Determination of Escitalopram in Biological Samples by Dispersive Liquid-Liquid Microextraction Combined with GC-MS/MS / 法医学杂志

Objective To establish a method for determination of escitalopram in biological samples by ultrasound-assisted ionic liquid-dispersive liquid-liquid microextraction combined with gas chromatography-tandem mass spectrometry （GC-MS/MS） and provide evidences for forensic determination of cases related to escitalopram. Methods The 1-hexyl-3-methylimidazolium hexafluorophosphate （[C6MIM][PF6]） was selected as an extract solvent to process biological samples. Ultrasound-assisted extraction was used on the samples. Then the samples were detected by GC-MS/MS. Results The linear range of escitalopram in blood and liver were 5.56-1 111.10 ng/mL and 0.025-5.00 mg/g, respectively. The correlation coefficient （r） were greater than 0.999, limit of detection （LOD） were 4.00 ng/mL and 2.00 μg/g, limit of quantitation （LOQ） were 14.00 ng/mL and 6.00 μg/g, respectively. The extraction recovery rates were all greater than 50%, the interday and intraday precision were less than 20%. Escitalopram was detected in blood and liver samples from the actual poisoning case by this method with a content of 1.26 μg/mL and 0.44 mg/g, respectively. Conclusion The ultrasound-assisted ionic liquid-dispersive liquid-liquid microextraction combined with GC-MS/MS is environment friendly, rapid, has good enriching effect and consumes less organic solvent and can be used for forensic determination of escitalopram related cases.

Application of Dispersive Liquid-Liquid Microextraction in Forensic Toxicological Analysis / 法医学杂志

Currently, the main sample pretreatment methods for forensic toxic analysis are liquid-liquid extraction （LLE） and solid-phase extraction （SPE）. As a simple, convenient, and low-cost LLE method, dispersion liquid-liquid microextraction （DLLME） has high enrichment factor and good extraction efficiency, and therefore has attracted the attention of many researchers in the field of toxicology analysis in recent years. As a multi-functional microextraction method, DLLME has been widely used in the analysis of pesticides, sleeping sedatives, drugs and heavy metal poisons in forensic toxic analysis. Meanwhile, it can also be used in combination with such a variety of analytical instruments as gas chromatography-electron capture detectors （GC-ECD）, high performance liquid chromatography-diode array detectors （HPLC-DAD）. As a sample pretreatment method, DLLME has the advantages of simple operation, less use of organic solvent, reliable results and good reproducibility, thus can meet the requirements of modern court toxic analysis.

Avaliação da presença de cocaína e anfetamina em amostras de sangue post mortem e de indivíduos vivos, utilizando técnica de microextração em fase líquida (HF-LPME) / Amphetamine, cocaine and tetrahydrocannabinol evaluation in blood samples of living people and post mortem blood samples using microextraction technique in liquid phase (HF-LPME)

Estima-se atualmente que mais de 5% da população mundial vem fazendo uso recreativo de algum tipo de substância psicoativa, sendo que o direito a esse uso é tema recorrente da sociedade contemporânea. Por apresentar riscos associados à saúde e a segurança das populações, o uso abusivo dessas substâncias tem instigado a toxicologia social na busca de respostas, com as quais se possa caracterizar, analisar e gerenciar esses riscos. Drogas de grande consumo no Brasil são a anfetamina, cocaína e Cannabis sativa. Esta tese desenvolveu uma nova metodologia para detectar e quantificar anfetamina, cocaína e tetrahidrocanabinol em sangue total, com uso de microextração em fase líquida via fibra de polipropileno (HF-LPME), seguida de cromatografia gasosa acoplada a espectrometria de massa (GC-MS). Trata-se de uma técnica que apresenta vantagens sobre as tradicionais, uma vez que demanda quantidades menores de solvente orgânico, diminuindo riscos e custos de processo. Também propôs um estudo com a aplicação dos métodos em 69 amostras de sangue de vivos e de post mortem, as quais foram obtidas por convênio com a superintendência da polícia técnica científica de São Paulo (SPTC/SP). Os métodos desenvolvidos foram validados de acordo com diretrizes internacionais de interesse forense. Como resultado da validação, os métodos desenvolvidos se mostraram precisos e exatos para anfetamina e cocaína. O limite de detecção da cocaína foi de 5 ng . mL-1 e o limite de quantificação de 10 ng . mL-1. Quanto a anfetamina, os limites de detecção e de quantificação foram de 5 ng . mL-1. A técnica de HF-LPME não foi aplicável ao tetraidrocanabinol (Δ9-THC). Como resultado da análise das amostras, 40% delas apresentaram resultados positivos para cocaína. Desses positivos, 35% foram oriundos das matrizes de sangue de vivos e 64% oriundos de sangue post mortem. Nenhuma delas apresentou resultado quantificável para anfetamina

It is currently estimated that more than 5% of the world's population has been doing recreational use of some kind of psychoactive substances and the legal right to such use is a recurring theme debated by contemporary society. Due to the risks associated with populations health and safety, the abusive use of these substances has been instigating by social toxicology to search for answers to characterize, analyze and manage these risks. Drugs of great consumption in Brazil are, amphetamine cocaine and marijuana. This thesis proposes to develop a new methodology to detect and quantify psychoactive drugs in whole blood with the use of liquid phase microextraction by polypropylene fiber (HFLPME), followed by gas chromatography coupled to mass spectrometry (GC-MS). It is a technique that presents advantages compared with traditional ones, because of the smaller amounts demands of organic solvent, reducing risks and process costs. It also proposes a study with 69 blood samples taken from living persons and post mortem blood samples, which were obtained by agreement with the Superintendency of São Paulo Scientific Technical Police (SPTC / SP). The methods developed were validated according to international guidelines of forensic interest. As a result of the validation, the methods developed were precise and accurate for amphetamine and cocaine. The limit of cocaine detection was 5 ng . mL-1 and the limit of quantification was 10 ng . mL-1. As for amphetamine, the limits of detection and quantification were 5 ng . mL-1. The HF-LPME technique was not applicable to tetrahydrocannabinol (Δ9-THC). As a result of the sample analysis, 40% of them presented positive results for cocaine. Of these, 35% were from blood samples taken from living persons and 64% from the post mortem blood samples. None of the samples presented quantifiable results for amphetamine

Validation of an analytical method for determination of 13 priority polycyclic aromatic hydrocarbons in mineral water using dispersive liquid-liquid microextraction and GC-MS

Dispersive liquid-liquid microextraction [DLLME] combined with gas chromatography-mass spectrometry [GC-MS] was used for the extraction and determination of 13 polycyclic aromatic hydrocarbons [PAHs] in mineral water samples. In this procedure, the suitable combination of extraction solvent [500 microL chloroform] and disperser solvent [1000 microL acetone] were quickly injected into the water sample [10.00 mL] by Hamilton syringe. After centrifugation, 500 microL of the lower organic phase was dried under a gentle stream of nitrogen, re-dissolved in chloroform and injected into GC-MS. Chloroform and acetone were found to be the best extraction and disperser solvent, respectively. Validation of the method was performed using spiked calibration curves. The enrichment factor ranged from 93 to 129 and the recovery ranged from 71 to 90%. The linear ranges for all the PAHs were 0.10-2.80 ngmL-1. The relative standard deviations [RSDs] of PAHs in water by using anthracene-d[10] as internal standard, were in the range of 4-11% for most of the analytes [n=3]. Limit of detection [LOD] for different PAHs were between 0.03 and 0.1 ngmL-1. The method was successfully applied to the analyze of PAHs in mineral water samples collected from Tehran

Desenvolvimento de métodos analíticos para a identificação de drogas facilitadoras de crime em amostras de urina / Developing analytical methods for identification of drug-facilitated crime in urine samples

As drogas facilitadoras de crime (DFC) são uma série de substâncias químicas que permitem o ato sexual e/ou roubo com pouca ou nenhuma resistência da vítima. Benzodiazepínicos, gama-hidroxibutirato (GHB), cetamina e etanol são clássicas DFC, porém outras substâncias também têm sido utilizadas. Devido às diferentes classes de DFC e a necessidade de métodos sensíveis, a determinação dessas substâncias é um desafio aos toxicologistas forenses. A proposta do estudo foi desenvolver métodos analíticos para determinação principais analitos alvos de DFC para benzodiazepínicos, cetamina e GHB em amostras de urina. Esta matriz biológica é considerada uma amostra não-invasiva e apresenta um período de detecção maior que o sangue. A preparação das amostras foi avaliada através de microextração em fase líquida (LPME) e extração líquido-líquido (LLE). A LPME é uma técnica de extração de drogas que utiliza menor quantidade de solventes orgânicos, maior praticidade e possibilidade de obtenção de altos valores de recuperação. Os analitos foram determinados por cromatografia gasosa acoplada à espectrometria de massas (GC-MS). A LPME validada para benzodiazepínicos e seus produtos de biotransformação exigiu uma combinação de solventes e dupla derivatização para atingir a sensibilidade exigida, enquanto o método para determinação de cetamina, norcetamina e deidronorcetamina utilizou óleo essencial de eucalipto como meio extrator, caracterizando-se um procedimento ecologicamente correto com alta sensibilidade. A extração de GHB foi efetiva por LLE com redução da quantidade de solvente e tempo de análise sem o prejuízo na sensibilidade. Em geral, os métodos desenvolvidos neste trabalho são sensíveis e confiáveis para todos os analitos relatados e conclui-se que a LPME é uma técnica de preparo de amostra eficiente, versátil de baixo custo. Estas condições permitem que sua implementação em qualquer laboratório de análises toxicológicas, podendo ser aplicada em situações de DFC ou de qualquer outra natureza

Drug-facilitated crime (DFC) are a series of chemicals that allow the sexual act and/or theft with little or no resistance from the victim. Benzodiazepines, gamma-hydroxybutyrate (GHB) and ketamine and ethanol are considered classic DFC, however other substances were also used as the DFC. Due to the different classes of DFC and the need for sensitive methods, the determination of these substances is a challenge to forensic toxicologists. The purpose of this study was to develop analytical methods for determination of the main target analytes of DFC for benzodiazepines, ketamine and GHB in urine samples. This biological matrix is considered a non-invasive sample and shows a larger window of detection than blood. Sample preparation was assessed using liquid phase microextraction (LPME) and liquid-liquid extraction (LLE). The LPME is a drug extraction technique that uses less organic solvents, greater practicality and possibility of obtaining high recovery values. The analytes were determined by gas chromatography - mass spectrometry (GC-MS). The validated LPME technique for benzodiazepines and their metabolites required a combination of solvents and double derivatization to achieve the required sensitivity, while the ketamine, norketamine and dehydronorketamine method used essential oil of eucalyptus as solvent, characterizing a green chemistry approach with high sensitivity. The extraction of GHB was effective by LLE with a reduced amount of solvent and the analysis time without loss in sensitivity. In general, the methods developed in this work using GC-MS are sensitive and reliable for all analytes reported and LPME technique showed to be an efficient sample preparation, versatile and low cost. These conditions allow LPME implementation in any laboratory of toxicological analysis and it can be applied in situations of DFC or any other kind of analysis

Redistribuição postmortem de antidepressivos e seus produtos de biotransformação em tecidos biológicos humanos / Postmortem redistribution of antidepressants and their metabolites in human biological tissues

Os antidepressivos pertencem a uma importante classe de medicamentos investigados na toxicologia forense. Em casos de amostras provenientes de cadáveres, o intervalo entre o óbito e a obtenção da espécie biológica pode proporcionar a redistribuição postmortem destes fármacos. Com o objetivo de elucidar esse fenômeno, métodos analíticos foram desenvolvidos e aplicados utilizando sangue total (ST), humor vítreo (HV) e fígado. Para as amostras de ST e HV, o método de extração escolhido e validado foi a microextração em fase líquida (LPME) trifásica. Fibras ocas constituídas de polipropileno, com a extensão de 8 cm cada, foram tratadas com o solvente orgânico dodecano (fase orgânica), resultando em um membrana com permeabilidade seletiva. No lúmen destas fibras, adicionou-se ácido fórmico 0,1 mol/L (fase aceptora). Em frasco de fundo chato com 5 mL de capacidade, pipetou-se 3,5 mL de NaOH 0,1 mol/L (fase doadora) e 0,5 mL de ST ou HV. Ao término da extração, as amostras foram introduzidas no GC-MS, sem a necessidade de reações de derivatização. O estudo com ST contemplou os antidepressivos amitriptilina (AMI), nortriptilina (NTR), imipramina (IMI), desipramine (DES), clomipramina (CLO), desmetilclomipramina (DMC), fluoxetina (FLU) e norfluoxetina (NFL). Os limites de quantificação para estas substâncias ficaram inferiores aos níveis terapêuticos (20 ng/mL). As médias dos coeficientes de variação intradia e interdia foram, respectivamente, de 9,7 e 9,8%. As curvas de calibração apresentaram linearidade entre as concentrações de 20 até 1200 ng/mL. A validação do parâmetro integridade da diluição assegurou a mensuração de quantidades superiores ao limite apresentado na curva de calibração. O método foi aplicado em sete amostras reais postmortem e em apenas um caso foi observada uma diferença significativa (300%) entre os valores quantificados no ST periférico e central. Os antidepressivos tricíclicos AMI, NTR, IMI e DES foram avaliados no HV e o efeito matriz foi detectado para os dois últimos analitos. O método foi otimizado e validado utilizando solução salina adicionada de AMI e NTR. O limite de detecção igual a 5 ng/mL, foi obtido com a redução da voltagem da fonte de íons do espectrômetro de massa para 50 eV. Coeficientes de variação foram inferiores a 15%. Os procedimentos validados foram aplicados em seis amostras reais de HV. A relação encontrada entre os valores obtidos no ST periférico e HV foi de aproximadamente 0,1. A extração acelerada por solvente (ASE) e, posteriormente, a extração em fase sólida (SPE) foram as técnicas de separação dos analitos da matriz fígado. Ao término das citadas extrações, os antidepressivos foram analisados no GC-MS. Para esta matriz sólida, são necessários mais estudos, pois os valores encontrados nos ensaios analíticos estão em desacordo com as diretrizes utilizadas na validação dos métodos

Antidepressants belong to an important class of drugs investigated in forensic toxicology. In cases of samples from corpses, the interval between death and obtaining the biological specimens can provide the postmortem redistribution of these drugs. Aiming to elucidate this phenomenon, analytical methods were developed and applied using whole blood (WB), vitreous humor (VH) and liver. For samples of WB and HV, the extraction method chosen and validated was the three-phase liquid phase microextraction (LPME). Hollow fibers consist of polypropylene, with a length of 8 cm each were treated with dodecane organic solvent (organic phase) resulting in a membrane with selective permeability. Into the lumen of these fibers was added formic acid 0.1 mol/ L (acceptor phase). In the vial containing 3.5 mL of NaOH 0.1 mol / L (donor phase) was spiked 0.5 ml of biological fluids (WB or VH). Subsequently, the samples were injected in GC-MS without derivatization reactions. The study of the ST included antidepressants amitriptyline (AMI), nortriptyline (NTR), imipramine (IMI), desipramine (DES), clomipramine (CLO), desmethylclomipramine (DMC), fluoxetine (FLU) and norfluoxetine (NFL). The quantification limits for these substances were below the therapeutic levels (20 ng / ml). The mean coefficients of variation and separate intradays were respectively 9.7 and 9.8%. The calibration curves showed linearity between concentrations of 20 to 1200 ng / mL. The validation of the integrity of the dilution parameter assured measurement higher than the limit shown in the calibration curve quantities. The method was applied to seven real postmortem samples and in one case a significant difference (300%) between the measured values in the peripheral and central ST was observed. The tricyclic antidepressants AMI, NTR, IMI and DES were evaluated in VH and the matrix effect was detected in the last two analytes. The method was optimized and validated using saline spiked AMI and NTR. The limit of detection (5 ng/ml) was obtained by reducing the voltage of the ion source of the mass spectrometer 50 eV. Coefficients of variation were below 15%. The procedures were validated in six real samples of HV. The relationship found between the values obtained in the peripheral ST and HV was approximately 0.1. Accelerated solvent extraction (ASE) and subsequently the solid phase extraction (SPE) were the techniques of separation of analytes liver matrix. At the end of the cited extractions, antidepressants were analyzed in GC-MS. To this solid tissue, further studies are needed, because the values found in the analytical tests were not in accordance with the guidelines used in the validation of the methods

Occupational exposure to mercury: air exposure assessment and biological monitoring based on dispersive ionic liquid-liquid microextraction

Exposure to mercury [Hg] as a heavy metal can cause health effects. The objective of this study was to assess occupational exposure to Hg in a chlor-alkali petrochemical industry in Iran by determining of Hg concentrations in air, blood and urine samples. The study was performed on 50 exposed subjects and 50 unexposed controls. Air samples were collected in the breathing zone of exposed subjects, using hopcalite sorbents. Analysis was performed using a cold vapor atomic absorption spectrophotometer [CV-AAS] according to NIOSH analytical method 6009. For all participants, blood and urine samples were collected and then transferred into sterile glass tubes. After micro-extraction with ionic liquid and back extraction with nitric acid, Hg concentrations in blood and urine samples were determined by CV-AAS. The mean concentration of air Hg was 0.042 +/- 0.003 mg/m[3]. The mean concentrations of Hg in blood and urine samples of exposed subjects were significantly higher than unexposed controls [22.41 +/- 12.58 versus 1.19 +/- 0.95 micro g/l and 30.61 +/- 10.86 versus 1.99 +/- 1.34 micro g/g creatinine, respectively]. Correlation of air Hg with blood Hg, urine Hg and blood Hg-urine Hg ratio were significant statistically [P< 0.05]. The values of Hg in blood and urine samples of chlor-alkali workers were considerably high. Correlation coefficients showed that blood Hg and blood Hg-urine Hg ratio are better indicators than urine Hg for assessing occupationally exposed workers in terms of current exposure assessment

Analysis of chlorpheniramine in human urine samples using dispersive liquid-liquid microextraction combined with high-performance liquid chromatography

A simple and environmentally friendly microextraction technique was used for determination of chlorpheniramine (CPM), an antihistamine drug, in human urine samples using dispersive liquid-liquid microextraction (DLLME) followed by high performance liquid chromatography with diode array detection (HPLC-DAD). In this extraction technique, an appropriate mixture of acetonitrile (disperser solvent) and carbon tetrachloride (extraction solvent) was rapidly injected into the urine sample containing the target analyte. Tiny droplets of extractant were formed and dispersed into the sample solution and then sedimented at the bottom of the conical test tube by centrifugation. Under optimal conditions, the calibration curve was linear in the range of 0.055-5.5 µg mL-1, with a detection limit of 16.5 ng mL-1. This proposed method was successfully applied to the analysis of real urine samples. Low consumption of toxic organic solvents, simplicity of operation, low cost and acceptable figures of merit are the main advantages of the proposed technique.

Utilizou-se uma técnica de microextração simples e ambientalmente amigável para a determinação de clorfeniramina (CPM), anti-histamínico, em amostras de urina humana, utilizando a microextração dispersiva líquido-líquido (DLLME), seguida por cromatografia líquida de alta eficiência com detecção por arranjo de diodos (HPLC-DAD). Nesse método de extração, mistura apropriada de acetonitrila (solvente dispersor) e tetracloreto de carbono (solvente de extração) foi injetada rapidamente na amostra de urina contendo o analito alvo. As pequenas gotículas de agente de extração foram formadas e dispersas na solução da amostra e, em seguida, sedimentadas no fundo do tubo cônico de ensaio por centrifugação. Em condições ótimas, a curva de calibração foi linear no intervalo entre 0,055 e 5,5 µg mL-1, com limite de detecção de 16,5 ng mL-1. O método proposto foi aplicado com sucesso na análise de amostras de urina reais. Baixo consumo de solventes orgânicos tóxicos, simplicidade de operação, baixo custo e figuras de mérito aceitáveis são as principais vantagens do método sugerido.

[Determination of furanic compounds from coffee powders in Tehran market using liquid phase microextraction followed by gas chromatography-mass spectrometry]

Furan was classified as "possibly carcinogenic to humans" by the International Agency for Research on Cancer in 1995. The joint FAO/WHO Committee set the maximum permissible furan at 2 mcg/kg body weight/day in 2010. The furan content of coffee is high as compared to other processed foods. Considering the increasing trend of coffee consumption in Iran, this study was initiated to determine the furan content of different kinds of coffee powder available in Tehran Market by headspace liquid-phase microextraction coupled with gas chromatography mass spectrometry [HS-LPMEGC-MS]. The e CCD mployed included 32 treatments at 5 levels for 4 factors with 8 replicates of center point. The furanic compounds from 66 different coffee samples were extracted by HSLPME atoptimal extraction conditions [salt 0 gram, stirrer rate 700 rpm, extraction temperature 45°C, and extraction time 15 min]. The effect of coffee brewing and coffee mix preparation on furanic compound content of coffee was also determined. The proposed method was validated by determining linearity, repeatability, recovery, enrichment factor, LOD, and LOQ. Determination of furan in coffee samples showed that there were significant differences [p<0.05] in furan concentration of different coffees [prepared by different methods] and that preparation method was the most important factor influencing the furan content of coffee. The coffee brewing and preparation of instant coffee and coffee mixes reduced furanic compounds concentration except furfural. The lowest and highest concentrations of furan in commercial coffee products were10 and 6320 ppb, respectively. In the present work, a simple, fast micro-extraction method [HS-LPME] for extraction and pre-concentration of furanic compounds in coffee samples was developed and validated. The advantages of this method are reduced solvent use, low-cost equipment use, simple experimental setup, acceptable precision and accuracy, a high enrichment factor, and no matrix interference. Differences detected in the furanic compound contents in the coffee samples are due to different green coffee bean species, coffee production process [roasting condition [time and temperature], time of degassing, and grade of grinding process]. Brewing coffee in open systems can result in decreases in the content of these compounds to an acceptable level

Extraction and determination of cyproheptadine in human urine by DLLME-HPLC method

Novel dispersive liquid-liquid microextraction [DLLME], coupled with high performance liquid chromatography with photodiode array detection [HPLC-DAD] has been applied for the extraction and determination of cyproheptadine [CPH], an antihistamine, in human urine samples. In this method, 0.6 mL of acetonitrile [disperser solvent] containing 30 micro L of carbon tetrachloride [extraction solvent] was rapidly injected by a syringe into 5 mL urine sample. After centrifugation, the sedimented phase containing enriched analyte was dissolved in acetonitrile and an aliquot of this solution injected into the HPLC system for analysis. Development of DLLME procedure includes optimization of some important parameters such as kind and volume of extraction and disperser solvent, pH and salt addition. The proposed method has good linearity in the range of 0.02-4.5 micro g mL[-1] and low detection limit [13.1 ng mL[-1]]. The repeatability of the method, expressed as relative standard deviation was 4.9% [n = 3]. This method has also been applied to the analysis of real urine samples with satisfactory relative recoveries in the range of 91.6-101.0%

Vortex-assisted ionic liquid microextraction coupled to flame atomic absorption spectrometry for determination of trace levels of cadmium in real samples

A simple and rapid vortex assisted ionic liquid based liquid-liquid microextraction technique [VALLME] was proposed for preconcentration of trace levels of cadmium. According to this method, the extraction solvent was dispersed into the aqueous samples by the assistance of vortex agitator. Cadmium preconcentration was mediated by chelation with the 8-hydroxyquinoline; [oxine] reagent and an IL, 1-octyl-3-methylimidazolium hexafluorophosphate [[Omim][PF[6]] was chosen as the extraction solvent to extract the hydrophobic complex. Several variables such as sample pH, concentration of oxine, volume of [Omim][PF[6]] and extraction time were investigated in details and optimum conditions were selected. Under the optimum conditions, the limit of detection [LOD] was 2.9 microg L[-1] for Cd [] and relative standard deviation [RSD%] for five replicate determinations of 125 microg L[-1] was 4.1%. The method was successfully applied to the determination of cadmium in tap water, apple and rice samples