Assessing the performance of various sorbents in micro-solid phase extraction cartridges for pesticide residue analysis in feed.

Newly designed micro-solid phase extraction cartridges are now available, reflecting the increasing shift towards laboratory automation, especially in the clean-up step for the analysis of pesticide residues in food and feed. In the present study, the introduction of different sorbents on the newly designed PAL µSPE CTC cartridges was investigated for the removal of matrix interferents and the recovery of pesticides. Eight cartridges containing different sorbent combinations and different amounts were used including EMR-lipid (not activated), Z-sep, chitin, C18, PSA, and GCB. The evaluation of co-extractive removal for each cartridge showed that the optimal choice for removing fatty acids was the cartridges containing PSA and Z-sep as clean-up sorbents. However, the presence of C18 and EMR-lipid was still required for the removal of sterols and tocopherols. Two grams of sample, fish feed (FF) and rapeseed cake (RSC) were extracted using QuEChERS citrate buffer, followed by a freeze-out step. The recoveries and repeatability of QuEChERS using µ-SPE clean-up were evaluated for 216 pesticide residues (112 compounds analyzed by GC-MS/MS and 143 compounds by LC-MS/MS, from which 39 compounds were analyzed using both techniques). The best results, with recovery between 70 and 120% and RSD <20%, were achieved when FF samples were cleaned-up with 15 mg EMR-lipid and 20 mg MgSO4. This was achieved for 94% of GC-amenable compounds and 86% of LC-amenable compounds. In the case of RSC, the best results were seen when samples were cleaned-up with the cartridge containing only 20 mg Z-sep and 20 mg MgSO4. This was achieved for 88% of GC-amenable compounds and 90% of LC-amenable compounds. Although these cartridges yielded optimal results in terms of recovery, their use could require more instrument maintenance, especially for GC-MS/MS, due to the lower removal of co-extractives.

Analysis of pesticides, veterinary drugs, and environmental contaminants in goat and lamb by the QuEChERSER mega-method.

Analysis of chemical residues in foods is a big challenge for developing countries due to lack of financial and professional resources needed to meet international quality standards for trade. However, the implementation of simple multiclass, multi-residue methods in monitoring programs can provide significant benefits to save cost, time, and labor. The aim of this project was to investigate the "quick, easy, cheap, effective, rugged, safe, efficient, and robust" (QuEChERSER) mega-method for the fatty muscle matrices of goat and lamb. To achieve wide analytical scope covering pesticides, environmental contaminants, and veterinary drugs, extracts were analyzed by both ultrahigh-performance liquid and low-pressure gas chromatography (UHPLC and LPGC) coupled with tandem mass spectrometry (MS/MS). The QuEChERSER mega-method was validated in ovine (goat) and caprine (lamb) muscles at four different spiking levels with 10 replicates per level for a total of 330 analytes and metabolites, consisting of 225 pesticides, 89 veterinary drugs, and 16 polychlorinated biphenyls (PCBs). In the case of LPGC-MS/MS (preceded by automated "instrument-top sample preparation"), 92% and 82% of the analytes met the data quality objectives of 70-120% recovery and <20% RSD for goat and lamb, respectively. For UHPLC-MS/MS, 95% and 92% of the analytes met the acceptable validation criteria in goat and lamb, respectively. Thus, the QuEChERSER mega-method has been demonstrated to be a useful streamlined approach to more efficiently replace multiple methods currently used to cover the same analytical scope.

Validation of the QuEChERSER mega-method for the analysis of pesticides, veterinary drugs, and environmental contaminants in tilapia (Oreochromis Niloticus).

Diverse food safety programmes around the world are designed to help ensure production of safe food. To meet this need, the development and implementation of more efficient and effective analytical methods to monitor residues (pesticides and veterinary drugs) and contaminants in food is important. In this study, we report the validation results for a simple high-throughput mega-method for residual analysis of 213 pesticides and veterinary drugs, including 15 metabolites, plus 12 environmental contaminants (polychlorinated biphenyls) in tilapia muscle for implementation in routine laboratory analyses. The generic sample preparation method and analytical approach are known as QuEChERSER (more than QuEChERS). A small portion of the initial extract (204 µL) is taken for analysis by ultrahigh-performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) covering 145 analytes, and the remaining extract undergoes a salting out step followed by an automated robotic instrument top sample preparation (ITSP) cleanup, also known as micro-solid-phase extraction (µSPE), plus fast low-pressure gas chromatography LPGC-MS/MS for 134 analytes (66 pesticides are targeted in both UHPLC-MS/MS and LPGC-MS/MS). The mega-method was validated in spiked tilapia samples at 5, 10, 15, and 20 ng/g with 10 replicates per level over two days (n = 80 overall), and 70-140% recoveries with RSDs ≤20% were achieved for 92% of the analytes in LC and 82% in GC. No significant matrix effects were observed for the analytes in LPGC-MS/MS, and only 5% of the analytes exceeded ±20% matrix effect in UHPLC-MS/MS. Analysis of standard reference materials (NIST SRMs 1946 and 1947) for contaminants in freeze-dried fish showed acceptable results, further demonstrating that the QuEChERSER mega-method can be implemented to expand analytical scope and increase laboratory efficiency compared to the QuEChERS method.

High-Throughput Mega-Method for the Analysis of Pesticides, Veterinary Drugs, and Environmental Contaminants by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Robotic Mini-Solid-Phase Extraction Cleanup + Low-Pressure Gas Chromatography-Tandem Mass Spectrometry, Part 1: Beef.

In this work, a new mega-method of sample preparation called "QuEChERSER" (more than QuEChERS) is being presented for the first time. Fast, efficient, and cost-effective analysis of chemical contaminants in meat is useful for international trade, domestic monitoring, risk assessment, and other purposes. The goal of this study was to develop and validate a simple high-throughput mega-method for residual analysis of 161 pesticides, 63 veterinary drugs, 24 metabolites, and 14 legacy environmental contaminants (polychlorinated biphenyls) in bovine muscle for implementation in routine laboratory analyses. Sample preparation of 2 g test portions entailed QuEChERS-based extraction with 10 mL of 4:1 (v/v) acetonitrile/water, and then 204 µL was taken, diluted, and ultracentrifuged prior to analysis of veterinary drugs and pesticides by ultra-high-performance liquid chromatography-tandem mass spectrometry. The remaining extract was salted out with 4:1 (w/w) anhydrous MgSO4/NaCl, and 1 mL was transferred to an autosampler vial for automated mini-cartridge solid-phase extraction (Instrument Top Sample Preparation) cleanup with immediate injection using fast low-pressure gas chromatography-tandem mass spectrometry analysis. The automated cleanup and both instruments were all operated in parallel in 13-15 min cycle times per sample. Method validation according to United States Department of Agriculture requirements demonstrated that 221 (85%) of the 259 analytes gave average recovery between 70 and 120% and interday relative standard deviation of ≤25%. Analysis of a certified reference material for veterinary drugs in freeze-dried bovine muscle was also very accurate, further demonstrating that the QuEChERSER mega-method can be implemented to save time, labor, and resources compared to current practices to use multiple methods to cover the same analytical scope.

High-Throughput Mega-Method for the Analysis of Pesticides, Veterinary Drugs, and Environmental Contaminants by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Robotic Mini-Solid-Phase Extraction Cleanup + Low-Pressure Gas Chromatography-Tandem Mass Spectrometry, Part 2: Catfish.

The goal of this study was to develop and validate a new method for simultaneous determination of 106 veterinary drugs and 227 pesticides and their metabolites plus 16 polychlorinated biphenyls (PCBs) at and below their regulatory levels established for catfish muscle in the European Union and U.S.A. To do this, two different QuEChERS-based methods for veterinary drugs and pesticides and PCBs were modified and merged into a single mega-method dubbed "QuEChERSER" (more than QuEChERS), which is presented here for the first time. The mega-method was validated in catfish at four different spiking levels with 10 replicates per level. Sample extraction of 2 g test portions was made with 10 mL of 4:1 (v/v) acetonitrile/water, and then an aliquot was taken for ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis of 106 veterinary drugs and 125 pesticides, including metabolites. The remaining extract after salting out was subjected to automated mini-solid-phase extraction cleanup (Instrument Top Sample Preparation) for immediate injection in low-pressure gas chromatography-tandem mass spectrometry (LPGC-MS/MS). The cleanup was conducted in parallel with the 10 min LPGC-MS/MS analysis for 167 PCBs, pesticides, and metabolites, which was conducted in parallel with the 10 min UHPLC-MS/MS analysis for 231 analytes to increase sample throughput (49 analytes were included in both techniques). In MS/MS, three ion transitions were monitored for nearly all targeted analytes to provide unambiguous identification as well as quantification. Satisfactory recoveries (70-120%) and relative standard deviations of ≤20% were achieved for 98 (92%) of the veterinary drugs and their metabolites and for 222 (91%) of pesticides, metabolites, and PCBs, demonstrating that the developed method is applicable for the analysis of these contaminants in fish as part of regulatory monitoring programs and other purposes.

Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function.

Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.