Toxicokinetics and Analytical Toxicology of Flualprazolam: Metabolic Fate, Isozyme Mapping, Human Plasma Concentration and Main Urinary Excretion Products

Abstract

An increasing number of benzodiazepine-type compounds are appearing on the new psychoactive substances market. 8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine (well known as flualprazolam) represents a potent ‘designer benzodiazepine’ that has been associated with sedation, loss of consciousness, memory loss and disinhibition. The aims of the present study were to tentatively identify flualprazolam metabolites using in vitro incubations with pooled human liver S9 fraction or HepaRG cells by means of liquid-chromatography-high resolution tandem mass spectrometry. Isozymes involved in phase I and II biotransformation were identified in vitro. Results were then confirmed using human biosamples of an 18-year old male who was admitted to the emergency department after suspected flualprazolam ingestion. Furthermore, the plasma concentration was determined using the standard addition method. Seven flualprazolam metabolites were tentatively identified. Several cytochrome P450 and UDP-glucuronosyltransferase isozymes, amongst them CYP3A4 and UGT1A4, were shown to be involved in flualprazolam biotransformation reactions, and an influence of polymorphisms as well as drug–drug or drug–food interactions cannot be excluded. Alpha-hydroxy flualprazolam glucuronide, 4-hydroxy flualprazolam glucuronide and the parent glucuronide were identified as most abundant signals in urine, far more abundant than the parent compound flualprazolam. These metabolites are thus recommended as urine-screening targets. If conjugate cleavage was performed during sample preparation, the corresponding phase I metabolites should be added as targets. Both hydroxy metabolites can also be recommended for blood screening. The flualprazolam plasma concentration determined in the intoxication case was as low as 8 μg/L underlining the need of analytical methods with sufficient sensitivity for blood-screening purposes.

Recent trends in drugs of abuse metabolism studies for mass spectrometry–based analytical screening procedures

“…The latter was also true for N-methoxybenzyl (NBOMe)-containing NPS. The phase I metabolites suitable for conjugation were produced by preincubation of the NPS with a single CYP isozyme previously found to catalyze the phase I metabolic reaction of interest [6,10,19]. After preincubation with CYP3A4, Wagmann et al detected two out of three flualprazolam glucuronides in UGT incubations compared to pooled HLS9 incubations [6].…”

A fluorine turns a medicinal benzodiazepine into NPS: the case of flualprazolam

“…One dihydroxylated and two monohydroxylated metabolites were formed through the hydroxylation at C 4 of the diazepine core and/or at α-C atom of the triazolo ring [14]. In another study [15], flualprazolam incubations with pHLS9 and HepaRG cells were used to study the metabolic pattern of flualprazolam. In total, six metabolites were detected, three derived through phase I and three through phase II reactions.…”

“…Phase I reactions included mono-and dihydroxylation, and α-hydroxyflualprazolam, 4-hydroxyflualprazolam and α,4-dihydroxyflualprazolam were identified (Fig. 3) [15]. Sofalvi et al [16] consider α-hydroxyflualprazolam as the major metabolite of flualprazolam.…”

“…In a plasma sample of a real case, only two (α-hydroxyflualprazolam and 4-hydroxyflualprazolam) out of the 7 metabolites and flualprazolam were detected, while in the respective urine sample, all seven metabolites and the parent compound were found. Due to their higher abundance in urine samples, the N-glucuronide of flualprazolam and the glucuronides of monohydroxylated metabolites are suggested to be used as screening targets for the toxicological analysis of urine samples [15]. There are no published data concerning the activities of flualprazolam metabolites.…”

Targeted delivery, drug release strategies, and toxicity study of polymeric drug nanocarriers

“… Renal glomeruli that hydrostatically filter small molecules with molecular weights smaller than 60 kDa through their pores. Near renal tubular cells and hepatocytes (liver cells) that actively transport chemicals from the blood into the renal tubules and bile ducts. Another excretory mechanism that plays an insignificant role in the excretion of chemical constituents involves the distribution and distribution of excretory pathways based on the amount of fat or acidity 98 …”