Determination of highly polar anionic pesticides in beehive products by hydrophilic interaction liquid chromatography coupled to mass spectrometry.

The analysis of highly polar pesticides is challenging due to their unique physicochemical properties, requiring specialized chromatographic techniques for their accurate and sensitive detection. Furthermore, the high level of co-extracted polar matrix components that can co-elute with the analytes can interfere with the analysis. Consequently, there is lack of pesticide monitoring data, as the European Food Safety Authority has pointed out. This article explores the overcoming of such difficulties in the analysis of these compounds. Analytical methodologies for the extraction, clean-up, and direct determination of 11 highly polar anionic pesticides, including glyphosate, glufosinate, ethephon, fosetyl-aluminium, and their related metabolites in complex food matrices such as honey and pollen by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry were successfully developed and validated. Solid-phase extraction and micro-solid-phase extraction employing strong anion exchange (SAX) cartridges were implemented for clean-up. The automation and miniaturization of SAX clean-up for these compounds were achieved for the first time. For method validation, SANTE/11312/2021 guideline was followed. Recoveries were between 70 and 120%, with RSDs below 20%. Limits of quantitation ranged from 0.005 to 0.020 mg kg-1. Linearity was evaluated from 0.002 to 0.200 mg kg-1. Matrix effects were assessed, showing medium to low signal suppression for most compounds. AMPA and glufosinate presented the highest signal suppression, but it was reduced after SAX clean-up. Analysis of real honey and pollen samples revealed the occurrence of the studied compounds in beehive products and showed the applicability of the validated methodologies for routine control of these complex samples.

Ion chromatography coupled to Q-Orbitrap for the analysis of formic and oxalic acid in beehive matrices: a field study.

There is an increasing concern about the use of synthetic acaricides to fight the ectoparasitic mite Varroa destructor. Natural products such as formic acid (FA) and oxalic acid (OA) have emerged as a possible alternative control strategy. However, given the difficulty of analysing these highly polar compounds and the lack of robust and reliable methods, there are very few studies of the concentration and distribution of these natural acaricides in the beehive compartments. We present a reliable and simple analytical methodology, based on sample extraction with modified quick polar pesticide (QuPPe) methods followed by ion chromatography coupled to a quadrupole Orbitrap mass analyser for the analysis of FA and OA in honeybees, honey, beeswax, and beebread. The developed methods have been used in a field study for the evaluation of the presence and distribution of FA and OA in the beehive products, as well as in adult bees and bee brood samples, before, during, and up to 3 months after the application of the treatments by the beekeeper. Beebread and honey samples presented the highest concentration levels of OA and FA, respectively, mainly due to their natural presence. As expected, the organic acids showed low persistence in wax after the treatments. The natural acaricides were found in adult and developing bees at concentration levels below the reported LD50 in all the cases; however, residue levels of OA in larvae during the treatment application were very close to the reported LD50.

Evaluation of automated clean-up for large scope pesticide multiresidue analysis by liquid chromatography coupled to mass spectrometry.

Clean-up step is essential during the multiresidue sample preparation process to remove undesired matrix components that may cause analytical interferences or suppression effect. However, its application generally by specific sorbents entails time-consuming work producing low recoveries for some compounds. Moreover, it usually needs to be adapted to the different co-extractives from the matrix present in the samples by using different chemical sorbents increasing the number of validation procedures. Therefore, the development of a more efficient and automated and unified clean-up procedure means a significant time reduction and laboratory work with improved performance. In this study, extracts from different matrices (tomato, orange, rice, avocado and black tea) were purified by manual dispersive clean-up (different procedures according to the matrix group) in parallel with an automated µSPE clean-up workflow, in both cases based on QuEChERS extraction. The latter procedure employed clean-up cartridges containing a mixture of sorbent materials (anhydrous MgSO4/PSA/C18/CarbonX) suitable for multiple matrices. All the samples were analysed by liquid chromatography mass spectrometry and the results obtained from both procedures have been compared in terms of the extract cleanness, performance, interferences, and sample workflow. At the levels studied, similar recoveries were achieved by both techniques (manual and automated) except for reactive compounds when PSA was used as the sorbent material producing low recoveries. However, the µSPE recoveries were between 70-120%. Furthermore, closer calibration line slopes were provided when µSPE was applied to the different matrix groups studied. It is important to note that up to 30% more samples per day can be analysed using an automated µSPE compared to the manual method (which requires shaking, centrifuging, then taking the supernatant and adding formic acid in ACN); it also provides good repeatability - an RSD (%) < 10%. Consequently, this technique is a very useful option for routine analyses, greatly simplifying the work of muti-residue methods.

Overcoming difficulties in the evaluation of captan and folpet residues by supercritical fluid chromatography coupled to mass spectrometry.

Serious difficulties in evaluating the fungicides captan and folpet by the usual chromatography systems coupled to mass spectrometry are well known. These compounds are highly prone to degradation due to different conditions into tetrahydrophthalimide (THPI) and phthalimide (PHI). Such an effect can be produced at different stages of the analytical procedure or during the growing crop, making their evaluation troublesome. As a consequence, the quantification of captan and folpet is typically performed through or together these metabolites. However, imide ring metabolites can be produced by other unknown sources, including other phthalimide derived pesticides enabling false positive results. For this reason, in the last decade, laboratories demand a robust method to quantify captan and folpet, that overcomes such a situation. In the present work, various operational parameters were optimized to ensure the no degradation of captan and folpet facilitated by supercritical fluid chromatography coupled to mass spectrometry (SFC-MS/MS). A direct comparison with reverse-phase LC-MS/MS and GC-MS/MS was conducted for comparative purposes. The representative commodities selected for this evaluation were pepper and tomato. Furthermore, possible oxidative degradation during the sample milling step was also evaluated and avoided by the application of crio-milling conditions and ascorbic acid addition. By the proposed procedure, captan and folpet were recovered in both matrices at the 84%-105% range and with an RSD below 8% at two concentration levels: 10 and 50 µg/kg. On the contrary, with GC-MS/MS, captan and folpet were not recovered, and, as a consequence, their evaluation was possible only by THPI and PI. In the case of LC-MS/MS a significant decrease in the sensitivity was observed compared to SFC-MS/MS. Other validation parameters evaluated were satisfactory. This new approach can assess the correct analysis of captan and folpet at low concentrations in fruits and vegetables.

Dual-channel chromatography a smart way to improve the analysis efficiency in liquid chromatography coupled to mass spectrometry.

Dual-channel chromatography was evaluated for pesticide residue analysis in fruits and vegetables and for unknown compounds detection. A dual-channel system was tested coupled to triple quadrupole and high-resolution mass spectrometry. The first part of the investigation was related to the improvement of the sample throughput with a 100 mm column. The dual-channel system provided the same analytical results as the single-channel system, however, with the throughput higher of about 70% (80 injections vs 137 injections in 24 h). Two types of calibration (in-channel and cross-channel) were checked. In the article, also solvent consumption is discussed. Six proficiency test samples were analysed to assess the quality of the results. Nor false positives neither false negatives were found. Calculated z-scores were typically <1. In the second part, a different approach was investigated. The 100 mm column was replaced by a 150 mm column keeping shorter run times than single channel system and 100 mm. The longer column improved the sensitivity and selectivity what was demonstrated in the target pesticide residue analysis. Additionally, the 150 mm column was compared with the 100 mm column in the analysis of unknown natural matrix compounds by high resolution mass spectrometry. The longer column allowed to detect up to 26% unknown compounds more than the shorter column.

Identification of unexpected chemical contaminants in baby food coming from plastic packaging migration by high resolution accurate mass spectrometry.

Plastic multilayers are widely used for baby food packaging. However, it is important to consider that migration of food contact materials (FCM) into the baby food can occur. The comprehensive identification of potential migrants, including intentionally added substances (IAS) and non-intentionally added substances (NIAS), is required to assess the safety of these packaging materials. In this study, high resolution accurate mass spectrometry (HRAMS) with a data-independent acquisition method of sequential mass windows enables the detection of substances with corresponding deconvoluted fragment mass spectra. The identification of unexpected migrants present in the food simulants and in real baby food was facilitated by filtering strategies and by an in-house library. This approach has allowed the identification of 42 migrants, including eight NIAS detected for the first time. Two oligomers were quantified by means of reference standard materials at concentration levels above 0.010 mg/kg, exceeding the maximum residue levels for baby food.

Miniaturized QuEChERS based methodology for multiresidue determination of pesticides in odonate nymphs as ecosystem biomonitors.

The impacts of the modern, agrochemicals based agriculture that threatens the overall systems sustainability, need to be monitored and evaluated. Seeking for agroecosystems monitors, the present article focus in the occurrence and abundance of aquatic macroinvertebrates, that have been frequently used as bioindicators of water quality due to their relationship with land use. Some of these organisms are on the top of the food chain, where bioaccumulation and biomagnification processes can be observed, and they can turn into secondary pollution sources of systems and terrestrial organisms as well. Odonate nymphs, which belong to the functional group of predators, were selected for this study. A methodology to determine 73 pesticide residues in odonate nymphs by LC-MS/MS and GC-MS/MS was developed. A QuEChERS sample preparation strategy was adapted. As it is complex to obtain samples especially in disturbed ecosystems, the method was minimized to a sample size of 200mg of fresh nymphs. The method was validated and good recoveries (71-120%) with RSDs below 20% for the majority of the studied pesticides at least at two of the assayed levels 1, 10 and 50µgkg-1 were obtained. For 32 analytes the limit of quantitation was 1µgkg-1 and 10µgkg-1 for the others. The lineal range was observed between 1-100µgkg-1 in matrix-matched and solvent calibration curves for most of the assessed pesticides. LC-MS/MS matrix effects were evaluated, 40% of the analytes presented low or no signal suppression. Only flufenoxuron presented high matrix effects. The obtained methodology is adequate for pesticide multiresidue analysis in aquatic macroinvertebrates (odonates) aiming to contribute to the ecological state evaluation of freshwater ecosystems.

Neonicotinoids transference from the field to the hive by honey bees: Towards a pesticide residues biomonitor.

The beehive as a quantitative monitor of pesticide residues applied over a soybean crop was studied through a semi field experiment of controlled exposure of honey bees to pesticides in macro tunnels. The distribution within exposed beehives of pesticides commonly used in soybean plantation, was assessed. Residue levels of insecticides in soybean leaves, honey bees, wax, honey and pollen were analyzed. The transference from pesticides present in the environment into the beehive was evidenced. The obtained results allow relating pesticide concentrations present in the environment with traces found in foraging bees. Therefore, pesticide transference ratios could be calculated for each detected compound (acetamiprid, imidacloprid and thiamethoxam) which showed a linear inverse trend with their 1-octanol/water partition coefficient (Kow). The least transferred pesticide to the hive (acetamiprid) has the highest vapor pressure (Vp). This study gives new insights on the usefulness of monitoring the environment through beehives aiming to evaluate if agroecosystems remain sustainable. It also contributes to generate valuable information for model building aiming to predict environmental quality through beehive's analysis.

QuEChERS Adaptability for the Analysis of Pesticide Residues in Beehive Products Seeking the Development of an Agroecosystem Sustainability Monitor.

Beehive products could be powerful monitors of pesticide residues originating in agroecosystems during production cycles. Their ready availability provides enough samples to perform analytical determinations, but their chemical complexity makes residue analysis a real challenge. Taking advantage of the plasticity of QuEChERS coupled to LC-MS/MS, validated methodologies were developed for bees, honey, beeswax, and pollen and applied to real samples for the simultaneous determination of 19 of the most employed pesticides in intensive cropping fields. Beehives placed in Uruguayan agroecosystems accumulated the pesticides thiacloprid, imidacloprid, methomyl, carbaryl, hexythiazox, azoxystrobin, pyraclostrobin, tebuconazole, and haloxyfop-methyl at 0.0001-0.01 mg/kg levels. The oscillations on the amount and occurrence of residue findings for specific apiaries were correlated statistically with the sampling season and the agroecosystem where the beehives were located, showing the potential of bees and bee products to record relevant information to survey the chemicals applied in their surroundings.