Benalaxyl Enantiomers Research Articles

An environmentally friendly method for the enantioseparation and determination of benalaxyl in tobacco and soil by ultra-performance convergence chromatography with tandem mass spectrometry.

For the purpose of chiral separation and determination of benalaxyl enantiomers in tobacco and soil, we developed a rapid, green, and sensitive method using ultra-performance convergence chromatography with tandem mass spectrometry. The samples were extracted and purified by the quick, easy, cheap, effective, rugged, and safe method before injection. The baseline separation was obtained on a chiral column in 5min with carbon dioxide and ethanol as mobile phase. Separation parameters were optimized for the best separation efficiency. Under optimal conditions, the recoveries of both enantiomers were 77.1-98.4% with relative standard deviations <5.0% at spiked level of 0.1, 2.0, and 5.0mg/kg in two matrices. Good coefficients of determination were achieved over the concentration range of 10-250ng/mL. The limit of detection and the limit of quantification for all enantiomers ranged from 0.43 to 0.72μg/kg and from 1.25 to 2.15μg/kg, respectively. The results show that ultra-performance convergence chromatography with tandem mass spectrometry provides a reliable, green, and rapid method for the separation and determination of benalaxyl enantiomers in tobacco and soil. This method has important theoretical significance for studying the enantioselectivity and bioactivity of benalaxyl in the environment and in organisms.

Enantioselective metabolism and enantiomerization of benalaxyl in mice

The enantiomerization and enantioselective metabolism of benalaxyl in mice after a single gavage administration were investigated in the present study. The pharmacokinetic result indicated that elimination of (−)-R-benalaxyl in plasma was slightly faster with the t1/2 of 26.65 h and 28.88 h for (−)-R- and (+)-S-benalaxyl, respectively. Consistent with this, elimination in tissues and excretion were also enantioselective, with (+)-S-benalaxyl enriched in all tissues, urine and feces. And formation of the metabolites also exhibited an enantioselective manner. Both benalaxyl and most of its metabolites exhibited a potent excretion to feces and urine. In addition, significant enantiomerization of benalaxyl enantiomers was observed in plasma, with a larger extent from R to S than S to R. Thus, this difference in enantiomerization may be one of the reasons to explain the enantioselective enrichment of (+)-S-benalaxyl in mice observed in this study. Data from this study proves that besides metabolic enzymes, enantiomerization could be another important factor that contributes to the enantioselective metabolism of a chiral pesticide. Thus, research at enantiomeric level is necessary for efficient risk assessment of chiral pesticides.

Stereoselective Behavior of the Fungicide Benalaxyl During Grape Growth and the Wine-Making Process.

Benalaxyl is widely applied as a fungicide during grape planting processing. In this experiment, the stereoselective behavior of benalaxyl was studied during the grape growth and wine-making process. A simple method based on high-performance liquid chromatography (HPLC) equipped with a chiral column and UV detector was established to separate and determine the enantiomers of benalaxyl. Stereoselective degradation of the two enantiomers of benalaxyl was found in grapes. The degradation of both enantiomers followed pseudofirst-order kinetics, and the degradation rate of R-(-)-benalaxyl was faster than S-(+)-benalaxyl. The half-life of R-(-)-benalaxyl was 27h, while the half-life of S-(+)-benalaxyl was 31h. The enantiomer fraction value decreased from 0.50 to 0.34 and finally only S-(+)-benalaxyl could be detected. In the fermentation process, both enantiomers of benalaxyl were hardly degraded, and no configuration interconversion was observed. Meanwhile, both enantiomers of benalaxyl showed little influence on the growth of the yeast, consumption of carbon sources, or production of alcohol. The result of this study might provide more sufficient data for the evaluation of food safety and potential risk. Chirality 28:394-398, 2016. © 2016 Wiley Periodicals, Inc.

A combined non-targeted and targeted metabolomics approach to study the stereoselective metabolism of benalaxyl enantiomers in mouse hepatic microsomes

Understanding of xenobiotic metabolism is necessary for risk assessment as well as toxicological research. In the present study, nanoLC/LTQ-Orbitrap mass based non-targeted metabolomics method coupled with ultra-performance liquid chromatography (UPLC)/triple quadrupole mass based targeted metabolomics method was carried out to investigate the stereoselective metabolism of benalaxyl in mouse hepatic microsomes. As a result, 7 metabolites of benalaxyl were identified, including 5 previously reported and 2 newly identified metabolites in present work. Hydroxylation, oxidation and esterolysis were major biotransformation reactions of benalaxyl in mouse hepatic microsomes. For stereoselective metabolism study, (−)-R-benalaxyl degraded much faster than its antipode with the t1/2 of 81.24 and 190.38 min for (−)-R- and (+)-S-benalaxyl, respectively. More importantly, stereoselectivity was also observed in the formation of the identified metabolites. In conclusion, the combined use of the mass spectrometry based targeted and non-targeted metabolomics provided a new approach to investigate stereoselective metabolism and identify novel metabolites of chiral pesticides. This study highlights the stereoselective metabolic profile of benalaxyl enantiomers and provides reliable data for benalaxyl toxicological risk assessment in mammal.

Stereoselective degradation and toxic effects of benalaxyl on blood and liver of the Chinese lizard Eremias argus

Benalaxyl as a xylem-systemic fungicide is usually direct sprayed on the soil surface, which is potential harm to the animals lived in the soil. However, the stereoselectivity of benalaxyl in reptiles have rarely been studied. In this study, Chinese lizards (Eremias argus) were firstly used to evaluate the stereoselectivity in biodegradation and toxicity of racemate and individual enantiomers of benalaxyl. A method for determining residues of the two enantiomers of benalaxyl in lizard blood and liver by high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC-MS/MS) was developed. The degradation followed pseudo first-order kinetics and the degradation of the (S)-(+)-benalaxyl was faster than its antipode in blood and liver (Half-time t1/2 of (R)-(−)-benalaxyl and (S)-(+)-benalaxyl were 5.08h and 3.75h in blood, 6.21h and 4.45h in liver, separately). Moreover, antioxidant defenses consisting of activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and lipid peroxide malondialdehyde (MDA) were determined in 24h acute exposure. Enantioselectivity of acute toxicity depended on the concentration and form of benalaxyl. In addition, cellular degeneration, decrease of cell number, clustering phenomena of cell nuclei and preliminary liver fibrosis were observed in pathological detection at the termination of 21-d subchronic exposure (20mg/kg−bw of racemate and individual enantiomers of benalaxyl). The enantiomer fractions (EFs) in racemate and individual enantiomer groups were approached both in blood and liver caused by the chiral conversion. The chiral conversion from (R)-(−)-benalaxyl to (S)-(+)-benalaxyl and (S)-(+)-benalaxyl to (R)-(−)-benalaxyl were the primary cause for no remarkable differences in toxicity between the enantiomers of benalaxyl.

The integration of in vivo and in vitro metabolism assays to investigate the stereoselective behaviors of benalaxyl in rainbow trout (Oncorhynchus mykiss)

The bioconcentration and elimination of racemic benalaxyl (BX) in trout liver microsomes and in juvenile rainbow trout (Oncorhynchus mykiss) were investigated to determine whether the fish can bioconcentrate and degrade this fungicide enantioselectively. Both enantiomers of BX were extracted with organic solvents and evaluated using high-performance liquid chromatography. In the microsomes, BX degradation followed first-order kinetics, and the S−(+) enantiomer of BX was eliminated twice as rapidly as the R−(−) enantiomer, resulting in residues enriched for R−(−)−BX. In vivo experiment, chiral analysis showed an obvious selective bioconcentration of BX based on statistically altered enantiomer fractions (EFs) in the fish compared with the values in the water. The R−(−)−BX was initially preferentially bioconcentrated by rainbow trout and then dissipated more slowly than its antipode. The mean half-lives for individual enantiomers were calculated as 31.6 h for R−(−)−BX and 20.3 h for the S−(+)−form. The results of the study showed that the degradation of BX enantiomers was stereoselective in rainbow trout.

Enantioselective toxic effects and biodegradation of benalaxyl in Scenedesmus obliquus

Enantioselectivity in ecotoxicity and biodegradation of chiral pesticide benalaxyl to freshwater algae Scenedesmus obliquus was studied. The 96h-EC50 values of rac-, R-(−)-, S-(+)-benalaxyl were 2.893, 3.867, and 8.441mgL−1, respectively. Therefore, the acute toxicities of benalaxyl enantiomers were enantioselective. In addition, the pigments chlorophyll a and chlorophyll b, antioxidant enzyme activities catalase (CAT) and superoxide dismutase (SOD) as well as lipid peroxide malondialdehyde (MDA) were determined to evaluate the different toxic effects. Chlorophyll a was induced by S-(+)-benalaxyl but inhibited by R-(−)-benalaxyl at 1mgL−1. Chlorophyll b were both induced at 1mgL−1, but S-(+)-form was fourfold higher than R-(−)-form. S-(+)-benalaxyl inhibited more CAT activities at 3mgL−1 and 5mgL−1, induced less SOD activity and MDA content at 5mgL−1 than R-(−)-benalaxyl. Based on these data, enantioselectivity occurred in anti-oxidative stress when S. obliquus response to benalaxyl. In the biodegradation experiment, the half-lives of S-(+)-benalaxyl and R-(−)-benalaxyl were 4.07d and 5.04d, respectively, resulting in relative enrichment of the R-(−)-form. These results showed that toxic effects and biodegradation of benalaxyl in S. obliquus were enantioselective, and such enantiomeric differences must be taken into consideration in pesticide risk.

Stereoselective metabolism of benalaxyl in liver microsomes from rat and rabbit

Benalaxyl (BX), methyl-N-phenylacetyl-N-2,6-xylyl alaninate, is a potent acylanilide fungicide and consist of a pair of enantiomers. The stereoselective metabolism of BX was investigated in rat and rabbit microsomes in vitro. The degradation kinetics and the enantiomer fraction (EF) were determined using normal high-performance liquid chromatography with diode array detection and a cellulose-tris-(3,5-dimethylphenylcarbamate)-based chiral stationary phase (CDMPC-CSP). The t(1/2) of (-)-R-BX and (+)-S-BX in rat liver microsomes were 22.35 and 10.66 min of rac-BX and 5.42 and 4.03 of BX enantiomers. However, the t(1/2) of (-)-R-BX and (+)-S-BX in rabbit liver microsomes were 11.75 and 15.26 min of rac-BX and 5.66 and 9.63 of BX enantiomers. The consequence was consistent with the stereoselective toxicokinetics of BX in vitro. There was no chiral inversion from the (-)-R-BX to (+)-S-BX or inversion from (+)-S-BX to (-)-R-BX in both rabbit and rat microsomes. These results suggested metabolism of BX enantiomers was stereoselective in rat and rabbit liver microsomes.

Enantioselective Acute Toxicity and Bioaccumulation of Benalaxyl in Earthworm (Eisenia fedtia)

The enantioselectivities of individual enantiomers of benalaxyl in acute toxicity and bioaccumulation in earthworm ( Eisenia fedtia ) were studied. The acute toxicity was tested by paper contact test. After 48 h of exposure, the calculated LC(50) values of the R-(-)-form, rac-form, and S-(+)-form were 4.99, 5.08, and 6.66 microg/cm(2), respectively. After 72 h of exposure, the calculated LC(50) values were 1.23, 1.73, and 2.45 microg/cm(2), respectively. Therefore, the acute toxicity of benalaxyl enantiomers was enantioselective. A method for determining residues of the two enantiomers of benalaxyl in earthworm tissue by high-performance liquid chromatography based on cellulose tri-(3,5-dimethylphenyl-carbamate) chiral stationary phase was developed. During the bioaccumulation experiment, the enantiomer fraction in earthworm tissue was maintained approximately at 0.6, whereas enantiomer fraction in spiked soil was maintained at 0.5; in other words, the bioaccumulation of benalaxyl was enantioselective in earthworm tissue. Peak-shaped accumulation curves were observed for both enantiomers, and the calculated biota to soil accumulations (kg dry kg(-1) wet weight) at steady state were below 1 for both enantiomers. During the elimination experiment, 79.0% of R-(-)-enantiomer and 89.6% of S-(+)-enantiomer in earthworm tissue were eliminated within 2 days.

Stereoselective degradation of benalaxyl in tomato, tobacco, sugar beet, capsicum, and soil

The stereoselective degradation of the racemic benalaxyl in vegetables such as tomato, tobacco, sugar beet, capsicum, and the soil has been investigated. The two enantiomers of benalaxyl in the matrix were extracted by organic solvent and determined by validated chiral high-performance liquid chromatography with a cellulose-tris-(3, 5-dimethylphenylcarbamate)-based chiral column. Rac-benalaxyl was fortified into the soil and foliar applied to vegetables. The assay method was linear over a range of concentrations (0.5-50 microg ml(-1)) and the mean recoveries in all the samples were more than 70% for the two enantiomers. The limit of detection for both enantiomers was 0.05 microg g(-1). The results in soil showed that R-(-)-enantiomer dissipated faster than S-(+)-enantiomer and the stereoselectivity might be caused by microorganisms. In tomato, tobacco, sugar, beet, and capsicum plants, there was significantly stereoselective metabolism. The preferential absorption and degradation of S-(+)-enantiomer resulted an enrichment of the R-(-)-enantiomer residue in all the vegetables.

Stereoselective degradation of fungicide benalaxyl in soils and cucumber plants

The enantioselective degradation of benalaxyl has been investigated to elucidate its behavior in several agricultural soils and plants (cucumber). Racemic benalaxyl was fortified into five types of agricultural soils and sprayed leaves of cucumber plants, respectively. The degradation kinetics and the enantiomer fraction (EF) were determined by normal-phase high-performance liquid chromatography (HPLC) with diode array detection (DAD) on the chiral column filled cellulose-tri-(3,5-dimethylphenylcarbamate)-based chiral stationary phase (CDMPC-CSP). The process of the degradation of benalaxyl enantiomers followed pseudo-first-order kinetics in cucumber plant. However, the dissipation phases of benalaxyl enantiomers in soils were biphasic ("slow-fast-slow" process). It has been shown that the degradation of benalaxyl was stereoselective. The results indicated that the (+)-S-benalaxyl showed a faster degradation in plants, while the (-)-R-benalaxyl showed a faster degradation in Soils 3, 4, and 5. No stereoselective degradation was observed in other soils.

Stereoselective determination of benalaxyl in plasma by chiral high‐performance liquid chromatography with diode array detector and application to pharmacokinetic study in rabbits

A chiral high-performance liquid chromatography method with diode array detector was developed and validated for stereoselective determination of benalaxyl (BX) in rabbit plasma. Good separation was achieved at 20 degrees C using cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase, a mixture of n-hexane and 2-propanol (97:3) as mobile phase at a flow rate of 1.0 ml/min. The assay method was linear over a range of concentrations (0.25-25 microg/ml) in plasma and the mean recovery was greater than 90% for both enantiomers. The limits of quantification and detection for both enantiomers in plasma were 0.25 and 0.1 microg/ml, respectively. Intra- and interday relative standard deviations (RSDs) did not exceed 10% for three-tested concentrations. The method was successfully applied to pharmacokinetic studies of BX enantiomers in rabbits. The result suggested that the pharmacokinetics of BX enantiomers was stereoselective in rabbits.

Direct chiral resolution and its application to the determination of fungicide benalaxyl in soil and water by high-performance liquid chromatography

A simple chiral high-performance liquid chromatography (HPLC) method with ultraviolet (UV) and circular dichroism (CD) detection was developed and validated for measuring benalaxyl enantiomers using ( R, R) Whelk-O 1 column. The effects of mobile phase composition and column temperature on the entioseparation were investigated. A CD detector was used to determine the elution order of the enantiomers. Excellent resolution was easily obtained using n-hexane-polar organic alcohols mobile phase. The chiral recognition mechanism was also discussed. Based on the developed chiral HPLC method, enantioselective analysis methods for this fungicide in environment matrix (soil and water) were developed and validated. Good linearities were obtained over the concentration range of 0.25–25 mg L −1 for both enantiomers. Liquid–liquid extraction and solid phase extraction (SPE) were used for the enrichment and cleanup of soil and water samples. Recoveries for the two enantiomers were 79–91% at 0.02, 0.04 and 0.2 mg kg −1 levels from soil, and 89–101% at 0.0025, 0.01 and 0.05 mg L −1 levels from water. Run-to-run and day-to-day assay precisions were below 10% for both enantiomers at concentrations of 0.5, 1 and 5 mg L −1. Individual detection limits of the two enantiomers were both 2 ng. Limits of detection (LOD) were 0.004 mg kg −1 in soil and 0.001 mg L −1 in water.