Unraveling biomarkers of exposure for tenuazonic acid through urinary metabolomics.

Mycotoxins are secondary metabolites produced by fungi such as Aspergillus, Alternaria, and Penicillium, affecting nearly 80% of global food crops. Tenuazonic acid (TeA) is the major mycotoxin produced by Alternaria alternata, a prevalent pathogen affecting plants, fruits, and vegetables. TeA is notably prevalent in European diets, however, TeA biomarkers of exposure and metabolites remain unknown. This research aims to bridge this knowledge-gap by gaining insights about human TeA exposure and metabolization. Nine subjects were divided into two groups. The first group received a single bolus of TeA at the Threshold of Toxicological Concern (TTC) to investigate the presence of TeA urinary biomarkers, while the second group served as a control. Sixty-nine urinary samples were prepared and analyzed using UPLC-Xevo TQ-XS for TeA quantification and UPLC-Orbitrap Exploris for polar metabolome acquisition. TeA was rapidly excreted during the first 13 h and the fraction extracted was 0.39 ± 0.22. The polar metabolome compounds effectively discriminating the two groups were filtered using Orthogonal Partial Least Squares-Discriminant Analysis and subsequently annotated (n = 122) at confidence level 4. Finally, the urinary metabolome was compared to in silico predicted TeA metabolites. Nine metabolites, including oxidized, N-alkylated, desaturated, glucuronidated, and sulfonated forms of TeA were detected.

Application of untargeted volatile profiling in inflammatory bowel disease research.

Inflammatory bowel disease (IBD) diagnosis depends on criteria based on histological, endoscopic, radiological, and clinical results. These studies show drawbacks as being expensive, invasive, and time-consuming. In this work, an untargeted metabolomic strategy based on the monitoring of volatile compounds in serum by headspace gas chromatography-mass spectrometry is proposed as a complementary, fast, and efficient test for IBD patient diagnosis. To develop the method and build a chemometric model that allows the IBD diagnosis, serum samples including IBD patients and healthy volunteers were collected. Analyses were performed by incubating 400 µL of serum for 10 min at 90 °C. For data processing, an untargeted metabolomic strategy was used. A total of 96 features were detected, of which a total of 10 volatile compounds could be identified and confirmed by means of the analysis of real standards. The chemometric treatment consisted of a discriminant analysis of orthogonal partial least squares (OPLS-DA) obtaining a 100% of classification rate, since all the analyzed samples were correctly classified.

Use of polypyrrole ferrite microparticles and liquid chromatography-mass spectrometry for testing natural grass contamination by multiclass mycotoxins.

An analytical methodology based on the combination of dispersive magnetic solid-phase extraction (DMSPE) and liquid chromatography-mass spectrometry (LC-MS) is proposed to explore the occurrence of 13 mycotoxins (aflatoxins B1, G1, B2, and G2; deoxynivalenol; T-2 toxin; ochratoxin A; HT-2 toxin; enniatins A, A1, B, and B2; and beauvericin) and their derivatives in natural grass samples. Magnetic microparticles (Fe3O4) coated with polypyrrole (PPy) polymer were used in DMSPE sample treatment as adsorbent phase, and Fourier-transform infrared spectroscopy, field emission scanning electron microscopy, and energy dispersive X-ray spectroscopy have been used for its characterization. The experimental parameters influencing the adsorption and desorption steps of DMSPE have been optimized. Method validation has been carried out obtaining limits of quantification between 0.07 and 92 µg kg-1 corresponding to enniatin B or A1 and DON, respectively. A total of 83 natural grass samples from 8 dehesa farms were analysed. Enniatin B was found in all the samples (0.29 to 488 µg kg-1 concentration range) followed by enniatin B1 (92.8% of the samples) with a 0.12-137 µg kg-1 concentration range. Moreover, co-occurrence of mycotoxins was studied and between 2 and 5 mycotoxins appeared simultaneously in 97.6% of the samples. Distribution of the contamination according to natural grass location was also investigated.

Spatial Distribution and Antioxidant Activity of Extracts from Citrus Fruits.

Citrus fruits are recommended components of the human diet because of their enriched composition in bioactive compounds and health benefits. Among their notable components are phenols, with a special emphasis on flavonoids, limonoids, and carboxylic acids. In this research, we have carried out a spatial metabolomics analysis for the characterization of these bioactive families in three citrus fruits, namely, lemons, limes, and mandarins. Sampling was undertaken, for which the juices and three fruit tissues, namely, albedo, flavedo, and segments, were analyzed. This characterization allowed for the determination of 49 bioactive compounds in all the samples. The composition of the different extracts was correlated with the antioxidant capacity measured by the DPPH radical scavenging activity and ß-carotene bleaching assays. Flavonoids, found in the albedo and flavedo at higher concentrations, were the main components responsible for DPPH radical scavenging activity. On the other hand, the combined action of flavonoids and limonoids contributed to explaining the antioxidant activity measured by the ß-carotene bleaching assay. Generally, the antioxidant capacity of juices was lower than that estimated for extracts from citrus tissues.

Analytical Platform for the Study of Metabolic Pathway of Propyl Propane Thiosulfonate (PTSO) from Allium spp.

The present work is focused on the development of an analytical platform to elucidate the metabolic pathway of PTSO from onion, an organosulfur compound well-known for its functional and technological properties and its potential application in animal and human nutrition. This analytical platform consisted of the use of gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography quadrupole with time-of-flight MS (UHPLC-Q-TOF-MS) in order to monitor volatile and non-volatile compounds derived from the PTSO. For the extraction of the compounds of interest, two different sample treatments were developed: liquid-liquid extraction (LLE) and salting-out assisted liquid-liquid extraction (SALLE) for GC-MS and UHPLC-Q-TOF-MS analysis, respectively. Once the analytical platform was optimised and validated, an in vivo study was planned to elucidate PTSO metabolisation, revealing the presence of dipropyl disulfide (DPDS) in liver samples with concentrations between 0.11 and 0.61 µg g-1. The DPDS maximum concentration in the liver was observed at 0.5 h after the intake. DPDS was also present in all plasma samples with concentrations between 2.1 and 2.4 µg mL-1. In regard to PTSO, it was only found in plasma at times above 5 h (0.18 µg mL-1). Both PTSO and DPDS were excreted via urine 24 h after ingestion.

Dispersive Magnetic Solid-Phase Extraction as a Novelty Sample Treatment for the Determination of the Main Aflatoxins in Paprika.

Dispersive magnetic solid-phase extraction (DMSPE) technique is proposed as a new sensitive and effective sample treatment method for the determination of aflatoxins in paprika samples. DMSPE was followed by ultrahigh-performance liquid chromatography and high-resolution mass spectrometry detection (UHPLC-HRMS) using a non-targeted acquisition mode for the detection of main aflatoxins (aflatoxin G1, G2, B1 and B2) and derivatives. DMSPE was based on the use of magnetic nanocomposite coated with polypyrrole (PPy) polymer and the main experimental parameters influencing the extraction efficiency in adsorption and desorption steps have been studied and optimized. Analyses were performed using 250 µL magnetic PPy nanocomposite into the sample solution, adsorbing the analytes in 30 min and desorbing them with ethyl acetate (2 mL) in 15 min. The method has been validated, obtaining quantification limits between 3.5 and 4.7 µg kg-1 and recoveries between 89.5-97.7%. The high recovery rate, wide detection range and the use for the first time of the reusable Fe3O4@PPy nanomaterial in suspension for solid food matrices, guarantee the usefulness of the method developed for adequate control of aflatoxins levels in paprika. The proposed methodology was applied for the analysis of 31 samples (conventional and organic) revealing the absence of aflatoxins in the samples.

A non-targeted metabolomic strategy for characterization of the botanical origin of honey samples using headspace gas chromatography-ion mobility spectrometry.

In this work, characterization of the botanical origin of honey was carried out using headspace gas chromatography coupled to ion mobility spectrometry (HS-GC-IMS). The proposed methodology was applied for the analysis of 89 samples from ten different botanical origins. A total of 15 volatile compounds could be identified, namely, 3-methyl-1-butanol, heptanal, valeraldehyde, octanal, trans-2-hexenal, nonanal, hexanal, benzaldehyde, 2-heptanone, 2-butanone, 2-hexanone, 6-methyl-5-hepten-2-one, 2-pentanone, ethyl acetate and linalool. The analytical method was characterized in terms of limits of detection and quantification, and precision, in order to quantify the identified compounds. Compounds were quantified using the sum of the protonated monomer and proton-bound dimer and logarithmic regression (R2 > 0.98), although the establishment of a concentration threshold that would allow creation of classification rules was not possible since there was variability within the group. Consequently, the establishment of chemometric models was necessary. A non-targeted strategy using 275 features is proposed. Orthogonal partial least squares-discriminant analysis (OPLS-DA) allowed the differentiation of five botanical origins: thousand flowers, rosemary, albaida, orange blossom, and "others" (rest of the investigated botanical origins, since a limited number of samples was available). A success validation rate of 100% allowed the classification of 14 honeys with unknown botanical origin.

Portable Raman Spectrometer as a Screening Tool for Characterization of Iberian Dry-Cured Ham.

Dry-cured Iberian ham is officially classified into different commercial categories according to the pig's breed and feeding regime. These reach very different prices, thus promoting labelling fraud and causing great damage to the food sector. In this work, a method based on Raman spectroscopy was explored as a rapid in situ screening tool for Iberian ham samples. A total of 110 samples were analyzed to assess the potential of this technique to differentiate purebred, crossbred, acorn-fed and feed-fed dry-cured Iberian ham. A continuous signal probably due to sample fluorescence was obtained, which hid the Raman scattering signal. Therefore, chemometric treatment was applied in order to extract non-apparent information. High validated classification rates were obtained for feeding regime (83.3%) and breed (86.7%). In addition, an interlaboratory study was carried out to confirm the applicability of the method with 52 samples, obtaining a validated rate above 80%.

Cellulose-ferrite nanocomposite for monitoring enniatins and beauvericins in paprika by liquid chromatography and high-resolution mass spectrometry.

Paprika is considered a high-quality product being one of the most consumed spices in the world. Contamination with mycotoxins may appear due to inappropriate practices during processing or resulting from invading mould in the final manufactured products. A sample treatment based on dispersive magnetic solid-phase extraction (DMSPE) has been proposed for emerging mycotoxin determination, enniatins (ENNs) and beauvericins (BEAs), in paprika. Different magnetic nanoparticles were tested, and cellulose-ferrite nanocomposite was selected for the extraction and preconcentration of the mycotoxins. Nanocomposite was characterised using field emission scanning electron microscopy and energy dispersive X-ray spectroscopy in terms of morphology and elemental composition. High-resolution mass spectrometry allowed the quantification of the five main emerging mycotoxins and the monitoring of unexpected members of this class of toxic fungal secondary metabolites. The method has been validated, obtaining limits of quantification between 9.5 and 9.9 µg kg-1 and testing its trueness through recovery studies, with satisfactory values of between 89.5 and 97.7%. Relative standard deviations were calculated to evaluate the intra- and inter-day precision and values lower than 8% were obtained in all cases. The analysis of 26 samples, including conventional and organic, demonstrated the presence of ENNB1 at 12.0 ± 0.6 µg kg-1 in one of the samples studied. Other analogues ENNs and BEAs were not detected.

Headspace Gas Chromatography Coupled to Mass Spectrometry and Ion Mobility Spectrometry: Classification of Virgin Olive Oils as a Study Case.

Due to its multiple advantages, ion mobility spectrometry (IMS) is being considered as a complementary technique to mass spectrometry (MS). The goal of this work is to investigate and compare the capacity of IMS and MS in the classification of olive oil according to its quality. For this purpose, two analytical methods based on headspace gas chromatography (HS-GC) coupled with MS or with IMS have been optimized and characterized for the determination of volatile organic compounds from olive oil samples. Both detectors were compared in terms of sensitivity and selectivity, demonstrating that complementary data were obtained and both detectors have proven to be complementary. MS and IMS showed similar selectivity (10 out of 38 compounds were detected by HS-GC-IMS, whereas twelve compounds were detected by HS-GC-MS). However, IMS presented slightly better sensitivity (Limits of quantification (LOQ) ranged between 0.08 and 0.8 µg g-1 for HS-GC-IMS, and between 0.2 and 2.1 µg g-1 for HS-GC-MS). Finally, the potential of both detectors coupled with HS-GC for classification of olive oil samples depending on its quality was investigated. In this case, similar results were obtained when using both HS-GC-MS and HS-GC-IMS equipment (85.71 % of samples of the external validation set were classified correctly (validation rate)) and, although both techniques were shown to be complementary, data fusion did not improve validation results (80.95% validation rate).

Ion mobility spectrometry and mass spectrometry coupled to gas chromatography for analysis of microbial contaminated cosmetic creams.

The most commonly used technique for monitoring microbial contamination in cosmetic products is plate counting. In this contribution, headspace - gas chromatography (HS-GC) coupled to mass spectrometry (MS) or ion mobility spectrometry (IMS) is proposed as a technique to evaluate rapidly and accurately the state of microbial colonies in cosmetic creams using the volatile organic compounds produced by microorganisms (MVOC). The work focuses on monitoring two of the microorganisms that most frequently occur in such creams, Candida albicans and Staphylococcus aureus. In addition, two different types of ingredient with antimicrobial properties (a chemical preservative and a natural preservative) were added to study the behaviour of these microorganisms under different conditions. The facial creams were elaborated and inoculated with the two above microorganisms, and then sampled weekly for 4 weeks, analysing the evolution of the MVOCs by HS-GC-MS and HS-GC-IMS. In addition, microbial contamination was determined by the classical plate counting method. The pH, colour, viscosity and water activity parameters were also measured. The use of chemometric tools is essential because of the large amount of data generated, and different models based on discriminant analysis with an orthogonal projection on latent structures (OPLS-DA) were constructed. The optimal models obtained by both analytical techniques allowed differentiation between contaminated and non-contaminated creams, with a validation success rate of 94.4%. In addition, MVOC monitoring also allowed assessment of the microbial concentration.

Untargeted headspace gas chromatography - Ion mobility spectrometry analysis for detection of adulterated honey.

The recognized properties of honey together with its price have, almost inevitably, led to economically motivated adulteration. In this work, headspace gas chromatography coupled to ion mobility spectrometry (HS-GC-IMS) is proposed for the differentiation of honey according to its purity and the level of adulteration by sugar cane or corn syrups. An easy and rapid sample treatment, consisting of incubating 1 g of honey at 100 °C for 15 min and then injecting 750 µL of the sample headspace into the GC-IMS system, is proposed. A 3-dimensional data map is obtained in 32 min. The proposed method was used for the analysis of 198 honey samples (56 pure honeys of different botanical origins, 71 honeys adulterated with sugar cane syrup and 71 adulterated with corn syrup). The influence of the adulterant on variations in the honey sample spectrum was studied. In order to obtain chemometric models for the detection of adulterated honey samples, the data obtained by HS-GC-IMS were processed selecting the significant markers of the spectrum fingerprint. OPLS-DA models were constructed using 80% of the samples, and the remaining 20% were used for method validation. The differentiation between pure and adulterated honeys had a validation success of 97.4%, and the assessment of adulterant content was obtained with a 93.8% validation success rate for both adulterant agents assayed. Nine commercial honey samples were analysed using the proposed methodology, and seven of them were classified as adulterated.