Hans G.J. Mol

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.

Determination of endocrine disruptors in water after derivatization with N-methyl-N(tert.-butyldimethyltrifluoroacetamide) using gas chromatography with mass spectrometric detection

The combined gas chromatographic determination of a number of hydroxyl-group containing endocrine disruptors, including 4-octylphenol, 4-nonylphenol, 2,4-dichlorophenol, pentachlorophenol, 4-tert.-butylbenzoic acid, bisphenol-A, 17beta-estradiol and 17alpha-ethynylestradiol, was investigated. Derivatization, required for sensitive determination of these compounds, was carried out using N-methyl-N-(tert.-butyldimethyltrifluoroacetamide). A number of parameters affecting the derivatization reaction, like temperature, time, matrix, solvent, and amount of reagent were studied in detail. Quantitative yields were obtained for real-life extracts after optimization, but the hormones were only mono-substituted. Both solid-phase extraction (SPE) and liquid-liquid extraction were studied as extraction methods, with emphasis on SPE material and effect of pH. Recoveries and RSD for analysis of surface water samples were 58-106 and 6-16% (n=4), respectively, when using SPE, and 109-117 and 6-14% (n=6) when using liquid-liquid extraction. The method developed allows routine analysis of surface water for traces of endocrine disruptors. The limits of detection of were 4-6 ng/l but higher for the hormones.

Determination of polar organophosphorus pesticides in vegetables and fruits using liquid chromatography with tandem mass spectrometry: selection of extraction solvent

A method based on liquid chromatography (LC)-mass spectrometry (MS)/MS was developed for sensitive determination of a number of less gas chromatography (GC)-amenable organophosphorus pesticides (OPs; acephate, methamidophos, monocrotophos, omethoate, oxydemeton-methyl and vamidothion) in cabbage and grapes. For extraction, several solvents were evaluated with respect to the possibility of direct injection, matrix-induced suppression or enhancement of response, and extraction efficiency. Overall, ethyl acetate was the most favourable solvent for extraction, although a solvent switch was required. For some pesticide/matrix combinations, reconstitution of the residue after evaporation required special attention. Extracts were analysed on a C18 column with polar endcapping. The pesticides were ionised using atmospheric pressure chemical ionisation on a tandem mass spectrometer in multiple reaction monitoring mode. The final method is straightforward and involves extraction with ethyl acetate and a solvent switch to 0.1% acetic acid/water without further cleanup. The method was validated at the 0.01 and 0.5 mg/kg level, for both cabbage and grapes. Recoveries were between 80 and 101% with R.S.D. < 11% (n = 5). The limit of quantification was 0.01 mg/kg and limits of detection were between 0.001 and 0.004 mg/kg.

Determination of polar organophosphorus pesticides in aqueous samples by direct injection using liquid chromatography-tandem mass spectrometry.

It was demonstrated that four out of six of the very polar organophosphorus pesticides (OPs), i.e. acephate, methamidophos, monocrotophos, omethoate, oxydemeton-methyl and vamidothion, could not be extracted from water using commonly available SPE cartridges. In addition, GC analysis on all six compounds was found to be troublesome due to their polar and thermolabile character. This initiated the development of an alternative highly sensitive and selective method for the determination of the above mentioned very polar OPs in water, based on LC-MS. Large volume (1 ml) water samples were directly injected onto an RP18 HPLC column with a polar endcapping. The latter was essential for obtaining retention and maintaining column performance under 100% aqueous conditions during the sampling. The compounds were ionized using atmospheric pressure chemical ionization and detected on a tandem mass spectrometer operated in multiple reaction-monitoring mode. The detection limits were in the range of 0.01-0.03 microg/l. Compared to conventional GC methods, the developed LC-MS procedure is very straightforward, fast and more reliable. This application demonstrates the applicability of LC-MS for analysis of polar OPs in surface, ground and drinking water, as a more favourable alternative to GC.

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.

Large-volume injection in gas chromatographic trace analysis using temperature-programmable (PTV) injectors

The use of programmed-temperature vaporising (PTV) injectors for large-volume injection in capillary gas chromatography is briefly reviewed. The principles and optimisation of large-volume PTV injection are discussed. Guidelines are given for selection of the PTV conditions and injection mode for specific applications. Relevant examples from the recent scientific literature serve as illustrations.

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.

Short-term transport of glyphosate with erosion in Chinese loess soil--a flume experiment.

Repeated applications of glyphosate may contaminate the soil and water and threaten their quality both within the environmental system and beyond it through water erosion related processes and leaching. In this study, we focused on the transport of glyphosate and its metabolite aminomethylphosphonic acid (AMPA) related to soil erosion at two slope gradients (10 and 20°), two rates of pesticide with a formulation of glyphosate (Roundup®) application (360 and 720 mg m(-2)), and a rain intensity of 1.0 mm min(-1) for 1 h on bare soil in hydraulic flumes. Runoff and erosion rate were significantly different within slope gradients (p<0.05) while suspended load concentration was relatively constant after 15 min of rainfall. The glyphosate and AMPA concentration in the runoff and suspended load gradually decreased. Significant power and exponent function relationship were observed between rainfall duration and the concentration of glyphosate and AMPA (p<0.01) in runoff and suspended load, respectively. Meanwhile, glyphosate and AMPA content in the eroded material depended more on the initial rate of application than on the slope gradients. The transport rate of glyphosate by runoff and suspended load was approximately 14% of the applied amount, and the chemicals were mainly transported in the suspended load. The glyphosate and AMPA content in the flume soil at the end of the experiment decreased significantly with depth (p<0.05), and approximately 72, 2, and 3% of the applied glyphosate (including AMPA) remained in the 0-2, 2-5, and 5-10 cm soil layers, respectively. The risk of contamination in deep soil and the groundwater was thus low, but 5% of the initial application did reach the 2-10 cm soil layer. The risk of contamination of surface water through runoff and sedimentation, however, can be considerable, especially in regions where rain-induced soil erosion is common.

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.