Chiral Metabolites Research Articles

Rapid chiral discrimination of oncometabolite dl-2-hydroxyglutaric acid using derivatization and field asymmetric waveform ion mobility spectrometry/mass spectrometry.

2-Hydroxyglutaric acid is a chiral metabolite whose enantiomers specifically accumulate in different diseases. An enantiomeric excess of the d-form in biological specimens reflects the existence of various pathogenic mutations in cancer patients, however, conventional methods using gas or liquid chromatography and capillary electrophoresis had not been used for large clinical studies because they require multiple analytical instruments and a long run time to separate the enantiomers. Here, we present a rapid separation method for dl-2-hydroxyglutaric acid using a chiral derivatizing reagent and field asymmetric waveform ion mobility spectrometry/mass spectrometry, which requires a single analytical instrument and <1 s for the separation. We compared three derivatization methods and found that a method using (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine enables the separation. In addition, we were able to detect dl-2-hydroxyglutaric acid in standard solution at lower concentrations than that previously reported for the serum. These results show the potential of the method to be used in clinical analysis.

Label-Free Ultrasensitive Detection of Abnormal Chiral Metabolites in Diabetes.

Homochirality is necessary for normal biochemical processes in humans. Abnormal amounts of chiral molecules in biofluids have been found in patients with diabetes. However, the detailed analysis of diabetes-related abnormal chirality in biofluids and its potential use for clinical applications have been hindered by the difficulty in detecting and monitoring the chiral changes in biofluids, due to their low molar mass and trace concentrations. Herein, we demonstrate the label-free detection of chiral molecules using only 10 μL with 107-fold enhancement in sensitivity compared with traditional plasmonic chiral metamaterials. The ultrahigh sensitivity and low sample consumption were enabled by microbubble-induced rapid accumulation of biomolecules on plasmonic chiral sensors. We have applied our technique on mouse and human urine samples, uncovering the previously undetectable diabetes-induced abnormal dextrorotatory shift in chirality of urine metabolites. Furthermore, the accumulation-assisted plasmonic chiral sensing achieved a diagnostic accuracy of 84% on clinical urine samples from human patients. With the ultrahigh sensitivity, ultralow sample consumption, and fast response, our technique will benefit diabetes research and could be developed as point-of-care devices for first-line noninvasive screening and prognosis of prediabetes or diabetes and its complications.

Toxicity and fate of chiral insecticide pyriproxyfen and its metabolites in zebrafish (Danio rerio).

Pyriproxyfen is a juvenile hormone analogue insecticide used worldwide. At present, the potential threat of pyriproxyfen to aquatic organism has not been well explored. In this work, the bioaccumulation, metabolic profile and toxicity of pyriproxyfen and its metabolites to zebrafish were studied, and the enantioselectivity of pyriproxyfen and the major chiral metabolites were also determined. Sixteen metabolites of pyriproxyfen in zebrafish were identified. Hydroxylation, ether linkage cleavage and oxidation in phase I metabolism, followed by sulfate and glucuronic acid conjugation. The bioconcentration factors ranged from 1175 to 1246. Hydroxylation metabolites of pyriproxyfen showed enantioselective behavior in zebrafish with enantiomer fractions (EFs) of 4'-OH- pyriproxyfen and 5″-OH- pyriproxyfen ranged from 0.50 to 0.71. Toxicological indexes including acute toxicity, joint toxicity and oxidative stress were tested. Among all the metabolites, 4'-OH- pyriproxyfen was found 2 folds more toxic to zebrafish than pyriproxyfen. (-)-Pyriproxyfen was found 2 folds more toxic than rac- and (+)-pyriproxyfen. Antagonistic effects were found in binary joint toxicity of pyriproxyfen and its hydroxylated metabolites. Pyriproxyfen and its metabolites also showed oxidative stress damage by inhibiting the activity of CAT and SOD and increasing MDA. This work provided deep insight into the metabolism and the potential risks of pyriproxyfen to aquatic organisms.

Ketamine and Norketamine: Enantioresolution and Enantioselective Aquatic Ecotoxicity Studies.

Ketamine is a chiral drug used for various clinical purposes but often misused. It is metabolized to norketamine, an active chiral metabolite. Both substances have been detected in environmental matrices, but studies about their enantioselective toxic effects are scarce. In the present study, the enantiomers of ketamine and norketamine were separated by a semipreparative enantioselective liquid chromatography method, and their toxicity was investigated in different aquatic organisms. The enantioseparation was achieved using a homemade semipreparative chiral column. Optimized conditions allowed the recovery of compounds with enantiomeric purity higher than 99%, except for (R)-ketamine (97%). The absolute configuration of the enantiomers was achieved by experimental electronic circular dichroism (ECD). The ecotoxicity assays were performed with the microcrustacean Daphnia magna and the protozoan Tetrahymena thermophila using Toxkit MicroBioTests. Different concentrations were tested (0.1-10 000 µg/L) to include environmental levels (~0.5-~100 µg/L), for racemates (R,S) and the isolated enantiomers (R or S) of ketamine and norketamine. No toxicity was observed in either organism at environmental levels. However, at greater concentrations, (R,S)-ketamine presented higher mortality for D. magna compared with its metabolite (R,S)-norketamine (85 and 20%, respectively), and the (S)-ketamine enantiomer showed higher toxicity than the (R)-ketamine enantiomer. In addition, (S)-ketamine also presented higher growth inhibition than (R)-ketamine for T. thermophila at the highest concentrations (5000 and 10 000 µg/L). Contrary to D. magna, growth inhibition was observed for both enantiomers of norketamine and in the same magnitude order of the (S)-ketamine enantiomer. The results showed that the 2 organisms had different susceptibilities to norketamine and that the toxicity of ketamine at high concentrations is enantioselective for both organisms. Environ Toxicol Chem 2022;41:569-579. © 2020 SETAC.

Regioselective Hydroxylation of Oleanolic Acid Catalyzed by Human CYP3A4 to Produce Hederagenenin, a Chiral Metabolite

Oleanolic acid (OA) is a pentacyclic triterpenoid widely found in plants and foods as an aglycone of triterpenoid saponins or as a free acid. OA exhibits beneficial activities for humans, including antitumor, antivirus, and hepatoprotection properties without apparent toxicity. The metabolites produced by the cytochrome P450 (P450) enzymes are critical for the evaluation of the efficacy and safety of drugs. In this study, the potential metabolites of OA were investigated by P450-catalyzed oxidation reactions. Among the various tested human P450s, only human CYP3A4 was active for the hydroxylation of OA. The major metabolite was characterized by a set of analyses using HPLC, LC–MS, and NMR. It was found to be 4-epi-hederagenenin, a chiral product, by regioselective hydroxylation of the methyl group at the C-23 position. These results indicated that CYP3A4 can hydroxylate an OA substrate to make 4-epi-hederagenenin. Possible drug–food interactions are discussed.

Popping candy-assisted dispersive liquid–liquid microextraction for enantioselective determination of prothioconazole and its chiral metabolite in water, beer, Baijiu, and vinegar samples by HPLC

To simultaneously determine the enantiomers of prothioconazole and its chiral metabolite prothioconazole-desthio in water, beer, Baijiu, and vinegar samples by HPLC, a simple, fast, environmentally-friendly popping candy-assisted dispersive liquid–liquid microextraction technique was developed. A green medium-chain fatty acid (decanoic acid) and popping candy could be used as the extractant and solid dispersant respectively to avoid the use of toxic organic solvents. Decanoic acid was collected after extraction by solidification at room temperature. The linear range of this technique was from 27.1 to 1000 µg L−1. The limits of detection and quantification were within the ranges of 8.1–11.2 μg L-1 and 27.1–37.3 μg L-1, respectively. The extraction recovery was 80.8% to 102.5% with the relative standard deviation ranged from 1.1 to 7.1%. This technique has been successfully applied to enantioselectively determine the residues of prothioconazole and prothioconazole-desthio in water, beer, Baijiu, and vinegar samples.

(S)-1-(5-(4-Methylpiperazin-1-yl)-2,4-dinitrophenyl)pyrrolidine-2-carboxylic acid as a derivatization reagent for ultrasensitive detection of amine enantiomers by HPLC-MS/MS and its application to the chiral metabolite analysis of (R)-1-aminoindan in saliva

(S)-1-(5-(4-Methylpiperazin-1-yl)-2,4-dinitrophenyl)pyrrolidine-2-carboxylic acid (Pro-PPZ) was employed as a chiral derivatization reagent (CDR) for the efficient enantioseparation and ultrasensitive mass spectrometric detection of chiral amines. Pro-PPZ was prepared from the one-step reaction of 1-(5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine (PPZ) and l-proline. Two amines and two amino acid methyl esters were selected as model chiral amines, which were easily labeled with Pro-PPZ under mild reaction conditions (35 °C for 10 min) generating Pro-PPZ-amine derivatives. The resulting diastereomers were completely separated by reversed-phase liquid chromatography (RP-LC) using an ODS column (Rs = 3.4–17.0 for amines). Ultrasensitive detection limits on femtomolar level were obtained for the tested amines using multiple reaction monitoring (MRM) chromatograms at a single monitoring ion, m/z 289 (0.1–5.0 fmol for amines). The practical metabolite analysis of (R)-1-aminoindan (R-AI) in saliva samples was performed by LC–MS/MS using the Pro-PPZ derivatization method. The method was validated in terms of precision, accuracy, and linearity. Using this method, R-AI concentrations in saliva were determined after a single oral administration of the drug rasagiline to healthy male and female subjects, but no (S)-1-aminoindan (S-AI) was detected, which suggesting that R-AI was not converted into S-enantiomer in the metabolic process. R-AI concentrations in four healthy volunteers ranged from 32.85 nM to 49.45 nM, with an average value of 43.76 nM. To date, there is no LC–MS (or MS/MS) method reported for the enantioselective determination of R-AI in human saliva samples.

Enantioselective analysis of venlafaxine and its active metabolites: A review on the separation methodologies.

Venlafaxine (VFX) is a serotonin and norepinephrine reuptake inhibitor chiral drug used in therapy as an antidepressant in the form of a racemate consisting of R- and S-VFX. The two enantiomers of VFX exhibit different pharmacological activities: R-VFX inhibits both norepinephrine and serotonin synaptic reuptake, whereas S-VFX inhibits only the serotonin one. R- and S-VFX are metabolized in the liver to the respective R- and S-O-desmethylvenlafaxine (ODVFX), R- and S-N-desmethylvenlafaxine (NDVFX), and R- and S-N,O-didesmethylvenlafaxine (NODVFX). The pharmacological profile of ODVFX is close to that of VFX, whereas the other two chiral metabolites (NDVFX and NODVFX) have lower affinity for the receptor sites. The pharmacokinetics of the VFX enantiomers appear stereoselective, including the metabolism process. In the past 20 years, several studies describing the enantioselective analysis of R- and S-VFX in pharmaceutical formulations and its chiral metabolites in biological matrices were published. These methods encompass liquid chromatography coupled with UV detection, mass spectrometry, or tandem mass spectrometry, and capillary electrophoresis. This paper reviews the published methods used for the determination of the individual enantiomers of VFX and its chiral metabolites in different matrices.

Enantiomeric separation of malathion and malaoxon and the chiral residue analysis in food and environmental matrix.

Malathion is a widely used chiral phosphorus insecticide, which has a more toxic chiral metabolite malaoxon. In this work, the enantiomers of malathion and malaoxon were separated by high-performance liquid chromatography-mass/mass (HPLC-MS/MS) with chiral columns using acetonitrile/water or methanol/water as mobile phase, and the chromatographic conditions were optimized. Based on the chiral separation, the chiral residue analysis methods for the enantiomers in soil, fruit, and vegetables were set up. Two pairs of the enantiomers were better separated on CHIRALPAK IC chiral column, and baseline simultaneous separations of malathion and malaoxon enantiomers were achieved with acetonitrile/water (40/60, v/v) as mobile phase at a flow rate of 0.5 mL/min. The elution orders were -/+ for both malathion and malaoxon measured by an optical rotation detector. The chiral residue analysis in soil, fruit, and vegetables was validated by linearity, recovery, precision, limit of detection (LOD), and limit of quantification (LOQ). The LODs and LOQs for the enantiomers of malathion were 1 μg/kg and 3-5 μg/kg and 0.08 μg/kg and 0.20-0.25 μg/kg for malaoxon enantiomers. Good linear calibration curves for each enantiomer in the matrices were obtained within the concentration range of 0.02-12 mg/L. The mean recoveries of the enantiomers of malathion and malaoxon ranged from 82.26% to 109.04%, with RSDs of 0.71-8.63%.The results confirmed that this method was capable of simultaneously determining the residue of malathion and malaoxon in food and environmental matrix on an enantiomeric level.

The enantioselective enrichment, metabolism, and toxicity of fenoxaprop-ethyl and its metabolites in zebrafish.

To investigate the impacts of the widely used chiral herbicide fenoxaprop-ethyl (FE) on aquatic organisms, the enrichment, metabolism, acute toxicity, and oxidative stress of fenoxaprop-ethyl and its main metabolites fenoxaprop (FA), ethyl-2-(4-hydroxyphenoxy)propanoate (EHPP), 2-(4-hydroxyphenoxy)propanoic acid (HPPA), and 6-chloro-2,3-dihydrobenzoxazol-2-one (CDHB) in zebrafish were studied. The enantioselectivity of fenoxaprop-ethyl and its chiral metabolites was also determined. Fenoxaprop-ethyl quickly degraded in zebrafish by aquatic exposure. FA, HPPA, and CDHB were the main metabolites that were enriched in the zebrafish. In the metabolism experiment, the half-lives of the metabolites were 0.92-1.72 days in zebrafish. The R-enantiomers of FA and HPPA were preferentially enriched and metabolized with enantiomeric fractions (EFs) of 0.65-0.85. According to the 96-h acute toxicity, FA, HPPA, EHPP, and CDHB were less toxic to zebrafish than FE, following the order of FE > CDHB > EHPP > FA > HPPA. The S-enantiomers of FE, FA, CDHB, and EHPP were more toxic than the R-enantiomers. FE and its metabolites could significantly increase catalase (CAT) and superoxide dismutase (SOD) activity and the malondialdehyde (MDA) content in gill and liver tissues, indicating their oxidative stress, and these effects were also enantioselective. This work could supply more information for evaluating the risks of fenoxaprop-ethyl on aquatic organisms concerning their metabolites and enantiomers.

Tissue Distribution, Accumulation, and Metabolism of Chiral Flufiprole in Loach (Misgurnus anguillicaudatus).

Flufiprole is an insecticide used in the rice field and may pose a potential threat to aquatic organisms including loach. To investigate the transformation products of flufiprole in loach, the accumulation, elimination, and tissue distribution in vivo as well as the metabolism in vitro at the enantiomeric level were studied. Flufiprole enantiomers rapidly accumulated and were metabolized to flufiprole sulfone, fipronil, and flufiprole amide in the tissues. Enantiomeric fractions showed the preferential accumulation and degradation of S-flufiprole. The residue of the chiral metabolite flufiprole amide was also enantioselective. The individual enantiomer treatment indicated that S-flufiprole was preferentially metabolized to flufiprole sulfone and R-flufiprole to fipronil. The metabolites were more persistent than flufiprole with longer half-lives. The metabolism in liver microsomes also reached consistent conclusions. The dietary risk assessment indicated that flufiprole would not cause unacceptable threats to human health. However, the metabolites of flufiprole should be considered in the risk evaluation.

Stereoselective bioactivity of the chiral triazole fungicide prothioconazole and its metabolite

Chiral triazole fungicides have played a significant role in plant pathogen control. Although their enantiomers often exhibit different bioactivity, the mechanism of the stereoselectivity has not been well studied. The stereoselective bioactivity and mechanisms of prothioconazole and its chiral metabolite against plant pathogenic fungi were investigated. The results indicated that the metabolite exerted more fungicidal activities than the activities of the parent compound. R-Prothioconazole and R-prothioconazole-desthio were 6-262 and 19-954 times more potent against pathogenic fungi than the S-enantiomers, respectively. The R-enantiomers were more effective than in inhibiting the biosynthesis of ergosterol and deoxynivalenol the S-enantiomer. Homology modeling and molecular docking suggested that the R-enantiomers of prothioconazole and prothioconazole-desthio possessed better binding modes than S-enantiomers to CYP51B. Moreover, exposure to prothioconazole and its metabolite enantiomers significantly changed the transcription levels of the CYP51 (CYP 51A, CYP51B, CYP 51C) and Tri (Tri5, Tri6, Tri12) genes. The results showed that application of the R-prothioconazole could require a smaller application amount to eliminate the carcinogenic mycotoxins and any environmental risks.

Stereoselective supercritical fluidic chromatography –mass spectrometry (SFC-MS) as a fast bioanalytical tool to assess chiral inversion in vivo and in vitro

The stereoisomers of a chiral drug can exhibit great differences in pharmacodynamic and/or pharmacokinetic properties. Chiral bioanalytical assays are necessary to measure the concentrations of individual enantiomers and/or their chiral metabolites in biological samples in order to assess the absorption, distribution, metabolism, excretion, and toxicity of the individual enantiomers. In addition, chiral assays are required to evaluate potential chiral inversion caused by the biotransformation process. While normal phase or reversed phase chromatography remains the common choices for chiral bioanalytical assays, supercritical fluid chromatography (SFC) demonstrated shorter analysis time without affecting separation efficiency, due to the lower viscosity and faster mass transfer properties of supercritical or subcritical carbon dioxide than traditionally used mobile phases. In this paper, we present examples for developing chiral SFC-multiple reaction monitoring (MRM) assays to measure the pharmacokinetic profiles of stereoisomers of drugs in three distinct cases. Furthermore, these assays can be used to investigate the potential chiral inversion of the small molecule drugs from in vivo or in vitro sources.

Efficient biocatalytic synthesis of D-tagatose 1,6-diphosphate by LacC-catalysed phosphorylation of D-tagatose 6-phosphate

D-tagatose 1,6-diphosphate is an important metabolite which can be formed via the classical tagatose 6-phosphate pathway or via the tagatose 1-phosphate pathway discovered more recently. The chiral metabolite naturally occurs as an intermediate in the galactose metabolism of various organisms, where it is formed by a reversible condensation of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate, whereas D-tagatose 6-phosphate has been synthesized chemically, no chemical synthetic method for further phosphorylation to the D-tagatose 1,6-diphosphate has been described so far. Therefore, a site-specific biocatalytic phosphorylation of the 1-position in D-tagatose 6-phosphate has been chosen. The lacC gene from Lactococcus lactis subsp. lactis was synthesized, cloned into an appropriate expression vector to allow for the production of LacC, the D-tagatose 6-phosphate kinase in Escherichia coli. Subsequently, an efficient biocatalytic synthesis has been developed for the phosphorylation of D-tagatose 6-phosphate at its C1-position by using the recombinant D-tagatose 6-phosphate kinase and the phosphoenolpyruvate/pyruvate kinase-system for ATP regeneration. This straightforward and scalable one-step biocatalytic synthesis of D-tagatose 1,6-diphosphate was successfully scaled up to the gram scale.

Facile synthesis of D-xylulose-5-phosphate and L-xylulose-5-phosphate by xylulokinase-catalyzed phosphorylation

The enantiomeric metabolites D-xylulose-5-phosphate and L-xylulose-5-phosphate are highly relevant in numerous important pathways from microbial to human metabolism. As these phosphorylated chiral metabolites are required for the analysis of enzyme activities, pathways and biological functions in health and disease, efficient and straightforward methodologies for their synthesis are needed. Biocatalytic phosphorylations of their corresponding monosaccharides offer single step synthetic routes. Therefore, D- and L-xylulokinases have been selected, the XKS1 gene from Saccharomyces cerevisiae and the lyx gene from Escherichia coli have been synthesized and cloned into an appropriate expression vector to allow for the production of the enzymes in E. coli. The expression has led to highly active xylulokinases in the soluble fractions, which could be directly used for the biocatalytic phosphorylation of D- and L-xylulose, using the phosphoenolpyruvate(PEP)/pyruvatekinase(PK)-system for the regeneration of the ATP cofactor. The time course of the phosphorylation reactions has been monitored by 31P-NMR and 1H-NMR spectroscopy and the monosaccharide substrates could be phosphorylated with nearly 100% conversion. After standard workup, both the D-xylulose-5-phosphate and the L-xylulose-5-phosphate have been obtained in high yield and purity.

Subcritical Fluid Chromatography at Sub-Ambient Temperatures for the Chiral Resolution of Ketamine Metabolites with Rapid-Onset Antidepressant Effects.

Chiral metabolites of ketamine exerting rapid-onset yet sustained antidepressant effects may be marketed directly in the future, but require chemo- and enantio-selective chromatographic methods for quality assurance and control. The chromatographic behavior of S-/R-ketamine, S-/R-norketamine, S-/R-dehydronorketamine, and (2R,6R)-/(2S,6S)-hydroxynorketamine in supercritical fluid chromatography (SFC) was investigated computationally and experimentally with the aim of identifying problematic pairs of enantiomers and parameters for chiral resolution. Retention on three different polysaccharide-based chiral stationary phases (Lux Amylose-2, i-Amylose-3, and i-Cellulose-5) provided new information on the significance of halogen atoms as halogen bond donors and hydrogen bond acceptors for enantioselectivity, which could be corroborated in silico by molecular docking studies. Modifiers inversely affected enantioselectivity and retention. Methanol yielded lower run times but superior chiral resolution compared to 2-propanol. Lower temperatures than those conventionally screened did not impair phase homogeneity but improved enantioresolution, at no cost to reproducibility. Thus, sub-ambient temperature subcritical fluid chromatography (SubFC), essentially low-temperature HPLC with subcritical CO2, was applied. The optimization of the SubFC method facilitated the chiral separation of ketamine and its metabolites, which was applied in combination with direct injection and online supercritical fluid extraction to determine the purity of pharmaceutical ketamine formulations for proof of concept.

Stereoselective endocrine-disrupting effects of the chiral triazole fungicide prothioconazole and its chiral metabolite

The wide use of chiral fungicides has generated interest in the stereoselectivity of their ecotoxicological effects. However, there are few studies about the potential endocrine-disrupting effects (EDEs) of chiral fungicides. This study evaluated the hormone receptor activities of the chiral triazole fungicide prothioconazole and its metabolite using reporter gene assays. The results indicated that prothioconazole and its metabolite possessed EDEs, and the metabolite exerted more activities than the activities of the parent compound, suggesting that the metabolic process is toxification. Stereoselective EDEs were observed, and the S-enantiomers possessed greater hormonal effects than those possessed by the R-enantiomers; the REC20 values ranged from 7.9 × 10−10 to 6.4 × 10−7 M for the thyroid hormone effects and from 3.2 × 10−9 to 7.8 × 10−8 M for the estrogenic effects. The molecular docking results revealed that the stereoselective EDEs of prothioconazole and its metabolite were partially attributed to enantiospecific receptor binding affinities. Overall, our results reveal that prothioconazole and its metabolite might disrupt the balance of the endocrine system by affecting the function of multiple nuclear hormone receptors and that they have the potential to affect the developmental and reproductive systems in humans.

Behavior of the Chiral Herbicide Imazamox in Soils: pH-Dependent, Enantioselective Degradation, Formation and Degradation of Several Chiral Metabolites.

Many pesticides show a pronounced biphasic degradation in soil, typically with a faster initial phase, followed by a slower decline. For chiral compounds, a biphasic decline of the total concentration may result from enantioselective degradation. In this study with the chiral herbicide imazamox, biphasic degradation was observed in most of the 18 soils investigated. In neutral soils, degradation was, in fact, enantioselective with faster degradation of (+)-imazamox. In slightly acidic soils, differences between enantiomers were not pronounced, and in strongly acidic soils, degradation was again enantioselective, but with reversed preference. Additional experiments with pure enantiomers indicated no interconversion. Enantioselective degradation thus contributed to the biphasic decline of the total concentration in certain soils. However, this was not the only factor since degradation of the individual enantiomers was biphasic in itself. In addition to the observed correlation between enantioselectivity and pH, degradation was generally faster in neutral than in acidic soils with half-lives ranging from only 2 to >120 days. Half-lives were also determined for two known metabolites and a further chiral metabolite, the structure of which was characterized by high resolution tandem mass spectrometry. As for the parent compound, half-lives of the metabolites varied considerably in the different soils.

Simultaneous determination of enantiomers of carfentrazone-ethyl and its metabolite in eight matrices using high-performance liquid chromatography with tandem mass spectrometry.

A reversed-phase simultaneous determination method for the enantiomers of carfentrazone-ethyl and its metabolite carfentrazone in agricultural and environmental samples was established using high-performance liquid chromatography-tandem mass spectrometry. A complete enantioseparation of carfentrazone-ethyl and its chiral metabolite carfentrazone enantiomers was obtained using a chiral column. The absolute configuration and specific optical rotation of carfentrazone-ethyl and carfentrazone enantiomers were first confirmed as peaks 1, 2, 3, and 4: S-(+)-carfentrazone, R-(-)-carfentrazone, S-(-)-carfentrazone-ethyl, and R-(+)-carfentrazone-ethyl, respectively. The specificity, matrix effect, linearity, precision, accuracy, and stability were surveyed to evaluate the feasibility of the method. The mean recovery was in the range of 77.5-102.8% with relative standard deviations of 0.4-9.8% for the samples. The limits of detection of the enantiomers were evaluated as 0.7 to 6.0 μg/kg, and the limits of quantification were 2.5 to 20 μg/kg. The stereoselective degradation of carfentrazone-ethyl and carfentrazone in rice plant was investigated, and there was no clear enantioselectivity for carfentrazone-ethyl. As for carfentrazone, the enantiomer fractions value reached 0.85 at 7 days after spraying. The developed method was simple and reliable enough for the corresponding risk assessment of carfentrazone-ethyl and its metabolite enantiomers in crop plants, cereal grains, and soil samples.

Enantioselective Distribution, Degradation, and Metabolite Formation of Myclobutanil and Transcriptional Responses of Metabolic-Related Genes in Rats.

Myclobutanil (MT), a chiral fungicide, can be metabolized enantioselectively in organisms. In this work, the associated absorption, distribution, metabolism and transcriptional responses of MT in rats were determined following a single-dose (10 mg·kg-1 body weight) exposure to rac-, (+)- or (-)-MT. The enantiomer fractions (EFs) were less than 0.5 with time in the liver, kidney, heart, lung, and testis, suggesting preferential enrichment of (-)-MT in these tissues. Furthermore, there was conversion of (+)-form to (-)-form in the liver and kidney after 6 h exposure to enantiopure (+)-MT. Enrichment and degradation of the two enantiomers differed between rac-MT and MT-enantiomers groups, suggesting that MT bioaccumulation is enantiomer-specific. Interestingly, the degradation half-life of MT in the liver with rac-MT treatment was shorter than that with both MT-enantiomer treatments. One reason may be that the gene expression levels of cytochrome P450 1a2 ( cyp1a2) and cyp3a2 genes in livers treated with rac-MT were the highest among the three exposure groups. In addition, a positive correlation between the expression of cyp2e1 and cyp3a2 genes and rac-MT concentration was found in livers exposed to rac-MT. Simultaneously, five chiral metabolites were detected, and the enantiomers of three metabolites, RH-9090, RH-9089, and M2, were separated. The detected enantiomers of (+)-MT metabolites were in complete contrast with those of (-)-MT metabolites. According to the results, a metabolic pathway of MT in male rats was proposed, which included the following five metabolites: RH-9089, RH-9090, RH-9090 Sulfate, M1, and M2. The possible metabolic enzymes were marked in the pathway. The findings of this study provide more specific insights into the enantioselective metabolic mechanism of chiral triazole fungicides.