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.

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.

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.

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.

Occurrence, Distribution, and Ecological Risk of Fluoroquinolones in Rivers and Wastewaters.

The use of fluoroquinolones for the treatment of infections in humans and animals has increased in Argentina, and they can be found in large amounts in water bodies. The present study investigated the occurrence and associated ecological risk of 5 fluoroquinolones in rivers and farm wastewaters of San Luis, Santa Fe, Córdoba, Entre Ríos, and Buenos Aires provinces of Argentina by high-performance liquid chromatography coupled to fast-scanning fluorescence detection and ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry detection. The maximum concentrations of ciprofloxacin, enrofloxacin, ofloxacin, enoxacin, and difloxacin found in wastewater were 1.14, 11.9, 1.78, 22.1, and 14.2 µg L-1 , respectively. In the case of river samples, only enrofloxacin was found, at a concentration of 0.97 µg L-1 . The individual risk of aquatic organisms associated with water pollution due to fluoroquinolones was higher in bacteria, cyanobacteria, algae, plants, and anurans than in crustaceae and fish, with, in some cases, risk quotients >1. The proportion of samples classified as high risk was 87.5% for ofloxacin, 63.5% for enrofloxacin, 57.1% for ciprofloxacin, and 25% for enoxacin. Our results suggest that the prevalence of fluoroquinolones in water could be potentially risky for the aquatic ecosystem, and harmful to biodiversity. Environ Toxicol Chem 2019;38:2305-2313. © 2019 SETAC.

Determination of glyphosate, AMPA and glufosinate in dairy farm water from Argentina using a simplified UHPLC-MS/MS method.

Argentina, together with the USA and Brazil, produces approximately 80% of the total worldwide glyphosate loadings. The development of a simplified ultra-high performance liquid chromatographic tandem mass spectrometric method (UHPLC-MS/MS) for the determination of glyphosate, aminomethylphosphonic acid (AMPA) and glufosinate in water is described, including studies of several alternatives of 9-fluorenylmethylchloroformate (FMOC-Cl) derivatization and pretreatment steps. The proposed method includes acidification and neutralization of a low sample volume (3 mL), 2 hours derivatization step, cleanup with dichloromethane, followed by reverse phase UHPLC-MS/MS determination of the analytes. Figures of merit were satisfactory in terms of linearity, selectivity, accuracy and intermediate precision (%REC 70-105% with RSD < 15%). Limits of quantification (LOQ) were suitable for monitoring purposes (0.6, 0.2, 0.1 µg/L for glyphosate, AMPA and glufosinate respectively). The validated methodology was applied for the analysis of livestock wells waters from 40 dairy farms located in the central region of Argentina. Glyphosate and AMPA were quantified in 15% and 53% of the analyzed samples with concentrations ranging from 0.6-11.3 µg/L and 0.2-6.5 µg/L respectively. Greater concentrations of glyphosate were also verified in waters from open-reservoir tanks, which are directly exposed to the farm environment. In these cases glyphosate and AMPA occurrence increased, being quantified in the 33% and 61% of the samples with values ranging 0.6-21.2 µg/L and 0.2-4.2 µg/L respectively. Also in this case glufosinate was found in 52% samples at

National short-term dietary exposure assessment of a selected group of pesticides in Argentina.

An evaluation of acute dietary exposure to pesticide residues, applying deterministic and stochastic methods, was performed for a selected group of pesticides in two representative age groups from Argentina. Thus, 28 active ingredients (a.i.) and 75 food items were evaluated for the group of 2-5-year-old children, while 9 a.i. and 59 food items were considered for the 10-49-year-old women group. A deterministic assessment was conducting following the Food and Agriculture Organization (FAO) and World Health Organization (WHO) procedure but using the national maximum residue limits (MRLs) as pesticide residue concentration data, while in the stochastic approach, a theoretical distribution modeled with the available information was used. Food consumption data were obtained from the 2004-2005 comprehensive national nutrition and health survey. The risk was estimated by comparing the short-term dietary exposure with the acute reference dose (ARfD) values for each pesticide-food combination evaluated. In the deterministic assessment, 173 (39.1%) and 40 (31.3%) combinations exceeded the ARfD thresholds for the 2-5-year-old children and 10-49-year-old women groups, respectively. This conservative study generated relevant information as a first stage of acute dietary risk assessment in Argentina.

Comprehensive estimate of the theoretical maximum daily intake of pesticide residues for chronic dietary risk assessment in Argentina.

A chronic dietary risk assessment for pesticide residues was conducted for four age groups of the Argentinian population following the procedure recommended by the WHO. The National Theoretical Maximum Daily Intake (NTMDI) for 308 pesticides was calculated for the first time, using the Maximum Residue Limits (MRLs) from several Argentinean regulations and food consumption data from a comprehensive National Nutrition and Health Survey. The risk was estimated by comparing the TMDI with the Acceptable Daily Intakes (ADI) identified by various sources. Furthermore, for each of the compounds with a TMDI >65% of the ADI, a probabilistic analysis was conducted to quantify the probability of exceeding the ADI. In this study 27, 22, 10, and 6 active ingredients (a.i.) were estimated to exceed the 100% of the ADI for the different population groups: 6-23 month-old children, 2-5 year-old children, pregnant women, and 10-49 year-old women, respectively. Some of these ADI-exceeding compounds (carbofuran, diazinon, dichlorvos, dimethoate, oxydemeton-methyl and methyl bromide) were found in all four of these groups. Milk, apples, potatoes, and tomatoes were the foods that contributed most to the intake of these pesticides. The study is of primary importance for the improvement of risk assessment, regulations, and monitoring activities.

Glyphosate loss by runoff and its relationship with phosphorus fertilization.

The aim of this study was to evaluate the relationship between glyphosate and phosphate fertilizer application and their contribution to surface water runoff contamination. The study was performed in Aquic Argiudoll soil (Tezanos Pinto series). Four treatments were assessed on three dates of rainfall simulation after fertilizer and herbicide application. The soluble phosphorus in runoff water was determined by a colorimetric method. For the determination of glyphosate and aminomethylphosphonic acid (AMPA), a method based on fluorenylmethyloxycarbonyl (FMOC) group derivatization, solid phase extraction (SPE) purification, and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was employed. The application of phosphorus fertilizer resulted in an increased loss of glyphosate by runoff after 1 day of application. These results suggest the need for further study to understand the interactions and to determine appropriate application timing with the goal of reducing the pollution risk by runoff.