Biodegradation of fipronil: current state of mechanisms of biodegradation and future perspectives.

Fipronil is a broad-spectrum phenyl-pyrazole insecticide that is widely used in agriculture. However, in the environment, its residues are toxic to aquatic animals, crustaceans, bees, termites, rabbits, lizards, and humans, and it has been classified as a C carcinogen. Due to its residual environmental hazards, various effective approaches, such as adsorption, ozone oxidation, catalyst coupling, inorganic plasma degradation, and microbial degradation, have been developed. Biodegradation is deemed to be the most effective and environmentally friendly method, and several pure cultures of bacteria and fungi capable of degrading fipronil have been isolated and identified, including Streptomyces rochei, Paracoccus sp., Bacillus firmus, Bacillus thuringiensis, Bacillus spp., Stenotrophomonas acidaminiphila, and Aspergillus glaucus. The metabolic reactions of fipronil degradation appear to be the same in different bacteria and are mainly oxidation, reduction, photolysis, and hydrolysis. However, the enzymes and genes responsible for the degradation are somewhat different. The ligninolytic enzyme MnP, the cytochrome P450 enzyme, and esterase play key roles in different strains of bacteria and fungal. Many unanswered questions exist regarding the environmental fate and degradation mechanisms of this pesticide. The genes and enzymes responsible for biodegradation remain largely unexplained, and biomolecular techniques need to be applied in order to gain a comprehensive understanding of these issues. In this review, we summarize the literature on the degradation of fipronil, focusing on biodegradation pathways and identifying the main knowledge gaps that currently exist in order to inform future research. KEY POINTS: • Biodegradation is a powerful tool for the removal of fipronil. • Oxidation, reduction, photolysis, and hydrolysis play key roles in the degradation of fipronil. • Possible biochemical pathways of fipronil in the environment are described.

Determination of fipronil and bixafen pesticides residues using gas chromatography mass spectroscopy with matrix matching calibration strategy after binary dispersive liquid-liquid microextraction.

The objective of this study was to determine bixafen and fipronil residues in domestic and industrial wastewater, soil and mint samples by binary dispersive liquid-liquid microextraction method (BDLLME) prior to gas chromatography-mass spectrometry (GC-MS). Extraction efficiency for the selected analytes was improved by optimizing the parameters such as solvent type, ratio and volume, dispersive solvent type/volume, mixing type and duration to increase overall analytical performance. Under the optimum chromatographic and extraction conditions, limits of detection values for bixafen and fipronil were determined as 7.3 and 6.1 µg L-1, respectively. Spiking experiments were performed for domestic and industrial wastewater, soil and mint samples to evaluate applicability and accuracy of the proposed method. Recovery results for the samples were calculated in the range of 89.4%-112.6% via matrix matching calibration strategy. It was determined that the detection power of GC-MS system was improved 7.8 times for bixafen and 119 times for fipronil over LOD comparisons of conventional GC-MS and B-DLLME-GC-MS systems.

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands.

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment-screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.

Development and validation of a method for the analysis of five diamide insecticides in edible mushrooms using modified QuEChERS and HPLC-MS/MS.

In this study, a new method for simultaneous determination of cyantraniliprole, chlorantraniliprole, tetrachlorantraniliprole, cyclaniliprole and flubendiamide in edible mushrooms by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) combined with a modified QuEChERS procedure. The samples were extracted using acetonitrile and then cleaned up by primary secondary amine (PSA) and octadecylsilane (C18). The determination of these insecticides was achieved in less than 5 min using an electrospray ionization source in positive mode (ESI+) for cyantraniliprole and chlorantraniliprole, while negative mode (ESI-) for tetrachlorantraniliprole, cyclaniliprole and flubendiamide. The linearities of the calibrations for all target compounds were acceptable (R2 ≥ 0.9922). The limits of detection and quantification were 0.05-2 µg kg-1 and 5 µg kg-1, respectively. Acceptable recoveries (73.5-110.2%) were acquired for these insecticides with RSDs less than 12.7%. The results demonstrated that the proposed method was effective and convenient for the determination of these insecticides in edible mushrooms.

Simultaneous determination of neonicotinoids and fipronils in tea using a modified QuEChERS method and liquid chromatography-high resolution mass spectrometry.

The widespread use of neonicotinoids (NEOs) and fipronils (FIPs) as insecticides in modern agriculture has been widely investigated because of their adverse effects on human health. This study aimed to develop an accurate quantitative approach to analyze NEOs and FIPs in tea by using a modified QuEChERS method based on dispersive solid-phase extraction procedure coupled with liquid chromatography-high resolution mass spectrometry. To minimize matrix interferences largely, we selected polyvinylpolypyrrolidone (PVPP) and strong cationic exchange adsorbent (PCX) as dispersive adsorbents to eliminate polyphenols and caffeine from tea extract, respectively. Under optimal conditions, a slight matrix effect was observed for NEOs and FIPs due to the highest reductions of polyphenols and caffeine (94% and 88%, respectively). The method was validated by the European Medicines Agency and Eurachem guidelines and was successfully applied to tea samples for NEOs and FIPs analysis. NEOs and FIPs were commonly detected in tea.

Quantitative bioanalytical assay for the selective RET inhibitors selpercatinib and pralsetinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

Selpercatinib and pralsetinib are potent and selective tyrosine kinase inhibitors targeting the rearranged during transfection (RET) receptor in various types of cancer. In this study, a bioanalytical assay was developed and fully validated for selpercatinib and pralsetinib in mouse plasma and partially in eight mouse tissue homogenates using liquid chromatograph-tandem mass spectrometry. Samples were pre-treated by protein precipitation with acetonitrile using erlotinib as internal standard. Separation of the analytes was performed on an ethylene bridged octadecyl silica C18 column by gradient elution using ammonium hydroxide (in water) and methanol. Analytes were detected by positive electrospray ionization in selected reaction monitoring mode. A linear concentration range of 2-2000 ng/ml was used for the validation of the assay for both inhibitors. The precision values (within-day and between-day) ranged between 3.4 and 10.2% for selpercatinib and 3.1-14.6% for pralsetinib in all matrices. Furthermore, data obtained for accuracy were between 91.7 and 109.3% and 85.1-114.1% for selpercatinib and pralsetinib, respectively. No significant matrix effects or extraction losses were observed and both analytes were stable under all investigated conditions. Finally, a pilot study for selpercatinib in mice was conducted employing this method, followed by a successful incurred sample reanalysis.

Metabolic Stability Assessment of Larotrectinib Using Liquid Chromatography Tandem Mass Spectrometry.

INTRODUCTION: Larotrectinib (VITRAKVI) is an orally potent tropomyosin receptor kinase (Trk) inhibitor that acts by competitive inhibition of all corresponding receptor kinases. It demonstrated a marked response rate (75%) and robust anticancer activity in Trk fusion-positive patients. This response is independent of cancer type, age and gender. METHODS: In this study, an efficient and accurate LC-MS/MS analytical method was developed for Larotrectinib (LRB) quantification in addition to evaluation of its metabolic stability. LRB and lapatinib (LTP) (which is chosen as an internal standard; IS) were eluted utilizing an isocratic mobile phase with a reversed phase elution system (C18 column). RESULTS AND DISCUSSION: The linearity range of the established method was 5-500 ng/mL (r 2 ≥ 0.9999) in the human liver microsomes (HLMs) matrix. Various parameters were calculated to validate the method sensitivity (limit of quantification was 5 ng/mL) and reproducibility (inter and intra-day accuracy and precision were below 3% in all samples) of our methodology. For evaluation of LRB metabolic stability in HLMs matrix, in vitro half-life (48.8 min) and intrinsic clearance (14.19 µL/min/mg) were computed. CONCLUSION: Accordingly, we can conclude that LRB is a moderate extraction ratio drug when compared with other tyrosine kinase inhibitors (TKIs). According to our knowledge, the discussed procedure in this study is the first LC-MS/MS analytical method for evaluating LRB metabolic stability.

Validated HPLC Method for Quantification of a Novel Trk Inhibitor, Larotrectinib in Mice Plasma: Application to a Pharmacokinetic Study.

Larotrectinib, is an orally active novel small molecule approved for the treatment of solid tumors in pediatrics and adult patients. It acts by inhibiting tropomyosin receptor kinase. In this paper, we report the development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of larotrectinib in mice plasma as per the FDA regulatory guideline. Plasma samples processing was accomplished through simple protein precipitation using acetonitrile enriched with internal standard (IS, enasidenib). The chromatographic analysis was performed using a gradient mobile phase comprising 10 mM ammonium acetate and acetonitrile at a flow-rate of 0.8 mL/min on an X-Terra Phenyl column. The UV detection wave length was set at λmax 262 nm. Larotrectinib and the IS eluted at 3.85 and 6.60 min, respectively with a total run time of 8.0 min. The calibration curve was linear over a concentration range of 0.20-5.00 µg/mL (r2=≥0.992). The intra- and inter-day precision and accuracy results were within the acceptable limits. Results of stability studies indicated that larotrectinib was stable on bench-top, in auto-sampler, up to three freeze/thaw cycles and long-term storage at -80°C. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.

Simultaneous determination of eight neonicotinoid insecticides, fipronil and its three transformation products in sediments by continuous solvent extraction coupled with liquid chromatography-tandem mass spectrometry.

Neonicotinoids (NEOs) and fipronil (FIP) are insecticides that are widely used in modern agriculture and have received considerable attention in recent years due to their adverse effects on non-target organisms in the environment. In the present study, a new method to simultaneously detect eight common NEO insecticides and FIP and its three transformation products (FIPs) in sediments was developed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based on a combined pretreatment of continuous solvent extraction (CSE) and solid phase extraction (SPE). Under optimized conditions, 5.0 g of freeze-dried sediment samples were initially extracted with methanol (20 mL)-methanol (15 mL)-water (20 mL) in sequence, and then the extract was cleaned with hydrophilic-lypophilic balance SPE cartridges, and HPLC-MS/MS analysis was conducted. The established method was validated to be sensitive, linear, accurate, and precise. The limits of detection (LOD) and limits of quantification (LOQ) of target compounds were 0.012-0.055 µg/kg d.w and 0.031-0.091 µg/kg d.w, respectively. Good linearity (R2 > 0.990) was observed between 4.0 × 10-2 and 20.0 µg/kg d.w. The recovery rates of all target insecticides were between 75.5% and 98.5%, and the relative standard deviations (RSD) were all less than 15.0% at the low, medium, and high spiked levels. Finally, the optimized method was applied to analyze 12 target insecticides in the sediments obtained from Jiaozhou Bay of China and its main inflow rivers. Acetamiprid, thiamethoxam, fipronil sulfide, and fipronil sulfone were detected in the river sediment samples at the concentration from

Fipronil in eggs. Is LC-MS/MS the only option? A comparison study of LC-MS/MS and GC-ECD for the analysis of fipronil.

Fipronil is a broad-spectrum insecticide belonging to the phenylpyrazole chemical family, classified by the U.S. EPA as a group C (possible human) carcinogen. It is highly toxic to crustaceans, insects and zooplankton as well as to termites, rabbits and certain groups of gallinaceous birds. In European Union and Greece, fipronil is authorized only for the control of termites and cockroaches and not as a plant protection product or as a veterinary drug. The definition of residue of fipronil comprises of the sum of fipronil and its sulfone metabolite expressed as fipronil and is set at 5 µg kg-1. In this study, a sensitive and reliable modified QuEChERS method, proposed by the European Reference Laboratory for animal origin products, has been validated and applied to the residue analysis of fipronil and its metabolites (sulfone and desulfinyl) in chicken eggs and other egg products by LC-MS/MS and GC-ECD analysis. The two analytical systems performed almost equally concerning validation process and with regard to the analysis of real samples, results from both systems were in agreement: out of 11 samples analysed, 4 samples showed measurable residues of fipronil sulfone with 3 samples exceeding the MRL limit.

In vivo investigation of pesticide residues in garlic using solid phase microextraction-gas chromatography-mass spectrometry.

In this study, a novel multiwalled carbon nanotubes/polyaniline-polypyrrole@polydimethylsiloxane (MWCNTs/PANI-PPy@PDMS) fiber was prepared by a facile electrodeposition strategy followed by polymer surface modification, and used as the sorbent of in vivo solid phase microextraction (in vivo SPME). The custom-made fiber exhibits better enrichment capacity than three commercial SPME fibers. Ultra-high enrichment factor (438-2659), satisfactory thermal and mechanical stability, excellent matrix-compatibility and anti-biofouling ability render the fiber attractive as in vivo sampling probe. Combining in vivo SPME with gas chromatography-mass spectrometry (GC-MS), a convenient and sensitive method was successfully developed for the rapid determination of pesticides (hexachlorobenzene, chlorothalonil, fipronil, chlorfenapyr) in garlic. Under the optimal conditions, the proposed method displays relatively wide linear range (three or four orders of magnitude) with a coefficient of determination above 0.9944 both in the standard solution and spiked homogenized garlic samples, respectively. Low detection limits from 0.38 to 1.90 ng g-1 were obtained in homogenized garlic samples. Relative standard deviations (RSDs) less than 15.5% and recovery between 84.0% and 108.2% indicate satisfactory precision and accuracy of the method. In summary, a promising non-lethal method based on in vivo SPME-GC-MS is developed in this study, which provides a green, efficient, economic and rapid strategy for the determination of trace pesticide residues in edible plants.

Photoelectrochemical determination of the activity of histone acetyltransferase and inhibitor screening by using MoS2 nanosheets.

The enzyme histone acetyltransferase (HAT) catalyzes the acetylation of a substrate peptide, and acetyl coenzyme A is converted to coenzyme A (CoA). A photoelectrochemical method is described for the determination of the HAT activity by using exfoliated MoS2 nanosheets, phos-tag-biotin, and ß-galactosidase (ß-Gal) based signal amplification. The MoS2 nanosheets are employed as the photoactive material, graphene nanosheets as electron transfer promoter, gold nanoparticles as recognition and capture reagent for CoA, and phos-tag-biotin as the reagent to link CoA and ß-Gal. The enzyme ß-Gal catalyzes the hydrolysis of substrate O-galactosyl-4-aminophenol to generate free 4-aminophenol which is a photoelectrochemical electron donor. The photocurrent increases with the activity of HAT. Under optimal conditions, the response is linear in the 0.3 to 100 nM activity range, and the detection limit is 0.14 nM (at S/N = 3). The assay was applied to HAT inhibitor screening, specifically for the inhibitors C646 and anacardic acid. The IC50 values are 0.28 and 39 µM, respectively. The method is deemed to be a promising tool for epigenetic research and HAT-targeted cancer drug discovery. Graphical abstract Histone acetyltransferase was detected using a sensitive photoelectrochemical method using MoS2 nanosheets as photoactive material.

Determination of 60 pesticides in hen eggs using the QuEChERS procedure followed by LC-MS/MS and GC-MS/MS.

An analytical method involving QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation, followed by LC-MS/MS and GC-MS/MS was developed and validated for the determination of 60 pesticides in eggs. Recoveries of 70-120% were achieved for selected pesticides and relative standard deviations <20% were obtained for most analytes at three concentrations. The limit of quantification was <10 µg kg-1 for 83% of the total pesticides. This method was used to analyze 58 egg samples and the residues of seven pesticides (disulfoton, fipronil sulfone, cyromazine, o,p-DDT, p,p-DDD, p,p-DDT, and permethrin) were quantified in 16 egg samples at levels of 5-10 µg kg-1, which was below the corresponding the maximum residue levels, as established by Korean Ministry of Food and Drug Safety. We demonstrated that LC-MS/MS and GC-MS/MS in combination with QuEChERS can be used to routinely monitor multiple pesticide residues in egg samples.

In-vitro metabolic profiling study of potential topoisomerase inhibitors 'pyrazolines' in RLMs by mass spectrometry.

Taking into consideration of the cytotoxicity and topo-IIα inhibitory activity of pyrazoline derivatives (1-3) against HCT15 cells, and known topo-IIα inhibitor, etoposide, respectively, the compounds were biotransformed in rat liver microsomes. LC-MS/MS and MALDI mass spectrometric techniques has been used for analysis. All three compounds were biotransformed into demethylated metabolites. Among three compounds, compounds 1 and 2 were biotransformed into mono-hydroxylated metabolites and compound 3 biotransformed into reduced and epoxidized metabolites. Reduced and reduced along with demethylation metabolites were identified from MALDI Orbitrap spectrometric analysis. Without NADPH or microsomes no compounds (1-3) were generated metabolites, it shows CYP450 enzymes involvement in the presence of NADPH in the metabolisms.

In vitroand in vivo investigation of metabolic fate of riociguat by HPLC-Q-TOF/MS/MS and in silico evaluation of the metabolites by ADMET predictor™.

Riociguat, a guanyl cyclase inhibitor, is one of its kind drug regimen approved for management of pulmonary arterial hypertension and chronic thromboembolism pulmonary hypertension. Extensive literature review indicates lack of comprehensive reports on its metabolic fate. The present study reports the in vivo and in vitro identification and characterization of metabolites of riociguat, using high-performance liquid chromatography-quadruple time-of-flight tandem mass spectrometry. In vitro studies were conducted by incubating the drug in human and rat liver microsomes in presence of respective cofactors. In vivo studies were undertaken by oral administration of suspension of drug to male Sprague-Dawley rats followed by collection of urine, feces and blood at specific intervals. A total of 18 metabolites were observed in in vivo and in vitro matrices which includes hydroxyl, N-oxide, desmethyl, defluorinated hydroxyl, glucuronides and N-acetyl cysteine conjugates. Presence of N-acetyl cysteine conjugates strongly points towards the formation of a reactive metabolite intermediate trapped through N-acetyl cysteine and can be considered a matter of concern as the reactive metabolites have been known to manifest toxicities. Their presence was mimicked in in vitro samples as well. The toxicological properties of drug and metabolites were evaluated by using ADMET Predictor ™ software.

Anticonvulsant evaluation of 2-pyrazolines carrying naphthyl moiety: An insight into synthesis and molecular docking study

A series of N-substituted-3-(napthalen-2-yl)-5-substituted phenyl-4,5-dihydropyrazole-1-carbothioamide derivatives (4a-n) were synthesized with the view of structural requirements of pharmacophore for potential anticonvulsant agents. The synthesized compounds were assayed intraperitoneally (i.p.) and subcutaneously (s.c.) in mice against seizures induced by MES and scPTZ methods, respectively.Neurologic deficit was evaluated by rotarod method. Among the tested compounds, 4g, 4i, 4j and 4n emerged as the most active molecule in the MES model at a dose of 30 mg/kg at 0.5h comparable to standardscarbamazepine and phenytoin. In the scPTZ test,4e and 4l were found to be most active compounds at the lowest dose of 30 mg/kg at 0.5h, in the management of the convulsive disorder. Molecular docking studies of the titled compounds were also donewith 3D crystal structure of human cytosolic branched chain amino transferase (hBCATc) enzyme and compound 4e was found to have five hydrogen bond interactions with the most important active site residues.In neurotoxicity studies, except compounds 4b, 4c, 4h and 4k, rest of the compounds showed no sign of toxicity.

Quantitative bioanalytical assay for the tropomyosin receptor kinase inhibitor larotrectinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

Larotrectinib is a promising tyrosine kinase inhibitor for solid tumors harboring tropomyosin receptor kinase gene fusions. A bioanalytical assay was developed for this drug in small volume samples using a 96-well format to efficiently support multiple mouse studies. The assay was completely validated for mouse plasma and partially for homogenates of eight different tissues: brain, heart, kidneys, liver, lungs, small intestine, spleen, and testes. Proteins in 10-µl samples were precipitated using acetonitrile containing momelotinib as internal standard. Chromatographic separation of analyte and internal standard from endogenous interferences was performed on an ethylene bridged octadecyl silica column using 0.1% (v/v) formic acid (in water) and methanol for gradient elution. Electrospray ionization and selected reaction monitoring on a triple quadrupole mass spectrometer were used for detection. In the range 1-2000 ng/ml the drug could be quantified in all 9 matrices with precisions (within-day and between-day) in the range 2.7-11.1% and accuracies in the range 87.4-101.4%. Compounds were sufficiently stable under all investigated conditions except for kidney homogenate. A pilot pharmacokinetic and tissue distribution study in mice demonstrated the applicability of the new presented assay for larotrectinib.

[Quick determination of fipronil and its metabolites in eggs by gas chromatography-triple quadrupole mass spectrometry].

A method was established for the simultaneous determination of fipronil and its metabolites in eggs by gas chromatography-tandem mass spectrometry coupled with QuEChERS. Fipronil and its metabolites were extracted from eggs with acetonitrile, and the solution was dehydrated with 150 mg anhydrous magnesium sulfate. The extracts were purified with 50 mg N-propyl ethylenediamine (PSA) and 50 mg C18, and a capillary column pesticide Ⅱ was used. The analytes were detected in timed selected reaction monitoring (timed-SRM) mode, and were quantified using external standard method with matrix correction standard curves. A good linearity in the range of 1.0-200 µg/L with correlation coefficients (R2)>0.999 was observed. The limits of quantification (LOQs) were between 0.5 and 1.0 µg/kg. The recoveries at three spiked levels (2.0, 5.0 and 10.0 µg/kg) were in the range of 87.8% to 111.5% with RSDs between 2.0% and 9.2% (n=3). This new method satisfied the related regulations of the European Union for the determination of fipronil and its metabolites in eggs.

Determination of fipronil and its metabolites in chicken egg, muscle and cake by a modified QuEChERS method coupled with LC-MS/MS.

An easy-to-use method for determining the levels of fipronil and its metabolites (fipronil-desulfinyl, fipronil-sulfone and fipronil-sulfide) in chicken egg, muscle and cake was developed and validated using a modified quick, easy, cheap, effective, rugged and safe (QuEChERS) approach coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The samples were extracted using acetonitrile, salted out with sodium chloride at -20°C, and then purified by combined PSA and C18 phases and anhydrous magnesium sulphate. The recoveries were 80.4-119% with relative standard deviations (RSDs) < 10% for the different matrixes. The validated method was used to analyse the target compounds in 214 real samples collected in Beijing. The metabolite fipronil-sulfone was detected in most of the samples and was identified as the main residue in the egg and cake. The method was validated using a proficiency test for fipronil in products of animal origin published by Wageningen University & Research in 2017.

Development and validation of a sensitive LC MS/MS method for the measurement of the checkpoint kinase 1 inhibitor prexasertib and its application in a cerebral microdialysis study.

LC MS/MS methods to measure prexasertib in mouse plasma and Ringer's solution containing 0.5% BSA (Ringer's/BSA) were developed and validated. Liquid-liquid extraction with tert-butyl methyl ether was used to extract prexasertib from mouse plasma and Ringer's/BSA. Reverse phase chromatography with gradient elution was performed to separate prexasertib from the endogenous interference in the matrix, followed by MS detection using positive ion MRM mode. The initial calibration curve for mouse plasma samples ranged from 1 to 500 ng/ml, and after validation of that curve and use in a preliminary study another calibration curve (0.2-200 ng/ml) was created to enable the quantitation of prexasertib at lower concentrations. The method described was precise and accurate with %CV in precision studies of ≤ 6.7% and accuracies within 95.0-110% of nominal target concentration across all concentrations tested for both matrices. This validated method was successfully applied in the analysis of prexasertib in mouse plasma and dialysate samples collected during a cerebral microdialysis study.