Ratnasekhar Ch

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.

Development, validation and comparison of two microextraction techniques for the rapid and sensitive determination of pregabalin in urine and pharmaceutical formulations after ethyl chloroformate derivatization followed by gas chromatography–mass spectrometric analysis

The present article reports first time the use of solid-phase microextraction (SPME) and dispersive liquid-liquid microextraction (DLLME) to extract pregabalin (PRG) from urine and pharmaceutical formulations followed by GC-MS analysis after ethyl chloroformate (ECF) derivatization. PRG is an antiepileptic and analgesic drug, which is a structural analogue of γ-amino-butyric acid (GABA). It is approved by Food and Drug Administration (FDA) for the treatment of central nervous system (CNS) disorders and neuropathic pain. Initially PRG was derivatized with ECF in the presence of pyridine at room temperature for 30s. Experimental parameters were investigated for derivatization, SPME and DLLME conditions. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.019 μg/ml and 0.063 μg/ml for SPME and 0.022 μg/ml and 0.075 μg/ml for DLLME respectively. The percentage recovery, in case of SPME was in the range of 83-98% while for DLLME it is in the range of 84-98%. The intra and inter-day precisions were found to be less than 6%. The developed methods after ECF derivatization were found to be simple, fast, efficient and inexpensive. DLLME has several advantages like lesser extraction time and cost effectiveness as compared to SPME. The developed methods may find wide application for the routine determination of PRG in biological as well as in quality control samples of pharmaceutical formulations.

Rapid and simultaneous determination of twenty amino acids in complex biological and food samples by solid-phase microextraction and gas chromatography-mass spectrometry with the aid of experimental design after ethyl chloroformate derivatization.

Amino acids play a vital role as intermediates in many important metabolic pathways such as the biosynthesis of nucleotides, vitamins and secondary metabolites. A sensitive and rapid analytical method has been proposed for the first time for the simultaneous determination of twenty amino acids using solid-phase microextraction (SPME). The protein samples were hydrolyzed by 6M HCl under microwave radiation for 120 min. Then the amino acids were derivatized by ethyl chloroformate (ECF) and the ethoxy carbonyl ethyl esters of amino acids formed were extracted using SPME by direct immersion. Finally the extracted analytes on the SPME fiber were desorbed at 260°C and analyzed by gas chromatography-mass spectrometer (GC-MS) in electron ionization mode. Factors which affect the SPME efficiency were screened by Plackett-Burmann design; most significant factors were optimized with response surface methodology. The optimum conditions for SPME are as follows: pH of 1.7, ionic strength of 733 mg, extraction time of 30 min and fiber of divinyl benzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS). The recovery of all the amino acids was found to be in the range of 89.17-100.98%. The limit of detection (LOD) of all derivatized amino acids in urine, hair and soybean was found to be in the range of 0.20-7.52 μg L(-1), 0.21-8.40 μg L(-1) and 0.18-5.62 μg L(-1), respectively. Finally, the proposed technique was successfully applied for the determination of amino acids in complex biological (hair, urine) and food samples (soybean). The method can find wide applications in the routine analysis of amino acids in any biological as well as food samples.

Optimization of UA-DLLME by experimental design methodologies for the simultaneous determination of endosulfan and its metabolites in soil and urine samples by GC–MS

A simple, economical, rapid and sensitive analytical method has been developed for the simultaneous determination of endosulfan (α- and β-) and its metabolites (endosulfan ether, endosulfan hydroxy ether, endosulfan lactone, endosulfan alcohol and endosulfan sulphate) in complex samples, such as soil and urine, based on ultrasound assisted dispersive liquid–liquid microextraction (UA-DLLME) followed by gas chromatography–mass spectrometric (GC–MS) analysis. The method parameters have been optimized using response surface design experiments. Trichloroethylene (TCE) and acetone were chosen as extraction and disperser solvents respectively. After UA-DLLME, the sediment phase obtained was directly analyzed by GC–MS without any further cleanup and preconcentration procedure. Several factors which can affect the UA-DLLME extraction were screened and optimized by 27–4 Plackett–Burman design (PBD) and central composite design (CCD) experiments respectively. Based on these experiments the optimized parameters for UA-DLLME extraction were as follows: extraction solvent, (TCE, 58 μL), disperser solvent (acetone, 1.27 mL) and ionic strength (Na2SO4, 7%, w/v). Intra- and inter-day precision were expressed as percent relative standard deviation (% RSD) and were found to be less than 6.33%. The limit of detection (LOD) of all the analytes in soil and urine were found to be in the range of 0.316–2.494 ng g−1 and 0.049–0.514 ng mL−1 respectively. The proposed method was successfully applied in the analysis of soil samples contaminated with endosulfan. The method may find wide application for the routine determination of endosulfan and its metabolites in environmental and biological samples.

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.

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.

Activity-guided chemo toxic profiling of Cassia occidentalis (CO) seeds: detection of toxic compounds in body fluids of CO-exposed patients and experimental rats.

Our prior studies have shown an association between the deaths of children and consumption of Cassia occidentalis (CO) seeds. However, the chemicals responsible for the CO poisoning are not known. Therefore, the present study was designed to identify the key moieties in CO seeds and their cytotoxicity in rat primary hepatocytes and HepG2 cells. Activity-guided sequential extraction and fractionation of the seeds followed by GC-MS analysis identified the toxic compounds in the CO seeds. These identified compounds were subsequently detected and quantified in blood and urine samples from CO-exposed rats and CO poisoning human study cases. GC-MS analysis of different fractions of methanol extracts of CO seeds revealed the presence of five anthraquinones (AQs), viz. physcion, emodin, rhein, aloe-emodin, and chrysophanol. Interestingly, these AQs were detected in serum and urine samples from the study cases and CO-exposed rats. Cytotoxicity analysis of the above AQs in rat primary hepatocytes and HepG2 cells revealed that rhein is the most toxic moiety, followed by emodin, aloe-emodin, physcion, and chrysophanol. These studies indicate that AQ aglycones are responsible for producing toxicity, which may be associated with symptoms of hepatomyoencephalopathy in CO poisoning cases.

Ultrasound assisted dispersive liquid–liquid microextraction followed by injector port silylation: a novel method for rapid determination of quinine in urine by GC–MS

BACKGROUND Silylation is a widely used derivatization method for the analysis of polar analytes by GC-MS. Ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) is an ecofriendly, rapid and simple microextraction method. For the first time, a novel approach has been developed and applied for the analysis of quinine in urine by combining UA-DLLME with injection port silylation. RESULTS The LOD and LOQ were found to be 5.4 and 18 ng/ml. The intra- and inter-day precisions were less than 5 and 8%, respectively. Mean recoveries of quinine were found to be in the range of 87 to 96%. CONCLUSION Ultrasound-assisted dispersive liquid-liquid microextraction is rapid, simple and consumes less reagent for the analysis of polar analytes such as quinine.

Superoxide mediated photomodification and DNA damage induced apoptosis by Benz(a)anthracene via mitochondrial mediated pathway

Benz(a)anthracene (BA) is an ubiquitous environmental pollutant of polycyclic aromatic hydrocarbons (PAHs) family. We showed superoxide (O2(-)) catalyzed BA photo modification and apoptosis in HaCaT keratinocytes under sunlight exposure. O2(-) generation was confirmed by quenching through superoxide dismutase (SOD). BA induced photocytotoxicity were investigated through MTT and NRU assay. We proposed DNA insults such as single and double strand breakage and CPDs formation which results in cell cycle arrest and apoptosis by photosensitized BA. BA induced apoptosis was caspase dependent and occurred through a mitochondrial pathway. Reduction of mitochondrial membrane potential, translocation of Bax to mitochondria and cytochrome c release favors involvement of mitochondria in BA phototoxicity. AO/EB double staining and TEM analysis also support apoptotic cell death. We propose a p21 regulated apoptosis via expression of Bax, and cleaved PARP under sunlight exposure. Thus, we conclude that it is imperative to avoid solar radiation during peak hr (between 11A.M. and 3P.M.) when the amount of solar radiation is high, in the light of DNA damage which may lead to mutation or skin cancer through photosensitized BA under sunlight exposure. Concomitantly, investigation is urgently required for the photosafety of BA photoproducts reaching in the environment through photomodification.

In matrix derivatization of trichloroethylene metabolites in human plasma with methyl chloroformate and their determination by solid-phase microextraction–gas chromatography-electron capture detector

Trichloroethylene (TCE) is a common industrial chemical that has been widely used as metal degreaser and for many industrial purposes. In humans, TCE is metabolized into dichloroacetic acid (DCA), trichloroacetic acid (TCA) and trichloroethanol (TCOH). A simple and rapid method has been developed for the quantitative determination of TCE metabolites. The procedure involves the in situ derivatization of TCE metabolites with methyl chloroformate (MCF) directly in diluted plasma samples followed by extraction and analysis with solid-phase microextraction (SPME) coupled to gas chromatography-electron capture detector (GC-ECD). Factors which can influence the efficiency of derivatization such as amount of MCF and pyridine (PYR), ratio of water/methanol were optimized. The factors which can affect the extraction efficiencies of SPME were screened using 2(7-4) Placket-Burman Design (PBD). A central composite design (CCD) was then applied to further optimize the most significant factors for optimum SPME extraction. The optimum factors for the SPME extraction were found to be 562.5mg of NaCl, pH at 1 and an extraction time of 22 min. Recoveries and detection limits of all three analytes in plasma were found to be in the range of 92.69-97.55% and 0.036-0.068 μg mL(-1) of plasma, respectively. The correlation coefficients were found to be in the range of 0.990-0.995. The intra- and inter-day precisions for TCE metabolites were found to be in the range of 2.37-4.81% and 5.13-7.61%, respectively. The major advantage of this method is that MCF derivatization allows conversion of TCE metabolites into their methyl esters in very short time (≤30 s) at room temperature directly in the plasma samples, thus makes it a solventless analysis. The method developed was successfully applied to the plasma samples of humans exposed to TCE.