Liquid chromatography-Orbitrap Tribrid high-resolution mass spectrometry using data dependent-tandem mass spectrometry with triple stage fragmentation as a screening tool to perform identification and risk assessment of unknown substances in food contact epoxy resin.

A new, fast, and automatic approach has been applied for the tentative identification of unknown substances released by food contact epoxy resin after performing a migration test with food simulant. This approach combines intelligent data acquisition with AcquireX linked to liquid chromatography-Orbitrap Tribrid high-resolution mass spectrometry using data dependent-tandem mass spectrometry with triple stage fragmentation coupled to Compound Discoverer™ software for automated data processing and compound identification. The identification of the observed features was performed using a set of identification criteria, including exact mass, isotope pattern, tandem mass spectrometry spectra match, and retention time. With these criteria, 263 substances were tentatively identified. Most of the identified compounds were additives, such as plasticisers, stabilizers, and antioxidants, used in different plastic applications. However, metabolites, biological constituents with pharmacological activity, and other substances with industrial applications were also detected. In order to perform a risk assessment of the food contact epoxy resin, threshold of toxicological concern approach was applied for the identified compounds. There was not risk associated with the migration of the identified substances.

In vitro skin penetration of bronidox, bronopol and formaldehyde from cosmetics.

The objective was to evaluate the influence of the formulation in the in vitro transdermal absorption through pig ear skin of three preservatives, bronopol, bronidox and formaldehyde as well as the absorption of formaldehyde from bronopol and dimethyloldimethyl hydantoin (DMDM hydantoin). An aqueous solution, an O/W emulsion and a hydrogel were assayed. Bronidox and bronopol absorption depends on the formulation. The O/W emulsion was the system that least promoted absorption of bronidox while the absorption of bronopol was lower from the hydrogel. The aqueous solution provided maximal transdermal absorption of both preservatives. Moreover, the transdermal absorption of formaldehyde released from bronopol also depends on the formulation, being the aqueous solution the system that allowed greater absorption. Transdermal absorption of formaldehyde, applied directly or released from DMDM hydantoin, is not conditioned by the excipients. The degree of transdermal absorption of all the preservatives tested is low and therefore the concentrations allowed by regulations are safely used. Nonetheless, since formaldehyde was detected in the receptor compartment after a long time exposure to bronopol and DMDM hydantoin it would be important to consider the possibility of limiting the use of these two preservatives to rinse off products as is the case of bronidox.

Determination of 60 Migrant Substances in Plastic Food Contact Materials by Vortex-Assisted Liquid-Liquid Extraction and GC-Q-Orbitrap HRMS.

A GC-HRMS analytical method for the determination of 60 migrant substances, including aldehydes, ketones, phthalates and other plasticizers, phenol derivatives, acrylates, and methacrylates, in plastic food contact materials (FCM) has been developed and validated. The proposed method includes migration tests, according to Commission Regulation (EU) 10/2011, using four food simulants (A, B, C, and D1), followed by vortex-assisted liquid-liquid extraction (VA-LLE) and GC-Q-Orbitrap HRMS analysis in selected ion monitoring (SIM) mode, with a resolving power of 30,000 FWHM and a mass accuracy ≤5 ppm. The method was validated, showing satisfactory linearity (R2 ≥ 0.98 from 40 to 400 µg L-1), limits of quantification (40 µg L-1), precision (RSD, 0.6-12.6%), and relative recovery (81-120%). The proposed method was applied to the analysis of field samples, including an epoxy-coated tin food can, a drinking bottle made of Tritan copolyester, a disposable glass made of polycarbonate, and a baby feeding bottle made of polypropylene, showing that they were in compliance with the current European regulation regarding the studied substances.

Determination of N-nitrosamines in cosmetic products by vortex-assisted reversed-phase dispersive liquid-liquid microextraction and liquid chromatography with mass spectrometry.

A new analytical method for the simultaneous determination of trace levels of seven prohibited N-nitrosamines (N-nitrosodimethylamine, N-nitrosoethylmethylamine, N-nitrosopyrrolidine, N-nitrosodiethylamine, N-nitrosopiperidine, N-nitrosomorpholine, and N-nitrosodiethanolamine) in cosmetic products has been developed. The method is based on vortex-assisted reversed-phase dispersive liquid-liquid microextraction, which allows the extraction of highly polar compounds, followed by liquid chromatography with mass spectrometry. The variables involved in the extraction process were studied to obtain the highest enrichment factor. Under the selected conditions, 75 µL of water as extraction solvent was added to 5 mL of n-hexane sample solution and assisted by vortex mixing during 30 s to form the cloudy solution. The method was successfully validated showing good linearity (0.5-50 ng/mL), enrichment factors up to 65 depending on the target compound, limits of detection values of 1.8-50 ng/g, and good repeatability (RSD < 9.8%). Finally, the proposed method was applied to different cosmetic samples. Quantitative relative recovery values (80-113%) were obtained, thus showing that matrix effects were negligible. The achieved analytical features of the proposed method, besides of its simplicity and affordability, make it useful to perform the quality control of cosmetic products to ensure the safety of consumers.

Vortex-assisted emulsification semimicroextraction for the analytical control of restricted ingredients in cosmetic products: determination of bronopol by liquid chromatography.

Vortex-assisted emulsification semimicroextraction is proposed as a one-step solution-extraction procedure for sample preparation in cosmetic products. The procedure allows rapid preparation based on dispersion of the sample in a mixture of 1 mL of n-hexane and 0.5 mL of ethanol, followed by the addition of 0.5 mL of water and centrifugation to obtain two separated phases. This procedure provides good sample clean-up with minimum dilution and is very useful for the determination of ingredients with restricted concentrations, such as bronopol. The procedure was applied to the determination of bronopol by liquid chromatography with UV detection. The best chromatographic separation was obtained by using a C18 column set at 40 °C and performing a stepwise elution with a mixture of ethanol/aqueous 1 % acetic acid solution as mobile phase pumped at 0.5 mL min(-1). The detection wavelength was set at 250 nm and the total run time required was 12 min. The method was successfully applied to 18 commercial cosmetic samples including creams, shampoos, and bath gels. Good recoveries and repeatability were obtained, with a limit of detection of 0.9 µg mL(-1), which makes the method suitable for the analytical control of cosmetic products. Moreover, it could be considered environmentally friendly, because water, ethanol, and only a low volume of n-hexane are used as solvents.

Determination of nitrofuran metabolites and nifurpirinol in animal tissues and eggs by ultra-high performance liquid chromatography-tandem mass spectrometry validated according to Regulation (EU) 2021/808.

In this work, an analytical method for the determination of eight non-allowed nitrofurans, including nifurpirinol and the metabolites of furazolidone, furaltadone, nitrofurantoin, nitrofurazone, nifursol, nitrovin, and nifuroxazide in animal tissues, including muscle (poultry, bovine, ovine, porcine, rabbit, and fish), kidney (bovine, ovine, porcine), and eggs, has been developed and validated according to Regulation (EU) 2021/808. The method was based on derivatization with 2-nitrobenzaldehyde in acid medium, followed by vortex-assisted liquid-liquid extraction and solid phase extraction for sample purification prior to ultra-high performance liquid chromatography-tandem mass spectrometry. Under selected conditions, the method was validated showing satisfactory relative matrix effects (CV ≤ 20 %), linearity (R2 ≥ 0.98), trueness (≤20 %, expressed as bias), accuracy (83-120 %), repeatability (1.7-19.9 %), reproducibility (1.9-25.7 %), specificity (blank signal ≤30 % at the LCL), and ruggedness. The decision limit for confirmation (CCα) for the target analytes ranged from 0.27 to 0.35 µg kg-1, all below the current reference point for action (RPA) of 0.5 µg kg-1 for the studied compounds. This validated method is currently accredited according to UNE-EN ISO/IEC 17025 by the Spanish National Accreditation Body (ENAC) to be implemented for official control analyses in the Public Health Laboratory of Valencia (Spain).

Analysis of microplastics in commercial vegetable edible oils from Italy and Spain.

In this work, assessment of microplastics (MPs) in commercial vegetable edible oils from Italy and Spain, including extra-virgin olive oil, olive oil, sunflower oil, and mixed seed oil, has been conducted for the first time. The method was based on sample dilution with ethanol:n-hexane (1:3, v/v), homogenization, vacuum filtration on macroporous silicon filters with 5 µm pore diameter to collect MPs, and automatic µ-FTIR spectroscopy for MPs detection and characterization. In the analysis of oil samples, a mean MPs abundance of 1140 ± 350 MPs/L was found. Observed MPs were characterized, being most of them fragments (81.2 %), with particle sizes < 100 µm (77.5 %), and mainly composed of polyethylene (50.3 %) and polypropylene (28.7 %), among others. Statistical analysis revealed that there were not significant differences (p-value > 0.05) in the abundance of MPs between oil samples or types.

Assessment of anthropogenic particles content in commercial beverages.

Microplastic (MPs) pollution is a current global concern that is affecting all environmental compartments and food sources. In this work, anthropogenic particles occurrence (MPs and natural and synthetic cellulosic particles), have been determined in 73 beverages packed in different containers. Overall, 1521 anthropogenic particles were found, being the lowest occurrence in water samples (7.2 ± 10.1 items·L-1) while beer had the highest (95.5 ± 91.8 items·L-1). Colourless/white particles were the most detected followed by blue and red colours. The highest mean size was 783 ± 715 µm in soft drinks. Cellulosic, both natural and semisynthetic particles, were the composition mostly found but regarding plastic polymers, it was polyester. Phenoxy resin particles from the can coatings were also identified in all metal containers which indicates that leaching from the packaging may be happening. The total estimated daily intake were 0.077 and 0.159 items·kg-1 body weight (b.w.)·day-1 for children and adult population, respectively.

Determination of 21 per- and poly-fluoroalkyl substances in paper- and cardboard-based food contact materials by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry.

In this work, a new analytical method was developed and validated for the determination of the total content of 21 per- and poly-fluoroalkyl substances (PFASs) in paper- and cardboard-based food contact materials (FCMs). This method is based on green ultrasound-assisted lixiviation followed by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). The method was validated in various paper- and cardboard-based FCMs, showing good linearity (R2 ≥ 0.99), limits of quantification (1.7-10 µg kg-1), accuracy (74-115%), and precision (RSD < 20%). The eco-friendly characteristics of the proposed analytical method were assessed according to the analytical eco-scale, demonstrating that it can be considered an excellent green analytical method (EcoScale score > 75). Finally, 16 field samples of paper- and cardboard-based FCMs, including pizza boxes, popcorn boxes, paper bags and cardboard boxes for potato fries, ice cream tubs, pastry trays, and cardboard-based packaging for cooked Spanish omelets, fresh grapes, frozen fish, and salads were analyzed, showing that they comply with the current European regulations regarding the studied PFASs. The developed method is currently accredited according to UNE-EN ISO/IEC 17025 by the Spanish National Accreditation Body (ENAC) to be implemented for official control analysis of FCMs in the Public Health Laboratory of Valencia, Generalitat Valenciana (Valencia, Spain).

Untargeted analysis and tentative identification of unknown substances in human tears by ultra-high performance liquid chromatography-high resolution mass spectrometry: Pilot study.

In this work, a new approach for the identification of unknown compounds in human tears has been developed and validated using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) linked to an intelligent data acquisition mode (AcquireX DS-dd-MS2) coupled to an automated data processing software (Compound Discoverer™ 3.2). As a pilot research study, four human tear samples from volunteers were analyzed. Data were acquired in both positive and negative ionization modes and exact mass, isotope pattern, and MS2 spectra match were used for the tentative identification. Following this approach, 58 substances were identified, 47 in positive mode and 11 in negative mode, with an estimated concentration ranging from 0.1 to 9000 ng mL-1. Most of them were amino acids, hormones, metabolites, and pharmaceuticals. In order to validate the proposed method, the system suitability was evaluated and 29 commercial analytical standards of the tentatively identified substances were analyzed, of which 28 were confirmed obtaining a high identification accuracy (96.6 %). These results confirm that the screening tool presented in this work can facilitate the discovery of new metabolites, novel potential biomarkers, and substances to which the person is exposed, such as pollutants.

Non-target screening and human risk assessment for adult and child populations of semi-volatile organic compounds in residential indoor dust in Spain.

In this work, an analytical strategy based on non-target screening of semi-volatile organic compounds and subsequent risk assessment for adult and child populations has been conducted for the first time in household indoor dust samples in Spain. The methodology was based on a microwave-assisted extraction followed by gas chromatography coupled to high resolution mass spectrometry determination, using a hybrid quadrupole-orbitrap analyzer. The procedure was applied to 19 residential indoor dust samples, collected in different Spanish regions (namely Galicia, La Rioja, Catalunya, the Balearic Islands, and the Valencian Region). From the generated data, 4067 features were obtained, of which 474 compounds were tentatively identified with a high level of identification confidence (probable structure by library spectrum match or confirmed by reference standard), using a restrictive set of identification criteria. Most of the identified chemicals were natural products, metabolites, additives, and substances with industrial applications in the field of foods, cosmetics, pharmaceuticals, pesticides, and plastics. Finally, risk assessment was carried out by applying the threshold of toxicological concern approach, showing that risk to adult and child populations associated with the presence of the identified substances in the indoor dust was not expected, although the existence of indoor environments with conditions of potential risk cannot be discarded under a worst-case scenario approach.

A green analytical method for the simultaneous determination of 30 tropane and pyrrolizidine alkaloids and their N-oxides in teas and herbs for infusions by LC-Q-Orbitrap HRMS.

A green analytical method for the simultaneous determination of 30 tropane and pyrrolizidine alkaloids and their N-oxides in dried teas and herbs for infusions has been developed and validated. The proposed method is based on QuEChERS procedure followed by LC-Q-Orbitrap HRMS analysis. The method includes a first screening analysis to assess the presence of alkaloids, followed by the quantification of suspected positive samples (cut-off level, 0.2-2.6 µg kg-1). The method was validated in five different tea and herb matrices showing satisfactory linearity (R2 ≥0.99), method limits of quantification (5 µg kg-1), accuracy (87-111 %), and precision (RSD <20 %). The greenness of the proposed method was evaluated according to the Analytical Eco-Scale, showing that it could be considered an excellent green analysis. Finally, eleven commercial field samples of tea and herbs for infusions, including rooibos, chamomile, red tea, black tea, green tea, white tea, linden, horsetail, and one infusion containing a mixture of herbs, were analyzed and the obtained results demonstrated that they were in compliance with the current European regulations regarding the studied substances.

A Fast and Automated Strategy for the Identification and Risk Assessment of Unknown Substances (IAS/NIAS) in Plastic Food Contact Materials by GC-Q-Orbitrap HRMS: Recycled LDPE as a Proof-of-Concept.

A fast and automated approach has been developed for the tentative identification and risk assessment of unknown substances in plastic food contact materials (FCM) by GC-Q-Orbitrap HRMS. The proposed approach combines GC-HRMS full scan data acquisition coupled to Compound Discoverer™ 3.2 software for automated data processing and compound identification. To perform the tentative identification of the detected features, a restrictive set of identification criteria was used, including matching with the NIST Mass Spectral Library, exact mass of annotated fragments, and retention index calculation. After the tentative identification, a risk assessment of the identified substances was performed by using the threshold of toxicological concern (TTC) approach. This strategy has been applied to recycled low-density polyethylene (LDPE), which could be used as FCM, as a proof-of-concept demonstration. In the analyzed sample, 374 features were detected, of which 83 were tentatively identified after examination of the identification criteria. Most of these were additives, such as plasticizers, used in a wide variety of plastic applications, oligomers of LDPE, and substances with chemical, industrial, or cosmetic applications. The risk assessment was performed and, according to the TTC approach, the obtained results showed that there was no risk associated with the release of the identified substances. However, complementary studies related to the toxicity of the unidentified substances and the potential mixture toxicity (cocktail effects) should be conducted in parallel using bioassays.

Stir bar sorptive-dispersive microextraction mediated by magnetic nanoparticles-metal organic framework composite: Determination of N-nitrosamines in cosmetic products.

A new analytical method based on the recently proposed stir bar sorptive-dispersive microextraction (SBSDME) technique has been developed to determine eight hazardous N-nitrosamines in cosmetic products. As previous step, a simple clean-up is carried out with hexane to remove those highly lipophilic compounds that disturb the SBSDME step. Subsequently, SBSDME is performed by using magnetic nanoparticles-metal organic framework composite, CoFe2O4/MIL-101(Fe), as sorbent to entrap the target analytes, which are later chemically desorbed and measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The experimental variables related to the SBSDME procedure were studied to achieve the highest analytical signal. Under the most favorable conditions, the proposed method was validated providing good linearity; enrichment factors up to 62 depending on the analyte; limits of detection from 3 to 13 µg kg-1; and good repeatability values (RSD < 17.0%). Finally, the method was successfully applied to cosmetic samples composed either of lipophilic or hydrophilic matrices. Quantitative relative recoveries (96-109%) were obtained by using standard addition calibration. The present work expands the applicability of the SBSDME technique, whereas its analytical features make it useful to carry out the quality control of cosmetic samples.

Determination of free formaldehyde in cosmetics containing formaldehyde-releasing preservatives by reversed-phase dispersive liquid-liquid microextraction and liquid chromatography with post-column derivatization.

An analytical method for the determination of traces of formaldehyde in cosmetic products containing formaldehyde-releasing preservatives has been developed. The method is based on reversed-phase dispersive liquid-liquid microextraction (RP-DLLME), that allows the extraction of highly polar compounds, followed by liquid chromatography-ultraviolet/visible (LC-UV/vis) determination with post-column derivatization. The variables involved in the RP-DLLME process were studied to provide the best enrichment factors. Under the selected conditions, a mixture of 500 µL of acetonitrile (disperser solvent) and 50 µL of water (extraction solvent) was rapidly injected into 5 mL of toluene sample solution. The extracts were injected into the LC-UV/vis system using phosphate buffer 6 mmol L-1 at pH 2 as mobile phase. After chromatographic separation, the eluate merged with a flow stream of pentane-2,4-dione in ammonium acetate solution as derivatizing reagent and passed throughout a post-column reactor at 85 °C in order to derivatize formaldehyde into 3,5-diacetyl-1,4-dihydrolutidine, according to Hantzsch reaction, which was finally measured spectrophotometrically at 407 nm. The method was successfully validated showing good linearity, an enrichment factor of 86 ±â€¯2, limits of detection and quantification of 0.7 and 2.3 ng mL-1, respectively, and good repeatability (RSD < 9.2%). Finally, the proposed analytical method was applied to the determination of formaldehyde in different commercial cosmetic samples containing formaldehyde-releasing preservatives, such as bronopol, diazolidinyl urea, imidazolidinyl urea, and DMDM hydantoin, with good relative recovery values (91-113%) thus showing that matrix effects were negligible. The good analytical features of the proposed method besides of its simplicity and affordability, make it useful to carry out the quality control of cosmetic products containing formaldehyde-releasing preservatives.

Determination of alternative preservatives in cosmetic products by chromophoric derivatization followed by vortex-assisted liquid-liquid semimicroextraction and liquid chromatography.

An analytical method for the simultaneous determination of phenethyl alcohol, methylpropanediol, phenylpropanol, caprylyl glycol, and ethylhexylglycerin, which are used as alternative preservatives in cosmetic products, has been developed. The method is based on liquid chromatography with UV spectrophotometric detection after chromophoric derivatization with benzoyl chloride and vortex-assisted liquid-liquid semimicroextraction. Different chromatographic parameters, derivatization conditions, and sample preparation variables were studied. Under optimized conditions, the limits of detection values for the analytes ranged from 0.02 to 0.06µgmL(-1). The method was validated with good recovery values (84-118%) and precision values (3.9-9.5%). It was successfully applied to 10 commercially available cosmetic samples. The good analytical features of the proposed method besides of its environmentally-friendly characteristics, make it useful to carry out the quality control of cosmetic products containing the target compounds as preservative agents.

Determination of hydroxytyrosol and tyrosol by liquid chromatography for the quality control of cosmetic products based on olive extracts.

An analytical method for the simultaneous determination of hydroxytyrosol and tyrosol in different types of olive extract raw materials and cosmetic cream samples has been developed. The determination was performed by liquid chromatography with UV spectrophotometric detection. Different chromatographic parameters, such as mobile phase pH and composition, oven temperature and different sample preparation variables were studied. The best chromatographic separation was obtained under the following conditions: C18 column set at 35°C and isocratic elution of a mixture ethanol: 1% acetic acid solution at pH 5 (5:95, v/v) as mobile phase pumped at 1 mL min(-1). The detection wavelength was set at 280 nm and the total run time required for the chromatographic analysis was 10 min, except for cosmetic cream samples where 20 min runtime was required (including a cleaning step). The method was satisfactorily applied to 23 samples including solid, water-soluble and fat-soluble olive extracts and cosmetic cream samples containing hydroxytyrosol and tyrosol. Good recoveries (95-107%) and repeatability (1.1-3.6%) were obtained, besides of limits of detection values below the µg mL(-1) level. These good analytical features, as well as its environmentally-friendly characteristics, make the presented method suitable to carry out both the control of the whole manufacture process of raw materials containing the target analytes and the quality control of the finished cosmetic products.