Evaluation of new safety decontamination approaches at lab scale for recycled highdensity polyethylene (rHDPE) intended for food contact.

BACKGROUND: The increasing use of plastic packaging materials generates concerns related to the environmental problem generated by their waste. As a result, the search for new recycling methodologies to extend the lifecycle of plastic packaging is becoming more important, without forgetting to ensure the safety of these materials. Currently, the use of recycled polyolefins as food contact materials is not widespread yet. This is because the decontamination processes currently available are insufficient to produce clean, safe materials suitable for such applications. This work is focused on the evaluation of the safety of recycled high-density polyethylene (rHDPE), and the search for strategies to achieve its decontamination. RESULTS: To this end, three batches of flakes and three batches of pellets of rHDPE coming from the mechanical recycling of post-consumer milk bottles were analyzed. The analysis of the volatile and semi-volatile compounds present in the samples was carried out using gas chromatography-mass spectrometry (GC-MS), finding a total of 67 compounds. The strategy to achieve the decontamination of flakes and pellets of this material has been based on the application of high temperature and vacuum at lab scale, obtaining a clear decrease in volatile compounds, below 50% of the initial value in most cases when applying 120 °C during 5 h. The migration test performed in the samples (treated and untreated) to different food simulants (10 % ethanol and 3 % acetic acid, 95 % ethanol) revealed also a clear decrease of concentrations of volatiles. SIGNIFICANCE: The findings are highly encouraging, demonstrating substantial progress toward the safe and effective use of rHDPE in specific food packaging applications. This indicates a significant step forward in the potential uses of rHDPE. Nevertheless, the lack of toxicity data for many migrants necessitates additional toxicological testing to obtain a more precise risk assessment.

Development of an analytical method for the determination of mineral oil aromatic hydrocarbons (MOAH) from printing inks in food packaging.

An analysis method was developed to detect chemical markers of mineral oil aromatic hydrocarbons (MOAH) from offset printing inks in food packaging materials. 16 aromatic hydrocarbons were used as target analytes and different solid phase extraction procedures (SPE) and gas chromatography coupled to mass spectrometry (GC-MS) were tested. The concentration range studied was 0.1-7.5 µg g-1 with R2 higher than 0.9963, intraday RSD values below 5 %, RSD values between days lower than 12 %, recoveries higher than 80 %, LOD and LOQ lower than 0.09 µg g-1. Ten of the target analytes were identified in offset printing inks at concentrations between 2.28 and 8.59 µg g-1. Nine of them were also identified in the food packages examined in concentrations ranging from 0.10 to 0.33 µg g-1. These compounds were: methylnaphthalene, 2-methylnaphthalene, biphenyl, 2,6-dimethylnaphthalene, acenaphthene, 3,3',5,5'-tetramethylbiphenyl, 4,6-dimethyldibenzothiophene, 1-methylpyrene, benzo(b)naphtha(1,2-d)thiophene and 9,9'-dimethylfluorene. Mineral oil in food packaging was previously analysed by GC with flame ionization detection (FID).

Atmospheric Solids Analysis Probe (ASAP) and Atmospheric Pressure Gas Chromatography (APGC) coupled to Quadrupole Time of Flight Mass Spectrometry (QTOF-MS) as alternative techniques to trace aromatic markers of mineral oils in food packaging.

The aim of this work was to select and identify the best markers of aromatic hydrocarbon mineral oil (MOAH) in food packaging. For this purpose, a series of mineral oils was initially analysed. Polycyclic Aromatic Hydrocarbons (PAHs) and the alkylated isomers of Methylnaphthalene (MNS), Diisopropylnaphtalene (DIPNs), Dibenzothiophenes (DBTS), Methyldibenzothiophene (MDBTs), Dimethyldibenzothiophenes (DMDBTs) and Benzonaphthiophenes (BNTS) were then explored. Their presence was confirmed by direct analysis of several mineral oils by Atmospheric Solids Analysis Probe Quadrupole-Time of Flight Mass Spectrometry (ASAP-QTOF-MS). Atmospheric Pressure Gas Chromatography Quadrupole-Time of Flight Mass Spectrometry (APGC-QTOF-MS) was used to confirm the markers in different samples of oils, recycled PET (rPET), recycled cardboard and packaging of couscous and semolina to confirm the contamination. 27 markers were found in the mineral oil samples, 22 of them in rPET, 8 in recycled board and no MOAH were found in packaging of couscous and semolina.

Determination of total plasma oxysterols by enzymatic hydrolysis, solid phase extraction and liquid chromatography coupled to mass-spectrometry.

The potential use of cholesterol esterases was tested to avoid alkaline hydrolysis for cleavage of plasma esterified oxysterols. The enzymatic hydrolysis was optimized by testing two sources of enzyme-Pseudomonas and bovine pancreas, presence of surfactants, incubation time and amount of enzyme. Free forms of 4ß-, 7-, 24-, 25- and 27-hydroxycholesterol (HC) as well 7-ketocholesterol (7-KC) were analyzed by liquid chromatography and mass-spectrometry using the deuterated internal standard, 25-HC(d6). Enzymatic hydrolysis was more effective using the Pseudomonas enzyme and in presence of surfactants. Compared to alkaline hydrolysis, it generated a cleaner chromatographic baseline and better recovery of the internal standard. Oxysterols were assayed with detection limits between 7 and 31 pg/mL. Interassay coefficients of variation were lower than 10% and extraction recovery efficiencies, higher than 90%. The procedure was used to characterize plasma levels of Cyp7b1-deficient rat, where it showed increased plasma levels of 7, 24 and 25-HC. Due to the low volume of sample required, it may be used in other animal models, particularly rodents, as well as in pediatric samples where sample amount is always a problem. Thus, the proposed new method offers mild enzymatic processing that greatly facilitates oxysterol determinations to delineate their role in physiopathology.

Toxic compounds from tobacco in placenta samples analyzed by UPLC-QTOF-MS.

Polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines (TSNAs) and aromatic amines are carcinogens present in cigarette smoke. These compounds are distributed in the human body and they could be transferred to the foetus during the pregnancy. Placenta is the main barrier to these toxic compounds and its study is the objective of this work. A method based on solid-phase extraction (SPE) with ultra-performance liquid chromatography-tandem quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS) has been examined and optimized for the analysis of 9 target analytes (4 tobacco-specific nitrosamines and some of their metabolites, 3 aromatic amines, nicotine and cotinine) in 26 placenta samples from smoking and non-smoking women. Limits of detection (LODs) were in the range of 3-27ng/g of placenta. Nicotine, cotinine, N-nitrosoanatabine (NAT) and 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK) metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) were detected in the placenta samples of smoking woman. Nicotine was detected in 3 out of 8 placentas from smoking women, always below the limit of quantification (88ng/g). This could be expected, as the half-life of nicotine in the body is limited to about 0.5-3h. Cotinine, the main metabolite from nicotine, was detected in all placentas from smoking women at concentrations between 17.2 and 61.8ng/g, reaching the highest values for those women that smoked the highest number of cigarettes. NAT and NNAL were detected in all placentas from smoking women, always below the limit of quantification (40ng/g and 33ng/g respectively).

Safety by design of printed multilayer materials intended for food packaging.

Printing inks are commonly used in multilayer plastics materials used for food packaging, and compounds present in inks can migrate to the food either by diffusion through the multilayers or because of set-off phenomena. To avoid this problem, the right design of the packaging is crucial. This paper studies the safety by design of multilayer materials. First, the migration from four different multilayers manufactured using polyethylene terephthalate (PET), aluminium (Al) and polyethylene (PE) was determined. The structural differences among materials such as the presence of inks or lacquer coatings as well as the differences in layers position allowed the study of a safety-by-design approach. Sixty-nine different compounds were detected and identified; 49 of them were not included in the positive list of Regulation EU/10/2011 or in Swiss legislation and 15 belong to Cramer class III, which means that they have a theoretical high toxicity. Some of the compounds related to ink composition were pyrene, a compound commercially used to make dyes and dye precursors and the antioxidant Irganox 1300. The application of external lacquers decreased the concentration of some migrants but also brought the potential for new migrants coming from its composition. A final risk assessment of the material allowed evaluating food safety for different food simulants and confirm it.

Direct screening of tobacco indicators in urine and saliva by Atmospheric Pressure Solid Analysis Probe coupled to quadrupole-time of flight mass spectrometry (ASAP-MS-Q-TOF-).

A new screening method has been explored for direct analysis of tobacco smoke biomarkers in biological matrices (i.e., saliva and urine). Single run analysis using Atmospheric pressure Solid Analysis Probe (ASAP) and high resolution mass spectrometry with quadrupole and time of flight detector has been applied directly to some biological samples (i.e., urine and saliva), providing a fast, efficient and sensitive method of identification. The method has been applied to saliva and urine samples from heavy tobacco smokers for exposure studies. Nicotine itself, nicotine metabolites (i.e., cotinine, trans-3'-hydroxycotinine, nicotine-N-glucuronide) and other related tobacco smoke toxic compounds (i.e., NNK 4-[methyl(nitroso)amino]-1-(3-pyridinyl)-1-butanone, anatabine) were found in the analyzed samples. The identification of compounds was confirmed by ultrahigh performance liquid chromatography with MS-triple quadrupole detector after sample treatment. Different temporal trends and biomarkers behavior have been found in time series related samples. Both methods are compared for screening of these biological matrices.

Atmospheric pressure solid analysis probe coupled to quadrupole-time of flight mass spectrometry as a tool for screening and semi-quantitative approach of polycyclic aromatic hydrocarbons, nitro-polycyclic aromatic hydrocarbons and oxo-polycyclic aromatic hydrocarbons in complex matrices.

A new screening and semi-quantitative approach has been developed for direct analysis of polycyclic aromatic hydrocarbons (PAHs) and their nitro and oxo derivatives in environmental and biological matrices using atmospheric pressure solid analysis probe (ASAP) quadrupole-time of flight mass spectrometry (Q-TOF-MS). The instrumental parameters were optimized for the analysis of all these compounds, without previous sample treatment, in soil, motor oil, atmospheric particles (ashes) and biological samples such as urine and saliva of smokers and non-smokers. Ion source parameters in the MS were found to be the key parameters, with little variation within PAHs families. The optimized corona current was 4 µA, sample cone voltage 80 V for PAHs, nitro-PAHs and oxo-PAHs, while the desolvation temperatures varied from 300°C to 500°C. The analytical method performance was checked using a certified reference material. Two deuterated compounds were used as internal standards for semi-quantitative purposes together with the pure individual standard for each compound and the corresponding calibration plot. The compounds nitro PAH 9-nitroanthracene and oxo-PAH 1,4-naphthalenedione, were found in saliva and urine in a range below 1 µg/g while the range of PAHs in these samples was below 2 µg/g. Environmental samples provided higher concentration of all pollutants than urine and saliva.

Online solid-phase extraction-liquid chromatography-mass spectrometry to determine free sterols in human serum.

An automated method for analyzing free non-cholesterol sterols in human serum using online solid phase extraction-liquid chromatography-mass spectrometry is proposed herein. The method allows the determination of three phytosterols (sitosterol, stigmasterol and campesterol) and two cholesterol precursors (desmosterol and lanosterol). The analysis of sterols in human serum is critical in the study of cholesterol-related disorders, such as inherited familial hypercholesterolemias. Special effort was made to isolate the analytes from the serum lipoproteins, their natural conveyance through the bloodstream. The sample treatment consisted of a Bligh-Dyer extraction followed by dilution of the extract. This treatment allowed the sample to be injected into the online system and ensured the correct detection of the analytes, while avoiding the matrix effects commonly related to serum samples. The analytical performance showed linear ranges that covered two orders of magnitude, with correlation coefficients above 0.99. Limits of detection and quantification ranged from 0.2 ng/mL to 13 ng/mL and from 1.0 ng/mL to 43 ng/mL, respectively. Recovery when spiking serum with a half or a tenth of the average concentration reported in human serum ranged from 99% to 111% and from 102% to 120%, respectively. Intra-day precision and inter-day precision were below 20%.

Characterization of wood plastic composites made from landfill-derived plastic and sawdust: volatile compounds and olfactometric analysis.

Application of wood plastic composites (WPCs) obtained from recycled materials initially intended for landfill is usually limited by their composition, mainly focused on release of volatile organic compounds (VOCs) which could affect quality or human safety. The study of the VOCs released by a material is a requirement for new composite materials. Characterization and quantification of VOCs of several WPC produced with low density polyethylene (LDPE) and polyethylene/ethylene vinyl acetate (PE/EVA) films and sawdust were carried out, in each stage of production, by solid phase microextraction in headspace mode (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). An odor profile was also obtained by HS-SPME and GC-MS coupled with olfactometry analysis. More than 140 compounds were observed in the raw materials and WPC samples. Some quantified compounds were considered WPC markers such as furfural, 2-methoxyphenol, N-methylphthalimide and 2,4-di-tert-butylphenol. Hexanoic acid, acetic acid, 2-methoxyphenol, acetylfuran, diacetyl, and aldehydes were the most important odorants. None of the VOCs were found to affect human safety for use of the WPC.

Development of a fast sample treatment for the analysis of free and bonded sterols in human serum by LC-MS.

The analysis of sterols in biological fluids allows the clinical study of cholesterol related diseases. This research is focused on reducing the sample processing time of the determination of free and bonded sterols in human serum. Ten sterols were studied: cholesterol precursors (desmosterol, lanosterol, and cholestanol); phytosterols (stigmasterol, campesterol, sitosterol, and sitostanol) and oxysterols (7-α-hydroxy-4-cholesten-3-one, 24-hydroxycholesterol, and 27-hydroxycholesterol). Ultrasound assistance was used to diminish the reaction time during the alkaline hydrolysis for determining total sterols. Different retention mechanisms of solid-phase extraction were compared, two reversed-phase sorbents DSC-18 and polymeric Oasis-HLB and a novel zirconia-coated silica phase. DSC-18 and zirconia-coated silica were the most suitable sorbents to analyze these metabolites. The resulting extracts were analyzed by liquid chromatography coupled to mass spectrometry. The analytical parameters were determined and better values were observed with DSC-18 cartridges for most sterols. LOQ were in the low ng/mL level. Recoveries were in the range 85-99%. Average intermediate precision was 15%. Accuracy for both cartridges was more than 92%. Zirconia-coated silica showed better performance for the oxysterols, with recoveries around 90%. The procedure allows the determination of free and bonded sterol precursors, phytosterols, and oxysterols in human serum.

Atmospheric pressure gas chromatography with quadrupole time of flight mass spectrometry for simultaneous detection and quantification of polycyclic aromatic hydrocarbons and nitro-polycyclic aromatic hydrocarbons in mosses.

Within the family of polycyclic aromatic hydrocarbons (PAHs), nitrated derivatives are of particular interest in environmental science because they have well-known carcinogenic and mutagenic effects. They are in fact more toxic than their parent PAHs. One valuable diagnosis of atmospheric pollution can be obtained using biomonitors such as mosses. These biomonitors can provide information about air pollution over long periods of time in wilderness areas. Thus, they can serve as monitors of the atmospheric transport of pollutants. In this study, atmospheric pressure gas chromatography coupled to a quadrupole hyphenated to a time of flight mass spectrometer (APGC-MS/Q-TOF) has been examined for the identification of target analytes (15 PAHs and 8 NPAHs) for subsequent use in the analysis of mosses. Working ranges in low µg g(-1) concentration levels were obtained with most correlation coefficients higher than 0.999. All LODs were in the 0.007-0.035µg g(-1) range and higher LODs (0.035µg g(-1)) were obtained for the less volatile PAHs with higher mass and retention times: benzo(g,h,i)perylene, dibenz(a,h)anthracene and indeno(1,2,3-c,d)pyrene. These LODs are of importance for the intended use, biomonitoring, especially taking into account that NPAHs are commonly found at very low concentration levels. Recoveries from mosses ranged from 75 to 98%. Intraday and interday precision ranged from 1.8 to 11.1% RSD and from 2.4 to 16.7% RSD, respectively. Very low concentrations of NPAHs were found in mosses compared to those of PAHs. All these data were used for pattern recognition of the pollutant source. The results are shown and discussed.

Behaviour of different lichen species as biomonitors of air pollution by PAHs in natural ecosystems.

Six different species of lichens (Parmelia sulcata Tayl., Evernia prunastri (L.) Ach., Ramalina farinacea, Pseudevernia furfuracea (L.) Zopf., Usnea sp. and Lobaria pulmonaria (Schreb.) Hoffm.) were collected in two mountain valleys in Central Pyrenees: the Aspe and Aragon valleys. Two multivariate techniques have been applied with different purposes, ANOVA and Discriminant Analysis (DA), to evaluate the data. The PAHs spatial distribution was studied in the three more abundant and widespread species in the area: P. sulcata, E. prunastri (L.) Ach. and R. farinacea in terms of total PAHs, PAHs related to the combustion processes and toxicity. Different behaviour of each lichen species to trap PAHs was found, being P. sulcata the best one to monitor the most persistent PAHs of pyrogenic origin and E. prunastri the most appropriate to provide information about pyrogenic and petrogenic PAHs. Traffic was the most relevant influence in PAHs bioaccumulation in lichen species.

VOC removal and deodorization of effluent gases from an industrial plant by photo-oxidation, chemical oxidation, and ozonization.

The efficiency of photo-oxidation, chemical oxidation by sodium hypochlorite, and ozonization for the industrial-scale removal of volatile organic compounds (VOCs) and odors from gaseous emissions was studied by applying these treatments (in an experimental system) to substances passing through an emission stack of a factory producing maize derivatives. Absorption and ozonization were the most efficient treatment, removing 75% and 98% of VOCs, respectively, while photo-oxidation only removed about 59%. The emitted chemical compounds and odors were identified and quantified by gas chromatography-mass spectrometry (in full-scan mode). In addition to presenting the results, their implications for selecting optimal processes for treating volatile emissions are discussed.

Lichens biomonitoring as feasible methodology to assess air pollution in natural ecosystems: combined study of quantitative PAHs analyses and lichen biodiversity in the Pyrenees Mountains.

The air quality in the Aragón valley, in the central Pyrenees, has been assessed by evaluation of lichen biodiversity and mapped by elaboration of the Index of Air Purity (IAP) based on observations of the presence and abundance of eight kinds of lichen with different sensitivity to air pollution. The IAP values obtained have been compared with quantitative analytical measures of 16 PAHs in the lichen Evernia prunastri, because this species was associated with a wide range of traffic exposure and levels of urbanization. Analyses of PAHs were carried out by the DSASE method followed by an SPE clean-up step and GC-MS analysis. The concentration of total PAHs found in lichen samples from the Aragón valley ranged from 692 to 6420 ng g(-1) and the PAHs profile showed predominance of compounds with three aromatic rings. The influence of the road traffic in the area has been shown because values over the median concentration of PAHs (>1092 ng g(-1)), percentage of combustion PAHs (>50%), and equivalent toxicity (>169) were found in lichens collected at places exposed to the influence of traffic. The combination of both methods suggests IAP as a general method for evaluating the air pollution referenced to PAHs because it can be correlated with the content of combustion PAHs and poor lichen biodiversity can be partly explained by the air pollution caused by specific PAHs.

Use of lichens as pollution biomonitors in remote areas: comparison of PAhs extracted from lichens and atmospheric particles sampled in and around the Somport tunnel (Pyrenees).

Lichens of the species Parmelia sulcata were collected from sites on both sides of the Somport tunnel (which links France and Spain) and atmospheric particles were collected by air samplers installed within and on either side of the tunnel. Polycyclic aromatic hydrocarbons (PAHs) in the lichen and particle samples were then extracted, identified, quantified, and compared to evaluate the potential utility of lichens as pollution biomonitors in remote areas. The origin of the PAHs was also assessed using the Phe/Ant, Flu/Pyr, Ant/Ant+Phe, Flu/Flu+Pyr, and BaA/BaA+Chr concentration ratios. The total concentration of 16 priority PAHs ranged from 6.79 to 23.3 microg/g in particles outside the tunnel, from 18.3 to 265.2 microg/g in particles inside the tunnel, and from 0.91 to 1.92 microg/g in the lichen samples. The PAH ratios found in the lichens and particulate matter indicate that they were of pyrogenic origin and that road traffic was a major contributor. Results from the lichen samples suggest that they may be excellent biomonitors of pollution in remote areas.

Determination of sterols in biological samples by SPME with on-fiber derivatization and GC/FID.

A new procedure for the determination of sterols in serum samples is proposed. The system consists of coating a Solid Phase Microextraction (SPME) microfiber in headspace mode with the derivatizing agent N,O-bis(trimethylsilyl)trifluoracetamide (BSTFA) and then applying this coated fiber to the simultaneous extraction and derivatization of three precursors in the cholesterol biosynthesis pathway (desmosterol, lathosterol and lanosterol) and two phytosterols (sitosterol and sitostanol) in serum samples. Optimization of the analytical procedure via the application of an experimental design, a study of matrix effects, and an analysis of serum pool samples are all described and discussed.

Determination of potential migrants in polycarbonate containers used for microwave ovens by high-performance liquid chromatography with ultraviolet and fluorescence detection.

The determination of several compounds present in a commercial polycarbonate container intended to be used in microwave ovens which could be considered as potential migrants has been carried out by reversed-phase high-performance liquid chromatography (HPLC) with both ultraviolet (UV) and fluorescence detectors. Total dissolution with dichloromethane and polymer reprecipitation with methanol have been used to evaluate 100% potential migration as the worst case. The extract consisted of a complex mixture containing monomers, oligomers, UV stabilizers, antioxidants, degradation products, and other additives. Phenol, Bisphenol A, 2,4-di-tert-butylphenol, Cyasorb UV5411, bis(2-ethylhexylphthalate), Irganox 1076, and Irgafos 168 were identified by both retention times and fluorescence-to-UV ratios. Additional confirmation was achieved by HPLC with diode array detection and gas chromatography-mass spectrometry. Recovery percentages were in the range of 73.8-94.4%, the lowest one being for the antioxidant Irgafos 168 due to its transformation into the phosphate form and 2,4-di-tert-butylphenol. The concentrations of the studied analytes present in the polycarbonate container ranged between 0.9 and 240 microg.g(-)(1). The total dissolution conditions that may affect the final concentration of analytes, mainly Bisphenol A, are discussed.