Development of a highly sensitive method based on QuEChERS and GC-MS/MS for the determination of polycyclic aromatic hydrocarbons in infant foods.

Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants that can be found in various food products, including those intended for infants. Due to their potential health risks, it is crucial to develop sensitive analytical methods for the accurate determination of PAHs in infant foods. This study describes the development and validation of a highly sensitive method for the quantification of European PAH markers, namely benzo[a]pyrene, benzo[a]anthracene, chrysene, and benzo[b]fluoranthene, using gas chromatography-tandem mass spectrometry (GC-MS/MS), in baby food samples. The first step was the optimization of the sample preparation procedure, performed using different methods based on the QuEChERS approach, also testing different extraction solvents. Several factors such as extraction efficiency, selectivity, and recovery were evaluated to choose the most effective procedure for sample preparation. Furthermore, the GC-MS/MS method was optimized, evaluating parameters such as linearity, sensitivity, accuracy, and robustness using spiked infant food samples. The method demonstrated excellent linearities with a correlation coefficient higher than 0.999 over a wide concentration range, and limits of detection and limits of quantification in the range 0.019-0.036 µg/kg and 0.06-0.11 µg/kg, respectively. Extraction recoveries were between 73.1 and 110.7%, with relative standard deviations always lower than 8%. These findings are compliant with the indications of the European Commission (Reg. 836/2011). To assess the applicability of the method to official control activities, a survey was conducted on commercially available infant food products. Four markers were determined in commercial samples belonging to different food categories for infants and young children. The outcome of this monitoring showed that PAH contamination, in all samples, was below the quantification limits. In conclusion, the developed GC-MS/MS method provides a highly sensitive and reliable approach for the determination of PAHs in baby foods. The optimized sample preparation, instrumental parameters, and validation results ensure accurate quantification of 4 PAHs even at trace levels. This method could contribute to the assessment of PAH exposure in infants and it could support regulatory efforts to ensure the safety and quality of infant food products with regular monitoring.

How new nanotechnologies are changing the opioid analysis scenery? A comparison with classical analytical methods.

Opioids such as heroin, fentanyl, raw opium, and morphine have become a serious threat to the world population in the recent past, due to their increasing use and abuse. The detection of these drugs in biological samples is usually carried out by spectroscopic and/or chromatographic techniques, but the need for quick, sensitive, selective, and low-cost new analytical tools has pushed the development of new methods based on selective nanosensors, able to meet these requirements. Modern sensors, which utilize "next-generation" technologies like nanotechnology, have revolutionized drug detection methods, due to easiness of use, their low cost, and their high sensitivity and reliability, allowing the detection of opioids at trace levels in raw, pharmaceutical, and biological samples (e.g. blood, urine, saliva, and other biological fluids). The peculiar characteristics of these sensors not only have allowed on-site analyses (in the field, at the crime scene, etc.) but also they are nowadays replacing the gold standard analytical methods in the laboratory, even if a proper method validation is still required. This paper reviews advances in the field of nanotechnology and nanosensors for the detection of commonly abused opioids both prescribed (i.e. codeine and morphine) and illegal narcotics (i.e. heroin and fentanyl analogues).

Multipurpose new gas chromatography column based on pillararenes functionalized with imidazolium ionic liquids.

BACKGROUND: Gas chromatography is worldwide recognized as one of the most important analytical techniques, due to its high versatility and reliability. The heart of a gas chromatograph is the column, that allows analyte peak separations and, consequently, accurate qualitative and qualitative analyses. New and more efficient columns are always requested to satisfy new and challenging analytical needs. RESULTS: In this work, imidazolium ionic liquids functionalized pillar [5] arenes have been used for the first time as gas chromatographic stationary phases, considering their highly symmetric pillar-shaped architecture with cavities rich in π-electrons. Four imidazolium ionic liquids functionalized pillar [5] arenes have been tested as stationary phases with numerous analytes and isomers. In particular, one of these showed superior performances if compared to commercial columns, enabling challenging isomeric separations of halogenated benzenes, aromatic aldehydes, and aromatic anilines. SIGNIFICANCE AND NOVELTY: To our knowledge, this is the first report on the use of the ionic liquid P[n]A as a stationary phase in chromatography, either in GC or liquid chromatography (LC) separations. This work demonstrates the promising potential of ionic liquid P[n]A stationary phases for chromatographic separations.

Efficient gas chromatographic separation of xylene and other aromatic isomers by using pillar[6]arene-based stationary phase.

The separation of aromatic isomers, in particular xylene isomers, represents a big issue in chemical and petroleum industries, owing to their similar molecular sizes and boiling points. In this work, the investigation ofpillar[6]arene derivative modified by long alkyl chains (P6A-C10) as a stationary phase for high-resolution gas chromatographic (GC) separations of xylene isomers is presented. Pillar[n]arenes are a new class of macrocyclic hosts that can accommodate specific guests due to their highly symmetrical and rigid pillar architectures with π-electron rich cavities. The P6A-C10 column showed high-resolution performance towards xylene isomers, with peculiar advantages if compared with the commercial HP-5, HP-35, DB-17, and PEG-20Mcolumns.A quantum chemistry calculation has been performed, showing a difference in non-covalent interactions with the P6A-C10 pillar framework, which leads to specific selectivity for xylene isomers.Furthermore, the P6A-C10 column exhibited good repeatability.

Chromatographic Separation of Aromatic Amine Isomers: A Solved Issue by a New Amphiphilic Pillar[6]arene Stationary Phase.

In this work, the fabrication, synthesis, and characterization of a new stationary phase based on an amphiphilic pillar[6]arene (P6A-C10-2NH2) for gas chromatographic analyses are reported. The gas chromatography (GC) column prepared with P6A-C10-2NH2 stationary phase exhibited a medium polarity, an efficiency of 3219 plates/m, and unmatched resolving capabilities toward chloroaniline, bromoaniline, iodoaniline, and toluidine isomers. Furthermore, the P6A-C10-2NH2 column showed excellent repeatability with maximum relative standard deviations equal to 0.02, 0.07, and 2.56% for run-to-run, day-to-day, and column-to-column, respectively, demonstrating a great potential as a new stationary phase in separation science.

A New Capillary Gas Chromatography Column Based on Poly(ethylene glycol) Methyl Ether-Functionalized Calix[4]arene.

In this work, a novel capillary column (C4A-mPEG) with a calixarene-based polymer stationary phase (poly(ethylene glycol) methyl ether-functionalized 4-tert-butylcalix[4]arene) was designed and used for gas chromatographic (GC) separations. The C4A-mPEG capillary column, prepared by the static coating method, showed moderate polarity and a column efficiency of 2332 plates/m, determined by 1-octanol at 120 °C. The separation features of C4A-mPEG stationary phase, resulting from its unique structure and multiple molecular recognition processes with analytes, including π-π, H-bonding, dipole-dipole, and van der Waals interactions, allowed to obtain high-resolution performances for a wide range of compounds and their isomers, especially benzaldehydes, phenols, and anilines. Moreover, compared with 4-tertbutyl calix[4]arene (C4A) and polyethylene glycol (PEG) stationary phases, a higher resolving capability was also observed for the separation of toluidine and xylidine isomers.

High-Resolution Micro-object Separation by Rotating Magnetic Chromatography.

The development of new technologies for the separation, selection, and isolation of microparticles such as rare target cells, circulating tumor cells, cancer stem cells, and immune cells has become increasingly important in the last few years. Microparticle separation technologies are usually applied to the analysis of disease-associated cells, but these procedures often face a cell separation problem that is often insufficient for single specific cell analyses. To overcome these limitations, a highly accurate size-based microparticle separation technique, herein called "rotating magnetic chromatography", is proposed in this work. Magnetic nanoparticles, placed in a microfluidic separation channel, are forced to move in well-defined trajectories by an external magnetic field, colliding with microparticles that are in this way separated on the basis of their dimensions with high accuracy and reproducibility. The method was optimized by using fluorescein isothiocyanate-modified polystyrene particles (chosen as a reference standard) and then applied to the analysis of cancer cells like Hep-3B and SK-Hep-1, allowing their fast and high-resolution chromatographic separation as a function of their dimensions. Due to its unmatched sub-micrometer cell separation capabilities, RMC can be considered a break-through technique that can unlock new perspectives in different scientific fields, that is, in medical oncology.

On the use of a 2D-carbon microfiber fractionation system to improve flow-injection QTOF-HRMS analysis in complex matrices: the case of Abelmoschus manihot flower extracts.

A two-dimensional microscale carbon fiber/active carbon fiber system combined with a quadrupole time of flight high-resolution mass spectrometry (2DµCFs-QTOF-HRMS) system is proposed for the rapid putative identification of polar, medium-polar and weakly polar constituents in complex matrices while strongly mitigating ionic suppression effects. The capabilities of 2DµCFs-QTOF-HRMS have been proven by analysing the composition of Abelmoschus manihot flower extracts, allowing, in a single run, the detection of 41 known substances and the presence of 6 compounds never revealed before in these samples. 2DµCFs-QTOF-HRMS has been compared with traditional HPLC-MS, showing higher versatility and a significant reduction of both analysis time (70 min to 5 min) and solvent consumption (35 mL to 1.5 mL). A comparison with the results obtained by direct flow-injection MS analyses demonstrated that 2DµCFs-QTOF-HRMS leads to a more comprehensive analysis and to improved detection sensitivity. The proposed method can be considered suitable for the rapid and comprehensive analysis of food, environmental and pharmaceutical complex samples. 2DµCFs-QTOF-HRMS can thus be considered a rapid, versatile, reliable, high-throughput and economical technique that allows for the collection of information on polar, semipolar, and weakly polar components in complex matrices.

Ex-situ and in-situ rapid and quantitative determination of benzene derivatives in seawater using nanoconfined liquid phase nanoextraction.

Benzene derivatives (BDs) constitute a class of environmental pollutants whose exposure poses a grave risk to human health. These compounds rapidly diffuse from the atmosphere to the marine ecosystem: for this reason, their monitoring in seawater is every day more compelling. In this work, nanoconfined liquid phase nanoextraction (NLPNE), a versatile extraction technique recently described, has been for the first time applied to the gas chromatographic mass spectrometry (GC/MS) analysis of BDs in seawater. Ex-situ and in-situ NLPNE procedures have been developed and optimized in terms of extraction capabilities, analysis time, precision, and accuracy. Compared to the traditional extraction procedures, based on solid-phase microextraction (SPME) and liquid-liquid extraction (LLE), the proposed NLPNE methods allowed a rapid on-site analysis of benzene compounds with low solvent consumption, higher enrichment factors, and improved automation grade. Determination coefficients ranging from 0.9929 to 0.9997 were obtained for all BDs in the range 0.10-500 ng mL-1 and 5.00-500 ng mL-1, for ex-situ and in-situ NLPNE, respectively. Ex-situ and in-situ limits of detection ranged from 0.2 to 7.6 ng mL-1 and 0.04-1.00 ng mL-1. Our results suggest that NLPNE coupled to GC-MS can be considered a powerful technique for high-throughput analyses of trace compounds in environmental, food and biological samples.

Data processing for fennel protein characterization by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS).

An untargeted shot-gun approach is described for the ultra-high-resolution analysis of fennel proteins by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) combined with a home-made Matlab search algorithm. The first step of the proposed bioinformatic strategy was the development of a custom-made fennel protein database, starting from the well-known, on-line available, protein NCBI database, under Foeniculum Vulgare organism, consisting of 231 total proteins. Partial and redundant forms of proteins, repeatedly included in the official NCBI database under different codes, were removed. In the final custom-made database, in addition to the 92 fennel specific non-redundant proteins, 10 proteins belonging to recognized allergenic sources associated with spice-mugwort-allergy syndrome (celery, carrot, parsley, birch, and mugwort) were also included. The second step was the in-silico enzymatic digestion, performed on all the 102 proteins, to obtain a theoretical list of m/z dataset of tryptic peptides. The Matlab processing data was the third and crucial step, necessary to search for in-silico mass calculated peptide sequences in the high resolution ICR mass spectra of the digested fennel extract. The final step was based on database searching in Peptide Mass Fingerprint (PMF) mode by using the matched m/z values as input data. The PMF search results confirmed the presence of 70 proteins (61 fennel specific and 9 allergenic proteins) inside the fennel extract.

Investigation of fennel protein extracts by shot-gun Fourier transform ion cyclotron resonance mass spectrometry.

A rapid shot-gun method by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is proposed for the characterization of fennel proteins. After enzymatic digestion with trypsin, few microliters of extract were analyzed by direct infusion in positive ion mode. A custom-made non-redundant fennel-specific proteome database was derived from the well-known NCBI database; additional proteins belonging to recognized allergenic sources (celery, carrot, parsley, birch, and mugwort) were also included in our database, since patients hypersensitive to these plants could also suffer from fennel allergy. The peptide sequence of each protein from that derived list was theoretically sequenced to produce calculated m/z lists of possible m/z ions after tryptic digestions. Then, by using a home-made Matlab algorithm, those lists were matched with the experimental FT-ICR mass spectrum of the fennel peptide mixture. Finally, Peptide Mass Fingerprint searches confirmed the presence of the matched proteins inside the fennel extract with a total of 70 proteins (61 fennel specific and 9 allergenic proteins).

Accurate glutamate monitoring in foodstuffs by a sensitive and interference-free glutamate oxidase based disposable amperometric biosensor.

L-Glutamate (L-Glu) is a well-known flavour enhancer that is present in several foodstuffs. Although L-Glu is generally recognized as safe, the use in foodstuffs remains controversial and then its fast and accurate monitoring represents an important issue. In this work a sensitive and interference-free disposable amperometric biosensor for glutamate monitoring in foodstuffs was developed. The biosensor was prepared by immobilizing glutamate oxidase through co-crosslinking with bovine serum albumin and glutaraldehyde onto a screen printed disposable platinum electrode modified with a permselective overoxidized polypyrrole film. The enzyme immobilization was optimized by using different experimental procedures. The optimized glutamate biosensor was integrated in a flow injection system and characterized in terms of linearity (0.005-1.0 mM, r2 = 0.992), limits of detection (1.8 µM) and quantitation (5.4 µM), repeatability (RSD < 3%) and stability of response under operational conditions (up to 50 h, over 400 analysis). The biosensor showed also excellent anti-interference characteristics towards the main electroactive interferents present in food matrices, and this allowed the application to the accurate monitoring of glutamate in different foodstuffs.

Enhancing online protein isolation as intact species from soy flour samples by actively modulated two-dimensional liquid chromatography (2D-LC).

In this study, an enhanced fully automated approach is described for the protein isolation from soy flour samples by two-dimensional liquid chromatography with active modulation interface. The use of two multi-port switching valves is proposed to on-line connect the first to the second dimension column, thus overcoming the problems associated with the re-mixing effects and incompatibility of eluent composition and pH. A 5-cm long C4 analytical column installed in the interface device allows to focus the proteins coming from the first column (size exclusion chromatography), before their selective elution in the second column (reversed-phase). A trap washing step was included in the total workflow, as a desalting step to remove buffer residues from the eluent of the first column and to enhance the chromatographic performances of the second column. The experimental conditions were optimized by analyses of mixed standard solutions of bovine serum albumin, glucose oxidase, immunoglobulin A, thyroglobulin and myoglobin. Then, the optimized 2D-LC method was applied to the protein analysis in extracts of soy flour, known worldwide as one of the major food allergen sources, with the final aim to recovery sufficient protein amounts for the molecular characterization and the assessment of the pattern of allergenic components.

Development of a screening analytical method for the determination of non-dioxin-like polychlorinated biphenyls in chicken eggs by gaschromatography and electron capture detection.

A sensitive and reproducible screening analytical method is here proposed for the determination of six non dioxin-like polychlorinated biphenyls (NDL-PCBs, congener 28, 52, 101, 138, 153, 180) in chicken eggs based on accelerated solvent extraction (ASE) procedure for the fat extraction and determination, a solid phase extraction (SPE) sample clean-up process, and a gas chromatography - electron capture detection (GC-ECD) analysis. The optimized chromatographic separation, in less than 25 min, returned good responses for the six NDL-PCBs in the range of 2.5-60.0 µg L-1, with correlation coefficients always higher than 0.9995. Instrumental limits of detection were between 0.08-0.35 µg L-1, corresponding to 0.05 and 0.23 ng g-1 fat in the matrix, while method detection limits, calculated on spiked egg samples, ranged from 1.6 to 3.5 ng g-1 fat. The method has been extensively validated in terms of selectivity, sensitivity, recovery, precision, ruggedness and measurement uncertainty, following the European Directives.

An automated food protein isolation approach on preparative scale by two-dimensional liquid chromatography with active modulation interface.

In this work, an automated 2D-LC approach for protein isolation from egg samples on preparative scale is proposed. The method is based on the use of a C18 guard column installed in a switching valve to focus the proteins coming from the first dimension column, before their elution in the second column. For the first dimension separation, a size-exclusion column, packed with 3 µm ultrapure silica particles was used. An RP column based on core-shell technology was used for the second dimension separation. A standard mixture of BSA, ß-lactoglobulin, and glucose oxidase, chosen as a protein model system, was used to optimize the chromatographic separation conditions. The fully automated workflow allowed to isolate, in a single-chromatographic analysis, a protein amount of 50 µg for each peak fraction, with a total time of 15 min for the first separation and additional 30 min of the second separation for each trapped protein. The final aim was the development of proper analytical tools for protein isolation from foodstuffs to be used for the molecular identification by MS, as well as for biotherapeutic uses, allergy testing, and large-scale investigations in biological systems.

Sensitive Screening Method for Determination of Pyrethroids in Chicken Eggs and Various Meat Samples by Gas Chromatography and Electron Capture Detection.

A sensitive and reproducible screening analytical method is described for the determination of six pyrethroids (phenothrin, permethrin, cyfluthrin, cypermethrin, deltamethrin, fenvalerate) in egg and meat samples by gas chromatography and electron capture detection (GC-ECD). A fast cleanup procedure, based on solid-phase extraction has been used, ensuring reduced solvent consumption and time and allowing the simultaneous preparation of multiple sample extracts. Under the optimal chromatographic conditions, an efficient separation was obtained with a total analysis time of less than 60 min, including the extraction-purification steps. Good responses for the six pyrethroids were obtained in a range of 50-500 µg L-1, with linear coefficients higher than 0.9992. Instrumental limits of detection were between 0.22 and 0.63 µg L-1, corresponding to 0.04 and 0.13 µg kg-1 in the matrix. Detection limits in chicken eggs and various meat samples, calculated on spiked samples, were in the range 0.05-0.25 µg kg-1 and 0.07-0.23 µg kg-1, respectively. The validation results confirmed that the proposed GC-ECD method can be used as a reliable screening tool for the determination of pyrethroids in official check analyses. The method was extensively validated following the European directives, demonstrating its conformity in terms of selectivity, sensitivity, recovery, precision, and measurement uncertainty.

Milk authenticity by ion-trap proteomics following multi-enzyme digestion.

The practice of adding adulterating substances in milk in order to raise profits is unfortunately worldwide. In addition, higher priced milk, coming from minor dairy species, is often illegally integrated with the lower priced cow milk. The presence of species-specific proteins, different from those declared in label, may be a serious problem for people with allergies. The development of proper analytical methods is therefore essential to protect consumer benefits and product authenticity. In this study, a proteomic approach for the detection of adulteration processes of specific milks in mixtures is proposed. Few microliters of milk samples have been digested with trypsin and chymotrypsin and analyzed by nanoLC-ESI-IT-MS/MS. A post-database processing was performed to obtain confident peptide sequence assignments, allowing the detection of milk adulteration at a level lower than 1%. Species-specific peptides from bovine ß-lactoglobulin and αS1 casein were identified as suitable peptide markers of milk authenticity.

Rapid method for the quantification of 13 sulphonamides in milk by conventional high-performance liquid chromatography with diode array ultraviolet detection using a column packed with core-shell particles.

In the present study, a column packed with core-shell particles was used for the separation and the quantification of 13 sulphonamides in milk by conventional high-performance liquid chromatography coupled with diode array ultraviolet detection (HPLC/UV-DAD). Preliminary experiments were carried out to investigate selectivity of different stationary phases. Best results were achieved using a C18 column packed with 2.6µm core-shell particles (diameter 4.6mm, length 75mm). A binary gradient elution based on acetate buffer solution at pH 4.50 and a mixture of methanol acetonitrile 50:50 (v/v) was employed at the flow rate of 1.2mLmin-1 with an injection volume of 6µL. These chromatographic conditions allowed the efficient separation of 13 sulphonamides in about 8min. To evaluate the suitability of the method for official control analysis, the most important validation parameters were investigated according to the European Decision 657/2002/EC as established for analysis of drug residues in food. Sulphonamides were recovered from milk samples by a simple and quick preparation procedure consisting of an extraction step with chloroform/acetone and a purification step with n-hexane. Mean recoveries from raw milk ranged between 55% and 86% at the Maximum Residual Limit of 100µgkg-1, and RSDs% resulted lower than Thompson and Horwitz RSD% reference values for all sulphonamides. The LOQ value (2.7-15µgkg-1) was low enough to satisfy legal limits suggested by European Regulation 37/2010/EC.

Combined use of peptide ion and normalized delta scores to evaluate milk authenticity by ion-trap based proteomics coupled with error tolerant searching.

A fundamental issue in proteomics is the peptide identification by database searching and the assessment of the goodness of fit between experimental and theoretical data. Despite the different number of ways to measure the quality of search results, the definition of a scoring criterion is still highly desirable in ion-trap based proteomics. Indeed, in order to fully take advantage of a low resolution MS/MS dataset, it is essential to strike a balance between greater information capture and reduced number of incorrect peptide assignments. In addition, the development of user-specified rules is a crucial aspect when very similar proteins of the same family are analyzed in order to infer the origin species. In this study, a post-processing validation scheme is provided for the evaluation of proteomic data in shot-gun ion-trap proteomics, when a flexible database searching based on the error tolerant mode is adopted in combination with a low-specificity enzyme to maximize sequence coverage. To validate peptide assignments, we used standard ß-casein digested with trypsin/chymotrypsin or trypsin alone and the popular search engine MASCOT to identify the relevant (known) peptide sequences. A linear combination between peptide ion score and normalized delta score (i.e. the difference between the best and the second best ion score, divided by the best score) is proposed to increase the accuracy in sequence assignments from low-resolution tandem mass spectra. Finally, the optimized post-processing database validation was successfully applied to the direct analysis of milk tryptic/chymotryptic digests of different origin, without resorting to two-dimensional electrophoresis that is usually performed for protein separation in ion-trap proteomics. The identification of species-specific amino acidic sequences among the validated peptide spectrum matches has allowed to fully discriminate between the animal species, so evaluating accurately the milk authenticity.

Nano-LC-MS/MS for the identification of proteins trapped in sorbent cartridges used for coupled plasma filtration-adsorption treatments of healthy pigs.

A dedicated proteomic approach based on nano-Liquid Chromatography coupled with tandem mass spectrometry in ion trap is proposed for the analysis of proteins trapped in sorbent resin cartridges used to remove inflammatory mediators from blood by coupled plasma filtration adsorption (CPFA). The final purpose of the proposed proteomic approach was to obtain a reference map of plasma proteins trapped in CPFA sorbents used for the extracorporeal blood purification of healthy pigs, with the potential impact to design new bio-filters able to control the inflammatory imbalance under pathological conditions, such as severe sepsis. The five main steps of the proteomics analysis, (i) protein extraction from resin cartridges, (ii) two-dimensional gel electrophoresis (2D-PAGE) for protein separation and profiling, (iii) in-gel proteolytic digestion, (iv) tandem mass analysis of peptides resulting from enzymatic cleavage and (v) bioinformatics, for protein identification and post-processing validation of MS/MS data sets, have been carefully evaluated. Prior to electrophoresis, the efficiency of different extraction solutions and procedures to recovery plasma proteins trapped into the sorbents were tested. Then, a rapid one-step procedure for protein extraction was optimized. Protein bands corresponding to the main plasma proteins, namely porcine serum albumin, serotransferrin and immunoglobulins, were identified. In addition, the presence of haptoglobin, hemopexin, α-1 acid glycoprotein and fetuin-A, that are known as acute-phase reaction proteins, was observed, suggesting that CPFA resins led to a non-specifically protein depletion from plasma, rather than targeting specific molecules.