LC-PDA-QTof-MS characterization of the stress degradation products of Zanubrutinib: A novel BTK inhibitor.

Zanubrutinib (ZAN) is an orally administered anti-cancer medication used for the treatment of Mantle cell lymphoma. Recently, it has also been approved by FDA for the treatment of chronic lymphocytic leukemia. Determination of impurities formed in drug substances/products as a result of manufacturing or storage forms an important aspect of drug life cycle management. The current study concentrated on understanding the stability of ZAN under various stress conditions as per the ICH Q1 (R2) guidelines. In total, ZAN produced thirteen degradation products under various hydrolytic (acid, base and neutral) and thermal stress conditions. The stress degradation products were separated by ultra-performance liquid chromatography, chemical structures of these products were characterized by MS/MS experiments combined with accurate mass measurements conducted on a LC-QTof-MS. The mechanism for the formation of these degradation products was also proposed. This study provides comprehensive information on the inherent stability of ZAN which will be useful in the drug development and manufacturing processes.

A green deep eutectic solvent based dispersive liquid-liquid microextraction for the quantitative analysis of 21 polychlorinated biphenyl metabolites in food of animal origin using injector port silylation-gas chromatography-tandem mass spectrometry.

An analytical method was developed for the quantitative determination of 21 polychlorinated biphenyls (PCBs) metabolites (17 were -OH, 1 -MeO, and 3 were MeSO2) in foods of animal origin using deep eutectic solvent (DES) based dispersive liquid-liquid microextraction followed by injector port silylation-gas chromatography-tandem mass spectrometry. The type of DES (thymol: camphor, 1:1 molar ratio) and optimum volume of DES (300 µL), pH (7.0), and disperser solvent (acetonitrile) were optimized to attain the maximum extraction efficiency. The limit of detection, limit of quantification, and percent recovery were found to be in the range of 0.12-0.23 ng/mL, 0.40-0.76 ng/mL, and 80.1-111.4%, respectively. The expanded uncertainty was observed to be in the range of 7.2-22.8% for the targeted analytes. The proposed method was applied to real food samples (milk, meat, fish, and egg) and the levels were found to be in the range of 0.64-32.14 ng/g. This is first of its kind method using green solvent based method for the analysis of PCB metabolites (-OH, MeO, and MeSO2) and will find extensive application in routine testing for foods of animal origin.

Development of liquid chromatography-triple quadrupole mass spectrometric method for the quantitative determination of a novel adjuvant, Imidazoquinoline gallamide in aluminum hydroxide gel-Imidazoquinoline gallamide and COVAXIN.

Imidazoquinoline gallamide is a toll-like receptor 7/8 agonist, belongs to the imidazoquinoline class, has the potential to activate antigen-presenting cells, and enhances immune response, primarily Th1 response. The COVAXIN is a whole virion inactivated Coronavirus disease 2019 vaccine formulated with this novel adjuvant called, aluminum hydroxide gel Imidazoquinoline gallamide, wherein, Imidazoquinoline gallamide is chemisorbed onto aluminum hydroxide gel. Herein, an analytical method based on liquid chromatography-tandem mass spectrometry was developed to identify and quantify Imidazoquinoline gallamide in aluminum hydroxide gel Imidazoquinoline gallamide and COVAXIN. The multiple reaction monitoring transitions were optimized for Imidazoquinoline gallamide quantification are [M+H]+ ions with 512.24→343.19 m/z (quantifier ion) and 512.24→360.22 m/z (qualifier ion). The developed method was validated as per the international conference on harmonization quality2 revison1 guidelines. The method was linear in the range of 0.025-10 µg/mL with a coefficient of determination of 0.9985 and the limit of quantification is 0.025 µg/mL. The accuracy was in the range of 82-121 % and intra- and inter-day precision was less than 7.1% and 5.39%, respectively. The expanded uncertainty results are 9.2% for Imidazoquinoline gallamide in the sample. The validated method was successfully applied to evaluate Imidazoquinoline gallamide concentration in every batch of COVAXIN.

Chemometric assisted natural DES based VA-DLLME-LC-MS/MS method for the quantitative determination of Garcinol in biofluids/tissues: A practical application to pharmacokinetics and biodistribution studies.

Garcinol (GAR) is a polyisoprenylated benzophenone obtained from Garcinia indica used as anti-oxidant and anti-inflammatory in traditional medicine and due to these activities, it possesses anticancer properties. It is considered to be a next generation epigenetic drug. A green solvent based analytical method which is efficient, sophisticated, and highly enriched has been developed for the quantitative analysis of GAR in biological samples (plasma, liver, kidney and spleen) with the use of deep eutectic solvent (DES) for its extraction. A series of 23 DESs were synthesized and out of which, Thymol (Th)-Terpeniol (T), 2:1 molar ratio with a more hydrophobic environment and high interaction efficiency between GAR and DES was identified for the better extraction from mice plasma and tissue samples. The Design of Experiment approaches like placket-burmann design and central composite design were used to optimize the method conditions. The method validation characteristics, such as limit of detection (0.193-0.237 ng/mL), limit of quantification (0.644-0.697 ng/mL), lower limit of quantification (0.5 ng/mL), broad range of linearity with R2 (0.9994-0.9997) with a percent recovery not less than 87% was observed, which are well within the acceptance criteria for a bioanalytical method. The enrichment factor is upto 53-60 folds, with high extraction efficiency (89-97%). The measurement uncertainty was estimated with an expanded uncertainty ranged between 10.9%-19.0%. The method developed and validated was effectively applied to examine the pharmacokinetic and biodistribution patterns for GAR in mice.

Chemical profiling of marine seaweed Halimeda gracilis using UPLC-ESI-Q-TOF-MSE and evaluation of anticancer activity targeting PI3K/AKT and intrinsic apoptosis signaling pathway.

Marine seaweeds are predominant for nutraceuticals and have been food source since ancient times and are currently being used in Chinese medicines and Japanese traditional medicines. In the present study, the chemical profile analysis of Halimeda gracilis was performed using UPLC-ESI-Q-TOF-MSE analysis and assessed for its anticancer activity. A cursory investigation of the total ion chromatograms of the both methanol (MHG) and ethyl acetate (EAHG) of H. gracilis extracts reveals that both extracts have different kind of metabolites including phenols and its derivatives, diterpenes, cinnamic acids derivatives etc. The in vitro anticancer activity of EAHG and MHG exhibiting significant activity and induced apoptosis against skin cancer cells by generating excessive ROS, damaging the mitochondrial membrane and nuclear components. Further, the western blot analysis showed that the EAHG and MHG downregulates the oncoproteins PI3K, AKT, p-AKT, and BcL2 and upregulates the apoptotic proteins Bax, Cyto C, p21, p53, Caspase 9 and Caspase 3. To best of our knowledge, this is the first report which implies the UPLC-ESI-Q-TOF-MSE based chemical profiling of H. gracilis and can be used for the treatment of cancer.

Phytometabolomic analysis of boiled rhizome of Nymphaea nouchali (Burm. f.) using UPLC-Q-TOF-MSE, LC-QqQ-MS & GC-MS and evaluation of antihyperglycemic and antioxidant activities.

Phytometabolomic analysis of Nymphaea nouchali (Burm. F.) boiled rhizome was carried out utilizing UPLC-Q-TOF-MSE, LC-QqQ-MS and GC-MS techniques and evaluated for antihyperglycemic and antioxidative stress potentials. Metabolomic analysis revealed presence of multiple antidiabetic and antioxidant compounds. Boiled rhizome powder exhibited potent antihyperglycemic activity against sugar-induced postprandial hyperglycemia in rats plausibly due to the presence of intestinal α-glucosidase inhibitory and augmenting cellular glucose uptake activities. It also prevented hyperglycemia-induced hemoglobin and insulin glycation. Rhizome displayed potent reducing power, effectively scavenged various reactive oxygen species. It displayed antioxidative stress potential in assuaging H2O2 induced erythrocyte hemolysis and antioxidant activity by inhibiting membrane lipid peroxidation. Boiled rhizome was also found to preserve the loss of cellular antioxidants under H2O2 induced oxidative stress and disturbances caused to mitochondrial membrane potential. This is the first research reporting boiled N. nouchali rhizome as an ideal food material to manage the cause of hyperglycemia and resultant oxidative stress.

Understanding the metabolic perturbations in Carica papaya Linn. due to different ripening practices/agents using gas chromatography-mass spectrometry based metabolomics.

The study of fruit-ripening mechanism is vital as it plays a key role in the maintenance of fruit quality. Use of various xenobiotics for quick ripening has been shown to impact the quality of fruit, which in turn affect human health. In the present study, we made an attempt to understand the metabolic perturbations in Carica papaya Linn. (papaya), which has been ripened either by the ripening practice (room temperature process as control) and/or ripening agents (calcium carbide and ethylene) using gas chromatography-mass spectrometry (GC-MS) based metabolomics. The partial least squares-discriminant analysis has revealed significant alternations in 13 metabolites mainly sugars, amino acids, fatty acids, and organic acids as well as disturbances in five metabolic pathways due to different ripening practice/agents. The individual comparison of calcium carbide with control and ethylene with control has found 13 and 11 metabolites, respectively, which are common to the PLS-DA of three ripening groups. The GC-MS-based metabolomics has been able to predict the metabolic perturbations in papaya resulting from the ripening practice/agents. The findings from the present analysis has a wide application in food quality and will help to address safety concerns.

Evaluating the metabolic perturbations in Mangifera indica (mango) ripened with various ripening agents/practices through gas chromatography - mass spectrometry based metabolomics.

Mangifera indica L. (mango) is said to be the king of fruits due to its rich nutritional properties and mainly originates from the Indian sub-continent. The consumption pattern of the mangoes has increased drastically, due to which, many ripening practices/agents were used to make it ready-to-eat fruit or juice for the consumers. The fruit quality and metabolic composition are said to be altered due to different ripening agents/practices. The present communication mainly deals to understand the metabolic perturbations in mango fruits due to different ripening practices/agents (room temperature ripening, ethylene, and calcium carbide) using gas chromatography - mass spectrometry based metabolomics. The partial least square-discriminant analysis has found 16 differential metabolites for different ripening agents/practices which are belong to the classes of amino acids, fatty acids, sugars, and polyols. Four metabolic pathways were found to alter in the fruit metabolome due to different ripening agents/practices. Fructose, glucose, and galactose were found to be significantly up-regulated due to calcium carbide ripening in comparison to other ripening agents/practices. Overall findings from the present study advocates that mass spectrometry based metabolomics can be valuable tool to understand the fruit quality and safety with respect to consumer health.

Saliva and urine metabolic profiling reveals altered amino acid and energy metabolism in male farmers exposed to pesticides in Madhya Pradesh State, India.

Globally, the human population is exposed to low doses of pesticides due to its extensive use in agriculture. The chronic exposure to pesticides can lead to cancer, depression, anxiety, Parkinson's and Alzheimer's diseases etc. Here, we have made an attempt to use mass spectrometry based metabolomics to investigate the metabolic perturbations induced by the pesticides in the urine and saliva samples of farmers from the Madhya Pradesh State of India. The study was aimed to establish non-invasive matrices like urine and saliva as alternative diagnostic matrices to the occupational exposure studies. Saliva and urine samples were collected from 51 pesticides applicators and acquired metabolic profiles of urine and saliva samples using gas chromatography-mass spectrometry (GC-MS). Multivariate pattern recognition and pathway analysis were used to analyze and interpret the data. Investigation of endogenous metabolic profiles revealed remarkable discrimination in both saliva and urine samples of the exposed population strongly suggesting the changes in metabolic composition within the identified metabolites (for urine samples: accuracy 0.9766, R2 = 0.9130, Q2 = 0.8703; for saliva samples, an accuracy of 0.9961, R2 = 0.9698, Q2 = 0.9637). Thirteen metabolites of urine samples and sixteen metabolites of saliva samples were identified as differential metabolites specific to pesticide exposure. Pathway analysis of differential metabolites revealed that amino acid metabolism, energy metabolism (glycolysis and TCA cycle) and glutathione metabolism (oxidative stress) were found to affect in pesticide exposed population. The present study suggested that GC-MS based metabolomics can help to reveal the metabolic perturbations in human population after pesticides exposure.

A Rapid Method for the Quantitative Determination of 34 Pesticides in Nonalcoholic Carbonated Beverages Using Liquid-Liquid Extraction Coupled to Dispersive Solid-Phase Cleanup Followed by Gas Chromatography with Tandem Mass Spectrometry.

An economical, rapid, and sensitive multiresidue method using liquid-liquid extraction (LLE) coupled with dispersive SPE (dSPE) cleanup was developed for the quantitative determination of 34 multiclass multiresidue (MCMR) pesticides (14 organochlorines, eight organophosphates, 10 synthetic pyrethroids, and two herbicides) in nonalcoholic carbonated beverages (cola, orange, lemon-lime, and citra) using GC with tandem MS. The procedure mainly involved LLE by dichloromethane and dSPE cleanup in the presence of magnesium sulfate, primary secondary amine, and C18. The RSD of the developed method was found to be less than 14%. The LOD and LOQ values for all the analyzed pesticides were found in the ranges of 0.001-0.027 µg/L and 0.004-0.088 µg/L, respectively. The LOQ levels of the pesticides analyzed were found to be well below the recommended limit by the European Union (0.1 µg/L in water). The mean recoveries of pesticides in different nonalcoholic carbonated beverages (cola, orange, lemon-lime, and citra) were found to be in the range of 79-111%, with RSDs less than 11%. The validation data prove that the method can be acceptable to regulatory agencies for the routine analysis of MCMR pesticides in nonalcoholic carbonated beverages.

Docosahexaenoic acid up-regulates both PI3K/AKT-dependent FABP7-PPARγ interaction and MKP3 that enhance GFAP in developing rat brain astrocytes.

The astrocyte marker, glial fibrillary acidic protein (GFAP), has essential functions in the brain, but may trigger astroglial scarring when expressed in excess. Docosahexaenoic acid (DHA) is an n-3 fatty acid that is protective during brain development. However, the effect of DHA on GFAP levels of developing brain remains unexplored. Here, we detected that treating developing rats with DHA-enriched fish-oil caused dose-dependent GFAP augmentation. We investigated the mechanism promoting GFAP, hypothesizing the participation of fatty acid-binding protein-7 (FABP7), known to bind DHA. We identified that DHA stimulated FABP7 expression in astrocytes, and FABP7-silencing suppressed DHA-induced GFAP, indicating FABP7-mediated GFAP increase. Further investigation proved FABP7 expression to be phosphatidylinositide 3-kinases (PI3K)/AKT and nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ)-dependent. We found that PI3K/AKT activated PPARγ that triggered FABP7 expression via PPARγ-responsive elements within its gene. Towards identifying FABP7-downstream pathways, we considered our previous report that demonstrated cyclin-dependent kinase-5 (CDK5)-PPARγ-protein-protein complex to suppress GFAP. We found that the DHA-induced FABP7 underwent protein-protein interaction with PPARγ, which impeded CDK5-PPARγ formation. Hence, it appeared that enhanced FABP7-PPARγ in lieu of CDK5-PPARγ resulted in increased GFAP. PI3K/AKT not only stimulated formation of FABP7-PPARγ protein-protein complex, but also up-regulated a FABP7-independent MAP-kinase-phosphatase-3 pathway that inactivated CDK5 and hence attenuated CDK5-PPARγ. Overall, our data reveal that via the proximal PI3K/AKT, DHA induces FABP7-PPARγ, through genomic and non-genomic mechanisms, and MAP-kinase-phosphatase-3 that converged at attenuated CDK5-PPARγ and therefore, enhanced GFAP. Accordingly, our study demonstrates a DHA-mediated astroglial hyperactivation, pointing toward a probable injurious role of DHA in brain development.

Assessing hazardous risks of indoor airborne polycyclic aromatic hydrocarbons in the kitchen and its association with lung functions and urinary PAH metabolites in kitchen workers.

BACKGROUND: Indoor air pollution is associated with decreased pulmonary function but the relative impact of pollution from kitchen sources on health risks in kitchen workers is not well-known or studied. A study was conducted to measure the kitchen indoor air quality including PAHs estimation and risk assessment based on reported PAHs in indoor air in a central kitchen at North India. METHODS: A cross sectional study was undertaken to assess the lung function status using spirometer and urinary PAH metabolite measurements using GC-MS/MS among 94 male kitchen workers and their corresponding controls. Assessment of the indoor air quality levels was evaluated using standard methods. RESULTS: All the indoor air pollutants were within the recommended guidelines except CO, TVOC and PAH emission in the kitchen. Incremental life time cancer risk (ICLR) based on indoor air PAH measurements indicates potential for carcinogenic risk. Significant lung function decline was observed among kitchen workers as compared to controls after adjusting for smoking habits. Urinary PAH metabolites were detected in kitchen workers and measured concentrations were comparatively higher than control subjects. CONCLUSION: The decline in lung functions after adjustment for confounders and detection of urinary PAH metabolites in kitchen workers can be associated with higher concentrations of PAHs, CO and TVOCs in kitchen indoor air.

Nucleation temperature-controlled synthesis and in vitro toxicity evaluation of L-cysteine-capped Mn:ZnS quantum dots for intracellular imaging.

Quantum dots (QDs), one of the fastest developing and most exciting fluorescent materials, have attracted increasing interest in bioimaging and biomedical applications. The long-term stability and emission in the visible region of QDs have proved their applicability as a significant fluorophore in cell labelling. In this study, an attempt has been made to explore the efficacy of L-cysteine as a capping agent for Mn-doped ZnS QD for intracellular imaging. A room temperature nucleation strategy was adopted to prepare non-toxic, water-dispersible and biocompatible Mn:ZnS QDs. Aqueous and room temperature QDs with L-cysteine as a capping agent were found to be non-toxic even at a concentration of 1500 µg/mL and have wide applications in intracellular imaging.

Imprinted nanospheres based on precipitation polymerization for the simultaneous extraction of six urinary benzene metabolites from urine followed by injector port silylation and gas chromatography-tandem mass spectrometric analysis.

In the present communication, uniformly sized molecularly imprinted polymer (MIP) as nanospheres were synthesized based on precipitation polymerization using dual-template imprinting approach and used it as sorbent for solid phase extraction of six urinary benzene metabolites (UBMs). This approach in combination with injector port silylation (IPS) has been used for the quantitative determination of these UBMs by gas chromatography-tandem mass spectrometry. The MIP was synthesized by using t,t-muconic acid (t,t-MA) and 1,2,4-trihydroxybenzene (THB) as templates, methacrylic acid (MAA) as a monomer, ethyleneglycoldimethacrylate (EGDMA) as crosslinker, acetonitrile and dimethylsulphoxide as a porogen and azobisisobutyronitrile (AIBN) as an initiator. The factors affecting the performance of polymer and IPS were investigated and optimized for the simultaneous determination of UBMs in urine. Binding study of imprinted and non-imprinted polymer (NIP) shows that, MIP possesses higher affinity in comparison to NIP for these analytes. Under the optimum conditions, the method developed was found to be linear with regression coefficients falls in the range of 0.9721-0.9988 for all the analyzed metabolites. The percent recovery of the metabolites analyzed in urine was found to be in the range of 76-89%, while the limit of detection and limit of quantification were found to be in the range of 0.9-9.1ngmL(-1) and 2.8-27ngmL(-1) respectively. The validated method was successfully applied to the real urine samples collected from different groups (kitchen workers, smokers and petroleum workers) and found that the developed method has been promising applications in the routine analysis of urine samples of benzene exposed population.

Determination of Urinary PAH Metabolites Using DLLME Hyphenated to Injector Port Silylation and GC-MS-MS.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and well-known carcinogens. Hydroxy derivatives of PAH are considered as biomarkers of PAH exposure, and there is a need to measure these metabolites at low concentrations. So, a precise and eco-friendly analytical method has been developed for rapid determination of PAH metabolites. For the first time, a new analytical method based on coupling of dispersive liquid-liquid microextraction (DLLME) with auto-injector port silylation (auto-IPS) followed by gas chromatography-tandem mass spectrometry (GC-MS-MS) analysis is reported for the analysis of seven urinary PAH metabolites. Factors affecting DLLME and IPS, such as type and volume of extraction and disperser solvent, pH, ionic strength, injector port temperature, volume of N,O-bis(trimethylsilyl)trifluoroacetamide and type of solvent were investigated. Under optimized conditions, the limit of detection and limit of quantification were found to be in the range of 1-9 and 3-29 ng/mL, respectively. Satisfactory recoveries of metabolites in urine samples in the range of 87-95% were found. The developed method has been successfully applied for the determination of PAH metabolites in urine samples of exposed workers. DLLME-auto-IPS-GC-MS-MS method is time, labor, solvent and reagent saving, which can be routinely used for the analysis of urinary PAH metabolites.

Isolation and functional analysis of a glycolipid producing Rhodococcus sp. strain IITR03 with potential for degradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT).

A 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) degrading bacterium strain IITR03 producing trehalolipid was isolated and characterized from a pesticides contaminated soil. The strain IITR03 was identified as a member of the genus Rhodococcus based on polyphasic studies. Under aqueous culture conditions, the strain IITR03 degraded 282 µM of DDT and could also utilize 10mM concentration each of 4-chlorobenzoic acid, 3-chlorobenzoic acid and benzoic acid as sole carbon and energy source. The catechol 1,2-dioxygenase enzyme activity resulted in conversion of catechol to form cis,cis-muconic acid. Cloning and sequencing of partial nucleotide sequence of catechol 1,2-dioxygenase gene (cat) from strain IITR03 revealed its similarity to catA gene present in Rhodococcus sp. strain Lin-2 (97% identity) and Rhodococcus strain AN22 (96% identity) degrading benzoate and aniline, respectively. The results suggest that the strain IITR03 could be useful for field bioremediation studies of DDT-residues and chlorinated aromatic compounds present in contaminated sites.

Molecularly imprinted polymer coupled with dispersive liquid-liquid microextraction and injector port silylation: a novel approach for the determination of 3-phenoxybenzoic acid in complex biological samples using gas chromatography-tandem mass spectrometry.

A novel analytical approach based on molecularly imprinted solid phase extraction (MISPE) coupled with dispersive liquid-liquid microextraction (DLLME), and injector port silylation (IPS) has been developed for the selective preconcentration, derivatization and analysis of 3-phenoxybenzoic acid (3-PBA) using gas chromatography-tandem mass spectrometry (GC-MS/MS) in complex biological samples such as rat blood and liver. Factors affecting the synthesis of MIP were evaluated and the best monomer and cross-linker were selected based on binding affinity studies. Various parameters of MISPE, DLLME and IPS were optimized for the selective preconcentration and derivatization of 3-PBA. The developed method offers a good linearity over the calibration range of 0.02-2.5ngmg(-1) and 7.5-2000ngmL(-1) for liver and blood respectively. Under optimized conditions, the recovery of 3-PBA in liver and blood samples were found to be in the range of 83-91%. The detection limit was found to be 0.0045ngmg(-1) and 1.82ngmL(-1) in liver and blood respectively. SRM transition of 271→227 and 271→197 has been selected as quantifier and qualifier transition for 3-PBA derivative. Intra and inter-day precision for five replicates in a day and for five, successive days was found to be less than 8%. The method developed was successfully applied to real samples, i.e. rat blood and tissue for quantitative evaluation of 3-PBA. The analytical approach developed is rapid, economic, simple, eco-friendly and possess immense utility for the analysis of analytes with polar functional groups in complex biological samples by GC-MS/MS.

Gas chromatography-mass spectrometry based metabolomic approach for optimization and toxicity evaluation of earthworm sub-lethal responses to carbofuran.

Despite recent advances in understanding mechanism of toxicity, the development of biomarkers (biochemicals that vary significantly with exposure to chemicals) for pesticides and environmental contaminants exposure is still a challenging task. Carbofuran is one of the most commonly used pesticides in agriculture and said to be most toxic carbamate pesticide. It is necessary to identify the biochemicals that can vary significantly after carbofuran exposure on earthworms which will help to assess the soil ecotoxicity. Initially, we have optimized the extraction conditions which are suitable for high-throughput gas chromatography mass spectrometry (GC-MS) based metabolomics for the tissue of earthworm, Metaphire posthuma. Upon evaluation of five different extraction solvent systems, 80% methanol was found to have good extraction efficiency based on the yields of metabolites, multivariate analysis, total number of peaks and reproducibility of metabolites. Later the toxicity evaluation was performed to characterize the tissue specific metabolomic perturbation of earthworm, Metaphire posthuma after exposure to carbofuran at three different concentration levels (0.15, 0.3 and 0.6 mg/kg of soil). Seventeen metabolites, contributing to the best classification performance of highest dose dependent carbofuran exposed earthworms from healthy controls were identified. This study suggests that GC-MS based metabolomic approach was precise and sensitive to measure the earthworm responses to carbofuran exposure in soil, and can be used as a promising tool for environmental eco-toxicological studies.

Simultaneous derivatisation and preconcentration of parabens in food and other matrices by isobutyl chloroformate and dispersive liquid-liquid microextraction followed by gas chromatographic analysis.

A simple, rapid and economical method has been proposed for the quantitative determination of parabens (methyl, ethyl, propyl and butyl paraben) in different samples (food, cosmetics and water) based on isobutyl chloroformate (IBCF) derivatisation and preconcentration using dispersive liquid-liquid microextraction in single step. Under optimum conditions, solid samples were extracted with ethanol (disperser solvent) and 200 µL of this extract along with 50 µL of chloroform (extraction solvent) and 10 µL of IBCF was rapidly injected into 2 mL of ultra-pure water containing 150 µL of pyridine to induce formation of a cloudy state. After centrifugation, 1 µL of the sedimented phase was analysed using gas chromatograph-flame ionisation detector (GC-FID) and the peaks were confirmed using gas chromatograph-positive chemical ionisation-mass spectrometer (GC-PCI-MS). Method was found to be linear over the range of 0.1-10 µg mL(-1) with square of correlation coefficient (R(2)) in the range of 0.9913-0.9992. Limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.029-0.102 µg mL(-1) and 0.095-0.336 µg mL(-1) with a signal to noise ratio of 3:1 and 10:1, respectively.

Ultra sound assisted one step rapid derivatization and dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometric determination of amino acids in complex matrices.

A rapid and economical method for the simultaneous determination of 20 amino acids in complex biological and food matrices (hair, urine and soybean seed samples) has been developed using ultrasound assisted dispersive liquid-liquid micro extraction (UA-DLLME). The method involves simultaneous derivatization and extraction followed by gas chromatography-mass spectrometric (GC-MS) analysis of amino acids. The parameters of UA-DLLME were optimized with the aid of design of experiments approach. The procedure involves the rapid injection of mixture of acetonitrile (disperser solvent), trichloroethylene (TCE) (extraction solvent) and ethylchloroformate (derivatization reagent) into the aqueous phase of sample extract containing pyridine. The Plackett-Burman design has indicated that, the factors such as volume of disperser and extraction solvents and pH were found to be significantly affects the extraction efficiency of the method. The optimum conditions of these factors based on central composite design were found to be 250µL of acetonitrile, 80µL of TCE and pH of 10. The limit of detection and limit of quantification were found to be in the range of 0.36-3.68µgL(-1) and 1.26-12.01µgL(-1) respectively. This is the first application of DLLME for the analysis of amino acids in any matrices. The advantages like (i) in situ derivatization and extraction of amino acids without any prior lyophilization and cleanup of sample, (ii) low consumption of extraction solvent, (iii) fast and simple, (iv) cost-effective and (iv) good repeatability make the method amenable for the routine analysis of amino acids in clinical, toxicological, nutritional and quality control laboratories.