RESUMEN
Alcohol abuse and addiction is a public health issue of global concern. Wastewater-based epidemiology (WBE) is a forceful and effective complementary tool for investigating chemical consumption. This study examined alcohol consumption in major cities of China via WBE and compared WBE estimates with other data sources. A simple and valid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of two alcohol metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS) in wastewater. The optimized method was applied to 62 sewage samples collected from wastewater treatment plants (WWTPs) in 31 provincial capital cities across China in the fourth quarter of 2020. The methodology established in this study was validated with the lower limit of quantification (LLOQ) up to 0.1 µg/L, good linearity in the range of 0.1-50 µg/L, intra-day and inter-day precision less than 5.58% and 5.55%, respectively, and the recoveries of the extracts were higher than 97.14%. The consumption range of alcohol estimated via WBE was 6.09 ± 4.56 ethanol/person/day (EPD) in the capital cities of China. Alcohol consumption varies significantly between cities in China, with WBE estimating lower alcohol consumption than WHO and lower than foreign countries. Investing in alcohol consumption based on WBE has great potential to accurately and efficiently estimate alcohol consumption.
Asunto(s)
Espectrometría de Masas en Tándem , Monitoreo Epidemiológico Basado en Aguas Residuales , Humanos , Cromatografía Liquida , Espectrometría de Masas en Tándem/métodos , Consumo de Bebidas Alcohólicas/epidemiología , Etanol/análisis , China/epidemiologíaRESUMEN
7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50-4000 ng/mg and 30-6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.
Asunto(s)
Síndrome de Smith-Lemli-Opitz , Biomarcadores , Colesterol , Deshidrocolesteroles , Cabello , Humanos , Lactante , Síndrome de Smith-Lemli-Opitz/diagnósticoRESUMEN
BACKGROUND: Smith-Lemli-Opitz syndrome is a birth defect caused by the deficiency of 7-dehydrocholesterol reductase in cholesterol biosynthesis pathway, which leads to accumulation of 7-dehydrocholesterol and reduction of cholesterol in body fluids. To effectively diagnose Smith-Lemli-Opitz syndrome and monitor therapy, a reliable method for simultaneous detection of 7-dehydrocholesterol and cholesterol is needed. METHODS: In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), 50 µL of human plasma were hydrolyzed at 70â for 40 min with 1 M potassium hydroxide in 90% ethanol, and then 7-dehydrocholesterol and cholesterol were extracted by 600 µL of n-hexane for three times. After microwave-assisted derivatization with 70 µL of N,O-bis(trimethylsilyl)trifluoroacetamide at 460 W for 3 min, the analytes were measured by gas chromatography-mass spectrometry. RESULTS: The limits of detection were 100 ng/mL for 7-dehydrocholesterol and 300 ng/mL for cholesterol. Good linearity was obtained in the range of 1-600 µg/mL for 7-dehydrocholesterol and 10-600 µg/mL for cholesterol, which completely covered the biochemical levels of Smith-Lemli-Opitz syndrome patients that have been reported. CONCLUSION: A time-saving and accurate gas chromatography with mass spectrometry based method was developed for the determination of 7-dehydrocholesterol and cholesterol in human plasma, which also serves as a useful tool for Smith-Lemli-Opitz syndrome diagnosis, treatment, and research.
Asunto(s)
Síndrome de Smith-Lemli-Opitz , Colesterol , Deshidrocolesteroles/análisis , Deshidrocolesteroles/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Humanos , Síndrome de Smith-Lemli-Opitz/diagnóstico , Síndrome de Smith-Lemli-Opitz/metabolismoRESUMEN
N-Ethylpentylone (NEP) is one of the most recent novel stimulants, and there is limited understanding of its toxicity. Here we employed zebrafish model for analyzing the effects of NEP on early embryos and cardiovascular and nervous systems at late developmental stages. We first observed multi-malformations in early embryos and larvae after NEP administration, together with significant deregulations of brain and heart development-associated genes (neurog1, her6, elavl3, nkx2.5, nppa, nppb, tnnt2a) at transcriptional level. Low-dosed NEP treatment induced an anxiety-like phenotype in zebrafish larvae, while higher doses of NEP exerted an inhibitory effect on locomotion and heart rate. Besides, the expression of th (tyrosine hydroxylase) and th2 (tyrosine hydroxylase 2), identifying dopamine (DA) release, were significantly increased during one-hour free swimming after effective low-dosed NEP administration, along with the upregulation of gene fosab and fosb related to stress and anxiety response. D1R antagonist SCH23390 and D2R antagonist sulpiride partially alleviated the aberrances of locomotion and heart rate, indicating dopaminergic receptors were involved in the bidirectional dosage-dependent pattern of NEP-induced performance. Meanwhile, sulpiride offset the upregulated expression of th, th2 and fosab in the group of 1.5 µM NEP, which highlighted the significant role of D2R in NEP-induced locomotive effects. This study systematically described the developmental, neuronal and cardiac toxicity of NEP in zebrafish, and identified the dopaminergic receptors as one of the downstream effectors of NEP administration.
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Benzodioxoles/toxicidad , Butilaminas/toxicidad , Sistema Cardiovascular/efectos de los fármacos , Agonistas de Dopamina/toxicidad , Dopamina/metabolismo , Sistema Nervioso/efectos de los fármacos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D2/agonistas , Proteínas de Pez Cebra/agonistas , Animales , Animales Modificados Genéticamente , Sistema Cardiovascular/embriología , Sistema Cardiovascular/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Frecuencia Cardíaca/efectos de los fármacos , Larva/efectos de los fármacos , Larva/metabolismo , Locomoción/efectos de los fármacos , Masculino , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Transcripción Genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Accurate determination of a person's blood alcohol concentration (BAC) is an important task in forensic toxicology laboratories because of the existence of statutory limits for driving a motor vehicle and workplace alcohol testing regulations. However, making a correct interpretation of the BAC determined in postmortem (PM) specimens is complicated, owing to the possibility that ethanol was produced in the body after death by the action of various micro-organisms (e.g., Candida species) and fermentation processes. This article reviews various ways to establish the source of ethanol in PM blood, including collection and analysis of alternative specimens (e.g., bile, vitreous humor (VH), and bladder urine), the identification of non-oxidative metabolites of ethanol, ethyl glucuronide (EtG) and ethyl sulfate (EtS), the urinary metabolites of serotonin (5-HTOL/5-HIAA), and identification of n-propanol and n-butanol in blood, which are known putrefaction products. Practical utility of the various biomarkers including specificity and stability is discussed.
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Nivel de Alcohol en Sangre , Etanol/análisis , Glucuronatos/análisis , Serotonina/metabolismo , Ésteres del Ácido Sulfúrico/análisis , 1-Butanol/sangre , 1-Propanol/sangre , Autopsia , Ionización de Llama , Toxicología Forense , Cromatografía de Gases y Espectrometría de Masas , Serotonina/orina , Manejo de EspecímenesRESUMEN
N-Ethylpentylone (NEP) is one of the most confiscated synthetic cathinones in the world. However, its pharmacology and pharmacokinetics remain largely unknown. In this study, the pharmacokentics of NEP in rat nucleus accumbens (NAc) was assessed via brain microdialysis after the intraperitoneal (ip) administration of NEP (20 or 50 mg/kg). The concentrations of dopamine (DA) and serotonin (5-HT) and their metabolites, including 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), and 5-hydroxyindoleacetic acid (5-HIAA), were simultaneously monitored to elucidate the pharmacological effect of NEP. In addition, the plasma levels of NEP were also assessed. The pharmacokinetics of NEP showed a dose-related pattern, with NEP rapidly passing through the blood-brain barrier and reaching a maximum concentration (Cmax ) at approximately 40-minutes postdose. Approximately 4% of plasma NEP was distributed to the NAc, and considering a homogeneous brain distribution, over 90% of plasma NEP was potentially distributed to the brain. High values of area under curve (AUC) and mean residence time (MRT) of NEP were observed in both the NAc and plasma, indicating large and long-lasting effects. NEP elicited dose-related increases in microdialysate DA and 5-HT and increased the concentration of 3-MT in a dose-related manner. However, the rate of DA converted into 3-MT was unaffected. NEP had a negative effect on the rates of which DA and 5-HT were transformed into DOPAC and 5-HIAA, respectively. In summary, NEP rapidly entered the NAc and showed a long-lasting effect. In addition, DA increased more significantly than 5-HT, indicating a large potential for NEP abuse.
Asunto(s)
Benzodioxoles/farmacología , Butilaminas/farmacología , Dopamina/metabolismo , Núcleo Accumbens/efectos de los fármacos , Psicotrópicos/farmacología , Serotonina/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Benzodioxoles/farmacocinética , Barrera Hematoencefálica/metabolismo , Butilaminas/farmacocinética , Cromatografía Liquida , Estado de Conciencia , Dopamina/análogos & derivados , Relación Dosis-Respuesta a Droga , Ácido Hidroxiindolacético/metabolismo , Masculino , Microdiálisis , Núcleo Accumbens/metabolismo , Psicotrópicos/farmacocinética , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en TándemRESUMEN
N-ethylpentylone (NEP), a new synthetic cathinone, has been rising to be one of the most popular cathinone derivatives in recent years. However, research on NEP is rather limited. In this study, locomotor stimulation and sensitization, as well as anxiety-like behavior induced by NEP were studied in Sprague-Dawley rats, using the open field and elevated plus maze respectively. Rats were administered NEP (5, 20 or 50 mg/kg, intraperitoneal), with saline as the negative control and methamphetamine (5 mg/kg) as a positive control. Acute administration of NEP at all the doses tested significantly promoted locomotor activity, presenting an inverted U-shaped dose-effect curve. The highest activity was observed at the 20 mg/kg dose group, with the average distance traveled 18 times higher than the saline group. Repeated administration of NEP enhanced locomotor activity only at the 5 mg/kg dose group. After a week's withdrawal, re-challenge of NEP failed to induce marked behavioral sensitization. In elevated plus maze experiments, both acute and repeated administration of 20 mg/kg NEP induced anxiolytic-like effects, while no significant alteration was observed in the 5 and 50 mg/kg dose groups. In summary, acute administration of NEP caused significantly enhanced locomotor activity in rats at all the tested doses, while repeated NEP administration enhanced locomotor activity only at a low dose (5 mg/kg), while a high dose (20 mg/kg) of NEP induced anxiolytic-like effects after both acute and repeated administration.
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Ansiedad/tratamiento farmacológico , Benzodioxoles/farmacología , Butilaminas/farmacología , Locomoción/efectos de los fármacos , Alcaloides/metabolismo , Alcaloides/farmacología , Animales , Ansiolíticos/farmacología , Ansiedad/metabolismo , Conducta Animal/efectos de los fármacos , Benzodioxoles/metabolismo , Butilaminas/metabolismo , Masculino , Metanfetamina/farmacología , Actividad Motora/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
N-Ethylpentylone (NEP) is a popular synthetic cathinone abused worldwide. To obtain more information about its pharmacokinetics and pharmacodynamics, a rapid, simple and sensitive liquid chromatography-tandem mass spectrometry method was developed for the determination of NEP, two important neurotransmitters, dopamine and serotonin, and their metabolites, including 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine and 5-hydroxyindole-3-acetic acid, in rat brain microdialysate. The analytes were separated on a Phnomenex Polar C18 column, with a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) under gradient elution to shorten the total chromatographic run time. A triple quadruple mass spectrometer coupled with an electrospray ionization source in both positive and negative ion mode was used to detect the analytes. This method showed excellent accuracy (87.4-113.5%) and precision (relative standard deviation <15%) at three quality control levels. The limits of detection were 0.2 ng/mL for NEP and 0.2-50 nm for the others and good linearity was obtained. This study pioneered a method to integrate exogenous drugs and endogenous neurotransmitters as the drugs act on the same determination system, which means that this innovation can provide support for further study of the addictive effects of NEP or other synthetic cathinones on extracellular levels of dopamine and 5-hydroxytryptamine.
Asunto(s)
Benzodioxoles/análisis , Butilaminas/análisis , Cromatografía Líquida de Alta Presión/métodos , Dopamina/análisis , Núcleo Accumbens/química , Serotonina/análisis , Animales , Benzodioxoles/administración & dosificación , Benzodioxoles/farmacocinética , Butilaminas/administración & dosificación , Butilaminas/farmacocinética , Dopamina/metabolismo , Límite de Detección , Modelos Lineales , Microdiálisis , Núcleo Accumbens/metabolismo , Ratas , Reproducibilidad de los Resultados , Serotonina/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Only sporadic data are available on hair concentrations of diazepam and some of its metabolites (nordazepam, oxazepam, and temazepam) following a single controlled dose. The aim of this study was to investigate the deposition of diazepam and its metabolites in human hair after eight healthy volunteers (four women and four men, ages 24-26, East Asian) consumed 10 mg of diazepam. Hair was collected from all volunteers 1 month after exposure, and also 2 months post-exposure from men and 10 months post-exposure from women. Diazepam and the complete metabolite profile, including oxazepam glucuronide and temazepam glucuronide, were measured by ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with limits of quantifications (LOQs) of 0.5-2.5 pg/mg for diazepam, nordazepam, oxazepam, and temazepam, and of 10 pg/mg for oxazepam glucuronide and temazepam glucuronide. There were no differences by gender in the amounts of diazepam or metabolites found. The concentration of the main metabolite nordazepam was consistently higher than that of diazepam at both 1 and 2 months after consumption. Oxazepam and temazepam traces were found in some volunteers' hair, but the glucuronides were not detected. Diazepam and nordazepam levels at 10 months post-exposure were extremely low (near the LOQ), indicating drug loss by personal hygiene and physical handling. To our knowledge, this is the first single-dose diazepam study using black hair and the first study to include measurements of oxazepam glucuronide and temazepam glucuronide in human hair.
Asunto(s)
Diazepam/análisis , Cabello/química , Hipnóticos y Sedantes/análisis , Adulto , Pueblo Asiatico , Cromatografía Liquida , Diazepam/administración & dosificación , Femenino , Voluntarios Sanos , Humanos , Hipnóticos y Sedantes/administración & dosificación , Masculino , Nordazepam/análisis , Oxazepam/análogos & derivados , Oxazepam/análisis , Espectrometría de Masas en Tándem , Temazepam/análogos & derivados , Temazepam/análisis , Adulto JovenRESUMEN
A novel and simple online solid-phase extraction liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of diazepam and its five metabolites including nordazepam, oxazepam, temazepam, oxazepam glucuronide, and temazepam glucuronide in human oral fluid. Human oral fluid was obtained using the Salivette(®) collection device, and 100 µL of oral fluid samples were loaded onto HySphere Resin GP cartridge for extraction. Analytes were separated on a Waters Xterra C18 column and quantified by liquid chromatography with tandem mass spectrometry using the multiple reaction monitoring mode. The whole procedure was automatic, and the total run time was 21 min. The limit of detection was in the range of 0.05-0.1 ng/mL for all analytes. The linearity ranged from 0.25 to 250 ng/mL for oxazepam, and 0.1 to 100 ng/mL for the other five analytes. Intraday and interday precision for all analytes was 0.6-12.8 and 1.0-9.2%, respectively. Accuracy ranged from 95.6 to 114.7%. Method recoveries were in the range of 65.1-80.8%. This method was fully automated, simple, and sensitive. Authentic oral fluid samples collected from two volunteers after consuming a single oral dose of 10 mg diazepam were analyzed to demonstrate the applicability of this method.
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Automatización , Diazepam/análisis , Saliva/química , Extracción en Fase Sólida , Cromatografía Liquida , Diazepam/metabolismo , Humanos , Espectrometría de Masas en TándemRESUMEN
A liquid chromatography with tandem mass spectrometry method was developed for the simultaneous screening of 34 drugs and poisons in forensic cases. Blood (0.5 mL, diluted 1:1 with water) or 1.0 mL of urine was purified by solid-phase extraction. Gastric contents (diluted 1:1 with water) were treated with acetonitrile, centrifuged, and supernatant injected. Detection was achieved using a Waters Alliance 2695/Quattro Premier XE liquid chromatography tandem mass spectrometry system equipped with electrospray ionization, operated in the multiple reaction monitoring modes. The method was validated for accuracy, precision, linearity, and recovery. The absolute recovery of drugs and toxic compounds in blood was greater than 51% with the limit of detection in the range of 0.02-20 ng/mL. The absolute recovery of drugs and toxic compounds in urine was greater than 61% with limit of detection in the range of 0.01-10 ng/mL. The matrix effect of drugs and toxic compounds in urine was 65-117% and 67-121% in blood. The limit of detection of drugs and toxic compounds in gastric content samples were in the range of 0.05-20 ng/mL. This method was applied to the routine analysis of drugs and toxic compounds in postmortem blood, urine, and gastric content samples. The method was applied to actual forensic cases with examples given.
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Cromatografía Liquida/métodos , Contenido Digestivo/química , Preparaciones Farmacéuticas/análisis , Espectrometría de Masas en Tándem/métodos , Humanos , Límite de Detección , Preparaciones Farmacéuticas/sangre , Preparaciones Farmacéuticas/orinaRESUMEN
A novel method for the screening of 151 drugs of abuse and toxic compounds in human whole blood has been developed and validated by online solid-phase extraction with liquid chromatography coupled to time-of-flight mass spectrometry. Analytes were extracted and separated by using a fully automated online solid-phase extraction liquid chromatography system with total chromatographic run time of 26 min. Time-of-flight mass spectrometry screening of 151 drugs of abuse and toxic compounds was performed in a full-scan (m/z 50-800) mode using an MS(E) acquisition of molecular ions and fragment ions data at two collision energies (one was 6 eV and another one was in the range of 5-45 eV). The compounds were identified based on retention times and exact mass of molecular ions and fragment ions. The limit of detection ranged from 1 to 100 ng/mL and the recovery of the method ranged from 6.3 to 163.5%. This method is proved to be a valuable screening method allowing fast and specific identification of drugs in human whole blood.
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Cromatografía Líquida de Alta Presión/métodos , Drogas Ilícitas/sangre , Drogas Ilícitas/aislamiento & purificación , Espectrometría de Masas/métodos , Extracción en Fase Sólida/métodos , Detección de Abuso de Sustancias/métodos , Automatización/métodos , HumanosRESUMEN
Ecgonine is suggested to be a promising marker of cocaine (COC) ingestion. A combined mass spectrometry (MS) and tandem MS (MS/MS) method was developed to simultaneously determine ecgonine and seven other metabolites of cocaine in human urine and whole blood with ultra-high-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The compounds were extracted from as little as 100 µL of sample by solid-phase extraction with a 96-well µElution solid-phase extraction plate. The protonated molecules or fragment ions at accurate mass acquired in MS mode were used to quantify specific analytes, following by dedicated MS/MS identification. The assay was linear in the range from 5 to 50-100 ng/mL for urine samples, except for ecgonine methyl ester (10-200 ng/mL) and ecgonine (40-400 ng/mL), and was linear from 1-2 to 50 ng/mL for whole blood samples, except for ecgonine methyl ester (20-1,000 ng/mL) and ecgonine (40-2,000 ng/mL). The correlation coefficients were all greater than 0.99. The limits of detection ranged from 0.2 to 16 ng/mL, and the lower limits of quantification ranged from 1 to 40 ng/mL. The repeatability and intermediate precision were 18.1% or less. The accuracy was in the range from 80.0 to 122.9%, process efficiencies were in the range from 8.6 to 177.4%, matrix effects were in the range from 28.7 to 171.0%, and extraction recoveries were in the range from 41.0 to 114.3%, except for ecgonine (12.8% and 9.3% at low and high concentrations, respectively). This method was highly sensitive in comparison with previously published methods. The validated method was successfully applied to the analysis of real samples derived from forensic cases, and the results verified that, on the basis of data from four positive samples, ecgonine is a promising marker of cocaine ingestion.
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Cromatografía Líquida de Alta Presión/métodos , Cocaína/análogos & derivados , Cocaína/sangre , Cocaína/orina , Espectrometría de Masas en Tándem/métodos , Cocaína/química , Humanos , Extracción en Fase SólidaRESUMEN
PURPOSE: This study aims to develop and validate a rapid, simple, and efficient bioanalytical method for the simultaneous quantification of phenobarbital and barbital in human whole blood using liquid-liquid extraction combined with direct analysis in real time (DART) and high-resolution mass spectrometry (HRMS). METHOD: Phenobarbital-d5 and aprobarbital were selected as internal standards (ISs) of phenobarbital and barbital, respectively. A mixed solvent of o-xylene and ethyl acetate at a ratio of 1:6 was used to extract analytes of interest and ISs from 100 µL of human whole blood samples. Phenobarbital and barbital were detected by DART-HRMS. The proposed method has been validated in accordance with United States Food and Drug Administration Guidelines for Bioanalytical Method Validation in terms of selectivity, linearity, accuracy, precision, matrix effect, recovery, stability, and dilution integrity. RESULTS: The lower limits of quantification (LLOQs) of phenobarbital and barbital were both 10 ng/mL. The linearities were in the range of 10-1000 ng/mL (R2 ≥ 0.99). The mean recovery values of phenobarbital and barbital were 99.7% and 88.1%, respectively. The interday and intraday precision values were less than 10.4%, and the interday and intraday accuracy values ranged from 87.6 to 106.7%. Furthermore, the validated method was applied to four cases of phenobarbital poisoning at the Shanghai Institute of Forensic Science. CONCLUSION: The developed and fully validated method enabled the simultaneous quantification of phenobarbital and barbital in human whole blood and was successfully applied to authentic cases.
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Barbital , Fenobarbital , Estados Unidos , Humanos , China , Espectrometría de Masas/métodos , Extracción Líquido-LíquidoRESUMEN
Alcohol is often found in the blood of the deceased. To cover up the true cause of victim's death, postmortem instillation of alcohol occurs in some criminal cases. Explaining the finding of alcohol is extremely vital in forensic practice. This study aims to evaluate whether ethyl glucuronide (EtG) and ethyl sulfate (EtS) in blood and vitreous humor (VH) can be used to distinguish alcoholic death and postmortem alcohol instillation. Saline or 12.6 g/kg ethanol (antemortem alcohol poisoning group) was introduced into rabbits' stomachs 2 h before sacrificed. Same amount of ethanol was introduced into rabbits' stomachs at 0 h, 0.5 h, 1 h and 2 h after death in four subgroups of postmortem alcohol instillation group, respectively. Cardiac blood and VH were collected at 10 min, 4 h, 10 h and 24 h after death in blank and antemortem alcohol poisoning group, and after instillation of alcohol in postmortem alcohol instillation group. Blood was also collected at 34 h. Ethanol and EtG levels in blood and VH and EtS in VH in antemortem alcohol poisoning group were overlapped with those in postmortem alcohol instillation group. The contents of EtG and EtS in blood in antemortem alcohol poisoning group (mean ≥ 7.833 µg/mL for EtG and ≥ 19.990 µg/mL for EtS) were much higher than those in postmortem alcohol instillation group (mean ≤ 0.118 µg/mL for EtG and ≤ 0.091 µg/mL for EtS), but apparent decomposition was observed in EtG, which might lead to misinterpretation. Blood EtS showed better stability and could be used to distinguish alcoholic death and postmortem alcohol instillation.
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Depresores del Sistema Nervioso Central , Conejos , Animales , Consumo de Bebidas Alcohólicas , Espectrometría de Masas en Tándem , Etanol , Glucuronatos , BiomarcadoresRESUMEN
Ethyl sulfate (EtS) and ethyl glucuronide (EtG) in urine are biomarkers to monitor ethanol consumption. Due to their high polarity, severe matrix effects have been observed during analysis of EtS and EtG in urine by liquid chromatography tandem mass spectrometry (LC-MS/MS), which can lead to a loss of sensitivity and accuracy. In the present study, a novel and simple sample preparation approach based on fast-dried urine spot was established to reduce the matrix effect of EtS and EtG in urine. 20 µL of urine was dropped on the Whatman 903# paper and was subsequently dried by microwave in one minute. After ultrasonic assisted extraction with 500 µL of methanol, the analysis was conducted using an LC-MS/MS system. Limits of detection were 5 ng/mL and linear ranges were 10 ng/mL-10 µg/mL for both EtS and EtG. Matrix effects were in the range of 99.3-107.8% for EtS and 86.7-91.0% for EtG at three QC levels. Matrix effects for EtS and EtG were compared between the current method and other sample preparation methods including protein precipitation, and solid-phase extraction. The results showed that this fast-dried urine spot-based extraction method could eliminate matrix effects significantly in analysis of urine EtS and EtG by LC-MS/MS.
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Cromatografía Liquida/métodos , Glucuronatos/orina , Ésteres del Ácido Sulfúrico/orina , Espectrometría de Masas en Tándem/métodos , Humanos , Límite de Detección , Modelos Lineales , Reproducibilidad de los Resultados , UrinálisisRESUMEN
BACKGROUND: Dopamine transporter (DAT) and serotonin transporter (SERT) are targets for many psychoactive substances. Functional assays including uptake inhibition and release assays often involve radiolabeled compounds like [3H]-dopamine and [3H]-serotonin to assess drug activity at transporters, which have high requirements on handling radioactive samples. AIMS: The aim of this study was to establish a label-free method to assess drug activity at DAT and SERT. METHODS: A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was established using transporter-transfected human embryonic kidney 293T (HEK293T) cells. This method was evaluated by testing the effects of amphetamine and cocaine in the assay procedure. RESULTS: The limits of detection of this method were 0.2 nM for both dopamine (DA) and serotonin (5-HT), with good linearities in the range of 0.5-160 nM. Amphetamine and cocaine's IC50 and EC50 on DAT and SERT determined by this method were consistent with previous reports. CONCLUSIONS: A rapid, reliable and label-free LC-MS/MS method for assessing drug activity was established, which affords an attractive alternative for those laboratories that do not have a radiation license or capabilities.
Asunto(s)
Cromatografía Liquida/métodos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Serotonina en la Membrana Plasmática/efectos de los fármacos , Espectrometría de Masas en Tándem/métodos , Anfetamina/administración & dosificación , Anfetamina/farmacología , Cocaína/administración & dosificación , Cocaína/farmacología , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores de Captación de Dopamina/administración & dosificación , Inhibidores de Captación de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Reproducibilidad de los Resultados , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismoRESUMEN
Identifying the source of ethanol in a decedent remained a complicated problem for forensic toxicologists because of postmortem ethanol formation. As ethanol's non-oxidative metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS) have the potential to distinguish between antemortem ethanol consumption and postmortem ethanol formation, due to their high sensitivity and selectivity. In the current study, a simple and quick liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the determination of EtG and EtS in human whole blood and vitreous humor (VH). A total of 20 µL of the sample was precipitated by methanol, and the analytes were detected by LC-MS/MS in a run of 6 min. This method achieved high sensitivity (limits of detection: 2 ng/mL for both EtG and EtS), with linearity in the range of 5-10,000 ng/mL in both whole blood and VH. Deviations in accuracy, inter- and intra-day precision were all lower than 15% at three quality control levels. Subsequently, this method was applied to 62 real forensic cases. Only blood samples were available in 52 cases. Paired blood and VH samples were present in 10 cases. The concentrations of EtG and EtS in blood were in the range of 0-22,264.8 ng/mL and 0-2,126.0 ng/mL, respectively. In one case with both blood and VH, the blood ethanol concentration was 1.22 mg/mL, with EtG and EtS both below limits of quantification (5 ng/mL) in VH, and no EtG and EtS found in whole blood. The results suggested that EtG and EtS were useful markers for the interpretation of ethanol resource in postmortem blood and VH.
Asunto(s)
Etanol , Espectrometría de Masas en Tándem , Consumo de Bebidas Alcohólicas , Biomarcadores , Cromatografía Liquida , Glucuronatos , Humanos , Ésteres del Ácido Sulfúrico , Cuerpo VítreoRESUMEN
A high-resolution mass spectrometric detection method is described for the identification of key metabolites in the selenium pathway in selenium enriched yeast. Iodoacetic acid (IAA) was used as the derivatizing reagent to stabilize the selenols. Oxidized forms of selenocysteine (Se-Cys), selenohomocystine (Se-HCys), selenoglutathione (Se-GSH), seleno-γ-glutamyl-cysteine (Se-Glu-Cys), N-(2,3-dihydroxy-1-oxopropyl)-selenocysteine (Se-DOP-Cys), N-(2,3-dihydroxy-1-oxopropyl)-selenohomocysteine (Se-DOP-HCys), selenomethionine (SeMet), seleno-S-adenosyl-homocysteine (Se-AdoHcy), the conjugate of glutathione and N-(2,3-dihydroxy-1-oxopropyl)-selenocysteine (GSH-Se-DOP-Cys), and the conjugate of glutathione and N-(2,3-dihydroxy-1-oxopropyl)-selenohomocysteine (GSH-Se-DOP-HCys) were found in the selenium enriched yeast certified reference material (SELM-1). Selenols were also derivatized with a mercury tag, p-hydroxymercurybenzoate (PHMB). The selenol-PHMB complexes showed the overlapped isotopic patterns of selenium and mercury, which provided supporting information for the identification of selenols. Both methods showed good agreement (<4 ppm difference) between the theoretical masses of the target compounds and the measured masses in the yeast matrix. The method using IAA as the derivatizing reagent was used to study the response of Saccharomyces cerevisiae to three forms of selenium, Se-Met, Na(2)SeO(3) (Se(IV)), and Na(2)SeO(4)·10H(2)O (Se(VI)) (concentration of Se: 100 mg/L). The production of selenocompounds observed over a 6 h period was high in the Se-Met treated group compared to the groups treated with Se(IV) and Se(VI).
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Selenio/metabolismo , Homocistina/análogos & derivados , Homocistina/química , Homocistina/metabolismo , Ácido Yodoacético/química , Mercurio/química , Redes y Vías Metabólicas , Compuestos de Organoselenio/química , Compuestos de Organoselenio/metabolismo , Oxidación-Reducción , Saccharomyces cerevisiae/metabolismo , Selenio/química , Compuestos de Selenio/química , Compuestos de Selenio/metabolismo , Selenocisteína/química , Selenocisteína/metabolismo , Selenometionina/química , Selenometionina/metabolismoRESUMEN
Problems of stability were found for biomarkers of alcohol consumption: ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanols (PEths), and fatty acid ethyl esters (FAEEs) in whole blood. The purpose of this study was to establish a method for the determination of these four kinds of ethanol's non-oxidative metabolites in dried blood spots (DBS) by liquid chromatography tandem mass spectrometry (LC-MS/MS), and to evaluate their stability. In this method, 50 µL of human blood was spotted onto a filter paper for DBS analysis. Samples were extracted by methanol, reconstituted by 2-propanol, and injected into the LC-MS/MS system. Limits of detection were among 0.5-50 ng/mL, and deviations in accuracy and precision were all lower than 15% at three quality control levels. The stability of the four kinds of ethanol non-oxidative metabolites in DBS was investigated during a 90-day range under three temperatures, -20 °C, 4 °C, and 25 °C. EtG and EtS showed a high level of stability in DBS in the 90-day range, regardless of the temperature. FAEEs were unstable after three days. PEths showed stability within 15 days in postmortem DBS and 60 days in antemortem DBS, respectively, at all temperatures.