, showing that fibre was a major contributor. Moreover, the complete data set of 27 feed samples and 25 feed ingredients x 13 PAH concentrations were analysed by PCA to find out what ingredients were controlling PAH pollution.

Feed Ingredients Mainly Contributing to Polycyclic Aromatic Hydrocarbon and Polychlorinated Biphenyl Residues

Animal nutrition constitutes an important issue for the animal production industry. Products intended for animal feed may contain undesirable substances which could endanger animal health or, because of their presence in livestock products, human health or the environment. In this sense, several incidents related with the presence of persistent organic pollutants, particularly with polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs), have happen in food and feed additives. Simple, fast, quantitative, and economic methods for determining PAHs and PCBs in potentially polluting feed ingredients were developed and evaluated. The methods involved liquid-liquid extraction (LLE), clean-up, and detection by liquid chromatography with fluorescence detection (LC-FD) for PAHs and gas chromatography (GC) coupled to electron capture detection (ECD) for PCBs, using tandem mass spectrometry (MS/MS) as confirmatory technique. The overall method quantification limits range from 0.020– 4.0 μg/Kg for PAHs and from 0.20–0.75 μg/Kg for PCBs, analyte recoveries are between 70 and 105% with relative standard deviations (RSD) lower than 20%. Once the method was optimized, it was applied to 25 feed ingredients considered as potential candidates for feedstuff. As expected, the samples of animal origin as shell powder and fish oil showed the highest PCB concentrations of 56 and 29 ng/g, which are equivalent to toxicological concentrations of 123 and 18 ng WHO-TEQDL-PCBs/Kg, respectively. Opposite results were obtained with PAHs and the highest PAH4 levels were detected alfalfa (47 μg/Kg), calcium phosphate (27 μg/Kg), and palm oil (23 μg/Kg) used as feed ingredients.