Detection of Mycobacterium tuberculosis complex field infections in cattle using fecal volatile organic compound analysis through gas chromatography-ion mobility spectrometry combined with chemometrics.

Bovine tuberculosis is considered a re-emerging disease caused by different species from the Mycobacterium tuberculosis complex (MTC), important not only for the livestock sector but also for public health due to its zoonotic character. Despite the numerous efforts that have been carried out to improve the performance of the current antemortem diagnostic procedures, nowadays, they still pose several drawbacks, such as moderate to low sensitivity, highlighting the necessity to develop alternative and innovative tools to complement control and surveillance frameworks. Volatilome analysis is considered an innovative approach which has been widely employed in animal science, including animal health field and diagnosis, due to the useful and interesting information provided by volatile metabolites. Therefore, this study assesses the potential of gas chromatography coupled to ion mobility spectrometry (GC-IMS) to discriminate cattle naturally infected (field infections) by MTC from non-infected animals. Volatile organic compounds (VOCs) produced from feces were analyzed, employing the subsequent information through chemometrics. After the evaluation of variable importance for the projection of compounds, the final discriminant models achieved a robust performance in cross-validation, as well as high percentages of correct classification (>90%) and optimal data of sensitivity (91.66%) and specificity (99.99%) in external validation. The tentative identification of some VOCs revealed some coincidences with previous studies, although potential new compounds associated with the discrimination of infected and non-infected subjects were also addressed. These results provide strong evidence that a volatilome analysis of feces through GC-IMS coupled to chemometrics could become a valuable methodology to discriminate the infection by MTC in cattle. IMPORTANCE Bovine tuberculosis is endemic in many countries worldwide and poses important concerns for public health because of their zoonotic condition. However, current diagnostic techniques present several hurdles, such as low sensitivity and complexity, among others. In this regard, the development of new approaches to improve the diagnosis and control of this disease is considered crucial. Volatile organic compounds are small molecular mass metabolites which compose volatilome, whose analysis has been widely employed with success in different areas of animal science including animal health. The present study seeks to evaluate the combination of fecal volatilome analysis with chemometrics to detect field infections by bovine tuberculosis (Mycobacterium tuberculosis complex) in cattle. The good robust performance of discriminant models as well as the optimal data of sensitivity and specificity achieved highlight volatilome analysis as an innovative approach with huge potential.

Comparing the potential of IR-spectroscopic techniques to gas chromatography coupled to ion mobility spectrometry for classifying virgin olive oil categories.

Virgin olive oil (OO) can be classified into three different categories: extra virgin, virgin and lampante. The official method for this classification, based on physicochemical analysis and sensory tasting, is considered useful and effective, although it is a costly and time-consuming process. The aim of this study was to assess the potential of some analytical techniques for classifying and predicting different OO categories to support official methods and to provide olive oil companies with a rapid tool to assess product quality. Thus, mid and near infrared spectroscopies (MIR and NIR) have been compared by using different instruments and with head-space gas chromatography coupled to an ion mobility spectrometer (HS-GC-IMS). High classification success rates in validation models were obtained using IR spectrometers (>70% and > 80% in average for ternary and binary classifications, respectively), although HS-GC-IMS showed greater classification potential (>85% and > 90%).

Feed Supplementation Detection during the Last Productive Stage of the Acorn-Fed Iberian Pig through a Faecal Volatilome Analysis.

The acorn-fed Iberian pig is known worldwide due to the quality of the resulting products commercialized after a natural and free grazing period of fattening in the dehesa agroforestry ecosystem. The quality regulation of the pig breed reserves "acorn" denomination for only those products obtained from animals exclusively fed grazing acorns and other natural resources; however, sometimes, feed supplementation of the pig's diet is fraudulently employed to reach an earlier slaughtering weight and to increase pig stocking rate, a strategy called postre (meaning "feed supplement"). In this sense, although many studies focused on Iberian pig diet have been published, the field detection of feed use for acorn-fed pig during the last finishing stage foraging in the dehesa, a practice which clashes with the official regulation, has not been explored yet. The present study employs a volatilome analysis (gas chromatography coupled to ion mobility spectrometry) of a non-invasive biological sample (faeces) to discriminate the grazing diet of only natural resources, that acorn-fed Iberian pigs are supposed to have, from those pigs that are also supplemented with feed. The results obtained show the suitability of the methodology used and the usefulness of the information obtained from faeces samples to discriminate and detect the fraudulent use of feed for acorn-fed Iberian pig fattening: a classification success ranging between 86.4% and 100% was obtained for the two chemometric approaches evaluated. These, together with the results of discriminant models, are discussed, in addition to the importance that the methodology optimized implies for the Iberian pig sector and market, which is also introduced. This methodology could be adapted to control organic farming animals or other upstanding livestock production systems which are supposed to be fully dependent on a natural grazing diet.

Review of the Methodologies for Measurement of Greenhouse Gas Emissions in Livestock Farming: Pig Farms as a Case of Study.

The global emission and accumulation of gases due to livestock farming is estimated to contribute to about 14.5% of the global warming effect due to greenhouse gases (GHG). Pig farming represents 9% of global livestock GHG emissions, without considering other activities of pork production process, such as feed production. Most of information about pig farms GHG emissions is based on theoretical calculations with not too much accuracy. Hence, there is a critical need to study the best sampling and analytical techniques (portable or not) that can be used to map their contribution to GHG emissions. The selection of the best analytical detection method becomes important for public policies on climate change, and in order to evaluate animal and manure handling practices to reduce GHG and to combat global warming. In this article, different techniques, which could be used to measure the emissions of GHG from livestock, are reviewed, showing the advantages and disadvantages of each technique, with special emphasis on those already used in studies about GHG from pig farms and those that allow the simultaneous determination of several species of gases. Open chambers equipped with photoacoustic multi-gas monitor have been the techniques most employed in intensive pig farms studies. Gas Chromatography coupled to different detectors has been only widely used in pig farms to monitor simultaneously several GHG species using previous sampling devices. However, there are no studies in the literature based on extensive pig farms. In these systems, micrometeorological techniques could be a promising strategy.

Authentication of the Montanera Period on Carcasses of Iberian Pigs by Using Analytical Techniques and Chemometric Analyses.

The potential of two complementary analytical techniques (near infrared spectroscopy, NIRS and gas chromatography-ion mobility spectrometry, GC-IMS) was used to establish the time that Iberian pigs have been fed on acorns and pasture and to verify their genetic purity. For both techniques it was neither necessary to carry out any chemical treatment in advance nor to identify individual compounds. The results showed that both the NIR spectrum and the spectral fingerprint obtained by GC-IMS were affected by the time that the Iberian pig feeds on natural resources. High percentages of correct classification were achieved in the calibration for both techniques: >98% for the days of montanera and >96% for the breed by NIRS and >99% for the days of montanera and >98% for the breed by GC-IMS. The results obtained showed that NIR spectra taken from intact samples is a quick classification method according to the time of montanera and breed.

Determination of several common disinfection by-products in frozen foods.

Disinfected water and/or disinfectants are commonly used by the freezing industry in such processes as sanitising, washing, blanching, cooling and transporting the final product. For this reason, disinfection by-products (DBPs) can be expected in frozen foods. This study focused on the presence of DBPs in a wide variety of frozen vegetables, meats and fish. For this purpose, the 14 halogenated DBPs more prevalent in disinfected water were selected (four trihalomethanes, seven haloacetic acids, two haloacetonitriles and trichloronitromethane). Up to seven DBPs were found in vegetables, whereas only four DBPs were present in meats and fish, and at lower concentrations, since their contact with disinfected water is lower than in frozen vegetables. It is important to emphasise that trichloronitromethane (the most abundant nitrogenous DBP in disinfected water) was found for the first time in foods. Finally, it was concluded that the freezing process can keep the compounds stable longer than other preservation processes (viz. sanitising, canning) and, therefore, frozen foods present higher DBP concentrations than other food categories (minimally processed vegetables, or canned vegetables and meats).

Simultaneous determination of 14 disinfection by-products in meat products using microwave-assisted extraction and static headspace coupled to gas chromatography-mass spectrometry.

This paper described the first analytical method to simultaneously determine 14 disinfection by-products (DBPs) in meat products using microwave-assisted extraction (MAE) and static headspace (SHS) followed by gas chromatography-mass spectrometry (GC-MS). The DBPs included were 4 trihalomethanes, 7 haloacetic acids, 2 haloacetonitriles and trichloronitromethane, which are commonly formed as a consequence of the disinfection process of water. The combination of the MAE and SHS techniques allows meat samples to be analysed in two sequential steps into the same HS vial in spite of the sample's complexity. Detection limits were obtained within the range of 0.06-0.70ng/g, and the average relative standard deviation was 7.4%. Recoveries throughout the whole process were between 86 and 95%. The SHS-GC-MS method was applied to determine DBPs in meat products with different industrial processing which could be contaminated through contact with disinfectants and/or treated water employed in the factory either for washing or for the cooking of meat. Up to 5 DBPs were found at ng/g levels in about 36% of the samples analysed, cooked ham being the most contaminated meat product because of the brine solutions employed in its manufacturing process.

Origin of disinfection by-products in cheese.

The disinfection of water, equipment and surfaces in a cheese factory is one of the factors that can originate disinfection by-products (DBPs) in cheese. This research has focused on studying cheese factories in order to evaluate the individual contribution of each step of the cheese-making process that can contribute to the presence of DBPs in cheese. Ten factories were selected according to their salting processes (brine or dry salting). Each factory was monitored by the collection of six representative samples (factory water supply, brine solution, milk, whey, curd and cheese) in which the concentrations of up to eight chemicals were detected. The study shows that contact with brine solutions containing significant levels of DBPs is the main source of these chemicals in cheese. A minor factor is the pasteurised milk used in their manufacture.

Control of disinfection by-products in canned vegetables caused by water used in their processing.

Canned vegetables come into contact with sanitizers and/or treated water in industry during several steps (namely washing, sanitising, blanching and filling with sauces or brine solutions) and therefore they can contain disinfection by-products - DBPs). This study focused on the occurrence of trihalomethanes (THMs) and haloacetic acids (HAAs) in a wide variety of canned vegetables (75 samples). For each vegetable, the edible solid and liquid phases of the package were separated and analysed individually. DBPs can be present in both solid (up to eight species) and liquid (up to 11 species) phases, their levels being higher in liquid ones. Volatile THMs predominate in the edible solid phase (up to four species), while HAAs do so in the liquid phase (up to five species) according to their ionic and non-volatile nature. The lowest concentrations of DBPs were found in tomatoes because they were often preserved in their own juice, without water.

Static headspace-gas chromatography-mass spectrometry for the simultaneous determination of trihalomethanes and haloacetic acids in canned vegetables.

Canned vegetables appear to be a possible exposure pathway for hazardous disinfection by-products due to the use of sanitizers and treated water by the canning industry in the preparation of these foods. This work reports on two static headspace-gas chromatography-mass spectrometry methods for the simultaneous determination of 10 trihalomethanes (THMs) and 13 haloacetic acids (HAAs) in both solid and liquid phases of the canned vegetables. Both methods carry out the whole process (including the leaching of target analytes from the vegetable), derivatization of HAAs and volatilization of THMs and HAA esters, in a single step within a static headspace unit. The methods proposed provide an efficient and simple tool for the determination of regulated disinfection by-products in canned vegetables. Average limits of detection for THMs and HAAs were 0.19 and 0.45µg/kg, respectively, in the solid phase of canned vegetables, and 0.05 and 0.09µg/L, respectively, in the liquid phase. Satisfactory recoveries (90-99%) and precision, calculated as relative standard deviations (RSD≤10%), were obtained in both phases of canned vegetables. The methods proposed were applied for the analysis of frequently-used canned vegetables and confirmed the presence of up to 3 THMs and 5 HAAs at microgram per kilogram or liter levels in both phases of the samples.

Detection of regulated disinfection by-products in cheeses.

Cheese can contain regulated disinfection by-products (DBPs), mainly through contact with brine solutions prepared in disinfected water or sanitisers used to clean all contact surfaces, such as processing equipment and tanks. This study has focused on the possible presence of up to 10 trihalomethanes (THMs) and 13 haloacetic acids (HAAs) in a wide range of European cheeses. The study shows that 2 THMs, (in particular trichloromethane) and 3 HAAs (in particular dichloroacetic acid) can be found at µg/kg levels in the 56 cheeses analysed. Of the two types of DBPs, HAAs were generally present at higher concentrations, due to their hydrophilic and non-volatile nature. Despite their different nature (THMs are lipophilic), both of them have an affinity for fatty cheeses, increasing their concentrations as the percentage of water decreased because the DBPs were concentrated in the aqueous phase of the cheeses.

Origin of haloacetic acids in milk and dairy products.

Haloacetic acids (HAAs) are formed during the process of water disinfection. Therefore their presence in foods can be correlated with the addition of or contact with treated water. To determine the origin of HAAs in milk and dairy products, firstly a chromatographic method was developed for their determination. The sample treatment involves deproteination of milk followed by derivatization/extraction of the HAAs in the supernatant. About 20% of the foods analyzed contained two HAAs - which in no case exceeded 2 µg L(-1), that can be ascribed to contamination from sanitizers usually employed in the dairy industry. The process of boiling tap water (containing HAAs) for the preparation of powdered infant formula did not remove them; therefore it would be advisable to prepare this type of milk with mineral water (free of HAAs). In addition, it is possible to establish if the milk has been adulterated with treated water through the determination of HAAs.

Simultaneous quantification of trihalomethanes and haloacetic acids in cheese by on-line static headspace gas chromatography-mass spectrometry.

Trihalomethanes (THMs) and haloacetic acids (HAAs) are the two most prevalent classes of disinfection by-products (DBPs) that are present in treated water. Four THMs and six HAAs are regulated by several countries in drinking waters but no regulation for these DBPs has been established in foods. THMs are volatile species that can easily be determined by static headspace (SHS)-GC-MS, but HAAs require a derivatisation step to make them suitable for GC due to their polar and hydrophilic nature. This paper describes the first analytical method that performs the simultaneous determination of 10 THMs and 13 HAAs (chlorinated, brominated and iodinated) in cheeses by SHS in one unique GC-MS run. Parameters controlling leaching, centrifugation, derivatisation and volatilisation were optimised taking into account the high volatility of THMs and the thermal instability of HAAs. To increase sensitivity, 3g of cheese was extracted with 10mL of water at pH 4.5-7.7, and after centrifugation the supernatant (∼8mL) was introduced into an HS vial for the derivatisation (HAAs) and volatilisation (HAA esters and THMs) of the species in an automatic SHS unit coupled to GC-MS. Detection limits within the range of 0.05-0.50 and 0.15-0.85µg/kg for THMs and HAAs, respectively, were obtained, and the relative standard deviation was lower than 10% for all the target analytes. Recoveries throughout the whole method were between 85-90% and 92-97% for THMs and HAAs, respectively. The SHS-GC-MS method was applied for the determination of THMs and HAAs in 3 groups of Spanish cheeses, which can be contaminated through contact with treated water during the manufacturing steps. Up to 2 THMs and 4 HAAs were found at µg/kg levels in the samples analysed.

Haloacetic acids content of fruit juices and soft drinks.

Water used in a food factory is frequently disinfected with chlorine, which originates disinfection by-products: haloacetic acids (HAAs) make up the second most prevalent class of these products. In this paper we propose the first static HS-GC-MS method developed for direct HAA determination in beverages; the method has higher sensitivity, simplicity and reliability than the only alternative available in the literature. From 150 beverages analysed, it is possible to conclude that at least 2 HAAs (dichloro- and trichloroacetic acids, DCAA and TCAA) are always present in beverages prepared with treated water, which remains constant for 2 or 3 months in the beverages. Moreover, beverages of 100% fruit juices and soft drinks prepared with mineral water (free of HAAs) do not contain any HAA at significant values. Therefore, DCAA and TCAA may indicate of the presence of treated water in beverages.

Eco-friendly microextraction method for the quantitative speciation of 13 haloacetic acids in water.

This paper describes the first micro liquid-liquid extraction (MLLE) gas chromatography-mass spectrometry (GC-MS) method for the speciation of emerging iodinated acetic acids, along with conventional chlorinated and brominated acids in water. The haloacetic acids (HAAs) were derivatised using 3 reagents for their methylation, both in aqueous and organic media. The acidic methanol derivatisation in aqueous medium provided the best efficiency, requiring minimal sample manipulation. The derivatisation yield was improved through the use of microwave energy that drastically reduced reaction time (2 min). The HAA methyl esters were finally extracted using 250 µL of methyl tert-butyl ether. This MLLE combined with the use of a large-volume sample injection coupled to a programmed temperature vaporiser-GC-MS improved the sensitivity of the method and minimised the generation of hazardous residues in accordance with the principles of "Green Chemistry". Detection and quantification limits (excepting tribromoacetic acid) within the range of 0.01-0.15 µg/L and 0.03-0.5 µg/L, respectively, were obtained and the relative standard deviation was lower than 10%. The eco-friendly method was applied to the speciation of the 13 HAAs in treated (chlorinated and chloraminated water) and untreated water. Up to 8 HAAs were found at detectable levels in treated water. The highly toxic monoiodoacetic acid was detected in almost all the chloraminated water.

Cotton-supported graphene functionalized with aminosilica nanoparticles as a versatile high-performance extraction sorbent for trace organic analysis.

Cotton fibers were functionalized, following the coating of fibers with graphene oxide (GO), the covalent attachment of aminosilica nanoparticles and the reduction to the cotton-GR-aminosilica material. Employing the cotton-supported graphene (GR)-aminosilica material, a novel and easily applicable extraction mode was put forward. Several groups of analytes were employed such as PAHs, phthalates, musks, phenolic endocrine disrupters and haloacetic acids to test the applicability of the functionalized cotton for extraction purposes. The extraction yields ranged from 76% to 96% corroborating the high degree of affinity of the material for the above groups of compounds while the limits of detection were between 0.06 and 1.10 µg/L using gas chromatography-electron capture detection for haloacetic acids and gas chromatography-mass spectrometry for the rest of the analytes. The accuracy of the method was evaluated in fortified lake water at a spiking level of three to ten times the lowest quantifiable concentration and the recoveries varied from 86% to 104% for all the studied analytes. The π-π and hydrophobic interactions, the hydrogen bonding between the functionalized cotton and analytes and the electron polarizability and ionizability of the chemical structures justify the extraction behavior.

Effect of the chlorinated washing of minimally processed vegetables on the generation of haloacetic acids.

Chlorine solutions are usually used to sanitize fruit and vegetables in the fresh-cut industry due to their efficacy, low cost, and simple use. However, disinfection byproducts such as haloacetic acids (HAAs) can be formed during this process, which can remain on minimally processed vegetables (MPVs). These compounds are toxic and/or carcinogenic and have been associated with human health risks; therefore, the U.S. Environmental Protection Agency has set a maximum contaminant level for five HAAs at 60 µg/L in drinking water. This paper describes the first method to determine the nine HAAs that can be present in MPV samples, with static headspace coupled with gas chromatography-mass spectrometry where the leaching and derivatization of the HAAs are carried out in a single step. The proposed method is sensitive, with limits of detection between 0.1 and 2.4 µg/kg and an average relative standard deviation of ∼8%. From the samples analyzed, we can conclude that about 23% of them contain at least two HAAs (<0.4-24 µg/kg), which showed that these compounds are formed during washing and then remain on the final product.

Development of a method for the quantitation of chloro-, bromo-, and iodoacetic acids in alcoholic beverages.

Chloroacetic, bromoacetic, and iodoacetic acids can be found in alcoholic beverages when they are used as preservatives/stabilizers or as disinfectants. As they are toxic components, their addition is not permitted under European Union and U.S. regulations. To date, no sensitive methods are available, and those proposed are very laborious. This paper describes a sensitive and straightforward method for the determination of the three monohalogenated acetic acids (m-HAAs) in wines and beers using static headspace extraction coupled with gas chromatography-mass spectrometry. Prior to extraction, the target analytes were esterified to increase their volatility, and all parameters related to the extraction/methylation process were optimized to achieve high efficiency (>90%). The study examined the influence both of the ethanol concentration on the headspace partitioning and of the primary acids present in wine on the derivatization reaction of the m-HAAs. The proposed method allows the determination of these compounds at microgram per liter levels in alcoholic beverages.