Development and Application of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Analysis of 20 Perfluoroalkyl Substances in Fruit and Vegetables at Sub-Parts-per-Trillion Levels.

In response to the European Food Safety Authority's establishment of a tolerable weekly intake (TWI) for the sum of PFOA, PFNA, PFHxS, and PFOS, a method was developed to quantify and confirm 20 PFASs at the sub-parts-per-trillion level in fruit and vegetables. Improved sensitivity was achieved by (i) increasing the sample intake, (ii) decreasing the solvent volume in the final extract, and (iii) using a highly sensitive mass spectrometer. Except for PFTrDA, target PFASs could be quantitatively determined with an apparent recovery of 90-119%, limits of quantitation down to 0.5 ng/kg, and a relative standard deviation under within-laboratory reproducibility conditions of <28%. The method was successfully applied to 215 fruit and vegetable samples obtained from local grocery stores and markets. Leafy vegetables prove to be the main vegetable category responsible to PFAS exposure, mainly of PFOA, followed by PFHpA and PFHxA.

Extended period of selection for antimicrobial resistance due to recirculation of persistent antimicrobials in broilers.

OBJECTIVES: Antimicrobials can select for antimicrobial-resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable, recirculation of subinhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens. METHODS: The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (unstable), doxycycline or enrofloxacin (stable). Antimicrobials were extracted from the faecal samples and were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism. RESULTS: Up to 37 days after treatment, doxycycline and enrofloxacin had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the amoxicillin treatment. The amoxicillin treatment showed a significant difference (P ≤ 0.01 and P ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic resistance and a significant difference (P ≤ 0.05 and P ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, enrofloxacin treatment resulted in a complete non-WT E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations. CONCLUSIONS: Based on our findings, we suggest that persistence of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.

Comparison of Compound Identification Tools Using Data Dependent and Data Independent High-Resolution Mass Spectrometry Spectra.

Liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS) is a frequently applied technique for suspect screening (SS) and non-target screening (NTS) in metabolomics and environmental toxicology. However, correctly identifying compounds based on SS or NTS approaches remains challenging, especially when using data-independent acquisition (DIA). This study assessed the performance of four HRMS-spectra identification tools to annotate in-house generated data-dependent acquisition (DDA) and DIA HRMS spectra of 32 pesticides, veterinary drugs, and their metabolites. The identification tools were challenged with a diversity of compounds, including isomeric compounds. The identification power was evaluated in solvent standards and spiked feed extract. In DDA spectra, the mass spectral library mzCloud provided the highest success rate, with 84% and 88% of the compounds correctly identified in the top three in solvent standard and spiked feed extract, respectively. The in silico tools MSfinder, CFM-ID, and Chemdistiller also performed well in DDA data, with identification success rates above 75% for both solvent standard and spiked feed extract. MSfinder provided the highest identification success rates using DIA spectra with 72% and 75% (solvent standard and spiked feed extract, respectively), and CFM-ID performed almost similarly in solvent standard and slightly less in spiked feed extract (72% and 63%). The identification success rates for Chemdistiller (66% and 38%) and mzCloud (66% and 31%) were lower, especially in spiked feed extract. The difference in success rates between DDA and DIA is most likely caused by the higher complexity of the DIA spectra, making direct spectral matching more complex. However, this study demonstrates that DIA spectra can be used for compound annotation in certain software tools, although the success rate is lower than for DDA spectra.

Development, validation, and application of a new method for the quantitative determination of monohydrogen-substituted perfluoroalkyl carboxylic acids (H-PFCAs) in surface water.

Per- and polyfluoroalkyl substances (PFASs) are a large and diverse class of chemicals. While some have been phased out internationally due to concerns over their human and environmental health risks, novel alternative PFASs continue to be manufactured and detected in environmental samples. The occurrence and fate of these alternatives remain poorly understood. The present study investigated the occurrence of an emerging class of PFAS alternative, the monohydrogen-substituted perfluoroalkyl carboxylic acids (H-PFCAs), in conjunction with the more well-known PFCAs. A weak anion exchange solid phase extraction-liquid chromatography tandem mass spectrometry method for quantitative determination of H-PFCAs in surface water was developed, validated, and applied on samples collected from the Netherlands. To improve chromatography, especially for short-chain (H-)PFCAs, an ion-pairing agent, tetrabutylammonium hydrogen sulphate, was used. The method was validated for linearity (R2 > 0.99), instrumental detection limits (0.01-0.09 ng/mL), method detection limits (0.03-0.75 ng/mL), matrix effects (<20%), percent absolute- and relative recovery (57-121%), trueness (130-80%), repeatability (<20%), and within-lab reproducibility (<20%). Eleven out of fourteen PFASs showed acceptable results. Application of the newly validated method to surface water throughout the Netherlands revealed trace levels of H-PFCAs (including two new H-PFCAs) and high concentrations of PFCAs.

Systematic assessment of acquisition and data-processing parameters in the suspect screening of veterinary drugs in archive matrices using LC-HRMS.

Monitoring strategies for veterinary drugs in products of animal origin are shifting towards a more risk-based approach. Such strategies not only target a limited number of predefined .substances but also facilitate detection of unexpected substances. By combining the use of archive matrices such as feather meal with suspect-screening methods, early detection of new hazards in the food and feed industry can be achieved. Effective application of such strategies is hampered by complex data interpretation and therefore, targeted data analysis is commonly applied. In this study, the performance of a suspect-screening data processing workflow using a suspect list or the online spectral database mzCloudTM was explored to facilitate detection of veterinary drugs in archive matrices. Data evaluation parameters specifically investigated for application of a suspect list were mass tolerance and the addition or omission of retention times. Application of a mass tolerance of 1.5 ppm leads to an increase in the number of false positives, as does omission of retention times in the suspect list. Different acquisition modes yielding different qualities of MS2 data were studied and proved to be a critical factor, where data-dependent acquisition is preferred when matching to the mzCloudTM database. Using this approach, it is possible to search for compounds on a dedicated suspect list based on the exact mass and retention times and, at the same time, detect unexpected compounds without a priori information. A pilot study was conducted and fourteen different antibiotics were detected (and confirmed by MS/MS). Three of these antibiotics were not included in the suspect list. The optimised suspect-screening method proved to be fit for the purpose of finding veterinary drugs in feather meal, which are not in the scope of the current monitoring methods and therefore, it gives added value in the perspective of a risk-based monitoring.

The Next Step to Further Decrease Veterinary Antibiotic Applications: Phytogenic Alternatives and Effective Monitoring; the Dutch Approach.

Antibiotics are used to control infectious diseases in both animals and humans. They can be life-saving compounds but excessive use in animal husbandry leads to the development of antibiotic resistance which can impact the public health. Since similar antibiotics are used in both animal and human healthcare, it is important to reduce the use of antibiotics in production animals. In the Netherlands policies have been developed aiming for a decrease of antibiotic usage in animals, and alternatives to antibiotics are investigated. Currently, a one-on-one relationship between farmer and veterinarian is successfully implemented and (national) registration of antibiotic usage is mandatory. Unfortunately, after a 70% decrease in antibiotic usage since 2009, this decrease is now stagnating in most sectors. Innovative strategies are required to facilitate a further reduction. One promising option is a focus on farm management and natural alternatives to antibiotics. The Dutch government has invested in the spread of knowledge of natural remedies and good animal management to support animal health via so called Barnbooks for farmers and veterinarians. Another option is the analysis of on-farm antibiotic use to prevent unregistered applications. New (bio)analytical strategies to monitor the correct and complete registration of antibiotic usage have been developed and trial-tested in the Netherlands. Such strategies support a risk-based monitoring and allow effective selection of high-risk (high antibiotic use or illegal antibiotic) users. Both effective monitoring and the availability and knowledge of alternatives is a prerequisite to achieve a further significant decrease in antibiotic veterinary usage.

A single method to analyse residues from five different classes of prohibited pharmacologically active substances in milk.

In the European Union, the use of veterinary drugs belonging to the A6 group is prohibited in food-producing animals according to Commission Regulation (EU) No. 2010/37. The aim of this study was to improve the analytical control strategy by developing a single method to analyse residues of prohibited pharmacologically active substances in milk. For this, a single method was developed to analyse 16 prohibited pharmacologically active substances belonging to five different substance classes at required or recommended levels: nitroimidazoles at 3 µg kg-1, nitrofurans at 0.5 µg kg-1, chloramphenicol at 0.1 µg kg-1, dapsone at 5 µg kg-1 and chlorpromazine at 1 µg kg-1. Milk sample preparation started with an acid hydrolysis combined with a derivatisation. These steps were followed by a clean-up consisting of a dispersive solid-phase extraction and a liquid-liquid extraction. Finally, the sample extracts were analysed by liquid chromatography combined with tandem mass spectrometry, operating alternately in the positive and negative mode. The method was fully validated according to Commission Decision 2002/657/EC for bovine milk and additionally validated for caprine milk. The validation proved that the method is highly effective to detect and confirm all 16 substances in bovine and caprine milk and, additionally to quantify 15 of these substances in bovine milk and 13 of these substances in caprine milk. This study resulted in a new multi-class method to detect, quantify and confirm the identity of 16 prohibited pharmacologically active substances belonging to five different substance classes in two types of milk.

A strategy to determine the fate of active chemical compounds in soil; applied to antimicrobially active substances.

Data on the fate of chemical substances in the environment after e.g. manure application is mandatory input for risk assessment in perspective of a more circular biobased economy. Such fate studies include a persistence study to determine a half-life value and a mobility study. It is recognized that not only the native substance should be considered, but that also degradation products should be included that might exert a similar effect as the native substance. We report a tiered fate study strategy that starts with a persistence study. For non-persistent substances a study is performed to determine if degradation products have a similar effect as the native compound. If so, a procedure using high resolution mass spectrometry is suggested to identify the potentially active degradation products. Based on the outcomes, substances are divided into three categories: (I) persistent, (II) degradable to inactive products or (III) degradable to active products. Even though the priority is with category I and III, for all substances and possible degradation products a mobility study is proposed. The fate strategy is successfully applied to ten antimicrobially active substances originating from the tetracyclines, sulfonamides, diaminopyrimidines, fluoroquinolones, macrolides and lincosamides. The fluoroquinolones, tetracyclines and trimethoprim were relatively persistent. The sulfonamides, macrolides and lincomycin (the latter also depending on soil type) degraded relatively quickly. Tylosin A proved to degrade to antimicrobially active degradation products which were tentitatively identified as tylosin C, tylosin A acid, tylosin B acid and tylosin C acid.

Non-targeted identification of per- and polyfluoroalkyl substances at trace level in surface water using fragment ion flagging.

The extent of unidentified Per- and Poly-fluoroalkyl substances (PFASs) found in environmental samples has led to the development of non-targeted screening methods. The study presented here reports the use of liquid chromatography hyphenated with high resolution mass spectrometry to detect and identify unknown and unexpected PFASs by fragment ion flagging (FIF). By exploring all ion fragmentation spectra for several characteristic fragments including CnF2n+1-, CnF2n-1-, CnF2n-3-, CnF2n-7-, CnF2n-11- and CnF2n+1O- the presence of widely different PFAS species can be anticipated without the need for targeted screening methods. These fragments are then related to their precursor ion by retention time matching and subsequently identified. With this methodology 40 PFASs were (tentatively) identified in four surface water samples sampled throughout the Netherlands. To the best of the authors' knowledge, four PFASs found through FIF are newly discovered species and have not been mentioned in any database or literature. This methodology eliminates the dependence on commonly reported full scan feature selection techniques such as mass defect filtering, homologous series detection and intensity threshold filtering, allowing the identification of PFASs at trace levels. Additionally, eight of the (tentatively) identified PFASs are not part of homologous series, stressing the shortcomings of commonly reported non-targeted PFASs screening methods and demonstrating the importance of more effective identification strategies such as FIF. Moreover, we like to emphasise that this approach is applicable to real-life environmental samples with PFASs at background concentration levels.

The effective design of sampling campaigns for emerging chemical and microbial contaminants in drinking water and its resources based on literature mining.

As well as known contaminants, surface waters also contain an unknown variety of chemical and microbial contaminants which can pose a risk to humans if surface water is used for the production of drinking water. To protect human health proactively, and in a cost-efficient way, water authorities and drinking water companies need early warning systems. This study aimed to (1) assess the effectiveness of screening the scientific literature to direct sampling campaigns for early warning purposes, and (2) detect new aquatic contaminants of concern to public health in the Netherlands. By screening the scientific literature, six example contaminants (3 chemical and 3 microbial) were selected as potential aquatic contaminants of concern to the quality of Dutch drinking water. Stakeholders from the Dutch water sector and various information sources were consulted to identify the potential sources of these contaminants. Based on these potential contamination sources, two sampling sequences were set up from contamination sources (municipal and industrial wastewater treatment plants), via surface water used for the production of drinking water to treated drinking water. The chemical contaminants, mycophenolic acid, tetrabutylphosphonium compounds and Hexafluoropropylene Oxide Trimer Acid, were detected in low concentrations and were thus not expected to pose a risk to Dutch drinking water. Colistin resistant Escherichia coli was detected for the first time in Dutch wastewater not influenced by hospital wastewater, indicating circulation of bacteria resistant to this last-resort antibiotic in the open Dutch population. Four out of six contaminants were thus detected in surface or wastewater samples, which showed that screening the scientific literature to direct sampling campaigns for both microbial and chemical contaminants is effective for early warning purposes.

Improving the chromatographic selectivity of ß-lactam residue analysis in milk using phenyl-column chemistry prior to detection by tandem mass spectrometry.

Analyte isobaric interferences can limit the development of a comprehensive analytical method for the quantitative liquid chromatography-tandem mass spectrometry profiling of an important cohort of veterinary drugs. In this work, a selective chromatographic separation was developed for the analysis of 32 ß-lactam antibiotic residues (12 penicillins, 14 cephalosporins, five carbapenems and faropenem) in milk samples. A range of analytical columns with different stationary phases and mobile phases were evaluated for retention and separation of the ß-lactam compounds. Results showed that, among the columns tested, only phenyl-hexyl could adequately separate ampicillin from cephalexin and amoxicillin from cefadroxil, which had shown isobaric interferences on a number of stationary phases. Chromatography was performed using a water/acetonitrile binary gradient with formic acid and ammonium acetate. The ß-lactam residues were extracted from the milk samples using a water:acetonitrile solution and purified by C18 dispersive solid-phase extraction (d-SPE) clean-up, followed by concentration under nitrogen and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) determination. Analytes were monitored in positive electrospray ionisation mode (ESI(+)). Possible interfering matrix effects were overcome by using 13 internal standards. The method was fully validated according to 2002/657/EC guidelines, showing satisfactory performance characteristics. Under within-laboratory reproducibility conditions, trueness and precision ranged from 91 to 130% and from 1.4 to 38.6%, respectively. Decision limits (CCα) were in the range 2.1-133 µg kg-1. Limits of detection (LODs) and quantitation (LOQs) ranged between 0.0090 and 1.5 µg kg-1 and from 0.030 to 5.0 µg kg-1, respectively.

The vertical transmission of antibiotic residues from parent hens to broilers.

Imprudent and superfluous use of antibiotics contributes to the selection of resistant bacteria, which is a large threat to human health. Therefore analytical procedures have been implemented in the poultry production sector to check if antibiotic treatments are registered, aiming to achieve more prudent use of antibiotics. These methods rely on the analysis of feathers, a matrix in which antibiotic residues persist. However, other routes besides direct administration, through which poultry feathers could contain antibiotic residues, should also be taken into account. In this research the vertical transmission from parent hen to broiler was investigated through a controlled animal study for the antibiotics enrofloxacin, doxycycline and sulfachlorpyridazine. Vertical transmission was observed for all antibiotics to both egg and egg shell. Also it is demonstrated that the transferred antibiotics from parent hen to chick are subsequently excreted via the chick's droppings. Through this route, the broilers' environment is contaminated. If eggs are hatched that were taken during treatment of the parent hen, this indirect route and/or the direct vertical transmission can eventually result in the detection of low concentrations of antibiotic residues in the broilers' feathers at greater age: <50 µg kg-1 for freely extractable residues and <10 µg kg-1 for non-freely extractable residues. No antibiotics were detected in the broilers' muscle or kidney from 4 weeks of age. This research provides relevant information regarding the possible amount of residues originating from vertical transmission when monitoring matrices such as feathers and broiler droppings in order to stimulate correct use and registration of antibiotics in the poultry sector.

A new extraction procedure to abate the burden of non-extractable antibiotic residues in manure.

Through agricultural soil fertilization using organic manure, antibiotic residues can accumulate in the environment. In order to assess the risks of environmental pollution by veterinary drugs, monitoring of manure for antibiotic residues is necessary. As manure is a complex matrix, extraction of antibiotics proved to be challenging. In this study, 24 extraction solvents were assessed for the extraction of residues from manure representing ten antibiotics from the antibiotic classes tetracyclines, quinolones, macrolides, lincosamides and sulfonamides. Especially for the tetracyclines and quinolones the extraction solvent selection is critical, due to high fractions of non-extractable residues especially when using aqueous solvents (62-77% and 90-95% respectively when using milli-Q water). In contrast, sulfonamides can effectively be extracted with aqueous solvents. Overall, 0.125% trifluoroacetic acid in acetonitrile in combination with McIlvain-EDTA buffer proved to be the most effective extraction solvent. A longitudinal study pointed out that most antibiotics bind to solid manure particles instantaneously after addition. Trimethoprim is an exception, but because by using the optimal extraction solvent, the optimum fraction of bound residues is desorbed, this does not hamper quantitative analysis when using spiked manure quality control samples. Based on these new insights, the current in-house multi-residue LC-MS/MS method for manure analysis, containing 48 antibiotics, was revised, additionally validated and applied to 34 incurred manure samples.

A study of the origin of chloramphenicol isomers in honey.

Due to the unexpected detection of chloramphenicol isomer residues in honey, we have studied the hypothesis of unauthorized or unintended use of unregistered veterinary drug preparations. First, we have investigated honey samples in which a discrepancy was observed between the results of the immunological screening methods and the confirmatory liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. In all samples, previously identified to be contaminated with the banned antibiotic chloramphenicol according to LC-MS/MS only, the presence of dextramycin (SS-para isomer of chloramphenicol) was detected by chiral LC-MS/MS. The source of dextramycin in honey was investigated by studying the preparations utilized in apiaries from which the above non-compliant honey samples have been received. In all these preparations (beehive strips applied against the mite Varroa destructor) chloramphenicol was detected in the concentrations ranging from 33 to 34,400 µg kg-1 . Chiral LC-MS/MS demonstrated the presence of chloramphenicol and dextramycin in different ratios, and it was concluded that these preparations can be the source of chloramphenicol and dextramycin residues in honey. These preparations were of foreign production and are not officially registered in accordance with current legislation.

Novel Selectivity-Based Forensic Toxicological Validation of a Paper Spray Mass Spectrometry Method for the Quantitative Determination of Eight Amphetamines in Whole Blood.

Paper spray tandem mass spectrometry is used to identify and quantify eight individual amphetamines in whole blood in 1.3 min. The method has been optimized and fully validated according to forensic toxicology guidelines, for the quantification of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methylamphetamine (MDMA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), para-methoxyamphetamine (PMA), para-methoxymethamphetamine (PMMA), and 4-fluoroamphetamine (4-FA). Additionally, a new concept of intrinsic and application-based selectivity is discussed, featuring increased confidence in the power to discriminate the amphetamines from other chemically similar compounds when applying an ambient mass spectrometric method without chromatographic separation. Accuracy was within ±15% and average precision was better than 15%, and better than 20% at the LLOQ. Detection limits between 15 and 50 ng/mL were obtained using only 12 µL of whole blood. Graphical abstract ᅟ.

Non-targeted workflow for identification of antimicrobial compounds in animal feed using bioassay-directed screening in combination with liquid chromatography-high resolution mass spectrometry.

A non-targeted workflow is reported for the isolation and identification of antimicrobial active compounds using bioassay-directed screening and LC coupled to high-resolution MS. Suspect samples are extracted using a generic protocol and fractionated using two different LC conditions (A and B). The behaviour of the bioactive compound under these different conditions yields information about the physicochemical properties of the compound and introduces variations in co-eluting compounds in the fractions, which is essential for peak picking and identification. The fractions containing the active compound(s) obtained with conditions A and B are selected using a microbiological effect-based bioassay. The selected bioactive fractions from A and B are analysed using LC combined with high-resolution MS. Selection of relevant signals is automatically carried out by selecting all signals present in both bioactive fractions A and B, yielding tremendous data reduction. The method was assessed using two spiked feed samples and subsequently applied to two feed samples containing an unidentified compound showing microbial growth inhibition. In all cases, the identity of the compound causing microbiological inhibition was successfully confirmed.

The analysis of tetracyclines, quinolones, macrolides, lincosamides, pleuromutilins, and sulfonamides in chicken feathers using UHPLC-MS/MS in order to monitor antibiotic use in the poultry sector.

In The Netherlands, all antibiotic treatments should be registered at the farm and in a central database. To enforce correct antibiotic use and registration, and to enforce prudent use of antibiotics, there is a need for methods that are able to detect antibiotic treatments. Ideally, such a method is able to detect antibiotic applications during the entire lifespan of an animal, including treatments administered during the first days of the animals' lives. Monitoring tissue, as is common practice, only provides a limited window of opportunity, as residue levels in tissue soon drop below measurable quantities. The analysis of feathers proves to be a promising tool in this respect. Furthermore, a qualitative confirmatory method was developed for the analyses of six major groups of antibiotics in ground chicken feathers, aiming for a detection limit as low as reasonably possible. The method was validated according to Commission Decision 2002/657/EC. All compounds comply with the criteria and, as a matter of fact, 58% of the compounds could also be quantified according to regulations. Additionally, we demonstrated that a less laborious method, in which whole feathers were analyzed, proved successful in the detection of applied antibiotics. Most compounds could be detected at levels of 2 µg kg-1 or below with the exception of sulfachloropyridazine, tylosin, and tylvalosin. This demonstrates the effectiveness of feather analysis to detect antibiotic use to allow effective enforcement of antibiotic use and prevent the illegal, off-label, and nonregistered use of antibiotics.

A global inter-laboratory study to assess acquisition modes for multi-compound confirmatory analysis of veterinary drugs using liquid chromatography coupled to triple quadrupole, time of flight and orbitrap mass spectrometry.

According to EU legislation a confirmatory method used for residue analysis should be able to confirm the identity of a compound beyond reasonable doubt. To provide an adequate instrumental set-up, Commission Decision 2002/657/EC introduced the concept of "identification points". A second aspect to assure unequivocal confirmation, is the establishment of ion ratio and retention time criteria. Currently, the gold standard for confirmatory analysis of most veterinary drug residues is liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) acquisition mode, isolating one precursor ion and monitoring two a priori selected product ions, yielding 4 identification points. We comprehensively evaluated the use of different low and high resolution LC-MS(/MS) techniques and acquisition modes with respect to the selectivity of 100 veterinary drugs in liver, muscle and urine extracts aiming to critically review the currently established identification points system. A comparison among MS/MS in SRM mode with high resolution mass spectrometry (HRMS) in full scan, all ion fragmentation and targeted MS/MS was made based on a unique inter-laboratory study, which comprises 21 laboratories from four different continents and equipment from all major vendors. In total 186 samples were analysed yielding results for 9282 analyte/matrix combinations. It was observed that the false positive rate approximately doubles if no ion ratio criterion is applied indicating that this criterion is important to prevent false positive results. Full scan HRMS analysis, only monitoring the molecular ion and allowing a ±5 ppm mass tolerance is, in general, less selective than low resolution MS/MS using SRM, and thus full scan alone is considered not sufficient for confirmatory analysis. Furthermore, even though the number of data on all ion fragmentation and targeted MS/MS at high resolution was limited, based on the data obtained, it was observed that the acquisition mode as well as the mass resolution needed, very much depend on the matrix and the compound itself. For complex matrix extracts and non-selective compounds (worst-case situation), only targeted MS/MS, monitoring the precursor ion and a single product ion in HR-MS using a maximum of ±5 ppm mass deviation, leads to comparable selectivity and false positive and negative rate as SRM monitoring two product ions in LR-MS. We conclude that the currently applied identification point system as established in commission decision 2002/657/EC should be revised with respect to the allocation of identification points.

Distribution of chloramphenicol to tissues, plasma and urine in pigs after oral intake of low doses.

Toxic effects of chloramphenicol in humans caused the ban for its use in food-producing animals in the EU. A minimum required performance level (MRPL) was specified for chloramphenicol at 0.3 µg kg(-1) for various matrices, including urine. In 2012, residues of chloramphenicol were found in pig urine and muscle without signs of illegal use. Regarding its natural occurrence in straw, it was hypothesised that this might be the source, straw being compulsory for use as bedding material for pigs in Sweden. Therefore, we investigated if low daily doses of chloramphenicol (4, 40 and 400 µg/pig) given orally during 14 days could result in residues in pig tissues and urine. A dose-related increase of residues was found in muscle, plasma, kidney and urine (showing the highest levels), but no chloramphenicol was found in the liver. At the lowest dose, residues were below the MRPL in all tissues except in the urine. However, in the middle dose, residues were above the MRPL in all tissues except muscle, and at the highest dose in all matrices. This study proves that exposure of pigs to chloramphenicol in doses occurring naturally in straw could result in residues above the MRPL in plasma, kidney and especially urine.

A critical assessment of the performance criteria in confirmatory analysis for veterinary drug residue analysis using mass spectrometric detection in selected reaction monitoring mode.

Besides the identification point system to assure adequate set-up of instrumentation, European Commission Decision 2002/657/EC includes performance criteria regarding relative ion abundances in mass spectrometry and chromatographic retention time. In confirmatory analysis, the relative abundance of two product ions, acquired in selected reaction monitoring mode, the ion ratio should be within certain ranges for confirmation of the identity of a substance. The acceptable tolerance of the ion ratio varies with the relative abundance of the two product ions and for retention time, CD 2002/657/EC allows a tolerance of 5%. Because of rapid technical advances in analytical instruments and new approaches applied in the field of contaminant testing in food products (multi-compound and multi-class methods) a critical assessment of these criteria is justified. In this study a large number of representative, though challenging sample extracts were prepared, including muscle, urine, milk and liver, spiked with 100 registered and banned veterinary drugs at levels ranging from 0.5 to 100 µg/kg. These extracts were analysed using SRM mode using different chromatographic conditions and mass spectrometers from different vendors. In the initial study, robust data was collected using four different instrumental set-ups. Based on a unique and highly relevant data set, consisting of over 39 000 data points, the ion ratio and retention time criteria for applicability in confirmatory analysis were assessed. The outcomes were verified based on a collaborative trial including laboratories from all over the world. It was concluded that the ion ratio deviation is not related to the value of the ion ratio, but rather to the intensity of the lowest product ion. Therefore a fixed ion ratio deviation tolerance of 50% (relative) is proposed, which also is applicable for compounds present at sub-ppb levels or having poor ionisation efficiency. Furthermore, it was observed that retention time shifts, when using gradient elution, as is common practice nowadays, are mainly observed for early eluting compounds. Therefore a maximum retention time deviation of 0.2 min (absolute) is proposed. These findings should serve as input for discussions on the revision of currently applied criteria and the establishment of a new, globally accepted, criterion document for confirmatory analysis. Copyright © 2016 John Wiley & Sons, Ltd.