Pressurized Liquid Extraction: A Powerful Tool to Implement Extraction and Purification of Food Contaminants

Pressurized liquid extraction (PLE) is considered an advanced extraction technique developed in the mid-1990s with the aim of saving time and reducing solvent with respect to traditional extraction processes. It is commonly used with solid and semi-solid samples and employs solvent extraction at elevated temperatures and pressures, always below the respective critical points, to maintain the solvent in a liquid state throughout the extraction procedure. The use of these particular pressure and temperature conditions changes the physicochemical properties of the extraction solvent, allowing easier and deeper penetration into the matrix to be extracted. Furthermore, the possibility to combine the extraction and clean-up steps by including a layer of an adsorbent retaining interfering compounds directly in the PLE extraction cells makes this technique extremely versatile and selective. After providing a background on the PLE technique and parameters to be optimized, the present review focuses on recent applications (published in the past 10 years) in the field of food contaminants. In particular, applications related to the extraction of environmental and processing contaminants, pesticides, residues of veterinary drugs, mycotoxins, parabens, ethyl carbamate, and fatty acid esters of 3-monochloro-1,2-propanediol and 2-monochloro-1,3-propanediol from different food matrices were considered

Microwave assisted saponification (MAS) followed by on-line liquid chromatography (LC)-gas chromatography (GC) for high-throughput and high-sensitivity determination of mineral oil in different cereal-based foodstuffs

A high throughput, high-sensitivity procedure, involving simultaneous microwave-assisted extraction (MAS) and unsaponifiable extraction, followed by on-line liquid chromatography (LC)-gas chromatography (GC), has been optimised for rapid and efficient extraction and analytical determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) in cereal-based products of different composition. MAS has the advantage of eliminating fat before LC-GC analysis, allowing an increase in the amount of sample extract injected, and hence in sensitivity. The proposed method gave practically quantitative recoveries and good repeatability. Among the different cereal-based products analysed (dry semolina and egg pasta, bread, biscuits, and cakes), egg pasta packed in direct contact with recycled paperboard had on average the highest total MOSH level (15.9mgkg(-1)), followed by cakes (10.4mgkg(-1)) and bread (7.5mgkg(-1)). About 50% of the pasta and bread samples and 20% of the biscuits and cake samples had detectable MOAH amounts. The highest concentrations were found in an egg pasta in direct contact with recycled paperboard (3.6mgkg(-1)) and in a milk bread (3.6mgkg(-1))

Monitoring and Occurrence of Heavy PAHs in Pomace Oil Supply Chain Using a Double-Step Solid-Phase Purification and HPLC-FLD Determination

peer reviewedPolycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental and processing contaminants generated by both spontaneous and anthropogenic incomplete combustion processes of organic matter. Contamination of PAHs in vegetable oils can result from several factors and processes, including environmental contamination, oil processing, and migration from food contact materials. The determination of PAHs in edible oil presents a challenge because of the complexity of the matrix. Since PAHs are present at lower levels than triglycerides, it is necessary to isolate the compounds of interest from the rest of the matrix. To this purpose, a new purification approach based on a double solid-phase extraction (SPE) step followed by high performance liquid chromatography–fluorometric detector (HPLC-FLD) analysis was developed. The method involves a first purification step by using a 5 g silica SPE cartridge, previously washed with dichloromethane (20 mL), dried completely, and then conditioned with n-hexane (20 mL). The triglycerides are retained by the silica, while the PAH-containing fraction is eluted with a mixture of n-hexane/dichloromethane (70/30, v/v). After evaporation, the residue is loaded on a 5 g amino SPE cartridge and eluted with n-hexane/toluene (70/30, v/v) before HPLC-FLD analysis. The focus was the evaluation of the contribution of the various phases of the pomace oil supply chain in terms of the heavy PAHs (PAH8) concentration. Data collected showed that pomace contamination increased (by 15 times) as storage time increased. In addition, the process of pomace drying, which is necessary to reduce its moisture content before solvent extraction of the residual oil, appeared to significantly contribute to the total heavy PAHs content, with increases in value by up to 75 times

Validation of the liquid chromatography-comprehensive multidimensional gas chromatography-time-of-flight mass spectrometer/flame ionization detector platform for mineral oil analysis exploiting interlaboratory comparison data

peer reviewe

Female Behaviour Drives Expression and Evolution of Gustatory Receptors in Butterflies

Secondary plant compounds are strong deterrents of insect oviposition and feeding, but may also be attractants for specialist herbivores. These insect-plant interactions are mediated by insect gustatory receptors (Grs) and olfactory receptors (Ors). An analysis of the reference genome of the butterfly Heliconius melpomene, which feeds on passion-flower vines (Passiflora spp.), together with whole-genome sequencing within the species and across the Heliconius phylogeny has permitted an unprecedented opportunity to study the patterns of gene duplication and copy-number variation (CNV) among these key sensory genes. We report in silico gene predictions of 73 Gr genes in the H. melpomene reference genome, including putative CO2, sugar, sugar alcohol, fructose, and bitter receptors. The majority of these Grs are the result of gene duplications since Heliconius shared a common ancestor with the monarch butterfly or the silkmoth. Among Grs but not Ors, CNVs are more common within species in those gene lineages that have also duplicated over this evolutionary time-scale, suggesting ongoing rapid gene family evolution. Deep sequencing (∼1 billion reads) of transcriptomes from proboscis and labial palps, antennae, and legs of adult H. melpomene males and females indicates that 67 of the predicted 73 Gr genes and 67 of the 70 predicted Or genes are expressed in these three tissues. Intriguingly, we find that one-third of all Grs show female-biased gene expression (n = 26) and nearly all of these (n = 21) are Heliconius-specific Grs. In fact, a significant excess of Grs that are expressed in female legs but not male legs are the result of recent gene duplication. This difference in Gr gene expression diversity between the sexes is accompanied by a striking sexual dimorphism in the abundance of gustatory sensilla on the forelegs of H. melpomene, suggesting that female oviposition behaviour drives the evolution of new gustatory receptors in butterfly genomes

Mineral oil hydrocarbons: development/optimization of analytical methods, investigation of migration from food packaging into semolina and egg pasta, and occurrence in human tissues.

Mineral oil is a complex mixture of compounds, primarily manufactured from crude petroleum through distillation processes and various refining steps, mainly consisting of MOSH (mineral oil saturated hydrocarbons), including n-alkanes, isoalkanes and cycloalkanes (naphtenes), and MOAH (mineral oil aromatic hydrocarbons), mostly alkylated. The MOSH fraction may include polyolefin oligomeric saturated hydrocarbons (POSH), oligomers of polyolefins, which can migrate from plastic bags, heat-sealable layers and other laminates as well as adhesives and plasticizers. Mineral oils hydrocarbons present in printing inks and recycled paper are sufficiently volatile (< n-C24) to migrate from the packaging to the food through the gas phase. Exposure to mineral oil hydrocarbons via packaging and some foods may pose a human health hazard. For mineral oil determination, sample extracts can be directly injected into the on-line liquid-gas chromatography (LC-GC), where a silica column retains fat and polar interferences, and fractionates MOSH and MOAH. As an alternative to the on-line method, MOSH and MOAH can be separated by off-line solid phase extraction (SPE) and then analyzed by GC with flame ionization detector (FID). Mineral oil extraction can be more or less demanding, depending on the food composition and source of contamination. Overnight extraction with hexane generally allows for the total extraction of mineral oil migrated from the packaging into dry foods such as pasta, but does not allow for the extraction of pre-existing contamination, firmly embedded into the solid structures. A more laborious and solvent consuming procedure has been proposed for the extraction of total contamination from wet-foods and pasta samples. This PhD work is divided into three main parts. The first one is dedicated to the investigation of the LC-GC system and extraction methods for mineral oil analysis. In particular, an application of a minor technical improvement on the LC-GC method proposed by Biedermann and co-workers is described. It allowed to largely improve the data throughput, to save time and solvent consumption and to increase sensitivity. In particular, a total time and solvent reduction of 34 % and 23 %, respectively, was obtained by speeding up the gas chromatographic run and reducing the liquid chromatographic reconditioning step. The band broadening occurring in the liquid chromatography column during stop flow in the multi-transfer mode was assessed by comparing the variances of the perylene peak width recorded in the stop-flow and normal modes. A band broadening directly proportional to the stop time of LC pumps was observed, however it did not affect the analysis reliability. In addition, a direct analytical comparison between two popular multidimensional LC-GC systems, namely the Y-interface (retention gap approach) and the syringe-based (programmed temperature vaporizer -PTV- approach) interface is reported. The two LC-GC methods developed were subjected to validation, in terms of linearity over the calibration range, analyte discrimination, precision, accuracy, limits of detection and quantification. Both LC-GC interfaces provided a satisfactory and comparable performance for the determination of MOSH contamination in food products. The present work also deals with the development/optimization of a comprehensive two-dimensional gas chromatographic method, with dual detection [FID and mass spectrometric (MS)], for the simultaneous identification and quantification of mineral-oil contaminants in a variety of food products. The quantitative results were compared with those obtained by performing a large volume injection, in a GC-FID system, after the same SPE process and by an on-line liquid-gas chromatography method, with very similar results observed. The presence of a series of unknown compounds, that appeared when using the off-line methods, was investigated using the mass spectrometric data, and were tentatively-identified as esterified fatty acids, most probably derived from vegetable oil based inks. Concerning the extraction approaches, a pressurized liquid extraction (PLE) method has been optimized for rapid mineral oil determination in cardboard and paper samples. The proposed method involves extraction with hexane (2 cycles) at 60 \ub0C for 5 min, and allows for the processing of up to 6 samples in parallel with minimal sample manipulation and solvent consumption. It gave good repeatability (coefficient of variation lower than 5 %) and practically quantitative extraction yield (less than 2 % of the total contamination found in a third separate cycle). The method was applied to different cardboards and paper materials intended for food contact. Furthermore, two different PLE methods, one for rapid determination of superficial contamination mostly coming from the packaging, the other for efficient extraction of total contamination from different sources, have been developed and optimized. The two methods presented good performance characteristics in terms of repeatability (relative standard deviation lower than 5 %) and recoveries (higher then 96 %). To show their potentiality, the two methods have been applied in combination on semolina pasta and rice packed in direct contact with recycled cardboard. The second part of this PhD thesis concerns the mineral oil migration into dry semolina and egg pasta packed in recycled and virgin paperboard and in plastic film at room temperature. In particular, migration kinetics has been monitored for up to 2 years, focusing on the influence of time, storage conditions, food packaging material and food characteristics. Mineral oil migration from packaging to food is a rapid phenomenon and reaches considerable levels already after 1 month from packaging time, in particular if recycled paperboard was used. A significant contribution to food contamination coming from packaging can be due to adhesives applied to close the boxes. Not only the packaging, but also the external environment contributed to the total food contamination. Food characteristics seem to influence mineral oil migration from packaging: the egg pasta reached higher levels of contamination than the semolina pasta, due to its higher fat content. Migration tests were performed also under accelerated conditions (40 and 60 \ub0C for paperboard and plastic film, respectively, up to 30 days) in order to find a correlation with the migration during the shelf-life of the product. The migration kinetic is accelerated with respect to that at room temperature, in particular for the heavier hydrocarbons, but the trend is very similar: the migration of volatile hydrocarbons up to C20 was very fast and complete in a short time. Finally, the third part of this work is dedicated to the investigation of mineral oil content in human tissues (fat tissue, mesenteric lymph nodes, spleen, liver, lungs, kidneys, heart and brain) focusing on its concentrations and molecular mass distribution. After the development of an appropriate extraction method, mineral oil contents found in human tissues have been correlated with some information on each individual\u2019s physical features and clinical history in order to identify the possible sources of contamination. For a quarter of the subjects (n = 37), a total amount of MOSH above 5g/body was calculated. The composition found in tissues seems determined not so much by the mineral oils the individuals are exposed to, but more so by the selectivity of the uptake, evaporation and metabolic elimination.Per \u201colio minerale\u201d si intende una complessa miscela di composti ottenuti principalmente dalla distillazione e raffinazione del petrolio, che comprendono idrocarburi saturi (MOSH, mineral oil saturated hydrocarbons) inclusi n-alcani, isoalcani e ciclo alcani (nafteni), e idrocarburi aromatici (MOAH, mineral oil aromatic hydrocarbons) che sono per la maggior parte alchilati. La frazione MOSH pu\uf2 includere anche oligomeri di poliolefine (POSH, polyolefin oligomeric saturated hydrocarbons), che possono migrare da materiali plastici, resine termosaldabili, adesivi e plasticizzanti. Gli oli minerali presenti negli inchiostri della stampa e nel cartone riciclato sono sufficientemente volatili (< n-C24) da migrare dall\u2019imballaggio all\u2019alimento. L\u2019esposizione agli idrocarburi di origine minerale dovuta agli imballaggi e al cibo rappresenta un pericolo per la salute umana. Per l\u2019analisi dell\u2019olio minerale, l\u2019estratto ottenuto dal campione in questione pu\uf2 essere direttamente iniettato in un sistema accoppiato LC-GC (liquid-gas chromatography), costituito da una colonna di silice che trattiene il grasso e gli interferenti polari e permette la separazione dei MOSH dai MOAH. In alternativa al metodo on-line, MOSH e MOAH possono essere separati off-line con un\u2019estrazione in fase solida (SPE) e analizzati poi con GC e rivelatore a ionizzazione di fiamma (FID, flame ionization detector). L\u2019estrazione dell\u2019olio minerale pu\uf2 essere un processo pi\uf9 o meno complicato a seconda della composizione della matrice e della fonte di contaminazione. Generalmente, un\u2019estrazione con esano per una notte permette il recupero totale dell\u2019olio minerale migrato dall\u2019imballaggio in alimenti secchi come la pasta, ma non permette un\u2019estrazione quantitativa di una contaminazione pre-esistente, intrappolata nella matrice. Una procedura pi\uf9 laboriosa \ue8 stata proposta per l\u2019estrazione della contaminazione totale di olio minerale da campioni \u201cumidi\u201d. Questa tesi di dottorato \ue8 divisa in tre parti. La prima parte \ue8 dedicata ad aspetti analitici e all\u2019estrazione dell\u2019olio minerale. In particolare, viene descritto un miglioramento tecnico, apportato al metodo gi\ue0 proposto da Biedermann e collaboratori, che permette di aumentare la sensibilit\ue0, di ridurre il tempo di analisi del 34 % e il consumo di solventi del 23 %, velocizzando la corsa cromatografica e riducendo la fase di ricondizionamento della colonna LC. E\u2019 stato valutato anche l\u2019allargamento della banda nella colonna LC durante l\u2019arresto del flusso (stop-flow) eseguito per trasferire pi\uf9 frazioni al sistema GC con un\u2019unica iniezione. Tale valutazione \ue8 stata fatta confrontando la varianza della larghezza del picco del perilene registrato utilizzando lo stop-flow e la modalit\ue0 normale. L\u2019allargamento di banda \ue8 risultato direttamente proporzionale al tempo in cui la pompa LC viene lasciata in stop, e tuttavia tale allargamento non sembra influenzare l\u2019affidabilit\ue0 dell\u2019analisi. Inoltre, \ue8 stato fatto un confronto tra due sistemi LC-GC con diversi tipi di interfaccia: uno strumento con la Y-interface basata sulla tecnica del retention gap e l\u2019altro con un vaporizzatore a temperatura programmata (PTV, programmed temperature vaporizer). I due diversi metodi sviluppati sono stati sottoposti a validazione in termini di linearit\ue0, discriminazione, precisione, accuratezza, limiti di quantificazione e di rilevabilit\ue0. Entrambe le interfacce hanno dato risultati soddisfacenti e confrontabili tra loro durante la determinazione di MOSH in prodotti alimentari. In questo lavoro \ue8 riportato anche lo sviluppo/ottimizzazione di una tecnica comprehensive GCxGC con doppio rivelatore (FID e spettrometria di massa) per una simultanea identificazione e quantificazione di olio minerale in prodotti alimentari. I risultati quantitativi sono stati confrontati con quelli ottenuti con analisi in GC-FID, previa separazione di MOSH e MOAH con SPE, e con il metodo on-line LC-GC, ottenendo valori del tutto simili. Con l\u2019utilizzo del metodo off-line SPE-GC-FID si \ue8 riscontrata la presenza di composti sconosciuti nel tracciato gas cromatografico, che sono stati poi identificati come esteri degli acidi grassi, molto probabilmente derivanti dall\u2019utilizzo di inchiostri a base di oli vegetali. Per quanto riguarda le tecniche di estrazione, \ue8 stato ottimizzato un metodo PLE (pressurized liquid extraction) per la determinazione dell\u2019olio minerale in campioni di carta e cartone. Il metodo proposto prevede un\u2019estrazione con esano (2 cicli) a 60 \ub0C per 5 minuti e permette di processare sei campioni contemporaneamente con una minima manipolazione del campione e un basso consumo di solvente. Tale metodo presenta una buona ripetibilit\ue0 (coefficiente di variazione inferiore al 5 %) e un\u2019estrazione praticamente quantitativa (meno del 2 % della contaminazione totale \ue8 stata ritrovata in un terzo ciclo separato). Il metodo \ue8 stato, quindi, applicato a differenti tipi di cartone e carta utilizzati per il contatto con alimenti. In aggiunta a ci\uf2, sono stati sviluppati anche due differenti metodi PLE per alimenti secchi con basso contenuto di grasso: uno per la determinazione di olio minerale superficiale, per la maggior parte proveniente da migrazione da imballaggio, e l\u2019altro per l\u2019estrazione della contaminazione totale proveniente da differenti fonti. I due metodi presentano una buona ripetibilit\ue0 (deviazione standard inferiore al 5 %) e ottimi recuperi (oltre il 96 %). Per sottolineare la potenzialit\ue0 di questi metodi, essi sono stati applicati in combinazione su campioni di riso e di pasta di semola tenuti a contatto diretto con cartone riciclato. La seconda parte di questa tesi riguarda la migrazione di olio minerale in pasta secca (di semola e all\u2019uovo) confezionata in imballaggi di cartone riciclato, in fibra vergine e in pacchi di film plastico, tenuti a temperatura ambiente. In particolare, le cinetiche di migrazione sono state monitorate nell\u2019arco di due anni, focalizzando l\u2019attenzione sull\u2019influenza del tempo, delle condizioni di stoccaggio, sul materiale dell\u2019imballaggio e sulle caratteristiche dell\u2019alimento. La migrazione dell\u2019olio minerale dall\u2019imballaggi all\u2019alimento \ue8 un processo rapido che pu\uf2 raggiungere considerevoli livelli di contaminazione gi\ue0 dopo un mese dal momento del confezionamento, soprattutto se viene utilizzato il cartone riciclato. La presenza di colla utilizzata per la chiusura delle confezioni sembra contribuire in modo significativo alla contaminazione totale dell\u2019alimento confezionato. Non solo l\u2019imballaggio, ma anche l\u2019ambiente di stoccaggio circostante al campione incide sulla contaminazione totale dell\u2019alimento. La quantit\ue0 di grasso dell\u2019alimento, inoltre, influenza la migrazione dell\u2019olio minerale dall\u2019imballaggio. Sono stati eseguiti anche test di migrazione accelerata a temperature di 40 \ub0C per imballaggi di cartone e 60 \ub0C per i pacchi in film plastico, della durata di 30 giorni, per cercare una correlazione con la migrazione durante la shelf-life del prodotto. Le cinetiche di migrazione sono ovviamente accelerate rispetto a quelle ottenute a temperatura ambiente, soprattutto per gli idrocarburi a pi\uf9 alto peso molecolare. In generale, per\uf2, l\u2019andamento rimane simile, con una migrazione degli idrocarburi fino al n-C20 molto rapida e completa in poco tempo. Infine, la terza parte di questa tesi \ue8 dedicata all\u2019indagine del contenuto di olio minerale in tessuti umani (tessuto adiposo, linfonodi mesenterici, milza, fegato, polmoni, reni, cervello e cuore) focalizzando l\u2019attenzione sulla sua concentrazione e distribuzione dei pesi molecolari. In seguito allo sviluppo di un appropriato metodo di estrazione, il contenuto di olio minerale ritrovato nei vari tessuti umani \ue8 stato correlato con informazioni personali e cliniche dell\u2019individuo a cui appartenevano, allo scopo di identificare una possibile fonte di contaminazione. Per un quarto dei soggetti analizzati (n = 37), sono stati trovati livelli di MOSH maggiori a 5 g per persona. La composizione della contaminazione ritrovata nei tessuti umani sembra determinata non tanto dall\u2019olio minerale a cui gli individui sono stati esposti, quanto dalla selettivit\ue0 dovuta a processi metabolici

Validation of the LC-GC×GC–MS/FID platform for mineral oil analysis through the comparison with the result of interlaboratory trials

The determination of mineral oil, a potentially toxic food contaminant, represents a real challenge from an analytical viewpoint. In fact, its quantification is characterized by a significant variability, caused for up to 20% by the chromatogram interpretation and integration, beside the additional variability due to the sample preparation methods. Recently the JRC and the DGF/ITERG organized two interlaboratory collaborative trials (ILCT) to validate a SOP for the analysis in infant formula and to update the official EN 16995:2017 method for oils and fats. We took advantage of these two ILCT to validate the novel fully integrated LC-GC×GC–ToFMS/FID platform and the software for 2D quantification that we have recently presented [1, 2]. The 2D data collected were compared with the results in 1D with the same platform and the entire results were put in perspective of the outcome of the ILCTs.MOHPlatfor

Characterisation of minor components in vegetable oil by comprehensive gas chromatography with dual detection

The profile of minor compounds, such as alcohols, sterols, free and alkyl fatty acids, waxes, etc., was investigated in different vegetable oils by a comprehensive gas chromatographic system, coupled with a simultaneous dual detection (flame ionisation detector and mass spectrometer) for quantitative and qualitative purposes. Such a system generated a unique two-dimensional chromatogram to be used as a chemical fingerprint. Multi-level information, due not only to a more "comprehensive" preparation technique, but also thanks to the exploitation of a more powerful and sensitive analytical determination allowed the extrapolation of diagnostic information from the minor components profile of different vegetable oils, along with their characteristic profile. Furthermore, an admixture of an extra virgin olive oil with a low amount of sunflower and palm oils was evaluated, attesting to the powerful diagnostic information provided by the proposed approach