Characterization of the designer benzodiazepine diclazepam and preliminary data on its metabolism and pharmacokinetics

Abstract

Designer benzodiazepines, first offered in online shops selling 'research chemicals' in 2012, provide an attractive alternative to prescription-only benzodiazepines as they are readily available over the Internet at a low price. However, as data regarding pharmacokinetic parameters, metabolism, and detectability in biological fluids are limited, they present a challenge for forensic laboratories. Most recently, diclazepam (other names: 2-chlorodiazepam, Ro 5-3448 or 7-chloro-5-(2-chlorophenyl)-1-methyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one) emerged as a new compound in this class of drugs. In this paper, this new designer benzodiazepine was characterized utilizing nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS) as well as liquid chromatography tandem mass spectrometry (LC-MS/MS) techniques. Furthermore, a self-experiment was performed to gain preliminary data on pharmacokinetic properties and to identify the main metabolites. For this purpose, one tablet of diclazepam (declared amount: 1 mg) was ingested by one of the authors, and serum as well as urine samples were collected for 14 and 21 days, respectively. Based on this study, diclazepam has an approximate elimination half-life of 42 h and is metabolized into the pharmacologically active benzodiazepines delorazepam, lorazepam, and lormetazepam which can be detected in urine for 6, 19, and 11 days, respectively, when applying the presented LC-MS/MS method. In serum, the consumption could be proven between 99 h post-intake targeting the parent compound and up to 10 days targeting the metabolite delorazepam. As immunochemical assays are applied for screening purposes quite often, detectability using this technique was assessed, especially since detection of low-dosed benzodiazepines can be sometimes problematic. However, only one of the utilized immunochemical assays was capable of detecting the intake of one tablet diclazepam, and the positive results were restricted to a few urine samples showing relatively high creatinine concentrations.

Detectability of designer benzodiazepines in CEDIA, EMIT II Plus, HEIA, and KIMS II immunochemical screening assays

“…[10] Cross-reactivity of etizolam in the CEDIA assay was found to be 144 %, which differed from our previous finding of 237%, [11] although both values were determined at about the same concentrations. [10] Cross-reactivity of etizolam in the CEDIA assay was found to be 144 %, which differed from our previous finding of 237%, [11] although both values were determined at about the same concentrations.…”

“…[6][7][8] However, immunoassays still hold their place in routine high-throughput urine drug screening and it has been observed that the new abused drugs may also cross-react in the existing commercial reagents. [10] In CEDIA, etizolam showed 237% cross-reactivity. [9] Knowledge about the detectability of designer benzodiazepines in commercial benzodiazepine immunoassays is limited.…”

Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines: An update comprising flunitrazolam, norflurazepam, and 4'‐chlorodiazepam (Ro5–4864)

“…4 mm column (Thermo Fisher Scientific, Idstein, Germany) using our routine screening gradient, as previously described. 4 mm column (Thermo Fisher Scientific, Idstein, Germany) using our routine screening gradient, as previously described.…”

“…1 Since the appearance of pyrazolam on the drug market in 2012, 2 internet shops continue to offer either modifications of popular, licensed benzodiazepines or metabolites thereof. 4,5 Appearance of highly potent triazolo-compounds such as flubromazolam as pure powder may lead to the abuse of such compounds in a drug-facilitated crime context and can easily lead to severe intoxications due to dosing errors. 3 In particular, offer of metabolites [eg, norflunitrazepam ('fonazepam') and 3-hydroxyphenazepam] or designer benzodiazepines metabolizing to licensed benzodiazepines (eg, diclazepam and cloniprazepam) pose challenges for correct interpretation of analytical findings.…”

“…This compound has been researched extensively because of its binding to the translocator protein (TSPO), also known as the peripheral benzodiazepine receptor. 25 The third compound investigated here was flunitrazolam (6-(2fluorophenyl)-1-methyl-8-nitro-4H- [1,2,4] assessed with a QSAR model. 24 However, it was also observed for this compound that it shows distinct differences in receptor binding among various species.…”