Bioanalytical studies of designer benzodiazepines

The fast appearance of benzodiazepine analogues, referred to as new psychoactive substance (NPS) or designer benzodiazepines, requires the continuous update of detection methods in order to keep up with the latest drugs on the recreational drug market. Moreover, as usually only limited information on toxicity and excretion patterns of these new drugs exists, this needs to be evaluated to report on adverse effects and to determine suitable targets for drug testing. Urine drug testing usually involves screening using immunoassay followed by confirmation of positive screening results using mass spectrometric (MS) methods. We studied the detectability of designer benzodiazepines in urine using commercial immunoassays and demonstrated that most designer benzodiazepines can be detected by immunoassay. It is thus important to update confirmation methods to include designer benzodiazepines. We also developed a liquid chromatographic–tandem MS (LC–MS/MS) confirmation method for designer benzodiazepines in urine using direct dilution of samples and hydrolysis of conjugates. Subsequently, a further improved screening and confirmation method using LC–high-resolution MS (LC–HRMS(/MS)) was developed. HRMS screening is performed in full scan and is a generic method that can easily include new analytes. LC–HRMS/MS confirmation only requires re-injection of the sample. Many samples from drug dependent patients with a positive immunoassay screening result for benzodiazepines but not containing prescription medicines detected a designer benzodiazepine instead. Comparable results were obtained for acute intoxication cases from emergency wards in the STRIDA project. In total, 28 designer benzodiazepines were covered by the analytical method and 17 of these were detected in the samples. Classification of a designer benzodiazepine as a narcotic substance generally meant that it was removed from the NPS market and replaced with another novel benzodiazepine. It was further demonstrated that intoxications by designer benzodiazepines might cause central nervous system depression. Studies on metabolic patterns of five designer benzodiazepines using urine samples from confirmed intoxication cases identified suitable analytical targets for urine drug testing, instead of or together with the parent compounds and both with and without hydrolysis of conjugated forms. In summary, the results demonstrated frequent use of designer benzodiazepines in Sweden and in cases of acute intoxication that they might cause serious adverse effects. This underlines the importance of including designer benzodiazepines and/or metabolites thereof in drug testing. Screening for designer benzodiazepines can be performed by immunoassay or LC–HRMS, and confirmation methods can make use of direct dilution of urine samples followed by hydrolysis and direct injection into LC–MS or LC–HRMS systems

In vitro metabolic fate of nine LSD-based new psychoactive substances and their analytical detectability in different urinary screening procedures

The market of new psychoactive substances (NPS) is characterized by a high turnover and thus provides several challenges for analytical toxicology. The analysis of urine samples often requires detailed knowledge about metabolism given that parent compounds either may be present only in small amounts or may not even be excreted. Hence, knowledge of the metabolism of NPS is a prerequisite for the development of reliable analytical methods. The main aim of this work was to elucidate for the first time the pooled human liver S9 fraction metabolism of the nine d-lysergic acid diethylamide (LSD) derivatives 1-acetyl-LSD (ALD-52), 1-propionyl-LSD (1P-LSD), 1-butyryl-LSD (1B-LSD), N6-ethyl-nor-LSD (ETH-LAD), 1-propionyl-N6-ethyl-nor-LSD (1P-ETH-LAD), N6-allyl-nor-LSD (AL-LAD), N-ethyl-N-cyclopropyl lysergamide (ECPLA), (2′S,4′S)-lysergic acid 2,4-dimethylazetidide (LSZ), and lysergic acid morpholide (LSM-775) by means of liquid chromatography coupled to high-resolution tandem mass spectrometry. Identification of the monooxygenase enzymes involved in the initial metabolic steps was performed using recombinant human enzymes and their contribution confirmed by inhibition experiments. Overall, N-dealkylation and hydroxylation, as well as combinations of these steps predominantly catalyzed by CYP1A2 and CYP3A4, were found. For ALD-52, 1P-LSD, and 1B-LSD, deacylation to LSD was observed. The obtained mass spectral data of all metabolites are essential for reliable analytical detection particularly in urinalysis and for differentiation of the LSD-like compounds as biotransformations also led to structurally identical metabolites. However, in urine of rats after the administration of expected recreational doses and using standard urine screening approaches, parent drugs or metabolites could not be detected