Detection of macrocyclic lactones in porcine liver,meat and fish tissue using LC–APCI–MS–MS

A selective and sensitive method for the simultaneous determination of five avermectins (abamectin, ivermectin, doramectin, emamectin and eprinomectin) and one milbemycin (moxidectin) in porcine liver, bovine meat and fish tissue was developed. The method involved extraction with acetonitrile and purification by C₁₈ solid-phase extraction. Detection was carried out using liquid chromatography coupled to multiple mass spectrometry (LC–MS²) equipped with APCI in the negative mode. This method was validated according to the requirements of Commission Decision EC/2002/657 (Implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off J Eur Commun. L221: 8–36.). In addition to the linear response (R ² between 59 and 97%), good repeatability (CV between 20 and 35%), reproducibility (CV between 20 and 35%) and detection (CCα) and quantification (CCβ) limits were obtained for all compounds in all matrices considered.     

Validation of a wide-spectrum microbiological tube test,the EXPLORER® test,for the detection of antimicrobials in muscle from different animal species

Explorer® is a simple and fast new kit for the detection of inhibitory substances in raw meat. The test, a 96-well microtitre plate, is based on the inhibition of microbial growth (Geobacillus stearothermophilus spores). It was validated in accordance with European Commission Decision 2002 European Commission. 2002. Commission Decision 2002/657/EC of 12 August 2002: implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off J Eur Comm, L221: 8–36.  [Google Scholar]/657/EC (2002). The specificity and detection capabilities for five compounds from major antimicrobial families and robustness were studied. The specificity of the test was assessed with four different animal species and was found to be very satisfactory (false-positive rates lower than 10%). The detection capabilities for amoxicillin (10 µg kg^?1) and tylosin (100 µg kg^?1) were at the maximum residue limit (MRL) level (50 and 100 µg kg^?1, respectively) for both, for doxycycline (200 µg kg^?1) and sulfathiazole (200 µg kg^?1) at twice the MRL (100 µg kg^?1 for each) and for cefalexin (500 µg kg^?1) at 2.5 times the MRL (200 µg kg^?1). Twenty-one samples were analysed in parallel with the Four Plate Test, the STAR protocol and the Explorer® test. One false-positive result and two false-negative results (samples containing oxytetracycline) were reported with the Explorer® test. In conclusion, the Explorer® test was shown to be robust and easily automated. Photometric reading allows informatic data storage and objective readings between technicians and days. The test can be used as a wide screening test because it enables detection of most of the antimicrobial families (penicillins, cephalosporins, tetracyclines, sulphonamides, and macrolides) in muscles from different animal species (porcine, bovine, ovine, poultry).     

Analytical strategy for the determination of non-steroidal anti-inflammatory drugs in plasma and improved analytical strategy for the determination of authorised and non-authorised non-steroidal anti-inflammatory drugs in milk by LC–MS/MS

A sensitive and selective method for the determination of six non-steroidal anti-inflammatory drugs (NSAIDs) in bovine plasma was developed. An improved method for the determination of authorised and non-authorised residues of 10 non-steroidal anti-inflammatory drugs in milk was developed. Analytes were separated and acquired by high performance liquid chromatography coupled with an electrospray ionisation tandem mass spectrometer (ESI–MS/MS). Target compounds were acidified in plasma, and plasma and milk samples were extracted with acetonitrile and both extracts were purified on an improved solid phase extraction procedure utilising Evolute? ABN cartridges. The accuracy of the methods for milk and plasma was between 73 and 109%. The precision of the method for authorised and non-authorised NSAIDs in milk and plasma expressed as % RSD, for the within lab reproducibility was less than 16%. The % RSD for authorised NSAIDs at their associated MRL(s) in milk was less than 10% for meloxicam, flunixin and tolfenamic acid and was less than 25% for hydroxy flunixin. The methods were validated according to Commission Decision 2002 European Commission Decision. 2002. Decision(2002/657/EC) of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and interpretation of results. Off J Eur Commun, L221: 8–36.  [Google Scholar]/657/EC.     

Polychlorinated dibenzo-p-dioxins,dibenzofurans and dioxin-like polychlorinated biphenyls in food and feed in Latvia in 2009–2011

During 2009–2011 a monitoring programme for 17 polychlorinated dibenzo-p-dioxins (PCDDs)/polychlorinated dibenzofurans (PCDFs) and 12 dioxin-like polychlorinated biphenyls (DL-PCBs) was conducted in the Latvian food and feed market. Using ISO 17025-accredited analytical methodology, investigation of 121 food (milk, dairy products, meat, eggs, fish, fish products) and 66 feed samples (fish meal and oil, compound and mineral feed, vegetable and animal fats) was performed. Most samples showed contamination below the European Commission (EC) Regulation No. 1881/2006 and Commission Directive 2006/13/EC limits. Average total toxicological equivalent (total-TEQ₍₁₉₉₈₎) concentrations within the food sample groups, except fish and fish products, ranged between 0.41 and 15.1 pg total-TEQ₍₁₉₉₈₎ g^?1 fat. Fish and fish products showed contamination levels from 0.18 to 46.0 pg total-TEQ₍₁₉₉₈₎ g^?1 fresh weight (f.w.). Fifty-seven per cent of cod liver samples were non-compliant. The most contaminated feed samples were fish meal and fish oil. A comparison with WHO-TEF₍₂₀₀₅₎ data is given.     

Silver migration from silver-modified activated carbon applied as a water filtration medium in classic cartridges of jug filter systems

A comprehensive study was undertaken in order to examine the possible adverse effect of jug filter systems (JFSs) on the quality of filtered water taking into account the released amounts of silver (Ag) into the filtered test water. Nine brands of JFSs (A–I) were investigated according to BS 8427:2004 using a validated ICP/MS method. Essential modification of BS 8427:2004 within the domain of the composite sample preparation was proposed and applied during the tests. The established grand mean concentrations of released Ag from A–H classic cartridges (containing Ag-modified activated carbon and ion exchange resins) and I classic cartridge (containing non-modified activated carbon and ion exchange resin) installed in corresponding JFSs were in the range of 2.6–13.1?µg?l^?1 and lower than 0.014?µg?l^?1, respectively. These values were applied for the estimation of the daily intakes of Ag connected with the consumption of water filtered using JFSs (ranging from?<?0.0004 to 0.374?µg?kg^?1?day^?1). After taking into account the grand mean concentrations of Ag established during the whole cycle of exploitation for nine JFSs and on the basis of available toxicological data for this element, no long-term risk for human health with respect to appearance of argyria (a condition caused by improper exposure to the Ag or Ag compounds) could be expected (the Hazard Quotient indices estimated as ratios of the daily intakes to the reference dose of Ag were equal or lower than 0.075). Ag-modified activated carbon is not included in the positive list of the authorised substances of the European Commission Regulation (EU) No. 10/2011. Additionally, this material has not been approved by European Food Safety Authority (EFSA). A part of water filtered by JFSs can be directly consumed as drinking water and additionally the remaining water can be applied for the preparation of food products (drinks, soups, etc.). In both cases the quality of water has to fulfil the requirements listed in Directive 98/83/EC (Regulation (EC) No. 178/2002 defines the quality of water intentionally incorporated into the food after the point of compliance as defined in Article 6 of Directive 98/83/EC). However, it should be underlined that point-of-use water treatment units (including JFSs) are not regulated under Directive 98/83/EC and additionally the parametric value for Ag is not included in this document. Therefore, a provisional migration limit for Ag leached from JFSs at the level of 25?µg?l^?1 was proposed. This value for Ag would limit intake to less than 13% of the human No Observed Adverse Effect Level (NOAEL) (0.39?mg person^?1?day^?1), using an assumption that each day 2?L of filtered water is consumed containing this metal at the provisional migration limit. All the JFSs tested meet this requirement.     

A novel method to determine valnemulin in feedingstuffs for several animal species by liquid chromatography-electrospray tandem mass spectrometry

A new method specific for the determination and unambiguous confirmation of the antibiotic valnemulin in feed for all animal species is described. Simple clean-up based on solvent extraction and liquid partition is carried out prior to determination by liquid chromatography coupled to electrospray tandem mass spectrometry on a hybrid QTRAP 4000 system, in multiple reaction monitoring mode. Valnemulin is licensed in the European Union as a veterinary drug for use in feed for pigs and rabbits, but unavoidable carryover in feed for non-target species should be monitored. The method is rapid and reliable, and was validated in-house at 10, 100 and 750 ng g^–1, evaluating the analytical performances according to Commission Regulation (EC) No. 882/2004. Good mean recoveries from 79.7% to 92.6%, and within-laboratory reproducibility RSDs, ranging from 6.2% to 12.1%, were measured; the limit of quantification at 10.0 ng g^–1 also allows for sensitive evaluation of undesirable carryover in feed for non-target species. The results of monitoring 24 compound feeds for several animal species are also reported.     

Determination of polyphosphates in products of animal origin: application of a validated ion chromatography method for commercial samples analyses

A reliable and accurate analytical method was developed and validated for the quantitative determination of polyphosphates (diphosphate, triphosphate, trimetaphosphate and tetrapolyphosphate) in products of animal origin (meat, dairy and fish products) by ion chromatography with suppressed conductivity detection. The chromatographic separations were accomplished by using an anion-exchange column eluted with a sodium hydroxide gradient. The method validation, performed according to Regulation 882/2004/EC and Decision 657/2002/EC, provided results conform with the European Directives with respect to linearity (R?>?0.996), specificity, precision (CV????4.5?%), recovery (ranging from 87.2 to 101.1?%), detection and quantification limits and ruggedness. The method reliability was confirmed evaluating the method measurement uncertainty, lower than 7.5?% and by proficiency test results. Finally, the method ability to discriminate samples treated from those not-treated with polyphosphates was verified by analyzing commercial samples containing polyphosphates (cooked ham, wurstel, corned beef, processed cheese and surimi) or treated in-house with polyphosphates (pangasius fillets, shrimps and cuttlefishes).     

Simultaneous analysis of coccidiostats and sulphonamides in non-target feed by HPLC-MS/MS and validation following the Commission Decision 2002/657/EC

Taking into consideration the maximum level (ML) for coccidiostats included in the European Regulation 574/2011 and the fact that the presence of residues of sulphonamides in non-target feed is forbidden, the aim of this article is to present an analytical method based on HPLC-MS/MS for the identification and quantification of sulphonamides and coccidiostats in non-target feeds. The method was validated following Commission Decision 2002/657/EC, and recovery, repeatability and reproducibility were within the limits established in the Decision. For coccidiostats, the decision limit and detection capability were calculated for the different species taking into account the ML allowed in Regulation 574/2011. The applicability of the method was investigated in 50 feed samples collected from dairy farms, 50 obtained from feed mills and 10 interlaboratory feed samples.     

Feed additives diclazuril and nicarbazin in egg and liver samples from Croatian farms

In total 307 egg and 275 liver samples were examined for nicarbazin and 365 eggs for diclazuril over a 30-month period. Enzyme-linked immunosorbent assay methods used for quantification were validated according to European Commission Decision 2002/657/EC. Non-compliant samples were confirmed by LC-MS/MS. Mean diclazuril concentrations in egg samples were 0.31?µg?kg^?1, which is below the MRL. In only one egg sample, 2.26?µg?kg^?1 was determined by enzyme-linked immunosorbent assay, although confirmation by LC-MS/MS gave a value of 1.6?µg?kg^?1. Mean nicarbazin levels determined were 1.85?µg?kg^?1 in egg and 21.1?µg?kg^?1 in liver samples. Four samples, one egg and three livers, yielded elevated concentrations of nicarbazin, but only in the egg sample the LC-MS/MS method confirmed nicarbazin (106?µg?kg^?1) above the MRL value.     

Comparison of migration from polyethersulphone and polycarbonate baby bottles

This work presents two analytical methods developed for measuring three components of polyethersulphone (PES) and applying them to the migration testing of 30 baby bottles made of PES. The study also provides migration results under the same conditions for bisphenol A (BPA) from 40 polycarbonate baby bottles using a well-established method adapted to low concentrations. For PES bottles, migration of diphenyl sulphone (DPS), 4,4′-dichlorodiphenyl sulphone (DCPS) and 4,4′-dihydroxydiphenyl sulphone (DHPS; also known as bisphenol S) was carried out using two different analytical methods with detection limits of 0.1–0.3?µg/kg, and, therefore, much below their respective European Commission Directive 2002/72/EC legislative migration limits of 50–3000?µg/kg, respectively. In parallel, 40 bottles made of polycarbonate were analysed for the migration of BPA using a method validated at EU level and modified to give a lower detection limit of 0.1?µg/kg. Migration tests were conducted into the simulant for milk 50% EtOH (as per Commission Regulation No. 321/2011 of 1 April 2011) according to the test conditions from the guidelines on test conditions for articles in contact with foodstuffs (with a focus on kitchenware) prepared by the EU Reference Laboratory and its network of National Reference Laboratories. None of the 30 bottles made of PES released any detectable amounts of DCPS or DHPS and only two bottles released a very low amount of DPS of ～1?µg/kg in the milk food simulant compared to a regulatory limit of 3000?µg/kg. For PC bottles, 32 bottles of 40 (80%) did not release BPA above the LOD of 0.1?µg/kg (in any of the three migration tests performed on each bottle). The other 20% of bottles exhibited only very minor migration, where the highest level in the first migration test was 1.83?µg/kg and most bottles did not release detectable BPA in the second and third test. Only one bottle, with a migration level of 1.08?µg/kg, in the first test still showed a detectable level in the last migration test (i.e. 0.42?µg/kg). It is important to note that the legal limit (European Commission Directive 2002/72/EC) was still 600?µg/kg for polycarbonate bottles at the time of purchase, preceding the precautionary ban taking effect from 1 June 2011 (Commission Directive 2011/8/EU; Commission Regulation No. 321/2011). This confirms that the likelihood of migration of BPA is very low and remains at very minute amounts. The results also suggest the absence of release from PES bottles based on the set of bottles investigated.     

The simultaneous determination of the ionophore antibiotics in animal tissues and eggs by tandem electrospray LC–MS–MS

A method has been developed for the simultaneous extraction and determination of the four most currently used Ionophore antibiotics (lasalocid, monensin, narasin and salinomycin) by LC–MS–MS from different animal tissues and eggs. Results show good repeatability, and mean spiked recoveries for lasalocid, monensin, narasin and salinomycin in animal livers are in the average range 93–103, 96–103, 93–102 and 97–106%, respectively, and in eggs the mean spiked recoveries are 101, 103, 98 and 102% for lasalocid, monensin, narasin and salinomycin, respectively. The detection limit is at 1 ng ml⁻¹ for all the named ionophorous compounds. A quantitation level of 50 ng g⁻¹ for lasalocid, monensin at 2.5 ng g⁻¹, and 10 ng g⁻¹ for narasin and salinomycin is achieved which represents half the action limit prescribed by the UK Regulation in compliance with the European Council Directive 96/23/EC. A high throughput of samples is achievable using this method which allows the analysis of 30–40 samples by one analyst in a day.     

Rapid multi-residue and multi-class qualitative screening for veterinary drugs in foods of animal origin by UHPLC-MS/MS

Multi-class UHPLC-MS/MS was developed for the analysis of more than 160 regulated or banned compounds of various classes: anthelmintics including benzimidazoles, avermectins and others; antibiotics including amphenicols, beta-lactams, macrolides, pyrimidines, quinolones, sulphonamides and tetracyclines; beta-agonists; corticosteroids; ionophores; nitroimidazoles; non-steroidal anti-inflammatory agents; steroids; and tranquillisers. Samples were extracted with acetonitrile, without any additional purification step, and analysed by using UHPLC-MS/MS. Validation was done in accordance with the guidelines laid down by European Commission Decision 2002/657/EC for qualitative screening methods. This simple method proved applicable to routine screening for residues in egg, honey, milk and muscle samples at half the maximum concentration permitted by the European Union for each drug. In most cases, the target value was set at 5?µg?kg^?1 for unauthorised compounds.     

Determination of tetracyclines in multi-specie animal tissues by pressurized liquid extraction and liquid chromatography–tandem mass spectrometry

A specific, sensitive and robust pressurized liquid extraction (PLE) and liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determining tetracycline, chlortetracycline, oxytetracycline and doxycycline in bovine, swine, poultry and lamb muscle tissues is presented. PLE was performed using an ASE^® 200 from Dionex and water as extractant, followed by solid-phase extraction (SPE) using an Oasis HLB cartridge. The method was validated for beef, chicken, pork and lamb meat in compliance with the requirements set by Commission Decision, 2002/657/EC [Commission Decision 2002/657/EC (2002). Implementing Council Directive 96/23/EC concerning the performance of analytical methods and interpretation of results. Official Journal of European Communities, L239, 66–98. (Available at: <http://europe.eu.int>)]. The average recoveries of the different meat samples, spiked with the four tetracyclines at three levels (1, 100 and 200 μg kg⁻¹ of each tetracycline), were always higher than 89% with intraday and interday precision lower than 15% and 17%, respectively. A good linearity was established for the four tetracyclines in the range from 5 to 10,000 μg kg⁻¹ with r > 0.995. The limits of quantification (LOQs) were between 0.5 and 1 μg kg⁻¹, which are well below the tolerance levels set by the European Union. The decision limit (CCα) and the decision capability (CCβ) were in the range 101–116 and 112–130 μg kg⁻¹, respectively. Compared with previous methods, sample preparation time required for the analysis and LOQs, are reduced. The method demonstrated its successful application for the analysis of 100 meat samples. Two samples of beef and one sample of chicken out of 25 of each type tested positive while none of 25 samples of either, lamb or pork, tested positive.     

Determination of flumequine, nalidixic acid and oxolinic acid in shrimps by high-performance liquid chromatography with fluorescence detection

A simple high-performance liquid chromatography (HPLC) method with fluorescence detection is presented for the determination of the quinolones flumequine, nalidixic acid and oxolinic acid in shrimps. The pharmaceuticals were extracted from the matrix with acetonitrile, purified by liquid–liquid extraction and analysed by HPLC with fluorescence detection at excitation wavelength of 312 nm and emission wavelength of 368 nm. The method has been validated according to the recommendations of the European Commission Decision 2002/657/EC under the consideration of specificity, trueness and precision as well as decision limit CCα and detection capability CCβ. The results of the validation demonstrate that the method is suitable to verify surely quinolone residues in shrimps and to decide with an estimated certainty if the investigated sample complies the European Council Regulation 2377/90/EEC or not. Furthermore, shrimp retail products of the years 2004 and 2005 were investigated with the developed HPLC-method.     

Multiresidue determination of carcinogenic polycyclic aromatic hydrocarbons in honey by solid-phase extraction and high-performance liquid chromatography

An analytical procedure based on solid-phase extraction, using ethyl acetate as the elution solvent, and high-performance liquid chromatography with fluorescence and diode array detection was developed for the identification and quantification of polycyclic aromatic hydrocarbons (PAHs) in honey. The method has been optimized and validated in accordance with Commission Regulation 333/2007 and Commission Decision 2002/657/EC. This method allows the identification of the 15 PAHs that should be monitored in food matrices, as proposed in 2002 by the Scientific Committee on Food and later by the European Union in the Commission Recommendation 2005/108/EC, because of their genotoxic and carcinogenic properties. The results of the validation study were in agreement with quality criteria described in European legislation in terms of sensitivity, accuracy, and ruggedness, and the method was applied to the analysis of 42 honey samples (21 from Spain and 21 from other regions). The honey samples were not contaminated by PAHs at detectable levels and thus could be marketed without health risk.     

Development of a real-time PCR protocol for the species origin confirmation of isolated animal particles detected by NIRM

At present, European legislation prohibits totally the use of processed animal proteins in feed for all farmed animals (Commission Regulation (EC) No. 1234/2003–extended feed ban). A softening of the feed ban for non-ruminants would nevertheless be considered if alternative methods could be used to gain more information concerning the species origin of processed animal proteins than that which can be provided by classical optical microscopy. This would allow control provisions such as the ban of feeding animals with proteins from the same species or intra-species recycling (Regulation (EC) No. 1774/2002). Two promising alternative methods, near-infrared microscopy (NIRM) and real-time polymerase chain reaction (PCR), were combined to authenticate, at the species level, the presence of animal particles. The paper describes the improvements of the real-time PCR method made to the DNA extraction protocol, allowing five PCR analyses to be performed with the DNA extracted from a single particle.     

Persistence of α-cypermethrin residues in milk of lactating donkeys (Equus asinus) using UHPLC-MS/MS

The aim of this study was to measure the persistence of residues of the pyrethroid insecticide α-cypermethrin (ACYP) in the milk of lactating donkeys following pour-on treatment. Milk was collected from animals (n = 7) before the treatment and at 12, 24, 36, 48, 60, 72 and 84 h post-treatment. The last sampling was taken 7 days post-treatment (168 h). Milk samples were analysed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The analytical method was validated following requirements of Commission Decision 2002/657/EC. All samples showed levels of ACYP below the maximum residue limit (MRL) of 20 μg kg^?1 established for bovine milk (Commission Regulation (EU) No. 37/2010). The results demonstrate that there is minimal partitioning of ACYP into milk in lactating donkeys from pour-on treatment.     

Determination of bixin and norbixin in meat using liquid chromatography and photodiode array detection

The development of an analytical method that enables routine analysis of annatto dye, specifically bixin and norbixin, in meat tissue is described. Liquid-solid extraction was carried out using acetonitrile. Analysis was by HPLC with photodiode array detection using two fixed wavelengths (458 and 486 nm). The possibilities of ion trap mass spectrometry (MS) were also assessed. Method performance characteristics, according to Commission Decision 2002/657/EC, were determined, with recoveries between 99 and 102% and calibration curves being linear in the 0.5–10 mg kg^?1 range. The limit of quantification was 0.5 mg kg^?1.     

Residue analysis of tetracyclines in poultry muscle: shortcomings revealed by a proficiency test.

A proficiency test for tetracycline drug residues in poultry muscle was organized according to the guidelines of International Laboratory Accreditation Cooperation (ILAC) ILAC-G13:2000 (2000). For the proficiency test, three test materials were prepared. The homogeneity and stability of the materials during the study were demonstrated. Sixteen laboratories accepted the invitation to participate in the proficiency test; 11 laboratories reported results within the time frame of the study. Most notably, only four of the participating laboratories complied with the definition of the maximum residue limit (MRL) concerning the inclusion of 4-epimers as stated in European Commission Regulation 281/96 (1996). Most participants reported values for the decision limit (CCalpha) and detection capability (CCbeta) and hence were already in compliance with European Commission 2002/657/EC (2002) for this aspect of method validation. However, some CCalpha and CCbeta values were not in agreement with the actual within-laboratory reproducibility calculated from the results reported in this proficiency test. Although most laboratories obtained satisfactory results, it is clear that an effort is needed to include 4-epiOTC, 4-epiTC and 4-epiCTC in the analytical methods. Moreover, reconsideration of values determined for CCalpha and CCbeta with respect to their accuracy may be necessary in some cases.     

Optimisation and validation of an HPLC method for determination of polycyclic aromatic hydrocarbons (PAHs) in mussels

A method for determination of 11 polycyclic aromatic hydrocarbons (PAHs) in mussels (Mytilus galloprovincialis), using HPLC coupled to a fluorescence detector, has been optimised and validated, according to European Community rules. Sample preparation involves alkaline digestion of mussel tissue, liquid–liquid extraction of organic compounds and solid phase clean-up. Accuracy and precision of the method were determined by a validation study, carried out to demonstrate that the method is useful for both screening purposes and confirmation. Commission Regulation (EC) No. 2007/333/EC stated the performance criteria for the analysis of the only benzo[a]pyrene (BaP) in food products of animal origin, since BaP is the most studied PAH. We extended the BaP analysis performance criteria to other 10 toxic PAHs (listed in Commission Recommendation (EC) No. 2005/108/EC) and the validation study was performed also in agreement with Commission Decision (EC) No. 2002/657/EC. The method was applied to investigate 27 mussel samples from actively producing shellfish plants located in Campania (Italy) and variable levels of PAHs were detected ranging from <0.2 to 16 μg/kg wet weight.