Quantitative Metabolism and Urinary Elimination Kinetics of Seven Neonicotinoids and Neonicotinoid-Like Compounds in Humans.

Reverse dosimetry, i.e., calculating the dose of hazardous substances that has been taken up by humans based on measured analyte concentrations in spot urine samples, is critical for risk assessment and requires metabolic and kinetic data. We quantitatively studied the metabolism of seven major neonicotinoid and neonicotinoid-like compounds (NNIs) after single oral doses in male volunteers and determined key kinetic parameters and urinary elimination for NNIs together with their metabolites. Complete and consecutive urine samples were collected over 48 h. All samples were analyzed by tandem mass spectrometry, following liquid or gas chromatographic separation. Single- and group-specific NNI metabolites were quantified, i.e., hydroxylated and N-dealkylated NNIs and NNI-associated carboxylic acids and their glycine derivatives. Large, substance-dependent variations of key toxicokinetic parameters were observed. Mean times of concentration maxima (tmax) in urine varied between 2.0 (imidacloprid) and 25.8 h (N-desmethyl-clothianidin), whereas mean urinary elimination half-times (t1/2) were between 2.5 (acetamiprid) and 49.5 h (sulfoxaflor). Mean 48 h excretion fractions (Fue's) were between 0.03% (2-chloro-1,3-thiazole-5-carboxylic acid glycine) and 84% (clothianidin). In contrast, the interindividual differences of Fue's between the volunteers for each of the NNIs and their metabolites remained low (below a factor of 2 between the maximum and minimum derived Fue with the exception of 6-chloronicotinic acid in the acetamiprid dose study). The obtained quantitative data enabled choosing appropriate biomarkers for exposure assessment and, at the same time, for risk assessment by reverse dosimetry at current environmental exposures, i.e., comparing the calculated doses that have been taken up to currently available acceptable daily intakes of NNIs.

Quantitation of 6-chloronicotinic acid and 2-chloro-1,3-thiazole-5-carboxylic acid and their glycine conjugates in human urine to assess neonicotinoid exposure.

Neonicotinoids and neonicotinoid-like compounds (NNIs) are widely used insecticides and their ubiquitous occurrence in the environment requires methods for exposure assessment in humans. The majority of the NNIs can be divided into 6-chloropyridinyl- and 2-chlorothiazolyl-containing compounds, suggesting the formation of the group-specific metabolites 6-chloronicotinic acid (6-CNA), 2-chloro-1,3-thiazole-5-carboxylic acid (2-CTA), and their respective glycine derivatives (6-CNA-gly, 2-CTA-gly). Here, we developed and validated an analytical method based on gas chromatography coupled to mass spectrometry (GC-MS/MS) to simultaneously analyze these four metabolites in human urine. As analytical standards for the glycine conjugates were not commercially available, we synthesized 6-CNA-gly, 2-CTA-gly, and their 13C2,15N-labeled analogs for internal standardization and quantitation by stable isotope dilution. We also ensured chromatographic separation of 6-CNA and its isomer 2-CNA. Enzymatic cleavage during sample preparation was proven unnecessary. The limits of quantitation were between 0.1 (6-CNA) and 0.4 µg/L (2-CTA-gly) and the repeatability was satisfactory (coefficient of variation was <19% over the calibration range). We analyzed 38 spot urine samples from the general population and were able to quantify 6-CNA-gly in 58% of the samples (median 0.2 µg/L). In contrast, no 6-CNA could be detected. The results are in line with well-known metabolic pathways specific in humans, that, compared to rodents, favor the formation and excretion of phase-II-metabolites (glycine derivatives) rather than phase-I metabolites (free carboxylic acids). Nevertheless, the exact source of exposure (i.e., the specific NNI) remains elusive in the general population, may even vary quantitatively between different NNIs, and also might be regional specific based on the respective use of individual NNIs. In sum, we developed a robust and sensitive analytical method for the determination of four group-specific NNI metabolites.

Rapid quantification of seven major neonicotinoids and neonicotinoid-like compounds and their key metabolites in human urine.

Neonicotinoids and neonicotinoid-like compounds (NNIs) are frequently used insecticides worldwide and exposure scenarios can vary widely between countries and continents. We have developed a specific and robust analytical method based on liquid chromatography-electrospray tandem mass spectrometry coupled to online-SPE (online-SPE-LC-ESI-MS-MS) to analyze the seven most important NNIs from a global perspective together with nine of their key metabolites in human urine. The method also includes the neonicotinoid-like flupyradifurone (FLUP), an important future substitute for classical neonicotinoids, and two of its major human metabolites, 5-hydroxy- and N-desfluoroethyl-FLUP. Validation of the method was carried out using pooled urine samples from low-dose human metabolism studies and spiked urine samples with a wide range of creatinine concentrations. Depending on the analyte, the limits of quantitation were between 0.06 and 2.1 µg L-1, the inter-day and intra-day imprecisions ≤6%, and the mean relative recoveries between 89% and 112%. The method enabled us to successfully quantify NNIs and their metabolites at current environmental exposures in 34 individuals of the German general population and 43 pregnant women from Brazil with no known occupational exposures to NNIs.

Human biomonitoring of neonicotinoid exposures: case studies after the use of a spray-agent to ornamental plants and a topical medication to pets.

Acetamiprid (ACE) and imidacloprid (IMI) are insecticides of global importance and are used as spray and watering agents for ornamental plants to control biting and sucking insects or as topical medications on pets to remove and control fleas. Human biomonitoring data on ACE and IMI exposures when applying these products are limited. We investigated exposures to ACE and IMI in male volunteers after the domestic application of either an ACE-containing agent or an IMI-containing spot-on medication. Complete and consecutive urine samples were collected for up to 56 h after application. Urine samples were analyzed for ACE, IMI, and their respective metabolites (N-desmethyl-ACE, IMI-olefin, and sum of 4-/5-hydroxy-IMI) by liquid chromatography-tandem mass spectrometry. Fairly uniform concentrations of N-desmethyl-ACE could be observed before and after orchid treatment, so that an ACE exposure associated with orchid treatment can most likely be excluded. In contrast, after the application of the IMI-containing medication, elevated concentrations of IMI, 4-/5-hydroxy-IMI, and IMI-olefin were quantified in urine samples post-20 h with maximum concentrations of 3.1, 14.9, and 8.0 µg/g creatinine, respectively, well above general background levels. Nevertheless, the IMI intake (10.6 µg/kg bw), calculated from the excreted amounts, was around five times below the current European acceptable daily intake. Based on the case results here, household exposures to ACE and IMI after spray treatment of ornamental plants and anti-flea treatment of dogs can be regarded as low and safe. However, people regularly applying neonicotinoid-containing formulations, such as professional gardeners and employees in animal shelters, should be studied in more detail.

Human metabolism and urinary excretion of seven neonicotinoids and neonicotinoid-like compounds after controlled oral dosages.

Few human data on exposure and toxicity are available on neonicotinoids and neonicotinoid-like compounds (NNIs), an important group of insecticides worldwide. Specifically, exposure assessment of humans by biomonitoring remains a challenge due to the lack of appropriate biomarkers. We investigated the human metabolism and metabolite excretion in urine of acetamiprid (ACE), clothianidin (CLO), flupyradifurone (FLUP), imidacloprid (IMI), sulfoxaflor (SULF), thiacloprid (THIAC) and thiamethoxam (THIAM) after single oral dosages at the currently acceptable daily intake levels of the European Food Safety Authority. Consecutive post-dose urine samples were collected up to 48 h. Suspect screening of tentative metabolites was carried out by liquid chromatography-high-resolution mass spectrometry. Screening hits were identified based on their accurate mass, isotope signal masses and ratios, product ion spectra, and excretion kinetics. We found, with the exception of SULF, extensive metabolization of NNIs to specific metabolites which were excreted next to the parent compounds. Overall, 24 metabolites were detected with signal intensities indicative of high metabolic relevance. Phase-I metabolites were predominantly derived by mono-oxidation (such as hydroxy-FLUP, -IMI, and -THIAC) and by oxidative N-desalkylation (such as N-desdifluoroethyl-FLUP and N-desmethyl-ACE, -CLO and -THIAM). IMI-olefin, obtained by dehydration of hydroxylated IMI, was identified as a major metabolite of IMI. SULF was excreted unchanged in urine. Previously reported metabolites of NNIs such as 6-chloronicotinic acid or 2-chlorothiazole-4-carboxylic acid and their glycine derivatives were detected either at low signal intensities or not at all and seem less relevant for human biomonitoring. Our highly controlled approach provides specific insight into the human metabolism of NNIs and suggests suitable biomarkers for future exposure assessment at environmentally relevant exposures.

Heroin-Related Compounds and Metabolic Ratios in Postmortem Samples Using LC-MS-MS.

Analysis of postmortem samples with the presence of morphine can sometimes be challenging to interpret. Tolerance complicates interpretation of intoxications and causes of death due to overlap in therapeutic and fatal concentrations. Determination of metabolites and metabolic ratios can potentially differentiate between abstinence, continuous administration, and perhaps time of administration. The purpose of this study was to (a) develop and validate a method for quantitation of morphine-3ß-D-glucuronide, morphine-6ß-D-glucuronide, normorphine, codeine-6ß-D-glucuronide, norcodeine, codeine, 6-acetylmorphine, and ethylmorphine in urine using liquid chromatography-tandem mass spectrometry; (b) apply the method to opiate related deaths; (c) compare metabolic ratios in urine in different causes of death (CoD) and after different drug intakes and (d) compare heroin intoxications in rapid and delayed deaths. Validation parameters such as precision, bias, matrix effects, stability, process efficiency, and dilution integrity were assessed and deemed acceptable. Lower limits of quantitation ranged from 0.01-0.2 µg/mL for all analytes. Autopsy cases (n=135) with paired blood and urine samples were analyzed. Cases were divided into three groups based on CoD; opiate intoxication, intoxication with other drugs than opiates, and other CoD. The cases were classified by intake: codeine (n=42), heroin (n=36), morphine (n=49), and ethylmorphine (n=3). Five cases were classified as mixed intakes and excluded. Heroin intoxications (n=35) were divided into rapid (n=15) or delayed (n=20) deaths. Parent drug groups were compared using metabolic ratio morphine-3ß-D-glucuronide/morphine and significant differences were observed between codeine vs morphine (p=0.005) and codeine vs heroin (p≤0.0001). Urine and blood concentrations, and metabolic ratios in rapid and delayed heroin intoxications were compared and determined a significant difference for morphine (p=0.001), codeine (p=0.009), 6-acetylmorphine (p=0.02) in urine, and morphine (p=0.02) in blood, but there was no significant difference (p=0.9) between metabolic ratios. Morphine-3ß-D-glucuronide results suggested a period of abstinence prior to death in 25% of the heroin intoxications.