RESUMEN
PURPOSE: We have developed and validated a high-sensitivity method to quantify lysergic acid diethylamide (LSD) and 2-oxo-3-hydroxy-LSD (OH-LSD) in oral fluid samples using liquid-liquid extraction and liquid chromatography-tandem mass spectrometry (LCâMS/MS). The method was applied to the quantification of both substances in 42 authentic oral fluid samples. METHODS: A liquid-liquid extraction was performed using 500 µL each of samples (oral fluid samples collected using Quantisal™ device) and dichloromethane/isopropanol mixture (1:1, v/v). Enzymatic hydrolysis was evaluated to cleave glucuronide metabolites. RESULTS: The limit of quantification was 0.01 ng/mL for both LSD and OH-LSD. The linearity was assessed between 0.01 and 5 ng/mL. Imprecision and bias were not higher than 10.2% for both analytes. Extraction recovery was higher than 69%. The analytes were stable in the autosampler at 10 °C for 24 h, and up to 30 days at 4 and -20 °C. The method was applied to the analysis of 42 oral fluid samples. LSD was detected in all samples (concentrations between 0.02 and 175 ng/mL), and OH-LSD was detected in 20 samples (concentrations between 0.01 and 1.53 ng/mL). CONCLUSIONS: A high-sensitive method was fully validated and applied to authentic samples. To our knowledge, this is the first work to report concentrations of LSD and OH-LSD in authentic oral fluid samples.
Asunto(s)
Dietilamida del Ácido Lisérgico , Espectrometría de Masas en Tándem , Cromatografía Liquida , Extracción Líquido-LíquidoRESUMEN
In recent years, experimental research on lysergic acid diethylamide (LSD) in humans has gained new momentum. In humans, LSD is metabolized rapidly into several metabolites but knowledge of the involved metabolizing enzymes is limited. The aim of the current study was to identify the cytochrome P450 (CYP) isoforms involved in the metabolism of LSD to 6-norlysergic acid diethylamide (nor-LSD) and 2-oxo-3-hydroxy-LSD (O-H-LSD) in vitro, in order to evaluate potential effects of enzyme polymorphisms or prescription drugs on LSD pharmacokinetics. Additionally, interactions of LSD and both metabolites with 5-hydroxytryptamine (5-HT) receptors were assessed. LSD was incubated with human liver microsomes over 4â¯h and the production of nor-LSD and O-H-LSD was quantified by liquid chromatography tandem mass spectrometry. Metabolism was inhibited by the addition of specific CYP inhibitors. Additionally, recombinant CYPs were used to verify the inhibition results obtained with microsomes and induction of metabolism was investigated in human hepatocyte-derived cells. Radioligand binding and calcium mobilization assays were used to determine 5-HT receptor affinities and activities, respectively. Human liver microsomes displayed minor metabolite formation (<1% metabolized) over 4â¯h. CYP2D6, 2E1, and 3A4 significantly contributed to the formation of nor-LSD, and CYP1A2, 2C9, 2E1, and 3A4 were significantly involved in the formation of O-H-LSD. These findings could be verified using recombinant CYPs. Enzyme induction with rifampicin distinctly increased the formation of both metabolites, whereas treatment with omeprazole only slightly increased formation of nor-LSD. LSD and nor-LSD were pharmacologically active at the 5-HT1A, 5-HT2A, 5-HT2B, and 5-HT2C receptors. Nor-LSD mainly differed from the parent compound by having a lower affinity to the 5-HT2C receptor. O-H-LSD displayed substantially weaker affinity and activity at serotonergic receptors in comparison to LSD. To conclude, human liver microsomes converted only small amounts of LSD to nor-LSD and O-H-LSD but several CYPs significantly contributed. Genetic polymorphisms and drug interactions could therefore influence pharmacokinetics and pharmacodynamics of LSD. Nor-LSD likely has hallucinogenic activity similar to LSD, whereas O-H-LSD is inactive. Drug-drug interaction studies in humans are required to further assess the clinical relevance of these findings.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Dietilamida del Ácido Lisérgico/análogos & derivados , Dietilamida del Ácido Lisérgico/metabolismo , Microsomas Hepáticos/metabolismo , Agonistas de Receptores de Serotonina/metabolismo , Animales , Interacciones Farmacológicas/fisiología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Dietilamida del Ácido Lisérgico/farmacología , Ratones , Microsomas Hepáticos/efectos de los fármacos , Células 3T3 NIH , Agonistas de Receptores de Serotonina/farmacologíaRESUMEN
Lysergic acid diethylamide (LSD) is administered in low dosages, which makes its detection in biological matrices a major challenge in forensic toxicology. In this study, two sensitive and reliable methods based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) were established and validated for the simultaneous determination of LSD and its metabolite, 2-oxo-3-hydroxy-LSD (O-H-LSD), in hair and urine. Target analytes in hair were extracted using methanol at 38°C for 15h and analyzed by LC-MS/MS. For urine sample preparation, liquid-liquid extraction was performed. Limits of detection (LODs) in hair were 0.25pg/mg for LSD and 0.5pg/mg for O-H-LSD. In urine, LODs were 0.01 and 0.025ng/ml for LSD and O-H-LSD, respectively. Method validation results showed good linearity and acceptable precision and accuracy. The developed methods were applied to authentic specimens from two legal cases of LSD ingestion, and allowed identification and quantification of LSD and O-H-LSD in the specimens. In the two cases, LSD concentrations in hair were 1.27 and 0.95pg/mg; O-H-LSD was detected in one case, but its concentration was below the limit of quantification. In urine samples collected from the two suspects 8 and 3h after ingestion, LSD concentrations were 0.48 and 2.70ng/ml, respectively, while O-H-LSD concentrations were 4.19 and 25.2ng/ml, respectively. These methods can be used for documenting LSD intake in clinical and forensic settings.
Asunto(s)
Cromatografía Liquida/métodos , Toxicología Forense/métodos , Cabello/química , Dietilamida del Ácido Lisérgico/análogos & derivados , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Adolescente , Cromatografía Liquida/instrumentación , Toxicología Forense/instrumentación , Humanos , Límite de Detección , Dietilamida del Ácido Lisérgico/análisis , Dietilamida del Ácido Lisérgico/orina , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
This work investigates, for the first time, the occurrence of 10 drugs of abuse, six metabolites, and three benzodiazepines in surface waters from the Jarama and Manzanares Rivers in the Madrid Region, the most densely populated area in Spain and one of the most densely populated in Europe. The results of this study have shown the presence of 14 out of the 19 compounds analyzed at concentrations ranging from 1.45 to 1020 ng L(-1). The most ubiquitous compounds, found in 100% of the samples, were the cocaine metabolite benzoylecgonine (BE), the amphetamine-like compound ephedrine (EPH), the opioids morphine (MOR), methadone (METH), and the METH metabolite 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), and the three investigated benzodiazepines alprazolam (ALP), diazepam (DIA) and lorazepam (LOR). Meanwhile, the largest concentrations observed corresponded to EPH (up to 1020 ng L(-1)), BE (823 ng L(-1)), EDDP (151 ng L(-1)), and LOR (167 ng L(-1)). The only not detected compounds were heroin (HER) and its metabolite 6-acetylmorphine (6ACM), lysergic acid diethylamide (LSD) and its metabolite 2-oxo-3-hydroxy-LSD (OH-LSD), and Δ(9)-tetrahydrocannabinol (THC). Overall, the levels measured are comparatively higher than those previously reported in Europe. Comparison of the results obtained for samples collected on different days (Thursday and Sunday) did not show meaningful differences between weekdays and weekends. The lack of (eco)toxicological data does not permit to predict or disregard potential adverse effects on wildlife. Risk assessment in humans would require further knowledge, not currently available, on exposure to these compounds through other routes like drinking water and/or food.