Detection of Carbamazepine and Its Metabolites in Blood Samples by LC-MS/MS / 法医学杂志

OBJECTIVES@#To establish a method for the detection of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS).@*METHODS@#The blood samples were treated with 1-butyl-3-methylimidazolium hexafluorophosphate as an extraction solvent. The samples were extracted by ultrasound-assisted extraction and separated by ZORBAX Eclipse Plus C18, 95Å column. The mobile phase A aqueous solution containing 0.1% formic acid and 10 mmol/L ammonium acetate, and mobile phase B mixed organic solvent containing acetonitrile/methanol (Vacetonitrile∶Vmethanol=2∶3) were used for gradient elution at the flow rate of 1.00 mL/min. An electrospray ion source in positive mode was used for detection in the multiple reaction monitoring.@*RESULTS@#The linearities of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples were good within the corresponding range, with correlation coefficients (r) greater than 0.995 6. The limits of detection were 3.00, 0.40 and 1.30 ng/mL, respectively. The limit of quantitation were 8.00, 1.00 and 5.00 ng/mL, respectively. The extraction recoveries ranged from 76.00% to 106.44%. The relative standard deviations of the intra-day and inter-day precisions were less than 16%. Carbamazepine and its main metabolite 10,11-dihydro-10,11-epoxycarbamazepine were detected in blood samples of death cases with a mass concentration of 2.71 μg/mL and 252.14 ng/mL, respectively.@*CONCLUSIONS@#This method has high sensitivity and good selectivity, which is suitable for the detection of carbamazepine and its metabolites in blood samples, and can be used for carbamazepine-related forensic identifications.

Uso da microextração por sorbente empacotado (MEPS) para preparo de amostras em análises toxicológicas envolvendo fármacos benzodiazepí­nico / Microextraction by packed sorbent (MEPS) for sample preparation in toxicological analyses involving benzodiazepines

A microextração por sorbente empacotado (MEPS) é uma técnica de preparo de amostras ainda pouco utilizada no âmbito da toxicologia, em que os mesmos princípios da extração em fase sólida convencional são adaptados para uma escala miniaturizada. As principais vantagens da técnica estão associadas ao pequeno volume de amostra e de solventes utilizados, à possibilidade de realizar múltiplas extrações com um mesmo cartucho e à facilidade de automação. Os benzodiazepínicos possuem grande relevância na toxicologia dada sua ampla utilização e seus efeitos que podem, por exemplo, comprometer a capacidade de dirigir, além do uso abusivo, e como drogas facilitadoras de crimes. Neste trabalho, um método de MEPS foi desenvolvido e otimizado para a determinação de sete benzodiazepínicos e seus produtos de biotransformação (diazepam, clonazepam, flunitrazepam, alprazolam, bromazepam, 7-aminoflunitrazepam e nordiazepam) utilizando 100 µL de amostra de sangue total post mortem. Após a extração, os eluatos foram analisados por cromatografia líquida em fase reversa acoplada a espectrometria de massas. O método foi validado de acordo com as recomendações do Scientific Working Group for Forensic Toxicology, apresentando linearidade adequada de 5 a 500 ng.mL-1 . Os valores de exatidão (90,4 a 109,5%), precisão intra-dia (2,5 a 10,7 %CV) e inter-dia (1,1 a 8,0 %CV) também foram satisfatórios. MEPS foi realizada mais de 60 vezes com a mesma fase extratora sem evidências de contaminação cruzada. Dez amostras reais fornecidas pelo Instituto Médico Legal de São Paulo foram analisadas. Foram quantificados diazepam, nordiazepam, clonazepam e bromazepam. Os resultados encontrados em cada uma das amostras foram comparados com dados da literatura

Microextraction by packed sorbent (MEPS) is a sample preparation technique still little used in toxicology, where the same principles of conventional solid phase extraction are adapted to a miniaturized scale. The main advantages of the technique are associated with the small volume of sample and solvents required, the possibility of performing multiple extractions with the same cartridge and ease process automation. Benzodiazepine drugs are relevant in toxicology because of their widespread use, and effects (which may, for example, compromise the ability to drive vehicles), abuse and records as crime-facilitating drugs. In this work, a MEPS method was developed and optimized for a determination of seven benzodiazepines and their metabolites (diazepam, nordiazepam, clonazepam, flunitrazepam, 7-aminoflunitrazepam, alprazolam, and bromazepam) using 100 µL of post mortem whole blood. After extraction, the eluates were analyzed by reversed-phase liquid chromatography coupled to mass spectrometry. The method was validated according to the recommendations of the Scientific Working Group for Forensic Toxicology, presenting adequate linearity from 5 to 500 ng.mL-1 . The values of accuracy (90.4 to 109.5%), intra-day precision (2.5 to 10.7 %CV) and inter-day (1.1 to 8.0 %CV) also presented satisfactory results. MEPS was performed more than 60 times with the same extractive phase without compromising the results with the evidence of carryover. Institute of Legal Medicine were submitted to analysis by MEPS-LC-MS/MS. In these samples, the following analytes were quantified: diazepam, nordiazepam, clonazepam and bromazepam. The results found in each of the samples were compared with data from the literature

A 2D-QSPR approach to predict blood-brain barrier penetration of drugs acting on the central nervous system

Drugs acting on the central nervous system (CNS) have to cross the blood-brain barrier (BBB) in order to perform their pharmacological actions. Passive BBB diffusion can be partially expressed by the blood/brain partition coefficient (logBB). As the experimental evaluation of logBB is time and cost consuming, theoretical methods such as quantitative structure-property relationships (QSPR) can be useful to predict logBB values. In this study, a 2D-QSPR approach was applied to a set of 28 drugs acting on the CNS, using the logBB property as biological data. The best QSPR model [n = 21, r = 0.94 (r² = 0.88), s = 0.28, and Q² = 0.82] presented three molecular descriptors: calculated n-octanol/water partition coefficient (ClogP), polar surface area (PSA), and polarizability (α). Six out of the seven compounds from the test set were well predicted, which corresponds to good external predictability (85.7%). These findings can be helpful to guide future approaches regarding those molecular descriptors which must be considered for estimating the logBB property, and also for predicting the BBB crossing ability for molecules structurally related to the investigated set.

Fármacos que atuam no sistema nervoso central (SNC) devem atravessar a barreira hematoencefálica (BHE) para exercerem suas ações farmacológicas. A difusão passiva através da BHE pode ser parcialmente expressa pelo coeficiente de partição entre os compartimentos encefálico e sanguíneo (logBB, brain/blood partition coefficient). Considerando-se que a avaliação experimental de logBB é dispendiosa e demorada, métodos teóricos como estudos das relações entre estrutura química e propriedade (QSPR, Quantitative Structure-Property Relationships) podem ser utilizados na previsão dos valores de logBB. Neste estudo, uma abordagem de QSPR-2D foi aplicada a um conjunto de 28 moléculas com ação central, usando logBB como propriedade biológica. O melhor modelo de QSPR [n = 21, r = 0,94 (r² = 0,88), s = 0,28 e Q² = 0,82] apresentou três descritores moleculares: o coeficiente calculado de partição n-octanol/água (ClogP), área de superfície polar (PSA) e polarizabilidade (α). Seis dos sete compostos do conjunto de avaliação foram bem previstos pelo modelo, o que corresponde a um bom poder de previsão externa (85,7%). Os resultados obtidos podem auxiliar de forma relevante em estudos futuros, orientando quais descritores moleculares devem ser considerados para estimar logBB e prever a passagem através da BHE de moléculas estruturalmente relacionadas às do conjunto investigado.

Prevalence of substance use among trauma patients treated in a Brazilian emergency room / Prevalência do uso de substâncias em pacientes com traumas em um pronto socorro brasileiro

OBJECTIVE: Although there is a considerable amount of data in the literature regarding the association between alcohol consumption and injuries treated in emergency rooms, little is known about the relationship between such injury and the use of other substances. The objective of this study was to estimate the prevalence of substance use in patients admitted to the emergency room for non-fatal injuries. METHOD: A prospective cross-sectional study assessing all patients admitted to the emergency room within 6 hours after a non-fatal injury was conducted over a three-month period. The following were used as measures of alcohol and drug use: a standardized World Health Organization questionnaire; a self-administered questionnaire related to drug consumption within the 24 hours preceding contact; the Drug Abuse Screening Test; urine screens for cannabis, cocaine and benzodiazepines; and determination of blood alcohol concentration. Descriptive analyses were performed and the confidence interval used was 95 percent. RESULTS: A total of 353 patients were included. Cannabis and cocaine screens were conducted for 242 patients and benzodiazepine screens were conducted for 166. Blood alcohol concentrations reached the level of positivity in 11 percent (n = 39), and 10 percent (n = 33) presented some degree of intoxication. Among the 242 patients screened, 13.6 percent (n = 33) tested positive for cannabis, and 3.3 percent (n = 8) tested positive for cocaine, whereas 4.2 percent (n = 7) of the 166 patients screened tested positive for benzodiazepines. CONCLUSIONS: Substance use was highly prevalent among these individuals. In this sample, the frequency for the use of cannabis (an illicit drug) was comparable to that of alcohol. More studies are needed in order to characterize such use among Brazilians and to develop proper approaches to such cases, with the aim of reducing substance use and its consequences.

OBJETIVO: Ainda que haja significativa literatura sobre a associação entre álcool e trauma, pouco se sabe sobre o uso de outras substâncias e trauma em pronto socorro. O objetivo do estudo foi estimar a prevalência do uso de substâncias em pacientes admitidos em um pronto socorro por trauma não fatal. MÉTODO: Um estudo prospectivo de corte transversal avaliando todos os pacientes admitidos dentro de 6 horas antes de trauma não fatal em pronto socorro durante um período de três meses. Um questionário padronizado pela Organização Mundial de Saúde, o auto-relato do consumo de drogas nas últimas 24 horas antes do contato; Drug Abuse Screening Test); screening de urina para maconha, cocaína e benzodiazepínicos e Concentração de Alcool no Sangue foram utilizados como medidas de avaliação do uso de álcool e drogas. Foram realizadas análises descritivas e o intervalo de confiança foi de 95 por cento. RESULTADOS: Foram incluídos 353 pacientes, tendo sido coletados screenings de maconha e cocaína de 242 pacientes e de 166 pacientes para benzodiazepínicos. A Concentração de Alcool no Sangue foi positiva em 11 por cento (n = 39) e 10 por cento (n = 33) apresentaram algum grau de intoxicação alcoólica. O teste de maconha foi positivo em 13,6 por cento (n = 33); respectivamente de cocaína em 3,3 por cento (n = 8) e de benzodiazepínicos em 4,2 por cento (n = 7). CONCLUSÕES: O uso de substâncias nesses indivíduos que sofreram trauma é altamente prevalente. Nesta amostra, a freqüência para maconha (uma droga ilícita) esteve próxima ao de álcool. Mais estudos são necessários a fim de identificar a realidade brasileira e elaborar identificações adequadas para estes casos, visando à redução do uso de substâncias e suas conseqüências.

Análisis farmacocinético del Loracepam en hombres y mujeres / Pharmacokinetics analysis of Lorazepam in men and women

Los informes de la evaluación cinética de los medicamentos desarrollados en los últimos 20 años documentan, prioritariamente, los resultados de ensayos realizados exclusivamente con varones; o bien, con muestras mixtas por cuyos resultados no se reagrupan ni analizan por la variable de sexo...

1H-NMR spectroscopic quantitative determination of clobazam

The 1H-NMR spectrum of clobazam showed a sharp singlet at 3.49 ppm assigned for the N-CH3, which was chosen for the quantitative measurements. The method involves comparing the integral of this signal to that of a sharp singlet at sigma 6.41 ppm assigned for maleic acid, used as internal standard for comparison. A standard curve was plotted the integration ratio Ic/Im and the amount of clobazam in mg. Good results were obtained on application of this method on authentic sample of clobazam. The mean percentage recovery is 100.12 +/- 1.133. Good recoveries were also obtained on application of this method on tablet form [Frisium] using standard addition technique. The mean percentage recoveries are 100.45 +/- 1.899 and 100.45 +/- 0.659. The results were compared with the reported spectrophotometric method. According to the variance ratio test [F test] and Student's t-test, there is no significant difference between the two methods, with respect to the precision and accuracy