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.

Multilaboratory Collaborative Study of a Nontarget Data Acquisition for Target Analysis (nDATA) Workflow Using Liquid Chromatography-High-Resolution Accurate Mass Spectrometry for Pesticide Screening in Fruits and Vegetables.

Nontarget data acquisition for target analysis (nDATA) workflows using liquid chromatography-high-resolution accurate mass (LC-HRAM) spectrometry, spectral screening software, and a compound database have generated interest because of their potential for screening of pesticides in foods. However, these procedures and particularly the instrument processing software need to be thoroughly evaluated before implementation in routine analysis. In this work, 25 laboratories participated in a collaborative study to evaluate an nDATA workflow on high moisture produce (apple, banana, broccoli, carrot, grape, lettuce, orange, potato, strawberry, and tomato). Samples were extracted in each laboratory by quick, easy, cheap, effective, rugged, and safe (QuEChERS), and data were acquired by ultrahigh-performance liquid chromatography (UHPLC) coupled to a high-resolution quadrupole Orbitrap (QOrbitrap) or quadrupole time-of-flight (QTOF) mass spectrometer operating in full-scan mass spectrometry (MS) data-independent tandem mass spectrometry (LC-FS MS/DIA MS/MS) acquisition mode. The nDATA workflow was evaluated using a restricted compound database with 51 pesticides and vendor processing software. Pesticide identifications were determined by retention time (tR, ±0.5 min relative to the reference retention times used in the compound database) and mass errors (δM) of the precursor (RTP, δM ≤ ±5 ppm) and product ions (RTPI, δM ≤ ±10 ppm). The elution profiles of all 51 pesticides were within ±0.5 min among 24 of the participating laboratories. Successful screening was determined by false positive and false negative rates of <5% in unfortified (pesticide-free) and fortified (10 and 100 µg/kg) produce matrices. Pesticide responses were dependent on the pesticide, matrix, and instrument. The false negative rates were 0.7 and 0.1% at 10 and 100 µg/kg, respectively, and the false positive rate was 1.1% from results of the participating LC-HRAM platforms. Further evaluation was achieved by providing produce samples spiked with pesticides at concentrations blinded to the laboratories. Twenty-two of the 25 laboratories were successful in identifying all fortified pesticides (0-7 pesticides ranging from 5 to 50 µg/kg) for each produce sample (99.7% detection rate). These studies provide convincing evidence that the nDATA comprehensive approach broadens the screening capabilities of pesticide analyses and provide a platform with the potential to be easily extended to a larger number of other chemical residues and contaminants in foods.

Cutting-edge approach using dual-channel chromatography to overcome the sensitivity issues associated with polarity switching in pesticide residues analysis.

A fast and sensitive method was validated for the analysis of pesticide residues in baby food. After an QuEChERS-based extraction, the samples were analysed with a dual-channel liquid chromatography instrument coupled to a triple quadrupole mass spectrometer. The method consisted of two independent injections per sample. In the first injection the sample was analysed with a mobile phase optimal for the positive polarity ionisation (water, methanol, formic acid, and ammonium formate), whereas in the second injection the mobile phase was optimised for the negative polarity ionisation (water, acetonitrile, acetic acid). The total number of pesticides was 264, out of which 238 were analysed with the methanol gradient and 26 with the acetonitrile gradient. The dual-channel instrument allowed for sample multiplexing. Thus, sample throughput was equivalent to that of a single-channel system. The limit of quantitation, determined according to the DG SANTE guidance document, was 0.003 mg/kg for 97% of the evaluated compounds. The validation study was followed by a real sample survey. In 42 samples, 16 positives were found.

Improving the simultaneous target and non-target analysis LC-amenable pesticide residues using high speed Orbitrap mass spectrometry with combined multiple acquisition modes.

The use of high-resolution mass spectrometry (HRMS) for the simultaneous target and non-target analysis of pesticide residues in food control is a subject that has been studied over the last decade. However, proving its efficacy compared to the more established triple quadrupole mass spectrometers (QQQ-MS2) is challenging. Various HRMS platforms have been evaluated, seemingly showing this approach not to be as effective as QQQ-MS2 for quantitative analysis, especially in routine food testing laboratories. The two main reasons are (i) the lower sensitivity especially in the case of the fragment ions produced and (ii) the lack of familiarity and an understanding of the most appropriate combination of HRMS acquisition modes to use. In fact, the number of different acquisition modes can appear as a puzzle to inexperienced users. This work was therefore focused on obtaining experimental data to gain a better understanding of the extended acquisition capabilities of a new Q-Orbitrap platform. Experimental data were obtained for 244 pesticides and their degradation products in commodities of varying matrix complexity (tomato, onion, avocado, and orange) using various combinations of acquisition modes. The best results for targeted analysis were obtained with a combination of full scan (FS), all-ions fragmentation (AIF) and target MS2 (tMS2) modes, and for non-target analysis using full scan (FS) and data-dependent MS2 (ddMS2) modes. All these acquisition modes (FS, AIF, tMS2, and ddMS2) could be applied simultaneously with cycle times ≤ 1 s. The tMS2 especially, proved to be a very powerful approach to increase sensitivity for MS2 fragments and identification rates. Overall, the results for the various pesticide-commodity combinations were fully satisfactory in terms of limit of quantitation (LOQ) repeatability and identification when considered against the SANTE EU Guideline criteria. In addition, the screening capabilities were evaluated for a non-target survey with the use of spectral libraries, the presence of non-target compounds was detected, thus proving the efficacy of the proposed approach. Another issue often overlooked is the optimization of use of spectral libraries, but in our experiments the compounds present in these libraries were not blindly sought in the screening analyses. To minimize the potential for false positives detects in our study, the extractability of the compounds present in the libraries, was also taken into account. The extractability of compounds using a QuEChERS acetonitrile procedure was estimated based on the physicochemical properties of target compounds. By removing compounds that will not be extracted, reduces the occurrences of false detects, reducing the time required for data processing and thus improving the efficiency of the overall screening workflow.

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.

A non-targeted metabolomic approach to identify food markers to support discrimination between organic and conventional tomato crops.

In the last decade, the consumption trend of organic food has increased dramatically worldwide. However, the lack of reliable chemical markers to discriminate between organic and conventional products makes this market susceptible to food fraud in products labeled as "organic". Metabolomic fingerprinting approach has been demonstrated as the best option for a full characterization of metabolome occurring in plants, since their pattern may reflect the impact of both endogenous and exogenous factors. In the present study, advanced technologies based on high performance liquid chromatography-high-resolution accurate mass spectrometry (HPLC-HRAMS) has been used for marker search in organic and conventional tomatoes grown in greenhouse under controlled agronomic conditions. The screening of unknown compounds comprised the retrospective analysis of all tomato samples throughout the studied period and data processing using databases (mzCloud, ChemSpider and PubChem). In addition, stable nitrogen isotope analysis (δ15N) was assessed as a possible indicator to support discrimination between both production systems using crop/fertilizer correlations. Pesticide residue analyses were also applied as a well-established way to evaluate the organic production. Finally, the evaluation by combined chemometric analysis of high-resolution accurate mass spectrometry (HRAMS) and δ15N data provided a robust classification model in accordance with the agricultural practices. Principal component analysis (PCA) showed a sample clustering according to farming systems and significant differences in the sample profile was observed for six bioactive components (L-tyrosyl-L-isoleucyl-L-threonyl-L-threonine, trilobatin, phloridzin, tomatine, phloretin and echinenone).

Evaluation of supercritical fluid chromatography coupled to tandem mass spectrometry for pesticide residues in food.

Supercritical fluid chromatography coupled to triple quadrupole mass spectrometry has been evaluated for pesticide residues in food. In order to check its advantages and limitations it was developed a method to identify and quantify 164 pesticides in three different matrices (tomato, orange and leek). A carbon dioxide gradient with methanol (containing 1 mM ammonium formate) was used allowing a flow rate of 1.5 mL/min that made the total run time of 12 min without any problem of overpressure. Addition of a post column flow 150 µL/min of Methanol with ammonium formate/formic acid was necessary to improve the ionization. The matrix effect study revealed that the percentages of pesticides with irrelevant matrix effect (suppression lower than 20%) was 99% in tomato, 87% in orange and 62% in leek, whereas significant suppression (higher than 50%) was not found in tomato and only 1% of the compounds in orange and 3% in leek.These results compare favorably with that typically obtained in LC-MS/MS. The absence of water in the mobile phase, also provided some important advantages regarding LC-MS/MS as (i) higher retention of polar compounds in the column, which elute with high sensitivity and good peak shape and (ii) a general increase of the sensitivity of the analysis, consequence of the high ionization and ion extraction efficiency. Pesticides evaluated were identified following the SANTE/11813/2017. At the spiking concentration of 5 µg/kg, 98% of the pesticides were identified in tomato, 98% in orange and 94% in leek, whereas for the concentration of 10 µg/kg all the compounds were identified in tomato and only spiromesifen was not identified in orange and leek. At the concentration of 20 µg/kg, spiromesifen was also identified in these two matrices. The linearity and reproducibility of the method were evaluated with results which guarantee high quality in the analytical measurements. Even though only 2 µL of final extract were injected, the sensitivity of the SFC method was enough to achieve stringent LOQs.Real samples, including 6 different fruits and vegetables, were analyzed by the SFC-MS/MS proposed method, the results being similar to those obtained by LC-MS/MS. The method was also applied to a proficiency test of fipronil in eggs with good results in all the cases. Carbon dioxide as mobile phase with methanol as modifier can represent a good alternative to LC-MS/MS with reduction of matrix effects and shorter run times.

Sensitive Detection of Neonicotinoid Insecticides and Other Selected Pesticides in Pollen and Nectar Using Nanoflow Liquid Chromatography Orbitrap Tandem Mass Spectrometry.

In this work, a new method based on nanoflow LC with high-resolution MS was developed for the determination of eight pesticides in pollen and nectar samples, including neonicotinoid insecticides and other selected pesticides commonly found in bees and beeswax. Detection was undertaken with a hybrid quadrupole-Orbitrap mass spectrometer (Q Exactive™) equipped with a commercial nanospray ion source. The extraction of pesticides from pollen samples was performed by a modified micro-QuEChERS method scaled down to Eppendorf tubes, whereas nectar samples were simply diluted with a water-methanol (95 + 5, v/v) solution. Good linearity (>0.999 in all cases) was obtained between 0.05 and 500 µg/kg and between 0.04 and 400 µg/kg for pollen and nectar, respectively. Recovery rates in pollen ranged from 85 to 97%, with RSDs <12%. Matrix effect was evaluated and showed negligible effects for all studied pesticides. The lowest concentration levels tested and validated were 0.5 and 0.4 µg/kg for pollen and nectar matrixes, respectively. In addition, selected incurred samples were studied, obtaining several positive findings in pollen and nectar samples, demonstrating the sensitivity and applicability of the proposed method.

Coupling Ion Chromatography to Q-Orbitrap for the Fast and Robust Analysis of Anionic Pesticides in Fruits and Vegetables.

Ion chromatography coupled to a quadrupole Orbitrap mass analyzer was used to develop a multiresidue method for the determination of highly polar pesticides and their metabolites (chlorate, perchlorate, fosetyl-aluminum, glyphosate, aminomethylphosphonic acid (AMPA), phosphonic acid, N-acetyl AMPA, and N-acetyl glyphosate) in fruits and vegetables. After extraction with methanol, samples were diluted 5× with water. No derivatization was applied. Pesticides were separated in an anion-exchange column. Water was used as the ion chromatography mobile phase. A gradient was created by increasing the concentration of KOH in the mobile phase. Ion chromatography provided good and stable retention and separation for all studied compounds. All investigated pesticides had an LOQ of 0.01 mg/kg and a linear range of 0.01-0.50 mg/kg. The ion ratio of the m/z ions produced was stable and adequate (deviation <30%) in all cases. The obtained mass errors (always in full-scan MS and MS2 mode) were <0.2 mDa. The high resolution (>100 000) provided by the Orbitrap analyzer with the low m/z ions obtained (e.g., m/z 80) was effective in obtaining low background matrix signals. The influence of postcolumn infusion of organic solvent on sensitivity was investigated. Acetonitrile was found to be more effective than methanol, increasing the sensitivity 3× with respect to water. The method was validated for five vegetable-based matrixes. Both the sample processing and the analytical measurement were very fast. Hence, the methodology is ideal for high-throughput work.

Evaluation of MS2 workflows in LC-Q-Orbitrap for pesticide multi-residue methods in fruits and vegetables.

LC-Q-Orbitrap efficiency was evaluated for pesticide multi-residue analysis by using three workflows involving simultaneous MS and MS2 analysis. They were as follows: data-dependent MS2 (dd-MS2), all-ion fragmentation (AIF) and variable data-independent analysis (vDIA). These MS2 workflows were tested for the main method validation parameters such as detection and identification capabilities, repeatability, linear range and quantitation. QuEChER acetonitrile extracts (blanks and spiked with 166 pesticides) of 11 different fruits and vegetables were used for this evaluation. Blank extracts were analysed to evaluate isobaric compounds and potential false identification. Spiked extracts (at 0.01 and 0.1 mg kg-1) were analysed to evaluate the false negatives potentially produced (considering a retention mass window of 0.2 min). At 0.01 mg kg-1, dd-MS2 had the highest identification rate (96-100%, depending on the matrix). In vDIA, it was 86-100% and in AIF 81-100%. But these two last workflows offered more possibilities for applying screening analysis. It was observed that application of the ion ratio criterion established in the SANTE Guidelines for identification can generate some artificial false negative results. It could be overcome by considering a mass error threshold (i.e. 5 ppm) as a selected criterion. Detection and quantitation were carried out in full-scan MS. MS2 data were used for identification. Dd-MS2 provided the highest number of points per chromatographic peak, and by that peak area, repeatability was the best (typically <10%). AIF and vDIA were characterised by longer cycle times; thus, the obtained peak area repeatability was slightly worse, but acceptable (<20%). All workflows showed very good linearity in the range 0.01-0.5 mg kg-1. The three MS2 workflows were applied to real samples with good results. Graphical abstract LC-Q-Orbitrap was used for pesticide residues analysis in fruit and vegetables. Three approaches to MS2 identificaton were evaluated.

Liquid chromatography Orbitrap mass spectrometry with simultaneous full scan and tandem MS/MS for highly selective pesticide residue analysis.

This paper describes the application of LC/Q-Orbitrap MS for the analysis of pesticide residues in fruit and vegetable commodities. LC/Q-Orbitrap MS working in full scan simultaneously with a single MS/MS scan was used to analyse 139 pesticide residues in QuEChERS extracts of tomato, pepper, orange and green tea. Full scan data were obtained at a resolution of 70,000 whereas MS/MS data were obtained at a resolution of 17,500. Quantitation and detection was carried out using full scan data while MS/MS data were used only for identification. MS/MS scans did not have a negative influence on quantitation under the applied conditions. Some peak area reproducibility problems were the consequence of the low sensitivity for some compounds (aldicarb, chlorpyriphos methyl, fenitrothion and fipronil) under the applied conditions. The relation between the operational parameters (viz. automatic gain control (AGC) target, maximum injection time (IT), underfill ratio, isolation window and apex trigger) and the number of automatically identified compounds was investigated. Mass error and minimal intensity of selected fragment ions were also studied. Various working modes were compared, such as full scan with single MS/MS scan and full scan with multiple MS/MS scans. In both cases, the number of automatically reported pesticides was the same. However full scan with single MS/MS scan ensured more points per peak in full scan mode and better peak area reproducibility. The evaluation of the identification and quantitation capabilities of the instrument was performed through the analysis of 100 real samples. The samples were also analysed by LC-QqQ MS/MS and the results of both analytical systems were compared. The comparison revealed that the two instruments were consistent with each other. They found the same pesticides and neither false positive nor false negatives were reported. Nevertheless the Q-Orbitrap MS allowed one to work in high resolution mass spectrometry, increasing the selectivity and, in full scan mode, permitting the retrospective analysis of the data feature that cannot be achieved with QqQ.

Large pesticide multiresidue screening method by liquid chromatography-Orbitrap mass spectrometry in full scan mode applied to fruit and vegetables.

The present work is focused on evaluating the main operational parameters for multiresidue screening of an Orbitrap mass spectrometer for pesticide residue analysis in fruits and vegetables. Operational parameters such as resolution, software for the automatic detection, mass tolerance and retention time extraction window, along with the analytical performance, were evaluated in an updated UHPLC-Orbitrap-mass spectrometer working in full scan mode. The evaluation was performed using QuEChERS extracts of tomato, pepper, orange and green tea. The extracts were spiked with 170 selected pesticides at four concentration levels (10µg/kg, 50µg/kg, 100µg/kg and 500µg/kg). Extracts were diluted 5 fold before injection. Three different resolution settings (17,500, 35,000 and 70,000) were evaluated at various concentration levels. At 10µg/kg, using a resolution of 17,500 and 5ppm of mass tolerance, the detected pesticide rates were from 91% in tomato, to 83% in green tea. These percentages increased at higher resolution values. A resolution of 70,000 was adequate for such analysis even when a small percentage of false detect at low concentration was obtained. The rates of detected compounds increased and were from 98% in tomato to 88% in green tea. Mass tolerance of 5ppm was the most adequate for screening purposes. The observed false negative detects were mainly a consequence of a lack of compound sensitivity exacerbated by ion suppression effects in the experimental conditions applied. With reporting limits of 10µg/kg, reproducibility improved with resolution levels of 35,000 or higher. Linearity was investigated in the 2-100ng/mL (equivalent to 10-500µg/kg in the sample) range. Particularly good automatic screening effectiveness was obtained using the selected settings in the analysis of real samples where no false negatives detects and 5% of false positives detects were obtained.

Evaluation of zirconium dioxide-based sorbents to decrease the matrix effect in avocado and almond multiresidue pesticide analysis followed by gas chromatography tandem mass spectrometry.

Two sorbents containing ZrO2 (Z-Sep and Z-Sep+) were tested as a d-SPE clean-up in combination with the QuEChERS and ethyl acetate multiresidue method in the pesticide residues extraction in avocado. All extracts were analysed using gas chromatography coupled with a triple quadrupole mass spectrometer working in multi-reaction monitoring mode. GC QToF was used to compare the amount of matrix compounds present in the final extracts, prepared according to different protocols. The highest number of pesticides with acceptable recoveries and the lowest amount of coextracted matrix compounds were provided by QuEChERS with Z-Sep. Subsequently, this method was fully validated in avocado and almonds. Validation studies were carried out according to DG Sanco guidelines including: the evaluation of recoveries at two levels (10 and 50 µg/kg), limit of quantitation, linearity, matrix effects, as well as interday and intraday precision. In avocado, 166 pesticides were fully validated compared to 119 in almonds. The method was operated satisfactorily in routine analysis and was applied to real samples.

Determination of pesticide residues in high oil vegetal commodities by using various multi-residue methods and clean-ups followed by liquid chromatography tandem mass spectrometry.

Several extraction methods were evaluated in terms of recoveries and extraction precision for 113 pesticides in avocado: QuEChERS with various d-SPE clean-ups (Z-Sep, Z-Sep+, PSA+C18 and silica), miniLuke and ethyl acetate. Extracts were analysed using liquid chromatography coupled with triple quadrupole mass spectrometer working in multi-reaction monitoring mode. Z-Sep and Z-Sep+ are new types of material for high lipid matrices - these two sorbents contain ZrO2, which improves fat removal from the extracts. The QuEChERS protocol with Z-Sep provided the highest number of pesticides with recoveries in the 70-120% range along with the lowest amount of coextracted matrix compounds. Subsequently, this method was validated in two matrices - avocado and almonds. In the validation recoveries at two levels - 10 and 50µg/kg - limit of quantitation, linearity, matrix effects, as well as the inter- and intraday precision were studied. In the avocado samples, 107 analytes had LOQs equal to 10µg/kg (signal to noise of quantitative transition was equal 20 or more). In the almond samples, 92 pesticides had LOQs equal to 10µg/kg (S/N≥20) and 2 pesticides at 50µg/kg. The validated method was employed in the analysis of real avocado and almond samples.

Comparison of three multiresidue methods to analyse pesticides in green tea with liquid and gas chromatography/tandem mass spectrometry.

The aim of the present work was to compare and choose the best method to extract pesticide residues from green tea. Three different multiresidue methods were tested on blank green tea samples fortified with 86 pesticides (insecticides, fungicides and herbicides) at the 100 µg kg(-1) level - modified QuEChERS (in order to limit the amount of coextractives, MgSO(4) was replaced with calcium chloride in the clean-up step); ethyl acetate extraction (not SweEt) and miniLuke. Due to matrix complexity, samples were diluted five times. The extracts were analysed by GC-MS/MS and LC-MS/MS. During the experiment recoveries, the precision and influence of coextracted compounds were compared. The study carried out on the three methods showed that the best results were achieved using QuEChERS. In terms of recoveries, QuEChERS showed the highest performance - successfully analysing 75 compounds (87% of the total number) with recoveries from 70 to 120% - whereas the miniLuke and ethyl acetate methods had 54 (63% of the total number) and 31 (36% of the total number), respectively. In addition, QuEChERS extracts contained the least amount of co-extracted matrix components. All three methods provided very good precision.

Pesticide analysis in teas and chamomile by liquid chromatography and gas chromatography tandem mass spectrometry using a modified QuEChERS method: validation and pilot survey in real samples.

This paper presents the validation of a modified QuEChERS method in four matrices - green tea, red tea, black tea and chamomile. The experiments were carried out using blank samples spiked with a solution of 86 pesticides (insecticides, fungicides and herbicides) at four levels - 10, 25, 50 and 100 µg/kg. The samples were extracted according to the citrate QuEChERS protocol; however, to reduce the amount of coextracted matrix compounds, calcium chloride was employed instead of magnesium sulphate in the clean-up step. The samples were analysed by LC-MS/MS and GC-MS/MS. Included in the scope of validation were: recovery, linearity, matrix effects, limits of detection and quantitation as well as intra-day and inter-day precision. The validated method was used in a real sample survey carried out on 75 samples purchased in ten different countries. In all matrices, recoveries of the majority of compounds were in the 70-120% range and were characterised by precision lower than 20%. In 85% of pesticide/matrix combinations the analytes can be detected quantitatively by the proposed method at the European Union Maximum Residue Level. The analysis of the real samples revealed that large number of teas and chamomiles sold in the European Union contain pesticides whose usage is not approved and also pesticides in concentrations above the EU MRLs.