Interlaboratory Study to Evaluate a Testing Protocol for the Safety of Food Packaging Coatings.

According to European regulations, migration from food packaging must be safe. However, currently, there is no consensus on how to evaluate its safety, especially for non-intentionally added substances (NIAS). The intensive and laborious approach, involving identification and then quantification of all migrating substances followed by a toxicological evaluation, is not practical or feasible. In alignment with the International Life Sciences Institute (ILSI) and the European Union (EU) guidelines on packaging materials, efforts are focused on combining data from analytics, bioassays and in silico toxicology approaches for the risk assessment of packaging materials. Advancement of non-targeted screening approaches using both analytical methods and in vitro bioassays is key. A protocol was developed for the chemical and biological screening of migrants from coated metal packaging materials. This protocol includes guidance on sample preparation, migrant simulation, chemical analysis using liquid chromatography (LC-MS) and validated bioassays covering endocrine activity, genotoxicity and metabolism-related targets. An inter-laboratory study was set-up to evaluate the consistency in biological activity and analytical results generated between three independent laboratories applying the developed protocol and guidance. Coated packaging metal panels were used in this case study. In general, the inter-laboratory chemical analysis and bioassay results displayed acceptable consistency between laboratories, but technical differences led to different data interpretations (e.g., cytotoxicity, cell passages, chemical analysis). The study observations with the greatest impact on the quality of the data and ultimately resulting in discrepancies in the results are given and suggestions for improvement of the protocol are made (e.g., sample preparation, chemical analysis approaches). Finally, there was agreement on the need for an aligned protocol to be utilized by qualified laboratories for chemical and biological analyses, following best practices and guidance for packaging safety assessment of intentionally added substances (IAS) and NIAS to avoid inconsistency in data and the final interpretation.

Analysis of ethylene oxide in ice creams manufactured with contaminated carob bean gum (E410).

Residues of ethylene oxide (EO), a banned fumigant in the EU, were found at amounts above the maximum residue limit (MRL) in carob (locust) bean gum (additive E410). The pesticide entered the food chain via stabiliser blends that are used as minor ingredients in the manufacture of ice cream. Consequently, all products that contained the non-compliant ingredient were withdrawn or recalled in several countries across the EU, in most cases irrespective of whether the pesticide residue was detectable or not in the final product. This is the first report of a reliable method to determine EO and its metabolite/marker compound 2-chloroethanol (2-CE), either together or independently in ice cream, with a limit of quantification at 0.01 mg EO/kg and recovery in the range of 87-104% across the levels investigated (0.01, 0.02 and 0.06 mg EO/kg). The method applies QuEChERS extraction and isotope dilution gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). High resolution mass spectrometry (HRMS) confirmed the specificity of low mass ions. Data on the stability of EO and 2-CE under thermal conditions revealed that 2-CE is relatively stable in an ice cream matrix (ca. 80% recovery of spiked material). Importantly, this study also demonstrates that not EO, but 2-CE is the predominant analyte detected in the contaminated samples, which is new information of significance in terms of the overall risk assessment of EO in foodstuffs.

Screening for chlorinated paraffins in vegetable oils and oil-based dietary supplements by planar solid phase extraction.

Extraction of chlorinated paraffins (CPs) in samples with high fat content is generally performed by a sulfuric acid treatment and liquid-liquid partitioning in n-hexane, followed by clean-up by column chromatography. Analysis and quantitation are commonly carried out via gas chromatography coupled with electron capture negative ionization mass spectrometry (GC/ECNI-MS) when low- and high-resolution MS (LRMS and HRMS) are used. In this work, we present a completely different and innovative approach for the determination of CPs by applying the planar solid phase extraction (pSPE) concept for a rapid and selective screening. pSPE offers the concentration of the analytes in a single target zone followed by the densitometric determination of the total CP content. After sulfuric acid treatment and liquid-liquid partition into n-hexane, pSPE was performed on silica gel plates employing a twofold development with cyclohexane/toluene (94:6, v/v) and methylene chloride/n-hexane (9:1, v/v) as mobile phases. CPs were quantified via the visual (VIS) absorption of the target zone after derivatization with o-tolidine, and amounts were calculated as the sum by means of a reference CP. Limits of detection and quantitation for the reference CP were 0.2 and 0.7 mg/kg oil, respectively, and recoveries from different vegetable oils were near 100%. The analysis of total CPs in dietary supplement samples by pSPE-VIS compared to GC/ECNI-HRMS proved the method as a reliable and suitable screening tool providing an appropriate alternative to existing time-consuming and complex methods.

N,N-dimethylpiperidinium (mepiquat): Part 1. Formation in model systems and relevance to roasted food products.

This preliminary study reports for the first time the formation of N,N-dimethylpiperidinium (also termed mepiquat, a plant growth regulator used widely on cereal crops) by Maillard-driven degradation of lysine under dry thermal conditions and in the presence of the naturally occurring alkaloid trigonelline. The heat treatment was carried out at 240°C, and the resulting samples were analysed by liquid chromatography coupled to a high-resolution mass spectrometer (LC-HRMS) operating based upon Orbitrap technology. Results confirmed that lysine undergoes cyclisation by decarboxylative deamination in the presence of a carbonyl source (e.g. reducing sugars) to yield piperidine. Moreover, methyl rearrangement in the presence of trigonelline and under typical temperatures of roasting generates detectable amounts of mepiquat, identified by detailed mass fragmentation studies. The essential role of lysine in the formation of mepiquat was confirmed in a model system using (13)C6-lysine, which showed the expected incorporation of five (13)C atoms into the mepiquat nitrogen heterocycle. These findings are relevant to the potential occurrence of mepiquat in roasted products such as coffee and cereals.

Quantitative high-throughput analysis of 16 (fluoro)quinolones in honey using automated extraction by turbulent flow chromatography coupled to liquid chromatography-tandem mass spectrometry.

A method making use of turbulent flow chromatography automated online extraction with tandem mass spectrometry (MS/MS) was developed for the analysis of 4 quinolones and 12 fluoroquinolones in honey. The manual sample preparation was limited to a simple dilution of the honey test portion in water followed by a filtration. The extract was online purified on a large particle size extraction column where the sample matrix was washed away while the analytes were retained. Subsequently, the analytes were eluted from the extraction column onto an analytical column by means of an organic solvent prior to chromatographic separation and MS detection. Validation was performed at three fortification levels (i.e., 5, 20, and 50 microg/kg) in three different honeys (acacia, multiflower, and forest) using the single-point calibration procedure by means of either a 10 or 25 microg/kg calibrant. Good recovery (85-127%, median 101%) as well as within-day (2-18%, median 6%) and between-day (2-42%, median 9%) precision values was obtained whatever the level of fortification and the analyte surveyed. Due to the complexity of the honey matrix and the large variation of the MS/MS transition reaction signals, which were honey-dependent, the limit of quantification for all compounds was arbitrarily set at the lowest fortification level considered during the validation, e.g., 5 microg/kg. This method has been successfully applied in a minisurvey of 34 honeys, showing ciprofloxacin and norfloxacin as the main (fluoro)quinolone antibiotics administered to treat bacterial diseases of bees. Turbulent flow chromatography coupled to LC-MS/MS showed a strong potential as an alternative method compared to those making use of offline sample preparation, in terms of both increasing the analysis throughput and obtaining higher reproducibility linked to automation to ensure the absence of contaminants in honey samples.

Multi-screening approach to monitor and quantify 42 antibiotic residues in honey by liquid chromatography-tandem mass spectrometry.

A multi-screening approach for monitoring potential chemical contaminants in honey by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed. A total of 42 veterinary drugs (5 tetracyclines, 7 macrolides, 3 aminoglycosides, 8 beta-lactams, 2 amphenicols and 17 sulfonamides) were surveyed with the ultimate goal of unambiguously confirmed and quantified these analytes at a concentration level of 20 microg/kg. A basic sample preparation including four subsequent liquid/liquid extraction steps was necessary to adequately extract the compounds of interest from the honey. The four extracts were injected into the LC-ESI-MS/MS using a stacking injection procedure. Validation of the entire procedure was carried out according to the European Union directive 2002/657/EC at three concentration levels, i.e., 10, 20 and 30 microg/kg. Good performance data were obtained for 37 analytes, out of the 42 studied. Limit of compliance and detection limit were calculated based on an internal limit set at 20 microg/kg for all the compounds and ranged between 24-30 and 27-80 microg/kg, respectively. A limited survey on honeys of different geographical origins has demonstrated that positive honey samples were often contaminated by more than one class of drugs, thus highlighting the usefulness of such multi-screening approach to ensure and warrants the quality of honey.

Evaluation of atmospheric pressure ionization interfaces for quantitative measurement of sulfonamides in honey using isotope dilution liquid chromatography coupled with tandem mass spectrometry techniques.

A comparison was made between electrospray, atmospheric pressure chemical and atmospheric pressure photospray ionizations to evaluate the MS/MS responses of standard sulfonamides and honey spiked samples. The sample preparation entails an acidic hydrolysis followed by a liquid/liquid extraction. Full method validation was realised by LC-APPI-MS/MS. Decision limit and detection capability were calculated for each analyte (at 50 microg/kg) and ranged between 53.6 and 56.9 and 57.5 and 63.2 microg/kg, respectively. Limits of detection and of quantification ranged, respectively, at 0.4-4.5 and 1.2-15.0 microg/kg. Precursor ion scan experiments of m/z 92 were also carried out as a survey experiment, linked with an enhanced product ion scan experiment to potentially identified additional sulfonamides via a library search.

Analysis of tetracyclines in honey by high-performance liquid chromatography/tandem mass spectrometry.

A confirmatory method coupling liquid chromatography to tandem mass spectrometry (LC/MS/MS) is described for the determination of tetracycline, oxytetracycline, doxycycline and chlortetracycline in honey. Demeclocycline, another tetracycline molecule not reported for its usage in honey, was used as internal standard to quantify the four analytes. The sample preparation entails a clean-up on an Oasis HLB solid-phase extraction cartridge and analyses were realised by LC/MS/MS in selected reaction monitoring mode. The stability of tetracyclines was checked under various storage conditions at -20, +4 and +20 degrees C (both under dark and light exposures). Indeed, tetracyclines are not stable molecules and the epimerisation phenomenon was evaluated in this work. Appropriate correction factors of the MS/MS responses of each epimer were studied for each of the four tetracyclines to accurately quantify them. Moreover, the matrix effects encountered during the LC/MS/MS analyses were also studied in spiked experiments from blank honey samples of various geographical origins and different flower types.

Glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency.

The glycan moiety of human recombinant gonadotrophins (r-hFSH, r-hLH, and r-hCG) produced in CHO cell lines has been characterized by a combination of chromatographic and mass spectrometric techniques, including both matrix-assisted laser desorption ionization and electrospray. Two glycan mapping methods have been developed for the three gonadotrophins that allow separation of the glycans according to either their charge or sialylation level or their antennarity. A method was also developed for r-hCG that permits the complete resolution of the N-glycan from the O-glycan species. Whereas the structure found for the N-glycans of the gonadotrophins was in agreement with the complex type model, the structure for an O-glycan of r-hCG, not yet described, has been unambiguously determined using nanoelectrospray ion trap mass spectrometry. Using these two glycan mapping methods, the high level of batch-to-batch consistency achieved for the glycosylation of the three recombinant gonadotrophins in commercial production has been shown. These data demonstrate the tight control that can be achieved in the manufacturing of complex recombinant therapeutic glycoproteins, which is a prerequisite to the delivering of a guaranteed dose of drug from vial to vial, and in turn to ensuring the clinical efficacy of the product.