RESUMO
BACKGROUND: Efavirenz (EFV) is a drug used to treat HIV. Low plasma concentrations of EFV result in suboptimal viral suppression, whereas high concentrations can cause adverse neuropsychiatric side reactions. Some studies have identified a correlation between the plasma concentrations of EFV metabolites and neurotoxicity. To our knowledge, no studies have investigated the metabolism of EFV in young children and its effect on treatment outcomes. Therefore, the aim of this study was to develop and validate a method for quantifying EFV and its metabolites in human plasma derived from children. METHODS: Sample preparation was performed using protein precipitation of 100 µL plasma. Thereafter, an aliquot of the supernatant was used to quantify EFV, 7-hydroxyefavirenz (7-OH-EFV), 8-hydroxyefavirenz (8-OH-EFV), and a newly discovered metabolite ("EFAdeg") associated with 8-OH-EFV. A second aliquot of the supernatant was hydrolyzed using ß-glucuronidase/arylsulfatase and used with the first aliquot to quantify phase II metabolites. The analyses were performed using a Dionex Ultimate 3000RS LC-system coupled with a Q Exactive Orbitrap mass spectrometer. RESULTS: The method has a measuring range of 100-50,000 ng/mL (EFV, 8-OH-EFV), 125-25,000 ng/mL (7-OH-EFV), and 200-10,000 ng/mL ("EFAdeg"). All criteria of the European Medicines Agency guidelines regarding precision, accuracy, and selectivity were met. Of note, carryover must be considered for 8-OH-EFV. Overall, the validated method was successfully applied to plasma samples obtained from children and confirmed the presence of the newly discovered metabolite, "EFAdeg." CONCLUSIONS: An LC-HRMS/MS method for the quantification of EFV and its phase I and II metabolites was developed and validated. This method is suitable for analyzing plasma samples from children. Furthermore, studies using this method identified an additional metabolite that may influence the concentration of 8-OH-EFV in patient samples.
Assuntos
Alcinos , Benzoxazinas , Ciclopropanos , Espectrometria de Massas em Tandem , Humanos , Benzoxazinas/sangue , Benzoxazinas/farmacocinética , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Criança , Fármacos Anti-HIV/sangue , Fármacos Anti-HIV/farmacocinética , Pré-Escolar , Infecções por HIV/tratamento farmacológico , Infecções por HIV/sangue , Reprodutibilidade dos TestesRESUMO
Scarce data are available on methylphenidate (MPH) plasma concentrations reached after doses higher than 180 mg. The interindividual and intraindividual variability in the exposure of MPH and ritalinic acid (RA) enantiomers was examined in 28 patients with ADHD and substance use disorders, with MPH daily doses between 30 and 600 mg (median 160 mg). MPH and RA plasma concentrations were analysed with an enantioselective LC-MS/MS method. d-MPH plasma concentration/dose varied 25-fold between subjects but was reasonably stable within an individual. Twelve subjects had quantifiable l-MPH plasma concentrations, which accounted for up to 48% of the total MPH plasma concentration. The less active l-MPH enantiomer could, in individuals with low carboxylesterase 1 (CES1) activity, contribute significantly to the total MPH plasma drug concentration and hamper the estimation of the exposure to the more active d-MPH enantiomer. However, the high correlation between the total (d + l) RA/MPH metabolic ratio and the d-RA/d-MPH metabolic ratio (rs = 0.94) indicates that the ratio based on non-enantioselective analysis could be used as a marker of CES1 activity. Whether this holds true for subjects with aberrant metabolism due to genetic variants or during concomitant treatment with inhibitors or inducers of the enzyme remains to be studied.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Estimulantes do Sistema Nervoso Central , Metilfenidato , Transtornos Relacionados ao Uso de Substâncias , Adulto , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Hidrolases de Éster Carboxílico , Estimulantes do Sistema Nervoso Central/uso terapêutico , Cromatografia Líquida , Humanos , Metilfenidato/uso terapêutico , Estereoisomerismo , Espectrometria de Massas em TandemRESUMO
Multiple new psychoactive substances (NPS) are released into the recreational drug market each year. One NPS drug class that has become more common in recent years is that of the benzodiazepines (designer benzodiazepines, DBZ). Several metabolism studies have been performed to improve their bioanalytical detection via the best target. These studies have shown the presence of parent glucuronides and, as polymorphisms have been noted for the catalyzing enzymes (UDP-glucuronyltransferases) responsible for glucuronide conjugation reactions, it is important to keep this in mind when interpreting DBZ cases in clinical and/or forensic toxicology. Therefore, the aim of this study was to determine the UDP-glucuronyltransferases (UGTs) responsible for parent compound conjugation of nine DBZ to facilitate interpretation of related cases. Clonazolam, deschloroetizolam, etizolam, flubromazolam, flunitrazolam, metizolam, nifoxipam, nitrazolam, and pyrazolam were incubated with pooled human liver microsomes (pHLM) or 13 different human UGTs. The samples were analyzed using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Glucuronide conjugates of flunitrazolam and nifoxipam were only detected in pHLM, suggesting that these reactions are performed by dimer complexes of several UGTs or complexes between UGTs and other metabolizing enzymes contained in pHLM. Nitrazolam or pyrazolam glucuronides were not detected. Glucuronidation of clonazolam, deschloroetizolam, etizolam, flubromazolam, and metizolam was catalyzed exclusively by UGT1A4. The conjugation of the majority of the DBZ was performed by the UGT isoform 1A4 for which polymorphisms have been described. This underlines the importance of taking glucuronidation polymorphism into consideration when interpreting intoxication cases.
Assuntos
Benzodiazepinas/metabolismo , Drogas Desenhadas/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Benzodiazepinas/análise , Benzodiazepinas/química , Cromatografia Líquida/métodos , Drogas Desenhadas/análise , Drogas Desenhadas/química , Glucuronídeos/química , Glucuronosiltransferase/química , Humanos , Insetos , Microssomos/metabolismo , Detecção do Abuso de Substâncias/métodosRESUMO
CONTEXT: In recent years, many unclassified benzodiazepines (BZD) have appeared through online sale as new psychoactive substances (NPS). This study describes bioanalytical and clinical data related to intoxications involving NPS BZD ("designer BZD") in the Swedish STRIDA project. STUDY DESIGN: Case series of consecutive patients with admitted or suspected intake of NPS presenting to hospitals all over Sweden for emergency treatment in 2012-2016. PATIENTS AND METHOD: Urine samples collected in the STRIDA project were analyzed for 28 NPS BZD, using immunoassay and liquid chromatography-high-resolution mass spectrometry . Data of patient's age, gender, reported substance exposure, clinical signs, and treatment were obtained from medical and Poisons Information Center (PIC) records. RESULTS: A total of fifteen different NPS BZD were analytically confirmed in 217 of 1913 (11%) cases involving patients (81% men) aged 15-66 (mean 28) years. The frequency of positive samples increased from 4% in 2012 to 19% in 2015. Etizolam (20 cases) was the first detected NPS BZD (January 2012), and it was followed by metizolam (four cases), estazolam (two), pyrazolam (33), flubromazepam (33), nifoxipam (five), diclazepam (four), meclonazepam (26), bromazepam (one), flubromazolam (92), deschloroetizolam (one), clonazolam (16), 3-hydroxyphenazepam (eight), ketazolam (one), and phenazepam (one). Most cases (89%) also involved other drugs. Use of NPS BZD was rarely (15%) reported during PIC consultation. In 24 patients exposed only to NPS BZD, CNS depression was the most prominent clinical sign, seven were observed in the intensive care unit, and they responded positively to flumazenil treatment. CONCLUSIONS: An increasing use of NPS BZD in Sweden was detected in acute intoxication cases, sometimes leading to intensive care monitoring and support needs.
Assuntos
Benzodiazepinas/intoxicação , Drogas Desenhadas/intoxicação , Adolescente , Adulto , Idoso , Benzodiazepinas/urina , Cromatografia Líquida de Alta Pressão , Serviços Médicos de Emergência , Feminino , Flumazenil/uso terapêutico , Moduladores GABAérgicos/uso terapêutico , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Transtornos Relacionados ao Uso de Substâncias/complicações , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Suécia/epidemiologia , Adulto JovemRESUMO
Over the past ~8 years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC-HRMS/MS system consisted of a YMC-UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using ß-glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono-hydroxy metabolites, and mono-hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono-hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2-19-fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites.
Assuntos
Benzodiazepinas/urina , Drogas Desenhadas/farmacocinética , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem/métodos , Benzodiazepinas/metabolismo , Cromatografia Líquida/métodos , Drogas Desenhadas/metabolismo , Glucuronídeos/metabolismo , Glucuronídeos/urina , Humanos , Hidrólise , Limite de DetecçãoRESUMO
Hundreds of new psychoactive substances (NPS) covering most drugs-of-abuse classes have been introduced to the recreational drug market in recent years. One class of NPS drugs that has become more common recently is "designer" benzodiazepines. Due to a close structural resemblance with prescription benzodiazepines, some of these substances may elicit a positive response (i.e. cross react) in immunoassay screening. Consequently, it is increasingly important to include NPS benzodiazepines during method confirmation to ensure accurate identification of closely-related compounds as well as detection of the benzodiazepines themselves. Here, we present our efforts to develop a screening and confirmation method for detection of 28 NPS benzodiazepines in urine using reversed-phase liquid chromatographic separation in combination with high-resolution mass spectrometry (LC-HRMS). MS was performed in positive electrospray mode on a Thermo Fischer Scientific Q Exactive Orbitrap instrument using either full scan (for screening) or parallel reaction monitoring (for confirmation). We found the lower quantification limit of the method to range from 5 to 50â¯ng/mL. Analytical precision and accuracy were ≤15% for both screening and confirmation for all except one analyte. The method was used to analyze patient urine samples from routine drug testing and samples from acute intoxication cases presenting in emergency wards. Altogether, 16 of the 28 benzodiazepines (i.e., clobazam, clonazolam, deschloroetizolam, diclazepam, estazolam, etizolam, flubromazepam, flubromazolam, flunitrazolam, 3-hydroxyflubromazepam, 3-hydroxyphenazepam, ketazolam, meclonazepam, metizolam, nifoxipam, and pyrazolam) were detected in the urine samples. The results from patient sample analysis indicate a high prevalence of NPS benzodiazepine use, emphasizing the importance of including novel drugs of abuse in drug testing menus.
RESUMO
The emerging new psychoactive substances made available for recreational drug use have recently started to include designer benzodiazepines. As a consequence, the routine immunoassay drug testing for benzodiazepines may become less effective, due to an increased occurrence of 'false negative' and 'false positive' results. This work aimed to extend the knowledge of analytical cross-reactivity of 13 designer benzodiazepines in the CEDIA, EMIT II Plus, HEIA, and KIMS II immunoassays. Urine standards were prepared by spiking blank urine with clonazolam, deschloroetizolam, diclazepam, estazolam, etizolam, flubromazepam, flubromazolam, flutazolam, 3-hydroxyphenazepam, meclonazepam, nifoxipam, phenazepam, and pyrazolam. Authentic urine samples from intoxication cases identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were also investigated. For the spiked standard samples, the 13 designer benzodiazepines generally showed a high cross-reactivity in all assays. This was further confirmed when investigating their detectability in authentic urine samples from cases of drug intake. The test responses also indicated additional reactivity from metabolites. The lowest detectability in spiked samples was observed for flutazolam, which shows the most divergent chemical structure compared with the other benzodiazepines. Overall, the KIMS II and CEDIA immunoassays, which both include enzymatic hydrolysis of conjugated forms, showed the highest, and EMIT II Plus the lowest degree of reactivity, for spiked parent substances and authentic urine specimens. The results of this study demonstrated that designer benzodiazepines can be detected in standard urine immunoassay drug screening and this should be taken into consideration when performing confirmation analysis. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Benzodiazepinas/urina , Drogas Desenhadas/farmacocinética , Imunoensaio/métodos , Detecção do Abuso de Substâncias/métodos , Benzodiazepinas/análise , Cromatografia Líquida/métodos , Drogas Desenhadas/análise , Humanos , Limite de Detecção , Espectrometria de Massas em Tandem/métodosRESUMO
New psychoactive substances (NPS) have become an increasing drug problem in the past decade. For detection of NPS, new analytical methods have to be developed, and the methods also have to be updated regularly. This study aimed at developing a multi-component LC-MS/MS method for detection and quantification of 11 NPS of the benzodiazepine sub-class ("designer benzodiazepines") in urine specimens. The method involves dilution of urine with internal standard and hydrolysis of any glucuronide conjugated forms. Separation of the compounds was achieved on a BEH Phenyl column, followed by MS/MS detection in positive electrospray mode. Method validation was performed following the EMA guideline. The method was applied to study the occurrence of designer benzodiazepines in Sweden in 2014-2015, by analysis of 390 samples retrieved from a routine drug testing laboratory. In 40% of these samples, selected based on a positive immunoassay benzodiazepine screening but a negative MS confirmation for the standard set of prescription benzodiazepines, intake of designer benzodiazepines was revealed. These results stress the importance of using and updating confirmation methods to include the increasing number of designer benzodiazepines appearing on the NPS market.