Paul Zomer

Toward a generic extraction method for simultaneous determination of pesticides, mycotoxins, plant toxins, and veterinary drugs in feed and food matrixes.

A fast and straightforward generic procedure for the simultaneous extraction of various classes of pesticides, mycotoxins, plant toxins, and veterinary drugs in various matrixes has been developed, for subsequent analysis by liquid chromatography with mass spectrometric detection. As a first step, four existing multianalyte procedures and three newly proposed methods were compared for a test set of 172 pesticides, mycotoxins, and plant toxins spiked to a feed matrix. The new procedures, which basically involved extraction/dilution of the sample with water and an acidified organic solvent (methanol, acetonitrile, or acetone), were most promising. The three new generic extraction methods were further tested for applicability to other matrixes (maize, honey, milk, egg, meat). Overall, the best recoveries were obtained for acetone, followed by acetonitrile. With respect to matrix effects, acetonitrile was the most favorable solvent and methanol was the worst. The occurrence of matrix effects decreased for the matrixes in the order of feed > maize > meat > milk > egg > honey. The extraction method selected as the default procedure (water/acetonitrile/1% formic acid) was also evaluated for applicability to multiple classes of veterinary drugs in all six matrixes, with satisfactory results. Finally, the generic extraction procedure was validated for 136 pesticides, 36 natural toxins, and 86 veterinary drugs in compound feed and honey at three levels (0.01, 0.02, and 0.05 mg/kg) using ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for analysis of the extracts. For over 80% of the analytes, recoveries were between 70 and 120% and precision (expressed as relative standard deviation) was mostly in the range of 5-10% (except for feed at 0.01 mg/kg; adequate recoveries for 62% of the analytes). The limits of detection were from <0.01 to 0.05 mg/kg for most analytes, which is usually sufficient to verify compliance of products with legal tolerances. The results clearly demonstrate the feasibility of the generic approach proposed. Application of the method in routine monitoring programs would imply a drastic reduction of both effort and time.

Direct analysis of dithiocarbamate fungicides in fruit by ambient mass spectrometry

Dithiocarbamates (DTCs) are fungicides that require a specific single-residue method for detection and verification of compliance with maximum residue limits (MRLs) as established for fruit and vegetables in the EU. In this study, the use of ambient mass spectrometry was investigated for specific determination of individual DTCs (thiram, ziram) in fruit. Two complementary approaches have been investigated for their rapid analysis: (i) direct analysis in real time (DART) combined with medium-high resolution/accurate mass time-of-flight mass spectrometry (TOFMS) and high-resolution/accurate mass Orbitrap MS, and (ii) desorption electrospray ionization (DESI) combined with tandem-in-time mass spectrometry (MS2). With both techniques, thiram deposited on a glass surface (DART) or Teflon (DESI) could be directly detected. With DART, this was also possible for ziram. Before the instrumental analysis of fruit matrix, an extract had to be prepared following a straightforward procedure. The raw extracts were deposited on a slide (DESI), or rods were dipped into the extracts (DART), after which thiram and ziram could be rapidly detected (typically 10 samples in a few minutes). In the case of thiram, the lowest calibration levels were 1 mg kg−1 (DART–TOFMS, DESI–MS2) and 0.1 mg kg−1 (DART–Orbitrap MS). For ziram, the achieved lowest calibration levels were 0.5 mg kg−1 (DART–TOFMS) and 1 mg kg−1 (DART–Orbitrap MS). In all cases, this was sufficiently low to test samples against EU-MRLs for a number of fruit crops. Using an internal standard, (semi)quantitative results could be obtained.

Screening of plant toxins in food, feed and botanicals using full-scan high-resolution (Orbitrap) mass spectrometry

A generic method based on LC with full-scan high-resolution (Orbitrap) mass spectrometry (MS) was systematically investigated for the simultaneous detection of a wide range of plant toxins in a variety of food and feed matrices. For a selection of 150 substances, representing various chemical classes, the limit of detection was established using fixed LC-MS conditions. Ion suppression effects and selectivity were evaluated using generic extracts from representative and relevant matrices (food supplement, honey, silage, compound feed). The majority of the substances could be measured as positive ions after electrospray ionisation (ESI+). Using a mass resolving power of 100,000 a reliable high mass accuracy was obtained despite the high abundance of co-extractants in the sample extracts. This enabled the use of ±5 ppm mass extraction windows, which in turn resulted in a high degree of selectivity. On the other hand, except for honey, strong ion suppression effects were frequently observed which adversely affected the detection limits. Nevertheless, for the majority of the substances the detection limits were in the range 0.01–0.05 mg kg−1. Since non-selective sample preparation and non-targeted data acquisition were performed, the presence of plant toxins initially not targeted for during data review can be subsequently investigated, which is a very useful option because for many known toxins no analytical reference standards are yet available. The applicability of the method was demonstrated by analysis of a variety of real-life samples purchased on the market or from cases of intoxication. These included honey, herbal tea, food supplements, poppy seeds, traditional Chinese medicines, compound feed, silage and herb-based feed additives. Plant toxins that were detected included various pyrrolizidine alkaloids, grayanotoxins, opium alkaloids, strychnine, ricinine (a marker for ricin), aconitine, aristolochic acid and cardiac glycosides (e.g. digitoxin, digoxin).

Identification in residue analysis based on liquid chromatography with tandem mass spectrometry: Experimental evidence to update performance criteria

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is one of the most widely used techniques for identification (and quantification) of residues and contaminants across a number of different chemical domains. Although the same analytical technique is used, the parameters and criteria for identification vary depending on where in the world the analysis is performed and for what purpose (e.g. determination of pesticides, veterinary drugs, forensic toxicology, sports doping). The rationale for these differences is not clear and in most cases the criteria are essentially based on expert opinions rather than underpinned by experimental data. In the current study, the variability of the two key identification parameters, retention time and ion ratio, was assessed and compared against requirements set out in different legal and guidance documents. The study involved the analysis of 120 pesticides, representing various chemical classes, polarities, molecular weights, and detector response factors, in 21 different fruit and vegetable matrices of varying degrees of complexity. The samples were analysed non-fortified, and fortified at 10, 50 and 200 μg kg(-1), in five laboratories using different LC-MS/MS instruments and conditions. In total, over 135,000 extracted-ion chromatograms were manually verified to provide an extensive data set for the assessment. The experimental data do not support relative tolerances for retention time, or different tolerances for ion ratios depending on relative abundance of the two product ions measured. Retention times in todays chromatographic systems are sufficiently stable to justify an absolute tolerance of ±0.1 min. Ion ratios are stable as long as sufficient response is obtained for both product ions. Ion ratio deviations are typically within ±20% (relative), and within ±45% (relative) in case the response of product ions are close to the limit of detection. Ion ratio tolerances up to 50% did not result in false positives and reduced the false negative rate for pesticides with product ions in the low S/N range to <5%. Without ion ratio criterion, two false positives were obtained in 105 non-fortified samples. Although the study has been conducted for pesticides residues in fruits and vegetables, the impact of these findings is believed to extend towards other application areas and possibly support adjustment or consolidation of criteria across other analytical domains.

Simultaneous quantitative determination, identification and qualitative screening of pesticides in fruits and vegetables using LC-Q-Orbitrap™-MS

A method based on QuEChERS extraction and LC-quadrupole-Orbitrap™ MS detection was established utilising an improved fully non-targeted way of data acquisition with and without fragmentation. A full-scan acquisition event without fragmentation (resolving power 70 000) was followed by five consecutive fragmentation events (variable data independent acquisition – vDIA; resolving power 35 000) where all ions from the full-scan range are fragmented. Compared with fragmentation in a single event (all-ion fragmentation – AIF), this improves both selectivity and sensitivity for the fragment ions, which is beneficial for screening performance and identification capability. The method was validated, using the data from the same measurements, for two types of analysis: quantitation/identification and qualitative screening. The quantitative validation, performed according to the guidelines in SANCO/12571/2013, tested the performance of the method for 184 compounds in lettuce and orange at two spiking levels: 10 and 50 ng g−1. The validation showed that the vast majority of the compounds met the criteria for trueness and precision set in the SANCO guidance document. In the qualitative validation the same 184 compounds were used to test the untargeted screening capabilities of the method. In this validation the compounds were spiked at three levels into 11 different fruit and vegetable matrices, which were measured twice on separate days. Taking all data from the qualitative validation together, an overall detection rate of 92% was achieved at the 10 ng g−1 level, increasing to 98% at 200 ng g−1. A screening detection limit (as defined in the SANCO guidelines) of 10 ng g−1 could be achieved for 134 compounds. For 39 and two pesticides the SDL was 50 and 200 ng g−1, respectively. For the other nine compounds no SDL could be established. The identification (ion ratio) criteria as recommended in the SANCO document could be met for 93% of the detected pesticide/matrix/concentration combinations. The outcome of both validations shows that the described method can be used to combine quantitative analysis and the identification of frequently detected pesticides (so far typically done using triple quadrupole MS/MS) with a qualitative screening to be used for a wide range of less frequent detected compounds in one measurement.

Determination of Glyphosate Levels in Breast Milk Samples from Germany by LC-MS/MS and GC-MS/MS

This study describes the validation and application of two independent analytical methods for the determination of glyphosate in breast milk. They are based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS), respectively. For LC-MS/MS, sample preparation involved an ultrafiltration followed by chromatography on an anion exchange column. The analysis by GC-MS/MS involved an extraction step, cleanup on a cation exchange column, and derivatization with heptafluorobutanol and trifluoroacetic acid anhydride. Both methods were newly developed for breast milk and are able to quantify glyphosate residues at concentrations as low as 1 ng/mL. The methods were applied to quantify glyphosate levels in 114 breast milk samples, which had been collected from August to September of 2015 in Germany. The mothers participated at their own request and thus do not form a representative sample. In none of the investigated samples were glyphosate residues above the limit of detection found.

Application of Fragment Ion Information as Further Evidence in Probabilistic Compound Screening Using Bayesian Statistics and Machine Learning: A Leap Toward Automation

In this work, we introduce an automated, efficient, and elegant model to combine all pieces of evidence (e.g., expected retention times, peak shapes, isotope distributions, fragment-to-parent ratio) obtained from liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS) data for screening purposes. Combining all these pieces of evidence requires a careful assessment of the uncertainties in the analytical system as well as all possible outcomes. To-date, the majority of the existing algorithms are highly dependent on user input parameters. Additionally, the screening process is tackled as a deterministic problem. In this work we present a Bayesian framework to deal with the combination of all these pieces of evidence. Contrary to conventional algorithms, the information is treated in a probabilistic way, and a final probability assessment of the presence/absence of a compound feature is computed. Additionally, all the necessary parameters except the chromatographic band broadening for the method are learned from the data in training and learning phase of the algorithm, avoiding the introduction of a large number of user-defined parameters. The proposed method was validated with a large data set and has shown improved sensitivity and specificity in comparison to a threshold-based commercial software package.

Influence of microplastic addition on glyphosate decay and soil microbial activities in Chinese loess soil

The intensive use of pesticide and plastic mulches has considerably enhanced crop growth and yield. Pesticide residues and plastic debris, however, have caused serious environmental problems. This study investigated the effects of the commonly used herbicide glyphosate and micrometre-sized plastic debris, referred as microplastics, on glyphosate decay and soil microbial activities in Chinese loess soil by a microcosm experiment over 30 days incubation. Results showed that glyphosate decay was gradual and followed a single first-order decay kinetics model. In different treatments (with/without microplastic addition), glyphosate showed similar half-lives (32.8 days). The soil content of aminomethylphosphonic acid (AMPA), the main metabolite of glyphosate, steadily increased without reaching plateau and declining phases throughout the experiment. Soil microbial respiration significantly changed throughout the entirety of the experiment, particularly in the treatments with higher microplastic addition. The dynamics of soil β-glucosidase, urease and phosphatase varied, especially in the treatments with high microplastic addition. Particles that were considerably smaller than the initially added microplastic particles were observed after 30 days incubation. This result thus implied that microplastic would hardly affect glyphosate decay but smaller plastic particles accumulated in soils which potentially threaten soil quality would be further concerned especially in the regions with intensive plastic mulching application.