Integrating a Multiple Isotopologue Reaction-Monitoring Technique and LC-MS/MS for Quantitation of Small Molecules: Ten Mycotoxins in Cereals as an Example.

Accurate quantification of mycotoxin in cereals is crucial for ensuring food safety and human health. However, the preparation of traditional multisample external calibration curves (MSCCs) is labor-intensive and error-prone. Here, a multiple isotopologue reaction-monitoring (MIRM)-LC-MS/MS method for accurate quantitation of ten major mycotoxins in cereals was successfully developed and validated, where a novel one-sample multipoint calibration curve (OSCC) strategy is used instead of MSCCs. The OSCC can be established by examining the correlation between the calculated theoretical isotopic abundances and the measured abundance across various MIRM channels. In comparison to the MSCC, the OSCC strategy exhibits outstanding performance including superior selectivity, accuracy (78.4-108.6%), and precision (<12.5%). Furthermore, the proposed OSCC-MIRM-LC-MS/MS method was successfully applied to investigate mycotoxin contamination in cereal samples in China. Considering the advantages of simplified workflows and improved throughput, the OSCC-MIRM-LC-MS/MS methodology holds great promise for accurately quantifying chemical contaminants in foods.

Advancing High-Throughput MS-Based Protein Quantification: A Case Study on Quantifying 10 Major Food Allergens by LC-MS/MS Using a One-Sample Multipoint External Calibration Curve.

The LC-MS-based method has emerged as the preferred approach for quantifying food allergens. However, the preparation of a traditional calibration curve (MSCC) is labor-intensive and error-prone. Here, a sensitive and robust LC-MS/MS method for quantifying 10 major food allergens was developed and validated, where the one-sample multipoint external calibration curve (OSCC) was employed instead of MSCC. By employing the multiple isotopologue reaction monitoring (MIRM) technique with only one spiked level in the blank, OSCC can be effectively established. Results demonstrate that the proposed method exhibits excellent performance in selectivity, sensitivity, accuracy, and precision, comparable to that of the traditional MSCC. Additionally, this strategy allows for isotope sample dilution by monitoring the less abundant MIRM channel. Moreover, the developed method was successfully applied to investigate the contamination of 10 food allergens in commercial food products. With its high throughput and robustness, the MIRM-OSCC-LC-MS/MS methodology has many potential applications, especially in the MS-based protein quantification analysis.

Facilitators and barriers of initiation and maintenance of physical activity among people with coronary heart disease: a qualitative study.

PURPOSE: We aimed to describe the facilitators and barriers of physical activity for patients with coronary heart disease. METHODS: A qualitative descriptive study using semi-structured interviews was conducted with 15 participants with coronary heart disease. The interview guide was developed based on a multi-theory model. Interviews were audio-recorded, transcribed verbatim, and analyzed using a thematic analysis. RESULTS: Two main themes were identified: facilitators of initiation and maintenance of physical activity (behavioral motivation, perceived benefits, behavioral confidence, supportive physical environment, positive emotional experience, self-regulation, supportive social environment, illness perception, and excellent self-control), barriers of initiation and maintenance of physical activity (perceived barriers, restricted physical environment, psychological distress, insufficient social support, and poor self-control). CONCLUSIONS: This study presents an in-depth theory-based exploration of facilitators and barriers to initiating and maintaining physical activity among people with coronary heart disease. Relevant factors should be taken into account to increase their effectiveness when designing the target interventions to encourage a physically active lifestyle in this population.

Before commencing cardiac rehabilitation, it is imperative to assess patients with coronary heart disease (CHD) to ascertain whether they have limited activity capacity, psychological distress, insufficient social support, and poor self-control.A customized cardiac rehabilitation plan should be meticulously devised for each patient with CHD.For patients in the early stage of initiating physical activity (PA), rehabilitation professionals should assist them in recognizing the severity of their condition and the advantages of engaging in PA.Rehabilitation professionals should also promote active utilization of social networks, stimulate CHD patients' motivation, and enhance their behavioral confidence.When guiding patients during the maintenance stage of PA, it is essential to regularly evaluate their psychological well-being, assist them in self-regulation based on their physical condition, and foster the development of self-control.Rehabilitation professionals should consistently provide social support to reinforce the patients' motivation to maintain their PA behavior.

An integrated calibration strategy for the development and validation of an LC-MS/MS method for accurate quantification of egg allergens (Gal d 1-6) in foods.

Accurate determination of egg allergens in food is vital for allergen management and labeling. However, quantifying egg allergens with mass spectrometry poses challenges and lacks validation methods. Here, we developed and validated an LC-MS/MS method for quantifying egg allergens (Gal d 1-6) in foods. Sample extraction, enzymatic digestion, purification, proteins/peptides selection, and calibration curves were optimized. VMVLC[+57]NR (Gal d 1) and GTDVQAWIR (Gal d 5) exhibited outstanding sensitivity and stability, serving as quantitation markers for egg white and yolk. Using a matrix-matched calibration curve with allergen ingredients as calibrants and labeled peptides as standards, we achieved highly accurate quantitation. Validation involved spiking egg protein into egg-free foods, showing excellent sensitivity (LOQ: 1-5 mg/kg), accuracy (62.4 %-88.5 %), and reproducibility (intra-/inter-day precision: 3.5 %-14.2 %/8.2 %-14.6 %). Additionally, we successfully applied this method to commercial food analysis. These findings demonstrate optimal allergen selection, peptides, and calibration strategy are crucial parameters for food allergen quantification via MS-based methods.

Mycotoxin contamination in the Arab world: Highlighting the main knowledge gaps and the current legislation.

Since the discovery of aflatoxins in the 1960s, knowledge in the mycotoxin research field has increased dramatically. Hundreds of review articles have been published summarizing many different aspects, including mycotoxin contamination per country or region. However, mycotoxin contamination in the Arab world, which includes 22 countries in Africa and Asia, has not yet been specifically reviewed. To this end, the contamination of mycotoxins in the Arab world was reviewed not only to profile the pervasiveness of the problem in this region but also to identify the main knowledge gaps imperiling the safety of food and feed in the future. To the best of our knowledge, 306 (non-)indexed publications in English, Arabic, or French were published from 1977 to 2021, focusing on the natural occurrence of mycotoxins in matrices of 14 different categories. Characteristic factors (e.g., detected mycotoxins, concentrations, and detection methods) were extracted, processed, and visualized. The main results are summarized as follows: (i) research on mycotoxin contamination has increased over the years. However, the accumulated data on their occurrences are scarce to non-existent in some countries; (ii) the state-of-the-art technologies on mycotoxin detection are not broadly implemented neither are contemporary multi-mycotoxin detection strategies, thus showing a need for capacity-building initiatives; and (iii) mycotoxin profiles differ among food and feed categories, as well as between human biofluids. Furthermore, the present work highlights contemporary legislation in the Arab countries and provides future perspectives to mitigate mycotoxins, enhance food and feed safety, and protect the consumer public. Concluding, research initiatives to boost mycotoxin research among Arab countries are strongly recommended.

Dietary exposure risk assessment of pyrethroids in fruits and vegetables: a national scale investigation.

The pyrethroids (PYRs) were extensively used to increase agriculture outputs. However, the cumulative exposures of PYRs would bring about potential risks through food intake. It is an urgent requirement to explore the cumulative exposures on the fruits and vegetables. In this study, a total of 1720 samples incorporating eight primary fruits and vegetables collected around China were investigated to assess the health risk for adults and children from eight PYRs. The relative potency factor (RPF) method was employed to reveal both chronic and acute cumulative exposure. As a result, the hazard index (HI) were 0.004 ~ 0.200% and 11.85 ~ 99.19% for chronic and acute cumulative dietary exposure, respectively. The national wide investigation indicated the cumulative assessments were not hazardous. Besides, the acute intake of pear, grape, and lettuce should be paid on more attention, particularly. This study provides compelling evidence to develop relative policy and regulation to improve the food quality and safety.

One sample multi-point calibration curve as a novel approach for quantitative LC-MS analysis: The quantitation of six aflatoxins in milk and oat-based milk as an example.

Preparing of calibration curves are critical steps for accurate quantitative LC-MS bioanalysis. Traditional multi-sample external calibration curve (MSCC) is labor-intensive and prone to error. In this study, a novel strategy of one sample multi-point calibration curve (OSCC) using multiple isotopologue reaction monitoring (MIRM) was proposed and validated using LC-MS for the quantitation of six aflatoxins in milk and oat-based milk samples. The developed MIRM-OSCC methodology is comprehensively validated and the results indicated that the established method exhibits good performance in selectivity, sensitivity, accuracy and precision. Furthermore, the OSCC could realize sample dilution by monitoring the MIRM channel with less intensity for samples beyond the upper limit of quantification, without the need of sample dilution, which improves the assay throughput. Considering the advantages of excluding the MSCC preparation and sample dilution in OSCC, this strategy can be widely applied in various fields such as drugs, food safety and environmental analysis.

Flavoromics Approach in Critical Aroma Compounds Exploration of Peach: Correlation to Origin Based on OAV Combined with Chemometrics.

It is essential to seek the critical aroma compounds to identify the origins of peach as well as provide a guidance for quality evaluation. In this study, the peach was characterized by HS-SPME/GC-MS. Subsequently, the odor activity value (OAV) was calculated to specify the primary aroma-active compounds. Afterwards, the chemometrics methods were employed to explore the potentially critical aroma on the basis of p value, fold change (FC), S-plot, jack-knifing confidence interval, variable importance for projection (VIP), and the Shared and Unique Structures (SUS) plots. As a result, five compounds (methyl acetate, (E)-hex-2-enal, benzaldehyde, [(Z)-hex-3-enyl] acetate, and 5-ethyloxolan-2-one) were considered as critical aromas. Moreover, the multi-classification model was developed with an outstanding performance (accuracy of 100%) using the five critical aroma. Moreover, the potential chemical basis of odors was sought through sensory evaluation. In addition, this study provides the theoretical and practical foundation for geographical origin traceability and quality evaluation.

Novel one-point calibration strategy for high-throughput quantitation of microcystins in freshwater using LC-MS/MS.

Precise quantification of microcystins (MCs) in freshwater is crucial for environmental monitoring and human health. However, the preparation of traditional multi-sample external calibration curve (MSCC) is time consuming and prone to error. Here, a novel one-point calibration strategy including one sample multi-point calibration curve (OSCC) and in sample calibration curve (ISCC) is proposed for the quantitation of eight MCs in freshwater lakes using liquid chromatography tandem mass spectrometry (LC-MS/MS). The multiple isotopologue reaction monitoring (MIRM) of MCs and its 15N-labelled internal standards were used for OSCC and ISCC, respectively. The isotopic abundance of each MIRM channel could be calculated and measured accurately. Additionally, this strategy was comprehensively validated and showed good performance in selectivity, sensitivity, accuracy and precision as the traditional MSCC. Interestingly, OSCC could realize sample dilution by monitoring the less abundant MIRM transitions, while ISCC remove blank matrixes and generate calibration curve in each study samples. Furthermore, the proposed methodology was successfully applied to analyze several freshwater lake samples contaminated by MCs. Considering the advantages of excluding the MSCC preparation, simplified workflows and improved throughput, OSCC and ISCC will be favored for MCs monitoring and as an emerging approach in environmental pollutant control and prevention.

Exploring critical metabolites of honey peach (Prunus persica (L.) Batsch) from five main cultivation regions in the north of China by UPLC-Q-TOF/MS combined with chemometrics and modeling.

The honey peach (Prunus persica (L.) Batsch) is the third most important worldwide fruit ranking after apples and pears. It is essential to seek the critical metabolites for identifying the origins as well as exploring potential valuable information for the honey peach. In this study, the honey peach from five main cultivation regions in the north of China were studied. Firstly, the metabolic profiling of honey peach was characterized by UPLC-Q-TOF/MS. Subsequently, the multivariate statistical techniques were performed to obtain critical metabolites. As a result, 58 metabolites were regarded as potential key markers that revealed the significant difference among the five groups. The screened critical metabolites of quercitrin, plantagoside, 3-p-coumaroylquinic acid, procyanidin, and quinic acid might have positive impacts on honey peach. Moreover, the partial least squares discriminant analysis (PLS-DA) model was developed with the critical metabolites. It gained acceptable predictive ability with the accuracy of 0.9444 for the validation set. Additionally, this study affords theoretical basis for the origin traceability of peach samples and provides the guidance for honey peach breeders for a long-range planting.

Characterization of honey peach (Prunus persica (L.) Batsch) aroma variation and unraveling the potential aroma metabolism mechanism through proteomics analysis under abiotic stress.

Honey peach (Prunus persica (L.) Batsch) is a climacteric fruit with short storage period. Generally, the low temperature storage (LTS) technology is implemented to lessen aroma loss and keep the quality. However, the LTS procedure brings about cold stress issues and affects the aroma metabolism. It is essential to unravel the primary aroma and the corresponding metabolism mechanism through key proteins under abiotic stress. In this study, the primary components were characterized under LTS at 1 °C during 0 to 40 days. Furthermore, the proteomics analysis was performed to acquire differentially expressed proteins to clarify the underlying metabolism mechanisms of the primary aroma and potential proteins. As a result, four proteins were considered as potential key proteins that associated with fatty acid and amino acid metabolism under cold stress. Additionally, this study provides theoretical cornerstones for regulating and improving the quality of honey peach.

Background ions into exclusion list: A new strategy to enhance the efficiency of DDA data collection for high-throughput screening of chemical contaminations in food.

Foods contaminated with hazardous compounds, could pose potential risks for human health. To date, there is still a big challenge in accurate identification. In this study, a novel data-dependent acquisition (DDA) approach, based on a combination of inclusion list and exclusion list, was proposed to acquire more effective MS/MS spectra. This strategy was successfully applied in a large-scale screening survey to detect 50 mycotoxins in oats, 155 veterinary drugs in dairy milk, and 200 pesticides in tomatoes. Compared with traditional acquisition modes, this new strategy has higher detection rate, particularly at ultra-low concentration by eliminating background influence, thereby generating the MS/MS spectra for more potential hazardous materials instead of matrix interference. Additionally, the obtained MS/MS spectra are simpler and more likely to be traced back than DIA. Moreover, this new strategy would be more comprehensively applied in food safety monitoring with the improvement of HRMS and post-acquisition techniques.

A novel calibration strategy based on isotopic distribution for high-throughput quantitative analysis of pesticides and veterinary drugs using LC-HRMS.

Preparation of calibration curves is a critical step for large-scale quantification. However, this procedure is time-consuming, labor intensive. Herein, a novel isotopologue multipoint calibration (IMC) strategy, was proposed and demonstrated for the simultaneous quantitation of 120 pesticides and 83 veterinary drugs in surface water samples using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). In this strategy, the natural isotopic distribution was used to generate external calibration curves, eliminating the need of analyst's adjustment and many sets of chemical standard solutions required in external calibration curves. Additionally, this strategy was comprehensively validated, and the results indicated this strategy had better performance in both accuracy and precision, fully meeting the requirements for the quantitative analysis. Interestingly, for the samples with high concentration beyond the upper limit of quantitation, the IMC strategy could avoid samples dilution by monitoring the less abundant isotopic channels. Furthermore, the IMC method was successfully applied in the surface water samples collected from Anhui province, China. Among which, sulfamethoxazole and imidacoprid were the main contributors. In conclusion, we present a promising LC-HRMS strategy for the accurate quantitation of small molecules, which has a potential application in food and environmental analysis.

Data-dependent acquisition based high-resolution mass spectrum for trace Alternaria mycotoxin analysis and sulfated metabolites identification.

Alternaria mycotoxins are food-related compounds that are mainly produced by Alternaria fungi species. However, it's difficult for Alternaria mycotoxins analysis, especially for conjugated metabolites in food safety surveillance. In this work, a novel data-dependent acquisition (DDA) full mass scan and products scan protocol was proposed for qualitative and quantitative analysis of five target mycotoxins in tomato samples using ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap). In total, 24 sulfated metabolites were detected with post-data analysis techniques, and two sulfated metabolites (AME-sulfated and AOH-sulfated) were identified in Alternaria fungi -inoculated tomatoes. In addition, a custom database was established, and it was successfully applied for Alternaria mycotoxins and sulfated metabolites screening in tomatoes. With the improvement in high-resolution mass spectrometry (HRMS) as well as post-data analysis techniques, DDA based HRMS method could be widely applied for compound analysis, identification, and screening in quantitative field.

Multiple antibodied based immunoaffinity columns preparation for the simultaneous analysis of deoxynivalenol and T-2 toxin in cereals by liquid chromatography tandem mass spectrometry.

Deoxynivalenol (DON) and T-2 toxin are major trichothecenes contaminated in cereals, which might bring harmful effects to humans. In this research, mixed anti-DON and anti-T-2 mAb were used for multiple immunoaffinity columns (mIACs) preparation. Under the optimal conditions, column capacities were tested at 1280 ng/mL for DON and 1160 ng/mL for T-2 toxin. Regeneration investigation showed mIACs capacities were over 510 ng/mL for DON and 440 ng/mL for T-2 toxin in 10 recycle usages. Good performances were obtained when applying mIACs purification coupled UHPLC-MS/MS for spiked samples with limit of detection at 3-13 µg/kg and mean recoveries at 79.0-97.6%. Applying to estimate the exposure of DON and T-2 toxin in commercial samples, maize samples were 100% DON positive and rice samples were 40% DON positive while T-2 toxin was negative in all tested samples. The proposed method is reliable and suitable for monitoring DON and T-2 toxin in cereal samples.

An integrated data-dependent and data-independent acquisition method for hazardous compounds screening in foods using a single UHPLC-Q-Orbitrap run.

Thousands of hazardous compounds that contaminate foods and feeds pose potential risks for human and animal health. However, it remains a challenge to perform a fast monitoring for safety surveillance. Herein we report a novel approach, integrated data-dependent and data-independent acquisition (DDIA) method, to efficiently screen for hundreds of chemicals in a single run using ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap). This method was successfully applied to analyze 180 veterinary drugs in milk, 220 pesticides in tomato and 50 mycotoxins in maize, respectively. Compared with the widely used approaches of data-dependent acquisition (DDA) or data-independent acquisition (DIA), the obtained results indicate that DDIA-based method combines the advantages of both DDA and DIA, since it achieves higher reproducibility of identification, lower false results for targeted compounds. Notably, the advantage of DDIA approach is that it enables better date retroactivity for untargeted compounds, such as metabolites and decomposition products. With the improvement in high-resolution mass spectrometry (HRMS) as well as data-mining techniques, we believe that DDIA data acquisition approach based on LC-HRMS will be widely applied in various fields in the near future, especially in compound screening and omics field, such as metabolomics and proteomics.

Toxicokinetics of α-zearalenol and its masked form in rats and the comparative biotransformation in liver microsomes from different livestock and humans.

Alpha-zearalenol (α-ZEL) and its masked form α-zearalenol-14 glucoside (α-ZEL-14G) have much higher oestrogenic activity than zearalenone. Owing to very limited toxicokinetic and metabolic data, no reference points could be established for risk assessment. To circumvent it, the toxicokinetic, metabolic profiles, and phenotyping of α-ZEL and α-ZEL-14G were comprehensively investigated in this study. As a result, the plasma concentrations of α-ZEL and α-ZEL-14G were all below LOQ after oral administration, while after iv injection, both could be significantly bio-transformed into various metabolites. A complete hydrolysis of α-ZEL-14G contributed to α-ZEL overall toxicity. Additionally, 31 phase I and 10 phase II metabolites of α-ZEL, and 9 phase I and 5 phase II metabolites were identified for α-ZEL-14G. For α-ZEL, hydroxylation, dehydrogenation, and glucuronidation were the major metabolic pathways, while for α-ZEL-14G, it was deglycosylation, reduction, hydroxylation, and glucuronidation. Significant metabolic differences were observed for α-ZEL and α-ZEL-14G in the liver microsomes of rats, chickens, swine, goats, cows and humans. Phenotyping studies indicated that α-ZEL and α-ZEL-14G were mediated by CYP 3A4, 2C8, and 1A2. Moreover, the deglycosylation of α-ZEL-14G was critically mediated by CES-I and CES-II. The acquired data would provide fundamental perspectives for risk evaluation of mycotoxins and their modified forms.

Occurrence of Neonicotinoids in Chinese Apiculture and a Corresponding Risk Exposure Assessment.

Neonicotinoids are the most widely used insecticides worldwide, but there is mounting evidence demonstrating that they have adverse effects on nontarget organisms. However, little is known about the extent of environmental neonicotinoids contamination in China. In this study, a total of 693 honey samples from across China, from both Apis melifera and Apis cerana, were analyzed to examine neonicotinoid concentrations and their geographical distribution, and correlation with the primary plant species from which the honey was obtained. Furthermore, chronic and acute exposure risk and risk ranking for humans eating honey were investigated, and risks to bees were also considered. The results revealed that 40.8% of the samples contained at least one of the five neonicotinoids tested. Honeys from commercial crops were found to be more frequently contaminated with neonicotinoids than those from noncommercial crops. Honey samples from Apis mellifera were more frequently contaminated than those from Apis cerana. The concentrations of neonicotinoids found in honey overlapped with those that have been found to have significant adverse effects on honeybee health. The dietary risk assessments indicated that the levels of neonicotinoids detected in honey were likely to be safe for human consumption.

Mycotoxin exposure assessments in a multi-center European validation study by 24-hour dietary recall and biological fluid sampling.

The European Food Consumption Validation (EFCOVAL) project includes 600 men and women from Belgium, the Czech Republic, France, the Netherlands, and Norway, who had given serum and 24-hour urine samples, and completed 24-hour dietary recall (24-HDR) interviews. Consumption, according to 24-HDR, was matched against the European Food Safety Authority (EFSA) databases of mycotoxin contaminations, via the FoodEx1 standard classifications, producing an indirect external estimate of dietary mycotoxin exposure. Direct, internal measurements of dietary mycotoxin exposure were made in serum and urine by ultra-performance liquid chromatography coupled to tandem mass spectrometry. For the first time, mycotoxin exposures were thoroughly compared between two 24-HDRs, and two 24-hour urine samples collected during the same days covered by the 24-HDRs. These measurements were compared to a single-time point serum measurement to investigate evidence of chronic mycotoxin exposure. According to 24-HDR data, all 600 individuals were exposed to between 4 and 34 mycotoxins, whereof 10 found to exceed the tolerable daily intake. Correlations were observed between two time points, and significant correlations were observed between concentrations in serum and urine. However, only acetyldeoxynivalenol, ochratoxin A, and sterigmatocystin were found to have significant positive correlations between 24-HDR exposures and serum, while aflatoxin G1 and G2, HT-2 toxin, and deoxynivalenol were associated between concurrent 24-HDR and 24-hour urine. Substantial agreements on quantitative levels between serum and urine were observed for the groups Type B Trichothecenes and Zearalenone. Further research is required to bridge the interpretation of external and internal exposure estimates of the individual on a time scale of hours. Additionally, metabolomic profiling of dietary mycotoxin exposures could help with a comprehensive assessment of single time-point exposures, but also with the identification of chronic exposure biomarkers. Such detailed characterization informs population exposure assessments, and aids in the interpretation of epidemiological health outcomes related to multi-mycotoxin exposure.

Gelsedine-type alkaloids: Discovery of natural neurotoxins presented in toxic honey.

The fascinating collection and evaluation of natural products with enormous structural and chemical diversity can contribute to ensure human health and inspire potential drug discovery. We reported the identification of 14-(R)-hydroxy-gelsenicine (HGE), a new component from poisonous honey, which has recently caused multiple serious intoxications and deaths up on consumption. The prevalence, toxicity, toxicokinetics and metabolic profile of HGE were evaluated through in vitro and in vivo analyses. HGE is a very toxic substance and shows significant gender difference with LD50 of 0.125 mg kg-1 and 0.295 mg kg-1 for the female and male mice, respectively. Toxicokinetics test indicates that HGE has good bioavailability in rats, and is metabolized extensively, in which hydroxylation, reduction, N-demethyl ether and glucuronication are the major metabolic pathways. Additionally, HGE shows specific neurotoxicity by enhancing the binding of γ-aminobutyric acid (GABA) to its receptors. We found that flumazenil, a selective antagonist of GABA receptor, could effectively increase the survival of the tested animals, which provides a potential therapy for future clinical applications.