RESUMO
Aiming towards simplifying sample preparation procedure, the present work explores use of unmodified laboratory filter paper as sorbent for extraction of nine basic drugs (five antidepressants, four benzodiazepines, and ketamine) from human blood samples and their analysis by gas chromatography-mass spectrometry (GC-MS). The procedure termed as cellulose paper sorptive extraction (CPSE) is straightforward. It involves adsorption of target analytes from deproteinized diluted blood samples on the unmodified cellulose paper followed by elution into 2 mL of methanol. Multivariate optimization, consisting of Placket-Burman design (PBD) and central composite design (CCD), was used to screen and optimize significant factors for CPSE. The proposed method follows the principles of green analytical chemistry (GAC), as the unmodified filter paper used as the sorbent is inexpensive and biodegradable. The technique is easy to perform and requires only 2 mL of MeOH during the entire extraction procedure. Under the optimized conditions, the limit of detection and quantification for the target analytes were estimated to be in the range of 0.003-0.035 and 0.010-0.117 µg mL-1, respectively. In contrast, the relative standard deviations were consistently below 10 %. The calibration curves were linear in the range of 0.015-2 µg mL-1 with a coefficient of determination (R2) in the range of 0.995-0.999.Satisfactory recoveries ranging from 87 to 99 % was achieved. As proof of concept, the analysis of nine drugs in blood samples from the patients was performed to demonstrate the potential application of the proposed method.
Assuntos
Celulose , Microextração em Fase Sólida , Humanos , Celulose/química , Microextração em Fase Sólida/métodos , Cromatografia Gasosa-Espectrometria de Massas , Limite de DetecçãoRESUMO
The increased incidence of diabetes to the magnitude of a global epidemic is attributed to non-traditional risk factors, including exposure to environmental chemicals. However, the contribution of xenobiotic exposure during the development of an organism to the etiology of diabetes is not fully addressed. Developing stages are more susceptible to chemical insult, but knowledge on the consequence of the same to the onset of diabetes is residual. In this context, by using Drosophila melanogaster having conserved Insulin/Insulin growth factor-like signaling (IIS) as well as glucose homeostasis as a model, we evaluated the potential of developmental exposure to dichlorvos (DDVP, an organophosphorus pesticide) or atrazine (herbicide) to cause diabetes in exposed organisms. Flies exposed to DDVP during their development display insulin deficiency or type 1 diabetes (T1D) while those exposed to atrazine show insulin resistance or type 2 diabetes (T2D), suggesting that exposure to these xenobiotics during organismal development can result in diabetes and that different mechanisms underlie pesticide mediated diabetes. We show that oxidative stress-mediated c-Jun N-terminal kinase (JNK) signaling activation underlies insulin resistance in flies exposed to atrazine during their development while DDVP-mediated T1D involves activation of caspase-mediated cell death pathway. Mitigation of oxidative stress through over-expression of SOD2 in atrazine (20µg/ml) exposed flies, revealed significantly decreased oxidative stress levels and reduced phosphorylation of JNK. Moreover, glucose and Akt phosphorylation levels in SOD2 over-expression flies exposed to atrazine were comparable to those in controls, suggesting restoration in insulin sensitivity. Therefore, exposure to xenobiotics during development is a common risk factor for the development of type 1 or type 2 diabetes. Accordingly, the present study cautions against the use of such diabetogenic pesticides. Also, mitigation of oxidative stress or anti-oxidant supplementation could be a potential therapy for xenobiotic mediated type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Resistência à Insulina/genética , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Atrazina/toxicidade , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diclorvós/toxicidade , Modelos Animais de Doenças , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Insulina/genética , Proteínas Quinases JNK Ativadas por Mitógeno , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/genética , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/genética , Xenobióticos/toxicidadeRESUMO
Rhamnolipid produced from a Lysinibacillus sphaericus IITR51 was characterized and its ability for dissolution of hydrophobic pesticides were evaluated. L. sphaericus produced 1.6â¯g/L of an anionic biosurfactant that reduced surface tension from 72â¯N/m to 52â¯N/m with 48% emulsification index. The biosurfactant was found stable over a wide range of pH (4.0-10.0), temperature (4-100⯰C), salt concentration (2-14%) and was identified as rhamnolipid. At the concentration of 90â¯mg/L rhamnolipid showed enhanced dissolution of α-, ß-endosulfan, and γ-hexachlorocyclohexane up to 7.2, 2.9, and 1.8 folds, respectively. The bacterium utilized benzoic acid, chlorobenzene, 3- and 4-chlorobenzoic acid as sole source of carbon and was found resistant to arsenic, lead and cadmium. Furthermore, the isolated biosurfactant showed antimicrobial activities against different pathogenic bacteria. The results obtained indicate the usefulness of rhamnolipid for enhanced dissolution and thereby increasing the bioavailability.
Assuntos
Bacillus/química , Glicolipídeos/química , Praguicidas/química , Interações Hidrofóbicas e Hidrofílicas , Solubilidade , Tensão Superficial , TemperaturaRESUMO
Adaptive behaviour of an organism has relevance towards developing better resistance in subsequent generations following xenobiotic exposures. Using a genetically tractable and functional insect model, Drosophila melanogaster, we aimed to examine the resistance of the organism against repeated exposures of benzene, an industrial and environmental-chemical and a class I human carcinogen. While 100â¯mM benzene exposure to one-day old flies for seven days caused â¼95% mortality (F0), its exposure to subsequent generations of flies led a significant decrease in mortality with maximum survival (â¼85%) as evident at F28 generation. While burden of benzene and its toxic metabolites was higher in initial generations, in latter generations (F24-F28), concentrations of less toxic metabolites were higher. In parallel, improved metabolism, less oxidative stress, less induction of hsp60 and hsp70 and higher induction of hsp26 and hsp27 along with increased gene dose ratio of three genes (cyp6g1, mrp1, and cyp12d1) were observed in latter generations of benzene exposed flies with maximum benefit accrued in F28 generation. The resistance developed in flies of F28 generation had a negative impact on reproduction which might be due to a cost against selection. The study demonstrates development of benzene resistance in Drosophila with permanent genetic changes.
Assuntos
Benzeno/toxicidade , Proteínas de Drosophila/genética , Drosophila melanogaster/efeitos dos fármacos , Resistência a Medicamentos/genética , Estresse Oxidativo/efeitos dos fármacos , Animais , Benzeno/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Choque Térmico/genética , Inativação Metabólica , Estresse Oxidativo/genética , Reprodução/efeitos dos fármacosRESUMO
Neurotransmitters are signaling molecules which play a key role in the central nervous system allowing signal transmission in the neuronal synapses. The role of these compounds is very crucial in the biological systems. A rapid, sensitive, economical and derivatisation free method has been developed for the analysis of 15 neurotransmitters in a single run on liquid chromatography tandem mass spectrometry. These 15 neurotransmitters are categorized into 5 groups on the basis of their fragmentation pattern. The proposed method "ionic liquid based ultrasound assisted dispersive liquid-liquid microextraction" hyphenated with tandem mass spectrometry is the first report for the analysis of neurotransmitters in cell samples along with other two matrices (rat brain and plasma). All the parameters that influence the extraction efficiency are optimized with aid of response surface methodology and desirability profile. Under these optimized conditions the developed method has been validated. The limit of detection was in the range of (1) 0.021-0.912⯵gâ¯L-1 for rat brain samples, (2) 0.028-0.978⯵gâ¯L-1 for plasma samples and (3) 0.025-0.945⯵gâ¯L-1 for cell samples with good linearity behavior for all analytes in the concentration range of 0.04-200⯵gâ¯L-1 in all the three matrices. The coefficient of determination for all the neurotransmitters was found in the range of (1) (R2)â¯≥â¯0.996 to 0.999 for rat brain samples and (2) (R2)â¯≥â¯0.991 to 0.999 for plasma and cell samples. The intra-day and inter-day variations were found less than (1) 1.78% and 8.94% for rat brain samples, (2) 1.83% and 8.37% for plasma samples and (3) 1.64% and 8.04% for cell samples respectively. The method has mean recoveries varied between (1) 81-128% for brain samples, (2) 88-107% for plasma samples and (3) 91-104% for cell samples at different spiking levels. The optimized and validated method was found free from matrix interferences and successfully applied for quantitative determination of 15 neurotransmitters in the rat brain, plasma and cell samples.
Assuntos
Química Encefálica , Líquidos Iônicos/química , Microextração em Fase Líquida/métodos , Neurotransmissores/análise , Neurotransmissores/sangue , Sonicação/métodos , Animais , Limite de Detecção , Masculino , Ratos Wistar , Espectrometria de Massas em Tandem/métodosRESUMO
Alpha-synuclein (α-synuclein) aggregation and impairment of the Ubiquitin proteasome system (UPS) are implicated in Parkinson's disease (PD) pathogenesis. While zinc (Zn) induces dopaminergic neurodegeneration resulting in PD phenotype, its effect on protein aggregation and UPS has not yet been deciphered. The current study investigated the role of α-synuclein aggregation and UPS in Zn-induced Parkinsonism. Additionally, levodopa (L-Dopa) response was assessed in Zn-induced Parkinsonian model to establish its closeness with idiopathic PD. Male Wistar rats were treated with zinc sulfate (Zn; 20 mg/kg; i.p.) twice weekly for 12 weeks along with respective controls. In few subsets, animals were subsequently treated with L-Dopa for 21 consecutive days following Zn exposure. A significant increase in total and free Zn content was observed in the substantia nigra of the brain of exposed groups. Zn treatment caused neurobehavioral anomalies, striatal dopamine decline, and dopaminergic neuronal cell loss accompanied with a marked increase in α-synuclein expression/aggregation and Ubiquitin-conjugated protein levels in the exposed groups. Zn exposure substantially reduced UPS-associated trypsin-like, chymotrypsin-like, and caspase-like activities along with the expression of SUG1 and ß-5 subunits of UPS in the nigrostriatal tissues of exposed groups. L-Dopa treatment rescued from Zn-induced neurobehavioral deficits and restored dopamine levels towards normalcy; however, Zn-induced dopaminergic neuronal loss, reduction in tyrosine hydroxylase expression, and increase in oxidative stress were unaffected. The results suggest that Zn caused UPS impairment, resulting in α-synuclein aggregation subsequently leading to dopaminergic neurodegeneration, and that Zn-induced Parkinsonism exhibited positive L-Dopa response similar to sporadic PD.
Assuntos
Levodopa/farmacologia , Doença de Parkinson Secundária , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos/efeitos dos fármacos , Ubiquitina/metabolismo , Zinco/toxicidade , alfa-Sinucleína/metabolismo , Animais , Masculino , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/metabolismo , Doença de Parkinson Secundária/patologia , Ratos , Ratos WistarRESUMO
The presence of pesticides in food items and beverages is a big threat to humankind, and their quantitative estimation with high precision and accuracy is always a challenge for analytical chemists. Hence, a simple and rapid method is proposed for the simultaneous determination of 30 pesticides in beverages (alcoholic and non-alcoholic drinks). The proposed method hyphenated with triple quadrupole liquid chromatography mass spectrometry has only 2 min chromatographic runtime for the analysis of all the pesticides. All the factors affecting the extraction yield have been optimized using an experimental design; and under optimized conditions, the developed method has been validated. The detection limits for all the pesticides were in the range of 0.001-0.348 µg/L with good linearity in the concentration range of 0.01-80.0 µg/L. The coefficient of determination was in the range of (R2 ) ≥ 0.977 to 0.999 for all the pesticides. The method was also checked for the precision of the relative standard deviation, which was below 4.75 (intra-day) and 8.96% (inter-day). The recovery of the method was 92-138%.
Assuntos
Cerveja/análise , Bebidas/análise , Sucos de Frutas e Vegetais/análise , Praguicidas/isolamento & purificação , Vinho/análise , Cromatografia Líquida , Espectrometria de Massas , Praguicidas/químicaRESUMO
Antibiotics that are used excessively and disposed of improperly are categorized as emerging pollutants. The determination of micropollutants in water with an accurate and precise method is always a big challenge. Hence, a simple, rapid, sensitive, economical and almost eco-friendly method is proposed for the quantitative determination of 19 antibiotics. The proposed method, ultrasound-assisted emulsification microextraction and solidified floating organic droplet coupled with liquid chromatography and triple quadrupole mass spectrometry, has only a 3 min chromatographic run time for the determination of the 19 antibiotics. We report for the first time the use of the developed method for the quantitative determination of the antibiotics in waste water samples with better results in terms of higher sensitivity, cost-effectiveness, better detection limits and a greener approach compared to the earlier reported methods. The limits of detection and quantification were in the range of 0.003-0.236 and 0.013-0.834 µg/L, respectively, with good linearity in the concentration range of 0.01-64.0 µg/L. The correlation coefficient was ≥0.987-0.99 for each analyte. The developed method has been successfully applied for the determination of antibiotics in water samples.