Solid-phase microextraction/gas chromatography–mass spectrometry method optimization for characterization of surface adsorption forces of nanoparticles

A complete characterization of the different physical chemical properties of nanoparticles (NPs) is necessary for the evaluation of their impact on health and environment. Among these properties, the surface characterization of the nanomaterial is the least developed and in many cases limited to the measurement of surface composition and Zeta potential. The biological surface adsorption index approach (BSAI) for characterization of surface adsorption properties of nanoparticles (NPs) has been recently introduced [1,2]. BSAI approach offers in principle the possibility to characterize the different interaction forces exerted between a nanomaterial surface and an organic –and by extension biological- entity. The present work develops further the BSAI approach of and optimizes a solid-phase microextraction – gas chromatography mass spectrometry (SPME/GC-MS) method, which is applied to measure the adsorption properties of different nanomaterials taking into account their specific surface area. This approach gives thus a better defined quantification of the adsorption properties on NPs. To optimize the SPME/GC-MS method, we investigated the various aspects of the process including: kinetics of adsorption of probe compounds on SPME fiber, kinetic of adsorption of probe compounds on NPs surface, and optimization of NPs concentration. The optimized conditions were then tested on 33 probe compounds and on Au NPs (15 nm) and SiO2 NPs (50 nm). The results demonstrated that this detailed optimization of the SPME/GC-MS method under various conditions is a critical factor and pre-requisite to the application of BSAI approach as a tool to characterize surface adsorption properties of NPs and therefore to any further conclusions on their potential impact on health.JRC.I.4-Nanobioscience

Report of the interlaboratory comparison organised by the EU Reference for Laboratory Food Contact Material - ILC02 2009- Bisphenol A in 50% aqueous ethanol (milk simulant): Laboratory performance and precision criteria of a harmonised method

The Institute for Health and Consumer Protection (IHCP) of the European Commission¿s Directorate-General Joint Research Centre hosts the Community Reference Laboratory for Food Contact Materials (EURL-FCM). One of its core tasks is to organize interlaboratory comparisons (ILCs) among appointed National Reference Laboratories (NRLs). This report presents the results of the second ILC exercise of the EURL-FCM for the year 2009, which focused on the determination of Bisphenol A (BPA) in 50% aqueous ethanol as a food simulant for milk. The general aim was to develop and perform the validation of a method for the analysis of Bisphenol A as model substance for a polycarbonate (PC), since it is a material that has typically been used as baby bottles and therefore typically in contact with mostly milk-type products. The strategy rose from the implementation of the new milk simulant 50% Ethanol (EtOH) in Commission Directive 2007/19/EC that current CEN standards for specific migration have not addressed yet. Four 50% ethanol solutions containing different concentrations of Bisphenol A were provided for analysis encompassing concentrations of relevance to exposure determination and compliance determination. The homogeneity and stability studies were performed by the EURL-FCM laboratory. Standard operating procedures (SOPs) for the two approaches were also written. There were 31 participants from twenty-five countries to whom samples were dispatched and 26 of which submitted results. The results of analyses were received and statistically interpreted. The assigned values were obtained as a consensus values after applying the robust statistics to the results obtained from the participants. Laboratory results were rated with z-scores in accordance with ISO 13528 [1]. Standard deviations for ILC comparison (also called target standard deviations) were set based on Horwitz equation and Horrat ratio 0.5. The results and preliminary report were discussed in the plenary of December 2009. The participation of the laboratories was regarded as satisfactory for the aim of the precision experiment with regards of the numbers of received results. As a conclusion of the precision exercise on the quantification of Bisphenol A in the new milk simulant 50% ethanol, this ILC showed that the validation of the method based on HPLC-FLD according to the description based mostly on the previous CEN standard TS 13130-13 was successful. The precision that can be suggested were of 15% reproducibility SD and 6% repeatability SD for the 0.0067 mg/kg level, 10% reproducibility SD and 4% repeatability SD for the 0.0.21 mg/kg level, 6% reproducibility SD and 2% repeatability SD for the 0.0.75 mg/kg level, 6% reproducibility SD and 0.2% repeatability SD for the 0.56 mg/kg level. With respect to the scarcity of data previously available in the validation performed as reported in the CEN standard TS 13130-13 (issued version of 2005), this validation also provides a great breadth of valuable detailed and traceable raw data, which should prove extremely relevant for the creation of an extension of the standard from CEN.JRC.DG.I.2-Chemical assessment and testin

Report of the interlaboratory comparison organized by the Community Reference Laboratory Food Contact Material: Plasticisers in Gaskets and Oil

The Institute for Health and Consumer Protection (IHCP) of the European Commission¿s Directorate-General Joint Research Centre hosts the Community Reference Laboratory for Food Contact Materials (CRL-FCM). One of its core tasks is to organize interlaboratory comparisons (ILCs) among appointed National Reference Laboratories (NRLs). This report presents the results of the first ILC of the CRL-FCM which focused on the determination of Plasticisers content in PVC Gasket and in Oil matrix. The test materials used in this exercise were virgin gasket lids coming from industrial sources for the proficiency exercise part A. For the second part of the exercise an industrial source of sunflower oil was used and spiked with several plasticisers by the CRL-FCM. There were 41 participants to whom samples were dispatched 34 of which submitted results for at least 1 analyte-material. 21 laboratories reported results for more than 10 analyte-material combination out of 14 required. The homogeneity studies were performed by the CRL-FCM laboratory. The assigned value and its uncertainty for part A, virgin gaskets, were obtained after applying the robust statistics to the results obtained from the participants. The assigned values for part B, oil samples, were those obtained based on formulation, from the gravimetric measurements used to spike the material. The uncertainty of the assigned values for oil samples was calculated combining the uncertainty of the spiking procedure with a contribution for the between-bottle homogeneity. Participants were invited to report four replicates measurements. This was done by most of the participants. Laboratory results were rated with z and z¿ scores in accordance with ISO 13528 [1] Standard deviations for proficiency assessment (also called target standard deviations) were set based on Horwitz equation for substances in the two oil samples. For the plasticisers in the two gasket samples the target standard deviation was set by the organizers to 15% in order to fulfil the required criteria for sufficient homogeneity of the sample within the lots.JRC.I.2-Chemical assessment and testin

Identification and quantification of the migration of chemicals from plastic baby bottles used as substitutes for polycarbonate

This report presents the results of the study on the analytical identification and quantification of migration of chemicals from plastics baby bottles found in the EU market made of materials that are now present as substitutes for polycarbonate. 449 baby bottles with a focus on 1st age or sets of bottles were purchased from 26 EU countries, Canada, Switzerland, and USA. From this collection, which contained several duplicates, a total of 277 baby bottles were analysed. The materials included different types of plastic such as polycarbonate (PC), polyamide (PA), polyethersulfone (PES), polypropylene (PP), but also silicone, and from the USA a copolyester marketed under the trade name Tritan™. The bottles were subjected to the conventional migration test for hot fill conditions i.e. 2 hours at 70°C. The simulant used was that of specified in the EU legislation 2007/19/EC for milk, i.e. 50% EtOH. In a first phase one migration was conducted since the scope of this investigation was a screening rather than a true compliance testing check. Second and third migrations were performed on selected articles when migrated substances exceeded limits specified in the legislation. In order to verify some materials, a portion of the bottle was cut to run a FT-IR fingerprint to confirm the nature of the polymer. The migration solutions in general showed low release of substances. Results showed that bottles made of polypropylene and silicones showed a greater number of substances in the migration solutions and in greater quantity. Chemicals from polypropylene included alkanes, which could be found in >65% of the bottles at levels up to 3500 µg/kg, and benzene derivatives in 17% of the baby bottles and found at levels up to 113 µg/kg. Some substances were found on a regular basis such as plasticisers, esters, and antioxidants (e.g.tris(2,4-di-tert-butylphenyl)phosphate, known as Irgafos 168. Some substances found were not included in the Community positive list, which means that those should not be found even in the first migration. Such substances included 2,6-di-isopropylnaphthalene (DIPN), found in 4% of the bottles at levels up to 25µg/kg, 2,4-di-tert-butyl phenol (in 90% of the bottles at levels up 400 µg/kg.). Moreover, Bisphenol A was detected and quantified in baby bottles made of polyamide, but limited to one brand and model specific (but labelled BPA-free). Results for baby bottles made of silicone also indicated the presence of components for example potentially coming from inks (benzophenone, diisopropyl naphtahalene – DIPN, which could come for example from and could be from the presence of instruction leaflets in the bottles). In the case of silicone, phthalates were also found in relevant concentrations, with levels for DiBP and DBP from the first migration test of 50-150 µg/kg. and DEHP at levels 25-50 µg/kg.JRC.I.1-Chemical Assessment and Testin

Solid-phase microextraction/gas chromatography–mass spectrometry method optimization for characterization of surface adsorption forces of nanoparticles

A complete characterization of the different physico-chemical properties of nanoparticles (NPs) is necessary for the evaluation of their impact on health and environment. Among these properties, the surface characterization of the nanomaterial is the least developed and in many cases limited to the measurement of surface composition and zetapotential. The biological surface adsorption index approach (BSAI) for characterization of surface adsorption properties of NPs has recently been introduced (Xia et al. Nat Nanotechnol 5:671–675, 2010; Xia et al. ACS Nano 5(11):9074–9081, 2011). The BSAI approach offers in principle the possibility to characterize the different interaction forces exerted between a NP's surface and an organic—and by extension biological—entity. The present work further develops the BSAI approach and optimizes a solid-phase microextraction gas chromatography–mass spectrometry (SPME/GC-MS) method which, as an outcome, gives a better-defined quantification of the adsorption properties on NPs. We investigated the various aspects of the SPME/GC-MS method, including kinetics of adsorption of probe compounds on SPME fiber, kinetic of adsorption of probe compounds on NP's surface, and optimization of NP's concentration. The optimized conditions were then tested on 33 probe compounds and on Au NPs (15 nm) and SiO(2) NPs (50 nm). The procedure allowed the identification of three compounds adsorbed by silica NPs and nine compounds by Au NPs, with equilibrium times which varied between 30 min and 12 h. Adsorption coefficients of 4.66 ± 0.23 and 4.44 ± 0.26 were calculated for 1-methylnaphtalene and biphenyl, compared to literature values of 4.89 and 5.18, respectively. The results demonstrated that the detailed optimization of the SPME/GC-MS method under various conditions is a critical factor and a prerequisite to the application of the BSAI approach as a tool to characterize surface adsorption properties of NPs and therefore to draw any further conclusions on their potential impact on health. [Figure: see text

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), 4,40-dichlorodiphenyl sulphone (DCPS) and 4,40-dihydroxydiphenyl sulphone (DHPS; also known as bisphenol S) was carried out using two different analytical methods with detection limits of 0.1–0.3 mg/kg, and, therefore, much below their respective European Commission Directive 2002/72/EC legislative migration limits of 50–3000 mg/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 mg/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 mg/kg in the milk food simulant compared to a regulatory limit of 3000 mg/kg. For PC bottles, 32 bottles of 40 (80%) did not release BPA above the LOD of 0.1 mg/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 mg/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 mg/kg, in the first test still showed a detectable level in the last migration test (i.e. 0.42 mg/kg). It is important to note that the legal limit (European Commission Directive 2002/72/EC) was still 600 mg/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.JRC.I.1-Chemical Assessment and Testin

Overall migration and kinetics of release of antioxidant compounds from citrus extract-based active packaging

Overall migration (OM) tests were conducted on an antioxidant active packaging prepared by coating plasma pre-treated and untreated polyethylene terepthalate (PET) trays with a Citrus extract. The release of antioxidant compounds into food simulants was measured to permit their subtraction from OM values in line with active packaging legislation. The results demonstrated the compliance of the packaging with the limit for OM for plastic material in contact with food. The validity of the procedure for OM in aqueous food simulants was questioned, with the loss of volatile compounds during evaporation of the simulant resulting in an underestimation of total compounds released. The study showed a total release of 75% of the Citrus extract coating into water and 25% into oil, which decreased to 45% and 12.5% respectively following the plasma pre-treatment of the trays.JRC.I.1-Chemical Assessment and Testin

Silver nanoparticles and metallic silver interfere with the Griess Reaction: reduction of the azo-dye formation via a competing Sandmeyer-like reaction

Silver (Ag) is the most common nanomaterial (NM) in consumer products. Much research has been focused on elucidating the potential impact of Ag-containing NMs on human health, e.g., cytotoxicity, genotoxicity, or proinflammatory responses. In the case of proinflammatory responses, a frequently used end point is the induction of nitric oxide (NO), which is indirectly quantified as nitrite (NO2-) with the Griess reaction. After preliminary studies in a macrophage-like cell culture system showed anomalous false negative results in the presence of silver nanoparticles (Ag NPs), we studied the influence of Ag on the detection of NO2- in a cell-free environment. Solutions containing a known concentration of NaNO2 were prepared in H2O, PBS, or complete cell culture medium (CCM) and analyzed using the Griess reaction in the presence of Ag in its metallic or ionic state. In Milli-Q H2O, the impact of salts on the detection was investigated using NaCl and KBr. After completion of the Griess reaction, the samples were analyzed spectrophotometrically or chromatographically. It was found that the presence of metallic but not ionic Ag interfered with the quantification of NO2-. The effect was more pronounced in PBS and H2O containing NaCl or KBr. The chromatographical analysis provided evidence of a competing reaction consuming the intermediate diazonium salt, which is critical to the Griess reaction. These findings demonstrate yet another substantial interference of NMs with a frequently used in vitro assay. If gone unnoticed, this interference might cause false negative results and an impaired hazard assessment of Ag NMs.JRC.F.2-Consumer Products Safet

Silver Nanoparticles and Metallic Silver Interfere with the Griess Reaction: Reduction of Azo Dye Formation via a Competing Sandmeyer-Like Reaction

Silver (Ag) is the most common nanomaterial (NM) in consumer products. Much research has been focused on elucidating the potential impact of Ag-containing NMs on human health, e.g., cytotoxicity, genotoxicity, or proinflammatory responses. In the case of proinflammatory responses, a frequently used end point is the induction of nitric oxide (NO), which is indirectly quantified as nitrite (NO₂^–) with the Griess reaction. After preliminary studies in a macrophage-like cell culture system showed anomalous false negative results in the presence of silver nanoparticles (Ag NPs), we studied the influence of Ag on the detection of NO₂^– in a cell-free environment. Solutions containing a known concentration of NaNO₂ were prepared in H₂O, PBS, or complete cell culture medium (CCM) and analyzed using the Griess reaction in the presence of Ag in its metallic or ionic state. In Milli-Q H₂O, the impact of salts on the detection was investigated using NaCl and KBr. After completion of the Griess reaction, the samples were analyzed spectrophotometrically or chromatographically. It was found that the presence of metallic but not ionic Ag interfered with the quantification of NO₂^–. The effect was more pronounced in PBS and H₂O containing NaCl or KBr. The chromatographical analysis provided evidence of a competing reaction consuming the intermediate diazonium salt, which is critical to the Griess reaction. These findings demonstrate yet another substantial interference of NMs with a frequently used <i>in vitro</i> assay. If gone unnoticed, this interference might cause false negative results and an impaired hazard assessment of Ag NMs