Comparison of filter membranes in the analysis of 183 veterinary and other drugs by liquid chromatography-tandem mass spectrometry.

Although filtration is one of the most common steps in sample preparation for chemical analysis, filter membrane materials can leach contaminants and/or retain some analytes in the filtered solutions. In multiclass, multiresidue analysis of veterinary drugs, it is challenging to find one type of filter membrane that does not retain at least some of the analytes before injection in ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). In this study, different filter membranes were tested for use in UHPLC-MS/MS analysis of 183 diverse drugs in bovine muscle, kidney, and liver tissues. Membranes evaluated consisted of polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polyethersulfone, nylon, and regenerated cellulose. Drug classes represented among the analytes included ß-agonists, ß-lactams, anthelmintics, macrolides, tetracyclines, sulfonamides, tranquilizers, (fluoro)quinolones, anti-inflammatories, nitroimidazoles, coccidiostats, phenicols, and others. Although the presence of a matrix helped reduce the binding of analytes on surface active sites, all of the filter types partially retained at least some of the drugs in the final extracts. In testing by flow-injection analysis, all of the membrane filters were also observed to leach interfering components. Ultimately, filtration was avoided altogether in the final sample preparation approach known as the quick, easy, cheap, effective, rugged, safe, efficient, and robust (QuEChERSER) mega-method, and ultracentrifugation was chosen as an alternative.

Production and certification of BOTS-1: bovine muscle-certified reference material for incurred veterinary drug residues.

A freeze-dried bovine muscle-certified reference material (CRM), known as BOTS-1 (DOI: https://doi.org/10.4224/crm.2018.bots-1 ), containing incurred residues of commonly used veterinary drugs was produced and certified for the mass fraction of eight veterinary drug residues. Value assignment was carried out using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods in conjunction with isotope dilution and standard addition approaches involving stable isotope internal standards. Data from the National Research Council of Canada (NRC), Canadian Food Inspection Agency (CFIA), United States Department of Agriculture (USDA), and the Federal Office of Consumer Protection and Food Safety in Germany (BVL) were used for value assignment. Results for two drug residues were also obtained through an international inter-laboratory comparison CCQM-K141/P178 organized under the auspices of the International Bureau of Weights and Measures (BIPM). Quantitative NMR (1H-qNMR) was used to characterize primary standards of all veterinary drugs certified. The certified mass fractions of the veterinary drug residues were 490 ± 100 µg/kg for chlorpromazine, 44 ± 4.4 µg/kg for ciprofloxacin, 3.3 ± 1.4 µg/kg for clenbuterol, 9.5 ± 0.8 µg/kg for dexamethasone, 57 ± 4.8 µg/kg for enrofloxacin, 3.0 ± 0.4 µg/kg for meloxicam, 12.4 ± 1.2 µg/kg for ractopamine, and 2290 ± 120 µg/kg for sulfadiazine with expanded uncertainties quoted (95% confidence) which include the effects due to between-bottle inhomogeneity, instability during long-term storage and transportation, and characterization.

Synthesis and spectroscopic characterization of 13 C4 -labeled 4-cyano-2-oxobutyraldehyde semicarbazone: A metabolite of nitrofurazone.

Nitrofurazone usage in food-producing animals is prohibited in most countries, including the United States. Regulatory agencies regularly monitor its use in domestic, export/import animals' food products by measuring the semicarbazide (SEM) metabolite as a biomarker of nitrofurazone exposure. However, the use of SEM is controversial because it is also produced in food naturally and thus gives false positive results. A cyano-metabolite, 4-cyano-2-oxobutyraldehyde semicarbazone (COBS), is proposed as an alternate specific marker of nitrofurazone to distinguish nitrofurazone from treated or untreated animals. A synthetic method was developed to produce COBS via metallic hydrogenation of nitrofurazone. The product was isolated and characterized by one- and two-dimensional nuclear magnetic spectroscopy (NMR) experiments, Fourier-transform infrared spectroscopy (FT-IR), and mass spectrometry. The developed synthetic procedure was further extended to synthesize isotopically labeled 4-[13 C]-cyano-2-oxo- [2, 3, 4-13 C3 ]-butyraldehyde semicarbazone. Labeled COBS is useful as an internal standard for its quantification in food-producing animals. Thus, the developed method provides a possibility for its commercial synthesis to procure COBS. This is the first synthesis of the alternate specific marker metabolite of nitrofurazone for possible usage in regulatory analysis to solve a real-world problem.

Stability study of selected veterinary drug residues spiked into extracts from different food commodities.

Analyte stability is more commonly a confounding factor in analytical chemistry than many analysts recognize. In this study, we assessed the stability of 31 common veterinary drugs in water and final extracts of bovine (milk and kidney/liver) and chicken (muscle and egg) matrices. Two different sample preparation methods were evaluated for one-month storage of the final extracts at typical room, refrigerator, and freezer temperatures. Liquid chromatography - mass spectrometry (LC-MS) by triple quadrupole and high-resolution techniques was used for analysis of the extracts spiked at different relevant concentrations for general regulatory purposes (10-1000 ng/g sample equivalent). Comparison of results between two labs demonstrated that stable drugs (≤20% loss) at all tested conditions consisted of danofloxacin, enrofloxacin, florfenicol, flubendazole, hydroxy-flubendazole, flumequine, flunixin, 5-hydroxy-flunixin, lincomycin, and meloxicam. The tested drugs found to be the most unstable (>20% loss at room temperature within a matter of days) consisted of the ß-lactams (ampicillin, cefalexin, cloxacillin, and penicillin G). Curiously, the following antibiotics (mostly macrolides) were apparently more stable in sample extracts than water: emamectin, erythromycin, ivermectin, lasalocid, monensin, tilmicosin, tulathromycin, and tylosin. Those and the other drug analytes (ciprofloxacin, doxycycline, florfenicol amine, 2-amino-flubendazole, oxytetracycline, sulfadiazine, sulfadimethoxine, sulfamethazine, and trimethoprim) were mostly stable for a month in refrigerated extracts, especially at higher concentrations, but not in all cases. In practice, freezer storage of extract solutions was found to be acceptable for at least a month, with a few exceptions.

Evaluation of a septumless mini-cartridge for automated solid-phase extraction cleanup in gas chromatographic analysis of >250 pesticides and environmental contaminants in fatty and nonfatty foods.

The QuEChERSER mega-method has recently been introduced to quantify and identify a wide range of chemical residues (pesticides, veterinary drugs, environmental contaminants, among others) in nearly all types of foods. The approach calls for taking a small amount of the initial extract to cover analytes amenable to liquid chromatography, and the remainder is salted out for analysis by gas chromatography (GC), both with mass spectrometry (MS) based detection. In the case of GC-MS(/MS), the extract undergoes automated robotic mini-cartridge solid-phase extraction (SPE) cleanup in a technique known as µSPE or instrument-top sample preparation (ITSP). In 2022, a septumless mini-cartridge for µSPE was introduced to improve upon the ITSP design. The new design houses a bed of 20 mg anhydrous MgSO4, 12 mg each of C18 and primary secondary amine sorbents, and 1 mg of graphitized carbon black, the latter substituting for CarbonX used in the ITSP product. The septumless µSPE mini-cartridge employs a different gripping mechanism with the syringe needle that allows leak-free operation at higher flow rates (e.g. 10 µL/s), whereas the ITSP design is limited to 2 µL/s. Based on cleanup and analyte elution profiles, the extract load volume and flow rate was increased in µSPE for QuEChERSER from 300 µL at 2 µL/s to 500 µL at 5 µL/s, which improved accuracy of results, sped the cleanup step, and obviated the need for micro-vial inserts in the receiving vials. The new design also reduced both the amount and consistency of dead (void) volumes in the mini-cartridges from 83 ± 14 µL to 52 ± 7 µL for 200-600 µL load volumes. Normalization of peak areas to internal standards led to recoveries between 80 and 120% with typical RSDs <5% in low-pressure GC-MS/MS of 227-242 out of 252 pesticides, polychlorinated biphenyls, polybrominated diphenyl ethers, and polycyclic aromatic hydrocarbons in hemp powder, spinach, whole milk, egg, avocado, and lamb meat.

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.

Comparison of analyte identification criteria and other aspects in triple quadrupole tandem mass spectrometry: Case study using UHPLC-MS/MS for regulatory analysis of veterinary drug residues in liquid and powdered eggs.

Ultrahigh-performance liquid chromatography (UHPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS) is one of the most powerful tools for the multiclass, multiresidue analysis of veterinary drugs, pesticides, mycotoxins, and other chemical contaminants in foods and other sample types. Until approximately 2010, commercial MS/MS instruments using multiple reaction monitoring (MRM) were generally limited to minimum dwell (and inter-dwell) times of 10 ms per ion transition. To achieve the needed accuracy and detection limits for hundreds of targeted analytes, older UHPLC-MS/MS methods typically acquired only two ion transitions per analyte (yielding only one ion ratio for qualitative identification purposes), which is still the norm despite technological advancements. Newer instruments permit as little as 1 ms (inter-)dwell times to afford monitoring of more MRMs/analyte with minimal sacrifices in accuracy and sensitivity. In this study, quantification and identification were assessed in the validation of 169 veterinary drugs in liquid and powdered eggs. Quantitatively, an "extract-and-inject" sample preparation method yielded acceptable 70-120% recoveries and < 25% RSD for 139-141 (82-83%) of the 169 diverse drug analytes spiked into powdered and liquid eggs, respectively, at three levels of regulatory interest. Qualitatively, rates of false positives and negatives were compared when applying three different regulatory identification criteria in which two or three MRMs/drug were used in each case. Independent of the identification criteria, rates of false positives remained <10% for 95-99% of the drugs whether 2 or 3 ions were monitored, but the percent of drugs with >10% false negatives decreased from 25-45 to 10-12% when using 2 vs. 3 MRMs/analyte, respectively. Use of a concentration threshold at 10% of the regulatory level as an identification criterion was also very useful to reduce rates of false positives independent of ion ratios. Based on these results, monitoring >2 ion transitions per analyte is advised when using MS/MS for analysis, independent of SANTE/12682/2019, FDA/USDA, or 2002/657/EC identification criteria. (Quant)identification results using all three criteria were similar, but the SANTE criteria were advantageous in their greater simplicity and practical ease of use.

Validation of a high-throughput method for analysis of pesticide residues in hemp and hemp products.

Hemp has been an agricultural commodity for millennia, and it has been undergoing a resurgence in interest and production due to its high content of cannabinoids, protein, fiber and other ingredients. For legal possession and use throughout the USA, hemp and hemp products must have delta-9-tetrahydrocannabinol (THC) concentration < 0.3%. As with most crops, pesticides may be applied when farming hemp, which need to be monitored in food, feed, and medicinal products. The aim of this work was to evaluate and validate the recently developed "quick, easy, cheap, effective, rugged, safe, efficient, and robust" (QuEChERSER) sample preparation mega-method to determine pesticide residues in hemp plants, flowers, powders, oils, and pellets. High-throughput analysis of final extracts for 106 targeted pesticides and metabolites from North American monitoring lists entailed: 1) ultrahigh-performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) with column back-flushing, and 2) instrument-top sample preparation + low-pressure gas chromatography (ITSP+LPGC-MS/MS). In QuEChERSER, 2 g sample is extracted with 10 mL 4/1 (v/v) acetonitrile/water by mechanical shaking for 10 min, followed by 3 min centrifugation. For LC, 0.2 mL of extract is taken and solvent exchanged into initial mobile phase followed by 5 min ultra-centrifugation prior to the 10 min analysis. For GC-amenable pesticides, the remaining initial extract is partitioned with 4/1 (w/w) anh. MgSO4/NaCl, and 1 mL is taken for automated ITSP cleanup in parallel with 10 min LPGC analysis. In the former case, the UHPLC column is back-flushed with 1/1 (v/v) methanol/acetonitrile for 3 min between each injection to keep the system clean and avoid ghost peaks. Multi-level, multi-day validation results achieved 70-120% recoveries with RSDs < 20% for more than 80% of the analytes in hemp protein powder, oil, pellets, and fresh plant (dried hemp plant and flower were too complex). Limits of quantification (LOQs) were < 10 ng/g were achieved for nearly all pesticides, yielding 2.8% false negatives among >13,000 analyte results in the spiked samples. The QuEChERSER method was demonstrated to meet the challenge for several complex hemp matrices.

Comparison of four different multiclass, multiresidue sample preparation methods in the analysis of veterinary drugs in fish and other food matrices.

In 2018, AOAC International issued Standard Method Performance Requirements (SPMR) 2018.010 - Screening and Identification Method for Regulated Veterinary Drug Residues in Food. In response, we compared 4 different multiresidue methods of sample preparation using the same analytical method entailing ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Tilapia was chosen for testing, and the analytes and monitoring levels were from SPMR 2018.010. The methods consist of efficient procedures with published validation results from the US Department of Agriculture (USDA), Food and Drug Administration (FDA), and Canadian Food Inspection Agency (CFIA), and an enhanced-matrix removal (EMR)-Lipid protocol from China. Each method was used to prepare 102 final extracts of tilapia spiked or not at different levels with the 78 targeted analytes plus metabolites. The same FDA/USDA rules of mass spectral identification were employed in all analyses to assess rates of false positives and negatives. Quantitative accuracy of the methods was also compared in terms of recoveries and reproducibility of spiked tilapia, incurred catfish, and spiked and certified reference material of bovine muscle. Each method yielded generally acceptable results for the targeted veterinary drugs, but the USDA "extract & inject" method was the fastest, simplest, and cheapest to achieve equally or more acceptable results for the widest scope of analytes for the tested food matrices.

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.

Extract-and-Inject Analysis of Veterinary Drug Residues in Catfish and Ready-to-Eat Meats by Ultrahigh-Performance Liquid Chromatography - Tandem Mass Spectrometry.

BACKGROUND: Validated analytical methods are needed to conduct regulatory monitoring of ready-to-eat meats and fish for food safety, risk assessment, and other purposes. The methods should be cost-effective, high-throughput, and meet acceptable performance standards for a wide scope of drugs and matrixes. OBJECTIVE: The goal of this study was to demonstrate the validity for possible implementation in the US National Residue Program of an efficient method for qualitative and quantitative analysis of 176 targeted drugs at levels as low as 10 ng/g in hot dogs, catfish and swai (Siluriformes), chicken tenders, fried bacon, and sausage using ultrahigh-performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS). METHODS: Sample preparation simply involved a 5 min extraction by shaking 2 g comminuted samples with 10 mL of 4/1 (v/v) acetonitrile/water followed by centrifugation and UHPLC-MS/MS analysis of 2 µL injections. For cleanup comparison purposes only, sausage extracts were also prepared using a cartridge-based EMR-Lipid method prior to analysis. RESULTS: Acceptable validation of 70-120% recoveries with <25% RSDs was met for 156-176 out of 186 drugs and quality control analytes without cleanup depending on the matrix. The EMR-Lipid method for sausage improved results for some analytes, such as mectin anthelmintics, due to reduction of indirectly interfering fats in the final extracts, but it also led to significantly worse results for several other drugs, resulting in 32 fewer analytes meeting the given validation criteria than without cleanup. CONCLUSIONS: The simple, high-throughput method was demonstrated to be valid to meet routine regulatory and other monitoring needs for many diverse targeted drugs in fish and ready-to-eat meat matrixes.

Critical review and re-assessment of analyte protectants in gas chromatography.

Despite nearly 80 years of advancements in gas chromatography (GC), indirect chemical matrix effects (MEs), known as the matrix-induced response enhancement effect, still occur to cause a high bias in the GC analysis of susceptible analytes, unless precautions are taken. Matrix-matched calibration is one common option used in GC to compensate for the MEs, but this approach is usually inconvenient, imprecise, and inefficient. Other options, such as the method of standard additions, surface deactivation techniques, chemical derivatizations, priming the GC, and/or use of internal standards, also have flaws in practice. When methods are accommodating, the use of analyte protectants (APs) can provide the best practical solution to not only overcome MEs, but also to maximize analyte signal by increasing chromatographic and detection efficiencies for the analytes. APs address the source of MEs in every injection by filling active sites in the GC inlet, column, and detector, particularly in GC-MS, rather than the analytes that would otherwise undergo degradation, peak tailing, and/or diminished response due to interactions with the active sites. The addition of an adequate amount of APs (e.g. sugar derivatives) to all calibration standards and final extracts alike often leads to lower detection limits, better accuracy, narrower peaks, and greater robustness than the other options to compensate for MEs in GC. This article consists of a critical review of the scientific literature, proposal of mechanisms and theory, and re-evaluation studies involving APs for the first time in GC-orbitrap and GC-MS/MS with a high-efficiency ion source design. The findings showed that 1 µg each of co-injected shikimic acid and sorbitol in the former case, and 1 µg shikimic acid alone in the latter case, led to high quality results in multi-residue analysis of pesticides and environmental contaminants.

Assessment of Test Portion Sizes after Sample Comminution with Liquid Nitrogen in an Improved High-Throughput Method for Analysis of Pesticide Residues in Fruits and Vegetables.

In this study, sample processing of bulk commodities using an efficient one-step comminution procedure with liquid nitrogen (LN2) was devised and assessed in the analysis of pesticide residues in fruits and vegetables. LN2 was added to the fresh samples from a tank by opening a valve, and the standard food chopper was kept in a laboratory hood to reduce safety risks. Test portions of four replicates each of 0.25, 0.5, 1, 2, 5, 10, and 15 g were taken from eight fruits and vegetables (tomato, squash, broccoli, apple, grape, peach, green bean, and cucumber) individually comminuted with LN2. For comparison without comminution, similar test portions of a reconstituted freeze-dried certified reference material of pesticides in cucumber were also analyzed by the same method. More than 100 pesticides were monitored by both ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and instrument top sample preparation (ITSP) + fast low-pressure gas chromatography-tandem mass spectrometry (LPGC-MS/MS). A new version of QuEChERS-based sample preparation was followed, in which 5 mL of 4:1 (v/v) acetonitrile/water per gram of sample is used for extraction and 200 µL of initial extract is quickly evaporated, reconstituted in water, and ultracentrifuged for UHPLC-MS/MS analysis. For ITSP+LPGC-MS/MS, another portion of the initial extract undergoes salt-out partitioning with 4:1 (w/w) anhydrous MgSO4/NaCl and the upper layer extract is transferred to an autosampler vial for automated cleanup and analysis in parallel. Quality control spikes were made during the comminution, extraction, cleanup, and analysis steps to isolate and estimate the individual and overall measurement uncertainties of the approach. The recommended test portion size is 2 g for routine monitoring by this approach, but results demonstrated that subsamples as low as 0.5 g typically gave overall biases and relative standard deviations of <10% for nearly all pesticides, commodities, and methods, which is 3-5% lower than previously evaluated sample processing and analytical methods. This approach can be used to improve data quality, laboratory efficiency, and sample throughput in routine monitoring programs for regulatory, risk assessment, and other purposes.

Less than one minute low-pressure gas chromatography - mass spectrometry.

Conventional gas chromatography - mass spectrometry (GC-MS) takes 20-40 min per sample, which is undesirably slow in any application if speed can be increased while still meeting analytical needs. In this study, we achieved reasonably good separations with full analysis cycle times of less than 1 min by combining for the first time low-pressure (LP) GC-MS with low thermal mass (LTM) resistive-heating for rapid temperature ramping and cooling of the capillary column. The analytical column is threaded into the LTM thin-walled metal tubing in an instrumental device known as "LTM Fast GC" that is mounted at the top of the gas chromatograph in a detector port. The column inlet and outlet are connected to the GC injector and MS transfer line as usual. For LPGC-MS, a 40 cm, 0.1 mm. i.d. uncoated flow restrictor capillary connected at the injector is coupled with a 2.6 m, 0.25 mm i.d., 0.25 µm film thickness analytical column leading to the MS. Thus, the inlet operates at normal GC pressures, but the analytical column is under vacuum, which increases the optimal helium carrier gas flow velocity thereby increasing speed of full range separations while maintaining acceptable quality of chromatography. This column configuration in LTM-LPGC-MS trades a 64-fold gain in speed of analysis vs. standard GC-MS for a 4-fold loss in chromatographic peak capacity, thereby converting analysis time from minutes into seconds in common applications. For example, jet fuel containing fatty acid methyl esters (akin to biofuel) was separated in 25 s with <1 min full analysis cycle time. An EPA Method 8270 mixture of 76 analytes was also analyzed in <1 min full cycle time by LTM-LPGC-MS. Other examples include very fast analysis of heroin in a street drug powder and elucidation of a new organic synthetic compound. In this report, we describe and discuss the several advantageous and practical features of LTM-LPGC-MS, as well as its trade-offs.

Cryogenic Sample Processing with Liquid Nitrogen for Effective and Efficient Monitoring of Pesticide Residues in Foods and Feeds.

With the monitoring of hundreds of pesticides in food and feed, the comminution step is equally crucial as any other to achieve valid results. However, sample processing is often underestimated in its importance and practical difficulty to produce consistent test portions for analysis. The scientific literature is rife with descriptions of microextraction methods, but ironically, sample comminution is often ignored or dismissed as being prosaic, despite it being the foundation upon which the viability of such techniques relies. Cryogenic sample processing using dry ice (-78 °C) is generally accepted in practice, but studies have not shown it to yield representative test portions of <1 g. Remarkably, liquid nitrogen has rarely been used as a cryogenic agent in pesticide residue analysis, presumably as a result of access, cost, and safety concerns. However, real-world implementation of blending unfrozen bulk food portions with liquid nitrogen (-196 °C) using common food processing devices has demonstrated this approach to be safe, simple, fast, and cost-effective and yield high-quality results for various commodities, including increased stability of labile or volatile analytes. For example, analysis of dithiocarbamates as carbon disulfide has shown a significant increase of thiram recoveries (up to 95%) using liquid nitrogen during sample comminution. This perspective is intended to allay concerns among working laboratories about the practical use of liquid nitrogen for improved sample processing in the routine monitoring of pesticide residues in foods and feeds, which also gives promise for feasible test sample size reduction in high-throughput miniaturized methods.

Characterization of MS/MS Product Ions for the Differentiation of Structurally Isomeric Pesticides by High-Resolution Mass Spectrometry.

Structural isomeric pesticides are used in agriculture and may be challenging to differentiate for accurate identification in pesticide monitoring programs. Due to structural similarity, isomeric pesticides are difficult to separate chromatographically, and thus, their accurate identification may rely solely on mass spectrometric analysis (MS). In this study, we challenged the ability of high-resolution quadrupole-orbitrap (Q-Orbitrap) mass spectrometry to produce and evaluate the tandem mass spectrometry (MS/MS) product ions for the selected five pairs of isomeric pesticides from different classes: Pebulate and vernolate, methiocarb and ethiofencarb, uniconazole and cyproconazole, sebuthylazine and terbuthylazine, and orbencarb and thiobencarb. The use of Q-Orbitrap instrument with a mass error <3 ppm allowed proposed elucidation of the product ion structures with consideration of the ion formulae, data interpretation, and literature searches. Product ions unique to pebulate, vernolate, methiocarb, ethiofencarb, and uniconazole were observed. Elucidation of the observed MS/MS product ion structures was conducted, and the fragmentation pathways were proposed. This information is valuable to increase selectivity in MS/MS analysis and differentiate isomeric pesticides, and thereby reduce the rates of false positives in pesticide monitoring programs.

Hits and misses in research trends to monitor contaminants in foods.

Monitoring of chemicals of toxicological concern in food is commonly needed for many purposes, which include (in part) food safety, regulatory enforcement, risk assessment, international food trade, label claims, environmental protection, industry needs, academic research, and consumer confidence. Chemicals of current concern include a variety of toxins, pesticides, veterinary drugs, growth promoters, environmental contaminants, toxic metals, allergens, endocrine disruptors, genetically modified organisms, melamine, acrylamide, furans, nitrosamines, food additives, packaging components, and miscellaneous other chemicals. In light of past crises, the potential harm from known or unknown chemicals not currently monitored are a source of additional concern by the food industry, regulators, scientists, and consumers. As global food trade has expanded and detection techniques have improved, chemical contaminant analysis of foods has also increased in importance and activity. This critical review article is aimed to highlight current trends in the literature, including neglected research needs, on the analysis of chemicals of toxicological concern in foods. Graphical abstract.