Residual Pattern of Chlorantraniliprole, Thiamethoxam, Flubendiamide and Deltamethrin in Tomato Fruit and Soil.

Tomato, Lycopersicon esculentum L. is grown widely as an important day-to-day demand vegetable. The crop is attacked by various polyphagous insect pests like tomato fruit borer, stink bug, cabbage looper, flea beetle, aphids, whitefly, two-spotted spider mite, etc., and oligophagous insects like leaf-miner, five-spotted hawkmoth, etc. To combat the damage and yield loss, various chemical insecticides were sprayed on tomatoes under field conditions. The residual pattern of insecticides like chlorantraniliprole, thiamethoxam, flubendiamide, and deltamethrin residues was studied following applications of chlorantraniliprole 18.5% SC (Coragen) @ 30 g a.i./ha, thiamethoxam 25% WG (Actara) @ 50 g a.i./ha, flubendiamide 39.35 M/M SC (Fame) @ 48 g a.i./ha and deltamethrin 2.8% EC (Decis 100) @ 12.5 g a.i./ha using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC). Fruit samples were collected at 0 (1 h after application), 1, 2, 3, 5, 7 days and at harvest time. All the residues of insecticides such as chlorantraniliprole (0.09 mg kg- 1), thiamethoxam (0.03 mg kg- 1), flubendiamide (0.02 mg kg- 1), and deltamethrin (0.01 mg kg- 1) were persisted up to 5th day. There were no residues found at harvest time. The residues of chlorantraniliprole and deltamethrin persisted up to 3rd day of spraying whereas the residues of flubendiamide and thiamethoxam were not detected on the same day in the soil.

Quality by design with design of experiments approach for development of a stability-indicating LC method for enzalutamide and its impurities in soft gel dosage formulation.

A novel ultra-performance liquid chromatographic (UPLC) method has been developed and approved for the quantitative determination of enzalutamide (ENZ) and its impurities in drug product dosage form by applying the quality by design with design of experiments approach. An efficient chromatographic separation was achieved on a Waters ACQUITY CSH C18 (100 × 2.1 mm × 1.7 µm) column in gradient elution mode. A mixture of potassium phosphate monobasic buffer and acetonitrile (10 mm, adjusted to pH 4.0 with 1% orthophosphoric acid) at a flow rate of 0.2 mL min-1 (column temperature at 40°C) under ultraviolet detection at 270 nm was used for quantitation. The peak resolution among ENZ and its impurities (Impurity-1, Impurity-2, Impurity-3, Impurity-4, Impurity-5, Impurity-6 and Impurity-7) was greater than 2.5. Regression analysis confers an R2 value (correlation coefficient) higher than 0.999 for the active substance and impurities. The detection level for ENZ impurities was at a level below 0.015% (0.12 µg/mL). The accuracy levels for different compounds were close to 100%. The inter- and intra-day precisions for ENZ and impurities were evaluated and their relative standard deviation (%) values were less than 3.5. Our results show that the UPLC-UV stability-indicating method will be an essential tool that could determine the drug product's impurities and be useful in regular quality control and stability studies of the ENZ drug product dosage form.

Revefenacin Absorption, Metabolism, and Excretion in Healthy Subjects and Pharmacological Activity of Its Major Metabolite.

Revefenacin inhalation solution is an anticholinergic indicated for the maintenance treatment of patients with chronic obstructive pulmonary disease. Mass balance, pharmacokinetics, and metabolism of revefenacin were evaluated after intravenous and oral administration of [14C]-revefenacin in healthy subjects. Pharmacological activity of the major revefenacin metabolite was also assessed. Adult males (n = 9) received 20 µg intravenously of approximately 1 µCi [14C]-revefenacin and/or a single 200-µg oral solution of approximately 10 µCi [14C]-revefenacin. Mean recovery of radioactive material was 81.4% after intravenous administration (54.4% in feces; 27.1% in urine) and 92.7% after oral dosing (88.0% in feces, 4.7% in urine). Mean absolute bioavailability of oral revefenacin was low (2.8%). Intact revefenacin accounted for approximately 52.1% and 13.1% of the total radioactivity in plasma after intravenous and oral administration, respectively. Two main circulating metabolites were observed in plasma. After an intravenous dose, a hydrolysis product, THRX-195518 (M2) was observed that circulated in plasma at 14.3% of total radioactivity. After an oral dose, both THRX-195518 and THRX-697795 (M10, N-dealkylation and reduction of the parent compound) were observed at 12.5% of total circulating radioactivity. THRX-195518 was the major metabolite excreted in feces and comprised 18.8% and 9.4% of the administered intravenous and oral dose, respectively. The major metabolic pathway for revefenacin was hydrolysis to THRX-195518. In vitro pharmacological evaluation of THRX-195518 indicated that it had a 10-fold lower binding affinity for the M3 receptor relative to revefenacin. Receptor occupancy analysis suggested that THRX-195518 has minimal contribution to systemic pharmacology relative to revefenacin after inhaled administration. SIGNIFICANCE STATEMENT: The major metabolic pathway for revefenacin was hydrolysis to the metabolite THRX-195518 (M2), and both revefenacin and THRX-195518 underwent hepatic-biliary and fecal elimination after oral or intravenous administration with negligible renal excretion. Pharmacological evaluation of THRX-195518 indicated that it had a 10-fold lower binding affinity for the M3 muscarinic receptor relative to revefenacin and that THRX-195518 has minimal contribution to systemic pharmacology after inhaled administration.

Effect of pH on the Hydrolytic Transformation of a New Mixture Formulation of Fomesafen and Quizalofop-ethyl in Water.

A hydrolytic transformation study was conducted in water of pH 4.0, 7.0 and 9.2 to evaluate the effect of pH on persistence of a new readymix formulation of fomesafen and quizalofop-ethyl. The water samples were fortified at 0.5 and 1 µg mL-1 levels and analysed at 0 (2 h), 1, 3, 7, 15, 30, 60, 90, 120, 150 days interval. Both the analytical methods were validated following SANTE guideline and found accurate based on average recovery of 80-100%, Relative standard deviation (RSD) < 20% and Coefficient of Determination (R2) 0.99. The dissipation of both the molecules was pH dependent and followed first order kinetics. Higher persistence of fomesafen was observed in alkaline pH as compared to neutral and acidic pH with half-life of 41.56-63.24 days, whereas higher stability of quizalofop-ethyl was observed in the water of acidic pH followed by neutral and alkaline pH with half-life of 1.26-8.09 days.

Low Cost Biomass Derived Biochar Amendment on Persistence and Sorption Behaviour of Flubendiamide in Soil.

Persistence and sorption behaviour of flubendiamide in two different Indian soils as affected by maize stalk biochar was studied. The persistence was more in West Bengal soil (178.6 days) than Sikkim soil (165.3 days) at 10 µg g-1 fortification level. Biochar amendment addition to soil at 5% enhanced the degradation process and half-life (T1/2) values were 103.5 and 117.4 days, respectively for biochar amended Sikkim and West Bengal soil. Sorption study through batch equilibrium method resulted the 4 h equilibrium time with adsorption 6.22% ± 0.16% and 5.26% ± 0.16% in Sikkim and West Bengal soil, respectively. Biochar addition at 5% increased the adsorption of flubendiamide to 8.12% ± 0.16% and 5.88% ± 0.16% indicating a greater influence in this process. The adsorption was more in biochar amended Sikkim soil than West Bengal soil. The values of desorption was slower than adsorption indicating a hysteresis effect having hysteresis coefficient (H1) ranges between 0.025 and 0.151 in two test soils.

Bioavailability, Biotransformation, and Excretion of the Covalent Bruton Tyrosine Kinase Inhibitor Acalabrutinib in Rats, Dogs, and Humans.

Acalabrutinib is a targeted, covalent inhibitor of Bruton tyrosine kinase (BTK) with a unique 2-butynamide warhead that has relatively lower reactivity than other marketed acrylamide covalent inhibitors. A human [14C] microtracer bioavailability study in healthy subjects revealed moderate intravenous clearance (39.4 l/h) and an absolute bioavailability of 25.3% ± 14.3% (n = 8). Absorption and elimination of acalabrutinib after a 100 mg [14C] microtracer acalabrutinib oral dose was rapid, with the maximum concentration reached in <1 hour and elimination half-life values of <2 hours. Low concentrations of radioactivity persisted longer in the blood cell fraction and a peripheral blood mononuclear cell subfraction (enriched in target BTK) relative to plasma. [14C]Acalabrutinib was metabolized to more than three dozen metabolites detectable by liquid chromatography-tandem mass spectrometry, with primary metabolism by CYP3A-mediated oxidation of the pyrrolidine ring, thiol conjugation of the butynamide warhead, and amide hydrolysis. A major active, circulating, pyrrolidine ring-opened metabolite, ACP-5862 (4-[8-amino-3-[4-(but-2-ynoylamino)butanoyl]imidazo[1,5-a]pyrazin-1-yl]-N-(2-pyridyl)benzamide), was produced by CYP3A oxidation.Novel enol thioethers from the 2-butynamide warhead arose from glutathione and/or cysteine Michael additions and were subject to hydrolysis to a ß-ketoamide. Total radioactivity recovery was 95.7% ± 4.6% (n = 6), with 12.0% of dose in urine and 83.5% in feces. Excretion and metabolism characteristics were generally similar in rats and dogs. Acalabrutinib's highly selective, covalent mechanism of action, coupled with rapid absorption and elimination, enables high and sustained BTK target occupancy after twice-daily administration.

Case report: relevance of metabolite identification to detect new synthetic opioid intoxications illustrated by U-47700.

Today, new psychoactive substances (NPS) producers increasingly appear to be targeting new synthetic opioids (NSOs), and the recent emergence of NSOs is causing considerable concern in North America and in Europe. For toxicologists, NSO detection in a forensic context presents three additional difficulties to the general NPS analytical detection challenge: (i) high frequency of new products, (ii) low concentrations (in µg/L range and under) in biological samples related to their high opioid potency, and (iii) extensive metabolism. In this context, the present work aims to highlight the relevance of NSO metabolite detection in potential intoxication cases. Illustration is given with U-47700, an emerging NSO, (i) that was identified in a powder recently collected in France and in a fatality case, (ii) whose metabolites were in vitro produced using human liver microsomes and their mass spectra (MS) added in our MS/MS and HRMS libraries, and (iii) for which metabolism data were compared to those of the literature: U-47700 was identified in the powder and at 3040 µg/L in peripheral blood in the fatality case. In addition, high amounts of several U-47700 metabolites, especially N-desmethyl-U-47700, were observed in urine. Even if metabolite formation may largely depend on the enzymatic activity as well as on the length of the survival time, confrontation of these results to data found in the literature strongly suggests that this metabolite is regularly a better blood and (mainly) urine biomarker of U-47700 intake than U-47700 itself. Indeed, in this fatality and in other previous reports, N-desmethyl-U-47700 produced the main observed chromatographic signal (i) systematically in vitro and (ii) commonly in vivo, especially in urines. N,N-Didesmethyl-U-47700 is also sometimes a better biomarker of U-47700 intake than U-47700 itself. Accordingly, we suggest adding N-desmethyl-U-47700 (and N,N-didesmethyl-U-47700) in mass spectrum databases used for toxicological screening in order to reduce the risk of false-negative results in intoxication cases involving U-47700.

A validated workflow for drug detection in oral fluid by non-targeted liquid chromatography-tandem mass spectrometry.

Oral fluid is recognized as an important specimen for drug testing. Common applications are monitoring in substance abuse treatment programs, therapeutic drug monitoring, pain management, workplace drug testing, clinical toxicology, and driving under the influence of drugs (DRUID). In this study, we demonstrate that non-targeted LC-MS/MS with subsequent compound identification by tandem mass spectral library search is a valuable tool for comprehensive detection and confirmation of drugs in oral fluid samples. The workflow developed involves solid-phase extraction and chromatographic separation on reversed phase materials. Mass spectrometric detection is accomplished on a quadrupole-quadrupole-time-of-flight instrument operated with data-dependent acquisition control. The workflow was optimized for 500 µl of neat oral fluid collected with the Greiner Bio-One saliva collection system. The fitness of the developed method was tested and proven by analyzing blank and spiked samples as well as 59 authentic patient samples. We could demonstrate that compounds with logP values in the range 0.5-5.5 are efficiently detected at low nanograms per milliliter concentrations. The true positive and true negative rates of automated library search were equal or close to 100%. The beauty of the non-targeted LC-MS/MS approach is the ability to detect compounds hardly included in routinely applied targeted assays, and this was demonstrated by detecting the synthetic opioid U-47700 in two patient samples. Graphical abstract ᅟ.

Magnetic solid-phase extraction of benzoylurea insecticides in tea samples with Fe3 O4 -hyperbranched polyester magnetic composite as sorbent.

In this work, a method for the analysis of benzoylurea insecticides, including hexaflumuron, flufenoxuron, lufenuron and chlorfluazuron, in tea samples by high-performance liquid chromatography with Fe3 O4 -hyperbranched polyester nanocomposite as the adsorbent for magnetic solid-phase extraction was developed. The magnetic nanocomposite was prepared and characterized by infrared spectroscopy, vibrating sample magnetometry, and scanning electron microscopy. The as-prepared nanocomposite was used as a sorbent for the extraction and preconcentration of pesticide residues in tea samples. The extraction and desorption conditions, including mass ratios of raw materials, amount of sorbent, pH value, extraction time, and desorption time, were investigated. Under the final conditions chosen for the analysis, good linearity was obtained for all the tested compounds, with R2 values of at least 0.9979. The limits of detection were determined in the range of 0.15-0.3 µg/L. The recovery obtained from the analysis of tea samples with various spiked concentrations was between 90.7 and 98.4%, with relative standard deviations (n = 4) lower than 4.1%. Furthermore, the present approach was successfully applied to the quantitative determination of residues of benzoylurea insecticides in real samples.

BJ-2266 ameliorates experimental autoimmune encephalomyelitis through down-regulation of the JAK/STAT signaling pathway.

CD4+ T cells differentiate into distinct effector subsets upon antigenic stimulation. Cytokines, and micro-environmental factors present during T-cell priming, direct differentiation of naïve CD4+ T cells into pro-inflammatory Th1 and Th17 cells. From extensive screening of 2,4,5-trimethylpyridin-3-ol derivatives with various functional groups at C(6)-position, BJ-2266, a 6-thioureido-derivative, showed potent inhibitory activity on in vitro T helper (Th)-cell differentiation. This compound inhibited IFN-γ and IL-17 production from polyclonal CD4+ T cells and ovalbumin (OVA)-specific CD4+ T cells that were activated by T-cell receptor (TCR) engagement. We assessed the inhibitory effect of BJ-2266 in experimental autoimmune encephalomyelitis (EAE). Our results suggest that BJ-2266 treatment significantly suppresses EAE disease progression with reduced generation of Th1 and Th17 cells. Notably, Th-cell differentiation was significantly suppressed by BJ-2266 treatment with no effect on apoptosis, activation and proliferation of activated T cells. Furthermore, adoptive transfer of BJ-2266 treated MOG-reactive Th1 and Th17 cells led to a lower EAE disease score and better clinical recovery from EAE. The underlying mechanism of BJ-2266 effect involved the inhibition of JAK/STAT phosphorylation that is critical for Th-cell differentiation. We conclude that BJ-2266 regulates the JAK/STAT pathway in response to cytokine signals and subsequently suppresses the differentiation of Th-cell responses.

Impact of fomesafen on the soil microbial communities in soybean fields in Northeastern China.

Fomesafen, a widely adopted residual herbicide, is used throughout the soybean region of northern China for the spring planting. However, the ecological risks of using fomesafen in soil remain unknown. The aim of this work was to evaluate the impact of fomesafen on the microbial community structure of soil using laboratory and field experiments. Under laboratory conditions, the application of fomesafen at concentrations of 3.75 and 37.5mg/kg decreased the basal respiration (RB) and microbial biomass carbon (MBC). In contrast, treatment with 375mg/kg of fomesafen resulted in a significant decrease in the RB, MBC, abundance of both Gram+ and Gram- bacteria, and fungal biomass. Analysis of variance showed that the treatment accounted for most of the variance (38.3%) observed in the soil microbial communities. Furthermore, the field experiment showed that long-term fomesafen application in continuously cropped soybean fields affected the soil bacterial community composition by increasing the relative average abundance of Proteobacteria and Actinobacteria species and decreasing the abundance of Verrucomicrobia species. In addition, Acidobacteria and Chloroflexi species showed a pattern of activation-inhibition. Taken together, our results suggest that the application of fomesafen can affect the community structure of soil bacteria in the spring planting soybean region of northern China.

Spectrofluorimetric determination of acotiamide hydrochloride trihydrate in the presence of its oxidative degradation product.

Acotiamide hydrochloride trihydrate is a novel gastroprokinetic drug which has been recently approved for the treatment of patients with functional dyspepsia. This study presents the first reported to investigate the fluorimetric behavior of acotiamide hydrochloride trihydrate in the presence of its oxidative degradation product. All variables that affect fluorescence intensity were studied and optimized. The described method involved the measurement of native fluorescence of the drug in ethanol at 404 nm after excitation at 326 nm. Calibration plot was found to be linear over the concentration range 0.1-0.9 µg/ml. The specificity of the method has been tested via selective determination of the studied drug in its synthetic mixtures with its degradation product. The proposed method has been successfully applied to the analysis of the drug in its new pharmaceutical dosage form and the results have been statistically compared with the reported HPLC method showing no significant differences by applying t-test and F-test.

Transfer of the Active Ingredients of Some Plant Protection Products from Raspberry Plants to Beehives.

Plant protection products (PPPs) have been found increasingly in the environment. They pose a huge threat to bees, contributing to honeybee colony losses and consequently to enormous economic losses. Therefore, this field investigation was designed to determine whether their active ingredients (AIs) were transferred from raspberry plants to beehives located in the immediate neighbourhood of the crop and to what extent they were transferred. Every week for 2 months, samples of soil, raspberry leaves, flowers and fruits, worker bees, honeybee brood, and honey were collected and analysed for the presence of propyzamide, chlorpyrifos, iprodione, pyraclostrobin, boscalid, cypermethrin, difenoconazole, azoxystrobin, and pyrimethanil residues. Five of these substances were found in the worker bee bodies. Chlorpyrifos, applied to only the soil through the irrigation system, also was detected in the brood. A small amount of boscalid was noted in the honey, but its residues did not exceed the maximum residue level. For chlorpyrifos, boscalid, and pyrimethanil, a positive correlation between the occurrence of PPPs in the crops and the beehives was found. Statistical methods confirmed that the application of PPPs on a raspberry plantation, as an example of nectar-secreting plants, was linked to the transfer of their AIs to beehives.

The transfer of active ingredients of insecticides and fungicides from an orchard to beehives.

This investigation was undertaken to determine whether active ingredients (AIs) of currently recommended plant protection products (PPPs) could be transferred to beehives from apple and pear trees. A field trial was carried out with apple trees of Ligol and Idared variety, and pear trees of Conference variety. For pest and diseases control of fungal origin, recommended PPPs were applied. Samples of flowers from the above-mentioned varieties of fruit trees, of bees, brood and honey from beehives located in their direct neighborhood were collected regularly and analyzed for the presence of lambda-cyhalothrin (an insecticide) and cyprodinil, captan, fluopyram, kresoxim-methyl, penthiopyrad and trifloxystrobin (fungicides). In samples of flowers of Ligol variety, fluopyram residues (on average 0.621 µg single flower-1) were at the highest levels, whereas in samples of pear flowers of Conference variety, and in flowers of Idared variety, captan residues (on average, respectively, 0.705 and 165.7 µg single flower-1). In samples of bees and honey, residues of five AIs were detected, and in brood six AIs, whereby in each case captan residues prevailed, respectively, up to 585.2, 51.52 and 126.5 µg kg-1 bees and honey. In the honey, significantly larger residues of captan were found out than maximum residue level (MRL) for this AI - 103.04% MRL. In the case of any AI, the daily intake did not exceed 0.002% acceptable daily intake (ADI).

Another fatal case related to the recreational abuse of U-47700.

The abuse of synthetic opioids has become a major threat in recent years. Several clinical reports and fatal case reports exist discussing life-threatening hypoventilation and fatal respiratory depression following the abuse of trans-3,4-dichloro-N-(2-(dimethylamino)cyclohexyl)-N-methylbenzamide (U-47700). The reported concentration of U-47700 in peripheral blood varies between 0.01 µg/mL and 1.46 µg/mL. These values depend on the mode of administration and whether the drug was used in combination with other drugs and/or pharmaceuticals. In the past, U-47700 was predominantly insufflated and not injected. The current study presents a non-targeted liquid chromatography/mass spectrometry (LC/MS)-based screening approach of urine and cerebrospinal fluid samples after intravenous injection of U-47700. Furthermore, quantitative values on U-47700 as obtained by liquid chromatography coupled to a linear ion trap (LC/ESI-QTRAPMS) are presented concerning femoral blood (0.29 µg/mL), urine (0.24 µg/mL), gastric contents (0.57 µg/mL), bile fluid (2.3 µg/mL), heart blood (1.25 µg/mL), liver (9.9 µg/g), cerebrospinal fluid (0.4 µg/mL), and hair (0.14 ng/mg). Thereof, concentrations in hair, gastric contents, bile fluid and cerebrospinal fluid have never been reported before. Drug paraphernalia were also analyzed by liquid chromatography coupled to a diode array detector (LC/DAD) and nuclear magnetic resonance spectrometer (NMR). The analyses show that the powder had a relatively high purity and was adulterated to a low degree. This is the first case report which lists concentration distributions of various specimens after intravenous injection. These findings as well as the U-47700 concentration are important to evaluate autopsy cases of U-47700 intoxication in the future.

Behavior of fluopyram and tebuconazole and some selected pesticides in ripe apples and consumer exposure assessment in the applied crop protection framework.

The supervised field trials were conducted in a commercial apple orchard in 2016. The trials were an attempt to determine a model for dissipation and toxicological evaluation of fluopyram, tebuconazole, captan, tetrahydrophthalimide (THPI), pirimicarb, spirodiclofen, and boscalid residues detected in fruit of Red Jonaprince, Lobo, and Gala varieties immediately before harvest. The analysis also covered amounts of pesticides still present in remnants of calyx in Lobo and Gala varieties immediately before harvest. Laboratory samples of ripe apples were collected within 14 days of the treatment. Levels of pesticide residues detected in the samples changed at a constant exponential rate, and the residue levels found in ripe apples of Red Jonaprince, Gala, and Lobo varieties immediately before harvest were below maximum residue levels (MRL). Overall, captan residues in remnants of calyx were at a level of 22.3% for the Gala variety and 9.3% for the Lobo variety. Likewise, the long-term daily intake of the detected substances by a Polish adult consumer was low, ranging from 0.02% ADI for pirimicarb to 0.72% ADI for captan.

Effects of aging process on adsorption-desorption and bioavailability of fomesafen in an agricultural soil amended with rice hull biochar.

Biochar has been introduced as an acceptable soil amendment due to its environmental benefits such as sequestering soil contaminants. However, the aging process in biochar amended soil probably decreases the adsorption capacity of biochar through changing its physico-chemical properties. Adsorption, leaching and bioavailability of fomesafen to corn in a Chinese soil amended by rice hull biochar after 0, 30, 90 and 180days were investigated. Results showed that the addition of 0.5%-2% fresh biochar significantly increases the adsorption of fomesafen 4-26 times compare to unamended soil due to higher SSA of biochar. Biochar amendment also decreases fomesafen concentration in soil pore water by 5%-23% resulting lower risk of the herbicide for cultivated plants. However, the aging process decreased the adsorption capacity of biochar since the adsorption coefficient values which was 1.9-12.4 in 0.5%-2% fresh biochar amended soil, declined to 1.36-4.16, 1.13-2.78 and 0.95-2.31 in 1, 3 and 6-month aged treatments, respectively. Consequently, higher desorption, leaching and bioavailable fraction of fomesafen belonged to 6-month aged treatment. Nevertheless, rice hull biochar was effective for sequestering fomesafen as the adsorption capacity of biochar amended soil after 6months of aging was still 2.5-5 times higher compared to that of unamended soil.

Development and validation of stability indicating assay method for cinitapride in bulk & tablets.

A simple stability indicating UV-spectrophotometric method has been developed and validated for the determination of cinitapride hydrogen tartrate (CHT) in bulk and solid pharmaceutical dosage form. Drug absorption was measured in different analytical mediums however; maximum absorption was seen in 0.1 N HCl at wavelength (λmax) of 266 nm. The calibration curve was found to be linear over the concentration range from 6 to14µg/mL with the correlation coefficient value (r) of 0.999. The LOD and LOQ were estimated to be 0.1019µg/mL and 0.309µg/mL respectively. The accuracy was evaluated by determining the percent drug recovery, performed at three different levels of 50%, 100% and 150%. The% recovery was found to be in the range of 99.96-100.64%. The precision of the method was determined by inter-day and intra-day variations. The % RSD value <0.5 indicates the underlying method is precise and accurate as well. The developed method was applied to characterize in vitro assay content of few brands of cinitapride (1 mg) available in local market. No interference of the formulation excipients with the drug absorption was observed during assay. Drug substance and drug product were exposed to various stressed conditions (acid, base, oxidative, thermal and photolysis). Forced degradation testing of drug product showed that the oxidation (20%) was found to be the major degradation pathway of the cinitapride. However; drug estimation was not influenced in presence of degradation moieties formed during acid, base, oxidation, thermal and photolytic breakdown. Overall, the investigated technique is robust and specific that would be successfully used to quantify the cinitapride hydrogen tartarate in pharmaceutical dosage and bulk form in future.

Development and evaluation of a highly reliable assay for SUMO-specific protease inhibitors.

SUMOylation, as a post-translational modification of proteins, plays essential regulatory roles in a variety of pathological conditions. In the dynamic process of SUMOylation and deSUMOylation, SENPs (SUMO-specific proteases), in charge of deconjugation of SUMO (small ubiquitin-related modifier) from substrate proteins, have recently been found to be potential therapeutic targets for cancer treatment. A reliable and practical assay is much needed to accelerate the discovery of SENPs inhibitors. We established a quantitative assay based on readily available SDS-PAGE-Coomassie system using RanGAP-SUMO as the substrate, thus avoiding the use of expensive fluorescent dyes or the error-prone fluorescent reporter. Its reproducibility and reliability were also evaluated in this report.

Pipette vial dispersive liquid-liquid microextraction combined with high-performance liquid chromatography for the determination of benzoylurea insecticide in fruit juice.

A simple, sensitive, and efficient method of using a pipette vial to perform dispersive liquid-liquid microextraction based on the solidification of floating organic droplets was coupled with high-performance liquid chromatography (HPLC) and a diode array detector for the preconcentration and analysis of four benzoylurea insecticides in fruit juice. In this method, 1-dodecanol was used as an extractant, and a snipped pipette was used as an experimental vial to simplify the procedure of collecting and separating solidified extractant. The experimental parameters were optimized using a Plackett-Burman design and one-factor-at-a-time method. Under the optimal conditions in the water model, the limits of detection for analytes varied from 0.03 to 0.28 µg/L, and the enrichment factors ranged from 147 to 206. Linearity was achieved for diflubenzuron and flufenoxuron in a range of 0.5-500 µg/L, for hexaflumuron in a range of 1-500 µg/L, and for triflumuron in a range of 5-500 µg/L. The correlation coefficients for the analytes ranged from 0.9986 to 0.9994 with recoveries of 91.4-110.9%. Finally, the developed technique was successfully applied to fruit juice samples with acceptable results. The relative standard deviations of the analytes at two spiking levels (50 and 200 µg/L) varied between 0.2 and 4.5%.