Development and validation of an analytical method for the simultaneous determination of 12 ergot, 2 tropane, and 28 pyrrolizidine alkaloids in cereal-based food by LC-MS/MS.

Alkaloids are naturally occurring compounds containing basic nitrogen atoms. They are biosynthesized mainly by plants but also by some fungi species. Many alkaloids are toxic to humans and animals, and they have been classified as food contaminants. Among them, ergot, tropane, and pyrrolizidine alkaloids have maximum levels in foods, established by the Commission Regulation (EU) 2023/915. In this study, an analytical method was successfully developed and validated for the simultaneous determination of 42 ergot, tropane, and pyrrolizidine alkaloids and their N-oxides in cereal-based food. The method includes QuEChERS-based extraction followed by liquid chromatography coupled with tandem mass spectrometry. The proposed method was validated providing recoveries ranging from 71 to 119 %, intra- and inter-day precision lower than 19 %, and limits of quantification between 0.5 and 1.0 µg kg-1. Finally, the analysis of reference materials coming from FAPAS proficiency tests demonstrated the suitability for purpose of the methodology (z-scores < 2). Nine cereal-based products samples were analyzed of which ergot alkaloids were detected in two of them, while one sample showed the presence of three pyrrolizidine alkaloids.

Assessment of exposure to pesticide mixtures in five European countries by a harmonized urinary suspect screening approach.

Humans are exposed to a mixture of pesticides through diet as well as through the environment. We conducted a suspect-screening based study to describe the probability of (concomitant) exposure to a set of pesticide profiles in five European countries (Latvia, Hungary, Czech Republic, Spain and the Netherlands). We explored whether living in an agricultural area (compared to living in a peri-urban area), being a a child (compared to being an adult), and the season in which the urine sample was collected had an impact on the probability of detection of pesticides (-metabolites). In total 2088 urine samples were collected from 1050 participants (525 parent-child pairs) and analyzed through harmonized suspect screening by five different laboratories. Fourty pesticide biomarkers (either pesticide metabolites or the parent pesticides as such) relating to 29 pesticides were identified at high levels of confidence in samples across all study sites. Most frequently detected were biomarkers related to the parent pesticides acetamiprid and chlorpropham. Other biomarkers with high detection rates in at least four countries related to the parent pesticides boscalid, fludioxonil, pirimiphos-methyl, pyrimethanil, clothianidin, fluazifop and propamocarb. In 84% of the samples at least two different pesticides were detected. The median number of detected pesticides in the urine samples was 3, and the maximum was 13 pesticides detected in a single sample. The most frequently co-occurring substances were acetamiprid with chlorpropham (in 62 urine samples), and acetamiprid with tebuconazole (30 samples). Some variation in the probability of detection of pesticides (-metabolites) was observed with living in an agricultural area or season of urine sampling, though no consistent patterns were observed. We did observe differences in the probability of detection of a pesticide (metabolite) among children compared to adults, suggesting a different exposure and/or elimination patterns between adults and children. This survey demonstrates the feasibility of conducting a harmonized pan-European sample collection, combined with suspect screening to provide insight in the presence of exposure to pesticide mixtures in the European population, including agricultural areas. Future improvements could come from improved (harmonized) quantification of pesticide levels.

A large scale multi-laboratory suspect screening of pesticide metabolites in human biomonitoring: From tentative annotations to verified occurrences.

Within the Human Biomonitoring for Europe initiative (HBM4EU), a study to determine new biomarkers of exposure to pesticides and to assess exposure patterns was conducted. Human urine samples (N = 2,088) were collected from five European regions in two different seasons. The objective of the study was to identify pesticides and their metabolites in collected urine samples with a harmonized suspect screening approach based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) applied in five laboratories. A combined data processing workflow included comprehensive data reduction, correction of mass error and retention time (RT) drifts, isotopic pattern analysis, adduct and elemental composition annotation, finalized by a mining of the elemental compositions for possible annotations of pesticide metabolites. The obtained tentative annotations (n = 498) were used for acquiring representative data-dependent tandem mass spectra (MS2) and verified by spectral comparison to reference spectra generated from commercially available reference standards or produced through human liver S9 in vitro incubation experiments. 14 parent pesticides and 71 metabolites (including 16 glucuronide and 11 sulfate conjugates) were detected. Collectively these related to 46 unique pesticides. For the remaining tentative annotations either (i) no data-dependent MS2 spectra could be acquired, (ii) the spectral purity was too low for sufficient matching, or (iii) RTs indicated a wrong annotation, leaving potential for more pesticides and/or their metabolites being confirmed in further studies. Thus, the reported results are reflecting only a part of the possible pesticide exposure.

Harmonized Quality Assurance/Quality Control Provisions for Nontargeted Measurement of Urinary Pesticide Biomarkers in the HBM4EU Multisite SPECIMEn Study.

A set of quality assurance/quality control (QA/QC) criteria for nontargeted measurement of pesticide exposure markers in a large-scale study of human urine has been proposed and applied across five laboratories within the HBM4EU project. Quality control material, including reference standards and fortified pooled urine samples (QC urine) were prepared in a centralized way and distributed across participants to monitor analytical performance and consistency of the liquid chromatography coupled to high-resolution mass spectrometry data generated with a harmonized workflow. Signal intensities, mass accuracy, and retention times of selected QA/QC markers covering a broad range of physicochemical properties were monitored across QC solvent standards, QC urine samples, study urine samples, and procedural blanks, setting acceptance thresholds for repeatability and accuracy. Overall, results showed high repeatability of the collected data. The RSDs of the signal intensities were typically below 20-30% in QC and study samples, with good stability of the chromatographic separation (retention time drift within 2-4 s intrabatch and 5 s interbatch) and excellent mass accuracy (average error < 2 ppm). The use of the proposed criteria allowed for the identification of handling errors, instrumental issues, and potential batch effects. This is the first elaboration of harmonized QA/QC criteria applied across multiple laboratories to assess the quality of data generated by nontargeted analysis of human samples.

A green analytical method for the simultaneous determination of 30 tropane and pyrrolizidine alkaloids and their N-oxides in teas and herbs for infusions by LC-Q-Orbitrap HRMS.

A green analytical method for the simultaneous determination of 30 tropane and pyrrolizidine alkaloids and their N-oxides in dried teas and herbs for infusions has been developed and validated. The proposed method is based on QuEChERS procedure followed by LC-Q-Orbitrap HRMS analysis. The method includes a first screening analysis to assess the presence of alkaloids, followed by the quantification of suspected positive samples (cut-off level, 0.2-2.6 µg kg-1). The method was validated in five different tea and herb matrices showing satisfactory linearity (R2 ≥0.99), method limits of quantification (5 µg kg-1), accuracy (87-111 %), and precision (RSD <20 %). The greenness of the proposed method was evaluated according to the Analytical Eco-Scale, showing that it could be considered an excellent green analysis. Finally, eleven commercial field samples of tea and herbs for infusions, including rooibos, chamomile, red tea, black tea, green tea, white tea, linden, horsetail, and one infusion containing a mixture of herbs, were analyzed and the obtained results demonstrated that they were in compliance with the current European regulations regarding the studied substances.

Biomonitoring of Phthalates, Bisphenols and Parabens in Children: Exposure, Predictors and Risk Assessment.

Exposure to emerging contaminants, such as phthalates, bisphenols and parabens in children has been associated with possible neurodevelopment and endocrine alterations. In the present study, the biomonitoring of biomarkers in children (5-12 years old) from the Valencia Region (Spain) have been implemented using urines from the BIOVAL program. More than 75% of the children studied (n = 562) were internally exposed (>LOQ) to bisphenols and parabens, and the whole population assessed (n = 557) were exposed to at least one phthalate. The geometric means (GM) of the concentrations of bisphenol A, methyl paraben and propyl paraben were 0.9, 1.4 and 0.39 ng/mL, respectively. Regarding phthalates, monoethyl phthalate GM was 55.0 ng/mL and diethyl hexyl phthalate (as the sum of five metabolites) GM was 60.6 ng/mL. Despite the studied population being widely exposed, the detection frequencies and concentrations were in general lower than in previous studies involving children in Spain and in other countries in recent years. Furthermore, the risk assessment study concluded that the internal exposure to phthalates, bisphenols and parabens is lower than the guidance values established, and, therefore, a health risk derived from the exposure to these compounds in the studied population is not expected.

Protein Biomarkers of Bovine Defective Meats at a Glance: Gel-Free Hybrid Quadrupole-Orbitrap Analysis for Rapid Screening.

An understanding of biological mechanisms that could be involved in the stress response of animal cattle prior to slaughter is critical to create effective strategies aiming at the production of high-quality meat. The sarcoplasmic proteome of directly extracted samples from normal and high ultimate pH (pHu) meat groups was studied through a straightforward gel-free strategy supported by liquid chromatography hybrid quadrupole-Orbitrap high-resolution mass spectrometry (LC-HRMS) analysis. A stepped proteomic pipeline combining rapid biomarker hunting supported by qualitative protein Mascot scores followed by targeted label-free peptide quantification revealed 26 descriptors that characterized meat groups assayed. The functional study of the proposed biomarkers suggested their relevant role in metabolic, chaperone/stress-related, muscle contractility/fiber organization, and transport activities. The efficiency, flexibility, rapidity, and easiness of the methodology proposed can positively contribute to the creation of innovative proteomic alternatives addressing meat quality assessment.

Primary care in the time of COVID-19: monitoring the effect of the pandemic and the lockdown measures on 34 quality of care indicators calculated for 288 primary care practices covering about 6 million people in Catalonia.

BACKGROUND: To analyse the impact of the COVID-19 epidemic and the lockdown measures on the follow-up and control of chronic diseases in primary care. METHODS: Retrospective study in 288 primary care practices (PCP) of the Catalan Institute of Health. We analysed the results of 34 indicators of the Healthcare quality standard (EQA), comprising different types: treatment (4), follow-up (5), control (10), screening (7), vaccinations (4) and quaternary prevention (4). For each PCP, we calculated each indicator's percentage of change in February, March and April 2020 respective to the results of the previous month; and used the T-Student test for paired data to compare them with the percentage of change in the same month of the previous year. We defined indicators with a negative effect those with a greater negative change or a lesser positive change in 2020 in comparison to 2019; and indicators with a positive effect those with a greater positive change or a lesser negative change. RESULTS: We observed a negative effect on 85% of the EQA indicators in March and 68% in April. 90% of the control indicators had a negative effect, highlighting the control of LDL cholesterol with a reduction of - 2.69% (95%CI - 3.17% to - 2.23%) in March and - 3.41% (95%CI - 3.82% to - 3.01%) in April; and the control of blood pressure with a reduction of - 2.13% (95%CI - 2.34% to - 1.9%) and - 2.59% (95%CI - 2.8% to - 2.37%). The indicators with the greatest negative effect were those of screening, such as the indicator of diabetic foot screening with a negative effect of - 2.86% (95%CI - 3.33% to - 2.39%) and - 4.13% (95%CI - 4.55% to - 3.71%) in March and April, respectively. Only one vaccination indicator, adult Measles-Mumps-Rubella vaccine, had a negative effect in both months. Finally, among the indicators of quaternary prevention, we observed negative effects in March and April although in that case a lower inadequacy that means better clinical outcome. CONCLUSIONS: The COVID-19 epidemic and the lockdown measures have significantly reduced the results of the follow-up, control, screening and vaccination indicators for patients in primary care. On the other hand, the indicators for quaternary prevention have been strengthened and their results have improved.

Risk assessment of exposure to phthalates in breastfeeding women using human biomonitoring.

In this study, we assessed the presence of 14 phthalate metabolites in the urine of 104 lactating mothers from Valencia (Spain) who took part in the human biomonitoring project BETTERMILK. Nine of the metabolites studied showed detection frequencies >80%, whereas the rest of the metabolites presented low detection frequencies (<5%). The concentrations ranged from

Primary Care Doctor Characteristics That Determine the Use of Teleconsultations in the Catalan Public Health System: Retrospective Descriptive Cross-Sectional Study.

BACKGROUND: eConsulta is a tele-consultation service involving doctors and patients, and is part of Catalonia's public health information technology system. The service has been in operation since the end of 2015 as an adjunct to face-to-face consultations. A key factor in understanding the barriers and facilitators to the acceptance of the tool is understanding the sociodemographic characteristics of general practitioners who determine its use. OBJECTIVE: This study aimed to analyze the sociodemographic factors that affect the likelihood of doctors using eConsulta. METHODS: A retrospective cross-sectional analysis of administrative data was used to perform a multivariate logistic regression analysis on the use of eConsulta in relation to sociodemographic variables. RESULTS: The model shows that the doctors who use eConsulta are 45-54 years of age, score higher than the 80th percentile on the quality of care index, have a high degree of accessibility, are involved in teaching, and work on a health team in a high socioeconomic urban setting. CONCLUSIONS: The results suggest that certain sociodemographic characteristics associated with general practitioners determine whether they use eConsulta. These results must be taken into account if its deployment is to be encouraged in the context of a public health system.

Target analysis and retrospective screening of veterinary drugs, ergot alkaloids, plant toxins and other undesirable substances in feed using liquid chromatography-high resolution mass spectrometry.

A comprehensive strategy combining a quantitative method for 77 banned veterinary drugs, mycotoxins, ergot alkaloids and plant toxins, and a post-target screening for 425 substances including pesticides and environmental contaminants in feed were developed using a QuEChERS-based extraction and an ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The quantitative method was validated after previous statistical optimisation of the main parameters governing ionisation, and presented recoveries ranging, in general, from 80 to 120%, with a precision in terms of Relative Standard Deviation (RSD) lower than 20%. The full-scan accurate mass data were acquired with a resolving power of 50000 FWHM and a mass accuracy lower than 5ppm. The method LOQ was lower than 12.5µgkg(-1) for the majority of the veterinary drugs and plant toxins and 20µgkg(-1) for ergot alkaloids. For post-target screening a customised theoretical database including the exact mass, the polarity of acquisition and the expected adducts was built and used for post-run retrospective screening. The analytical strategy was applied to 32 feed samples collected from farms of the Valencia Region (Spain). Florfenicol, zearalenone and atropine were identified and quantified at concentrations around 10µgkg(-1). In the post-target screening of the real samples, Sulfadiazine, Thrimetoprin and Pirimiphosmethyl were tentatively identified.

Combined target and post-run target strategy for a comprehensive analysis of pesticides in ambient air using liquid chromatography-Orbitrap high resolution mass spectrometry.

A comprehensive strategy for the analysis of current airborne pesticides has been developed using liquid chromatography coupled to high resolution mass spectrometry. The methodology includes both quantitative target analysis and post-run target screening analysis. The quantitative method was validated after a previous statistical optimisation of the main factors governing the ion source ionization and a study of the single-stage Orbitrap fragmentation through the HCD cell. The quantitative method presented recoveries ranging from 73 to 116%, with precision (RSD) lower than 20%, for the 35 substances in the scope of the target method. The full-scan accurate mass data were acquired with a resolving power of 50000 FWHM (scan speed, 2 Hz), and alternating two acquisition events, ESI+ without fragmentation and ESI+ with fragmentation. The method-LOQ was 6.5 pg m(-3) for most of the target pesticides. For post-target screening a customized theoretical database, that included pesticides, metabolites and other substances such as emerging flame retardants was built up. For identification, accurate exact mass with less than 5 ppm, and some diagnostic ions including isotopes and/or fragments were used. The strategy was applied to ten samples collected in a rural area of Valencia (Spain). Four pesticides, namely carbendazim, metalaxyl, myclobutanil and terbuthylazine, were detected in concentrations from 16 pg m(-3) to 174 pg m(-3). Some pesticides and metabolites (endothal, fenfuram, terbuthylazine-2-OH), in addition to two flame retardants were tentatively identified in the post-run target screening analysis.

Qualitative screening of 116 veterinary drugs in feed by liquid chromatography-high resolution mass spectrometry: potential application to quantitative analysis.

Veterinarian and human pharmaceuticals may be intentionally added to animal feed to enhance animal production. Monitoring these substances is necessary for protecting the consumers. In this work, a screening method covering 116 human and veterinary drugs has been developed and validated in five types of animal feed at 0.02 and 0.2 mg kg(-1). After a simple extraction and dilution, the samples were analysed by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). Nearly all compounds tested were detected at 0.02 mg kg(-1), based on the presence of the accurate-mass (de)protonated molecule. However, the identification using a second accurate-mass ion was more problematic at this level. Finally, the procedure was applied to 22 feed samples, where trimethoprim, robenidine, or α- and ß-nandrolone were detected and identified. The potential applicability of the method to quantitative analysis of the compounds detected in the samples was also evaluated.

Evaluation of mycotoxins and their metabolites in human breast milk using liquid chromatography coupled to high resolution mass spectrometry.

Humans can be exposed to mycotoxins through the food chain. Mycotoxins are mainly found as contaminants in food and could be subsequently excreted via biological fluids such as urine or human breast milk in native or metabolised form. Since breast milk is usually supposed as the only food for new-borns, the occurrence of mycotoxins in thirty-five human milk samples was evaluated by a newly developed method based on QuEChERS extraction and UHPLC-HRMS detection. The method described here allows the detection of target mycotoxins in order to determine the quality of this initial feeding. The method has been fully validated, with recoveries ranging from 64% to 93% and relative standard deviations (RSD, %) being lower than 20%. Using the method described, non-metabolised mycotoxins such as ZEA, NEO, NIV, ENA, ENA1, ENB, ENB1 and metabolites, such as ZEA metabolites, HT-2, DOM and T-2 triol were detected in human milk samples. Results obtained help to estimate the exposure of mothers and infants to mycotoxins. Moreover, to the best of our knowledge, this is the first work describing the simultaneous detection, quantification and screening of mycotoxins and their metabolites in human mature milk.

Wide-range screening of banned veterinary drugs in urine by ultra high liquid chromatography coupled to high-resolution mass spectrometry.

In this work, an ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) methodology is proposed for the multi-class multi-residue screening of banned and unauthorized veterinary drugs in bovine urine, using an Orbitrap Exactive™ analyzer working at a resolving power of 50,000 FWHM in full scan, both in positive and negative mode. The method currently covers 87 analytes belonging to different families such as steroid hormones, ß-agonists, resorcylic acid lactones (RAL), stilbens, tranquillizers, nitroimidazoles, corticosteroids, NSAIDs, amphenicoles, thyreostatics and other substances such as dapsone. A database including the elemental composition, the polarity of acquisition, retention time and expected adducts was built for the targeted analysis, and a high mass accuracy (<5 ppm) was set as one of the identification criteria. After comparing different sample preparation procedures, QuEChERS was selected as the most appropriate methodology. An efficient separation of analytes was achieved using ultra high performance liquid chromatography with a column packed with sub-2 µm particles. The performance of the method has been evaluated in accordance with the EU guidelines for the validation of screening methods for the analysis of veterinary drugs residues. The screening target concentrations were established between 0.2 µg/l and 20 µg/l, demonstrating the usefulness of UHPLC-HRMS as an ideal tool for compliance monitoring in regulatory laboratories.

Development of a method for the analysis of seven banned azo-dyes in chilli and hot chilli food samples by pressurised liquid extraction and liquid chromatography with electrospray ionization-tandem mass spectrometry.

An automated, confirmatory and sensitive procedure has been developed and validated for the determination of Sudan (I-IV), Sudan Orange G, Sudan Red 7B and Para Red in hot chilli food samples. The proposed method includes pressurised liquid extraction (PLE) with acetone, gel permeation chromatography (GPC) clean-up and detection by liquid chromatography (LC) coupled to electrospray ionization in positive mode tandem mass spectrometry (ESI-MS-MS). The main parameters affecting the performance of the different ionization sources and PLE parameters were previously optimised using statistical design of experiments (DOE). The method was in-house validated on chilli powder and chilli meat. Linear calibrations were obtained with correlation coefficients R(2)>0.999. The limits of detection (LOD) and quantification (LOQ) of the method were in the ranges of 0.002-0.012 ng g(-1) and 0.006-0.036 ng g(-1), respectively for chilli powder. The decision limit and detection capability were between 0.005-0.022 ng g(-1) and 0.007-0.026 ng g(-1), respectively for chilli meat. Recoveries ranged from 94% to 105%. The applicability of the method to the determination of azo-dyes in hot chilli products was demonstrated.

Determination of 3-MCPD by GC-MS/MS with PTV-LV injector used for a survey of Spanish foodstuffs.

3-Monochloropropane-1,2-diol (3-MCPD) is the most common chemical contaminant of the group of chloropropanols. It can occur in foods and food ingredients at low levels as a result of processing, migration from packaging materials during storage and domestic cooking. A sensitive method for determination of 3-MCPD in foodstuffs using programmable temperature vaporization (PTV) with large-volume injection (LVI) gas chromatography (GC) with tandem mass spectrometry detection (MS/MS) has been developed and optimized. The optimization of the injection and detection parameters was carried out using statistical experimental design. A Plackett-Burman design was used to estimate the influence of resonance excitation voltage (REV), isolation time (IT), excitation time (ET), ion source temperature (IST), and electron energy (EE) on the analytical response in the ion trap mass spectrometer (ITMS). Only REV was found to have a statically significant effect. On the other hand, a central composite design was used to optimize the settings of injection temperature (T(inlet)), vaporization temperature (T(vap)), vaporization time (t(vap)) and flow (Flow). The optimized method has an instrumental limit of detection (signal-to-noise ratio 3:1) of 0.044 ng mL(-1). From Valencian, Spain, supermarkets 94 samples of foods were surveyed for 3-MCPD. Using the optimized method levels higher than the limit established for soy sauce by the European Union were found in some samples. The estimated daily intake of 3-MCPD throughout the investigated foodstuffs for adults and children was found about 0.005 and 0.01%, respectively, of the established provisional tolerable daily intake.

Development of a pressurised liquid extraction and liquid chromatography with electrospray ionization-tandem mass spectrometry method for the determination of domoic acid in shellfish.

Amnesic shellfish poisoning (ASP) is a potentially lethal human toxic syndrome which is caused by domoic acid (DA) that originates in marine phytoplankton belonging to the Pseudonitzschia genus. A confirmatory and sensitive procedure has been developed and validated for the determination of DA in shellfish. The proposed method includes pressurised liquid extraction (PLE) with methanol/acetone (9:1), florisil clean-up purification inside the PLE extraction cell and detection by liquid chromatography (LC) coupled to electrospray ionization in positive mode tandem mass spectrometry (ESI-MS-MS). Comparison of ionization sources (ESI, atmospheric pressure ionization (APCI) atmospheric pressure photoionization (APPI) and combined APCI/APPI) were carried out in order to improve the analytical signal. The main parameters affecting the performance of the different ionization sources and PLE parameters were previously optimised using statistical design of experiments (DOE). Linear calibrations were obtained using mussel tissue extracts 0.05-5 microg DA/ml (R2>0.999). The limits of detection (LOD) and quantitation (LOQ) of the method were 0.2 and 0.5 microg/g respectively and recoveries ranged from 81 to 95%. This method was successfully applied to determine DA levels in 46 shellfish samples collected from Valencian (Spain) supermarkets, showing high sample throughput.