Drug Testing and Analysis
Research article Received: 18 November 2013
Revised: 24 January 2014
Accepted: 27 January 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1628
Characterization of the designer benzodiazepine diclazepam and preliminary data on its metabolism and pharmacokinetics Bjoern Moosmann,a,b Philippe Biselc and Volker Auwärtera* 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)-1methyl-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 selfexperiment 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. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: LC-MS/MS; benzodiazepines; designer drugs; diclazepam; metabolism
Introduction Recently, online retailers selling ‘research chemicals’ started offering diclazepam (other names: 2-chlorodiazepam, Ro 5-3448 or 7-chloro5-(2-chlorophenyl)-1-methyl-1,3-dihydro-2H-1,4-benzodiazepin-2one) (Figure 1) in addition to the two designer benzodiazepines pyrazolam[1,2] and flubromazepam,[3] as alternatives to prescriptiononly benzodiazepines. Similar to its predecessors, the synthesis of this compound was published in an article on pharmaceutical drug candidate research in the 1960s,[4] and the compound is not approved for medical use anywhere in the world. Regarding the potency of this new drug, only limited data can be found in the literature. One study conducted by Babbini et al. showed that diclazepam is approximately 4–8 times more potent than diazepam in terms of reducing motor activity and conflict behavior in rats[5] and studies carried out by Sternbach et al. found diclazepam to be approximately equally potent as diazepam with regard to muscle relaxant and sedative effects in mice and twice as potent than diazepam investigating the same effects in cats.[6] The Ki value at recombinant wild-type α1ß2γ2 γ-aminobutyric acid (GABAA) receptors demonstrated a 30 times higher binding affinity for diclazepam than for diazepam.[7] However, Bradley et al. found that diclazepam given in the same doses as diazepam showed no differences in effects on the behavioral activity of monkeys.[8]
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Due to the structural similarity to diazepam, demethylation, and hydroxylation can be anticipated as the main metabolic reactions which would lead to the benzodiazepines delorazepam, lorazepam, and lormetazepam, all of them being pharmacologically active. In the present study, preliminary data regarding pharmacokinetic properties and metabolism in humans as well as its window of detection in serum and urine are presented. Furthermore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the quantification of diclazepam and its three main metabolites in serum and urine samples. Additionally, different immunochemical assays were assessed for their capability of detecting uptake of a low dose of diclazepam.
* Correspondence to: V. Auwärter, Institute of Forensic Medicine, Forensic Toxicology Department, University Medical Center Freiburg, Albertstr. 9, 79104 Freiburg, Germany. E-mail: [email protected] a Institute of Forensic Medicine, Forensic Toxicology Department, University Medical Center Freiburg, Albertstr. 9, 79104 Freiburg, Germany b Hermann Staudinger Graduate School, University of Freiburg, Hebelstr. 27, 79104 Freiburg, Germany c Institute for Pharmaceutical Sciences, University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany
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Drug Testing and Analysis
B. Moosmann, P. Bisel and V. Auwärter
Figure 1. Molecular structures of diclazepam, diazepam and the two ‘designer-benzodiazepines’ flubromazepam and pyrazolam.
Experimental Materials and chemicals Ammonium formate (99.995%), ethanol (analytical grade), as well as ethyl acetate (analytical grade) were purchased from Sigma Aldrich (Steinheim, Germany). Methanol (HPLC grade) was obtained from Th. Geyer (Renningen, Germany) and acetonitrile (HPLC grade) from J.T. Baker (Deventer, the Netherlands). Formic acid (Rotipuran® ≥ 98%, p.a.) was purchased from Carl Roth (Karlsruhe, Germany) and 1-chlorobutane (LiChrosolv®) from Merck (Darmstadt, Germany). Acetic acid (AnalaR NORMAPUR 100%) was from VWR International (Darmstadt, Germany) and deuterated chloroform (CDCl3) from Euriso-Top (Saint-Aubin, France). Deionized water was prepared using a cartridge deionizer from Memtech (Moorenweis, Germany). Lorazepam-D4, Delorazepam (both 100 μg/mL), Diazepam-D5, Nordazepam-D5, Temazepam-D5, Lorazepam and Lormetazepam (all 1.0 mg/mL) were obtained from LGC Standards GmbH (Wesel, Germany). Diclazepam was extracted from tablets and the filtrate was evaporated to dryness (purity: 96 %, as determined by GC-MS and nuclear magnetic resonance (NMR)). The pure substance was exactly weighed in and dissolved in methanol leading to a 1 mg/mL stock solution. β-Glucuronidase (Escherichia coli, 140 U/mg at 37 °C) and βglucuronidase/arylsulfatase (Helix pomatia, β-glucuronidase 5.5 U/mL and arylsulfatase 2.6 U/mL at 38 °C) were from Roche Diagnostics (Mannheim, Germany). Diclazepam tablets (declared amount: 1 mg per tablet) were ordered from an online shop selling research chemicals. Human blank serum and urine samples were provided by volunteers and stored at -20 °C prior to use. Borate buffer (pH 9) was prepared as previously described.[1,3]
SP isocratic pumps, SIL-20 AC autosampler, CTO-20 AC column oven, DGU-20A3 degasser and CBM-20A controller (Shimadzu, Duisburg, Germany). For data acquisition Analyst® software version 1.5.2 was used (ABSciex, Darmstadt, Germany). Separation was performed as described elsewhere,[1] In brief, gradient elution was applied on a Synergi 4u Polar RP column (150 x 2 mm, 4 μm) with a Polar RP guard column (4 x 2 mm) (Phenomenex, Aschaffenburg, Germany). Mobile phase A consisted of 0.1% HCOOH and 1 mM ammonium formate in deionized water and mobile phase B of 0.1% HCOOH in methanol. The gradient increased from 20% mobile phase B to 95% mobile phase B in 10 min, was held for 1.5 min at 95% mobile phase B, decreased to 20% mobile phase B within 0.5 min and held for 3 min prior to the injection of the next sample. The flow rate was set to 0.4 mL/min and the injection volume was 20 μL. Column oven temperature and autosampler temperature were 40 °C and 4 °C, respectively. A GC-MS mass spectrum was recorded in electron impact ionization (EI) mode using a 6890 series GC system with a 5973 series mass selective detector and 7683 B series injector as well as Chemstation G1701GA version E.02.00.493 software (Agilent, Waldbronn, Germany). GC parameters and MS conditions were as follows and described elsewhere.[1] Splitless injection with an injection port temperature of 270 °C, separation on an HP-5-MS capillary (30 x 0.25 mm i.d., 0.25 μm film thickness (Agilent, Waldbronn, Germany)) with helium as carrier gas and a flow rate of 1 mL/min. Oven temperature was set to 100 °C for 3 min, increased to 310 °C at 30 °C/min and held at 310 °C for 10 min. Transfer line heater and ion source temperature were 280 °C and 230 °C, respectively, and the ionization energy was set to 70 eV. Self-administration study
Identification of diclazepam For identification and further characterization of diclazepem, NMR, LC-MS/MS and gas chromatography mass-spectrometry (GC-MS) techniques were applied after extraction of the compound from the tablets with ethanol. 1D-1H-NMR at 400 MHz, 13C-NMR at 100 MHz as well as selective 2D 1H/13C HSQC, 1H/1H COSY and 1H/13C HMBC spectra were recorded in CDCl3 utilizing a DRX 400 instrument (Bruker BioSpin GmbH, Rheinstetten, Germany). For LC-MS/MS characterization, an enhanced product ion (EPI) spectrum of the ions with m/z 319.0 Da was recorded applying collision energy spread (CES) (collection of data at 20, 35 and 50 eV in one EPI spectrum) and an ion trap fill time of 20 ms and a scan rate of 4000 Da/s on a QTRAP 4000 triple-quadrupole linear ion trap instrument (ABSciex, Darmstadt, Germany) equipped with a TurboIonSpray interface and coupled to a Shimadzu Prominence HPLC system consisting of two LC-20 AD
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To gain preliminary data on pharmacokinetic parameters and investigate the metabolism of diclazepam, one of the authors (43 years, 73 kg) ingested one diclazepam tablet (declared amount: 1 mg diclazepam). Next to a serum and urine sample obtained prior to the intake, 13 serum samples over 14 days and 46 urine samples over 21 days were collected and stored at -20 °C until analysis. In Germany, approval by an ethics committee is not required for self experiments. Identification of the main metabolites For identification of the main metabolites various serum samples (0; 1; 2; 3; 6; 12, 25, 36, 50, and 72 h post ingestion) and urine samples (0; 3; 6; 8; 10; 14; 21, 25, 45, 54, 58, 69, and 93 h post ingestion) were screened for potential metabolites applying LC-MS/MS EPI scan and precursor ion scan experiments utilizing the instrument and conditions described above. In case of the EPI experiments hypothetic masses of potential phase I
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Diclazepam: a new designer benzodiazepine metabolites including mono-, di- and trihydroxylation, demethylation, dehalogenation, methoxylation, formation of an aminobenzophenone or quinazoline-2-one as well as combinations and potential phase II metabolites (glucuronidation or sulfation) of the above, reflecting common metabolic steps for benzodiazepines,[9–12] were implied. For precursor ion experiments characteristic fragments of delorazepm, diclazepam, lorazepam and lormetazepam were included as precursor masses. All samples were analyzed after alkaline liquid-liquid extraction with 1-clorobutane and pretreatment with ß-glucuronidase/ arylsulfatase as well as after precipitation with ice-cold acetonitrile. Analysis of serum, urine and tablet samples Serum and urine sample preparation
A 100 μL aliquot of the sample was spiked with 20 μL of internal standard (IS) solution (2 μg/mL Diazepam-D5, Lorazepam-D4, Nordazepam-D5, Temazepam-D5). Subsequent, 500 μL of phosphate buffer (pH 6) and 50 μL of ß-glucuronidase for enzymatic hydrolysis were added to the urine samples and these samples were incubated for 2 h at 45 °C. Afterwards, liquid-liquid extraction under alkaline conditions, using 1 mL of 1-chlorobutane and 900 μL of borate buffer (pH 9), was performed for both the urine and serum samples. Finally, the supernatant was, after transfer into an HPLC vial and evaporation to dryness under a stream of nitrogen, reconstituted in 100 μL of mobile phase (A/B 80:20) and analyzed using LC-MS/MS. Quantification of diclazepam tablets
For quantification of diclazepam in the purchased tablets, 13 tablets were dissolved separately in 1 mL ethanol, centrifuged and the supernatant transferred into an HPLC vial. Afterwards, the remnant was extracted twice more and the respective supernatants combined. After evaporation to dryness under a stream of nitrogen the samples were reconstituted in 1 mL of methanol and 1:20 000 dilutions in mobile phase (A/B 80:20) were quantified using LC-MS/MS and a five point calibration curve (10–100 ng/mL) in mobile phase. Calibration range, limit of detection and long-term stability
Calibration curves were prepared by spiking blank urine and serum with delorazepam, diclazepam, lorazepam, and lormetazepam in the concentration range of 0.25–100 ng/mL. To account for heteroscedasticity a weighted calibration model (1/x2) was applied. Long-term stability was assessed as recommended by the Society of Toxicological and Forensic Chemistry (GTFCh),[13] by fortifying two pooled blank serum samples and dividing into 12 aliquots each at low (2 ng/mL) and high concentrations (8 ng/mL) of diclazepam, delorazepam, lorazepam, as well as lormetazepam. One set of six samples per concentration was analyzed on the day of preparation (control). The second set was stored at -20 °C for 14 days prior to analysis. Stability was assumed if the mean concentration of the stored samples were within 90–110% of the mean concentration of the control samples. Furthermore, the 90% confidence interval of the stored samples had to fall within 80–120% of the mean concentration of the control samples. As semi-quantitative results were sufficient for gaining preliminary pharmacokinetic data, a full method validation was not carried out. LC-MS/MS analysis
LC-MS/MS instrumentation consisted of an API 5000 triple-quadrupole instrument equipped with a TurboIonSpray interface (ABSciex,
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Darmstadt, Germany) coupled to a Shimadzu HPLC system (two LC-10 AD VP pumps, SCL-10A controller, CTO-10 AC column oven (Shimadzu, Duisburg, Germany), ERC-3415a degasser (ERC, Rimerling, Germany), HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland) equipped with a 100 μL syringe (Hamilton, Reno, NV, USA)). For data acquisition Analyst® software version 1.5.2 was used (ABSciex, Darmstadt, Germany). LC-MS/MS conditions and the applied column were identical, except from storage of the samples at room temperature in the autosampler, to the ones described for the identification of the compound. The MS was operated in multiple reaction monitoring (MRM) mode, including the MRM transitions listed in Table 1 with a minimum dwell time of 20 ms for each transition. Declustering potentials, collision energies and cell exit potentials were optimized for all internal standards, as well as for delorazepam, diclazepam, lorazepam, and lormetazepam. Ion source temperature and ion source voltage were set at 400 °C and +2000 V, respectively. Curtain gas (N2) pressure was 40 psi, ion source gas 1 and 2 (compressed air) pressure were 60 and 70 psi and collision gas (N2) pressure was set to 2 psi. Immunochemical assays
All the serum samples obtained within the first 50 h of the study were tested on a Konelab® 30 instrument (Thermo Fisher Scientific, Dreieich, Germany) with nitrazepam calibrators at 0, 300, and 800 ng/mL and a cut-off at 0 ng/mL nitrazepam equivalents (Microgenics GmbH, Passau, Germany) [cloned enzyme donor immunoassay (CEDIA)]. Furthermore, all urine sample obtained during the study were tested on an Olympus AU400 instrument (Beckman Coulter GmbH, Krefeld, Germany) with the same antibody kit as described above and calibrators at 0, 50, and 200 ng/mL and a cut-off at 50 ng/mL. Additionally the same urine samples were retested on an Axsym® 4602 instrument with nordazepam calibrators at 0, 200, 400, 800, 1200, and 2400 ng/mL
Table 1. Multiple reaction monitoring transitions and mass spectrometry parameters of diclazepam, delorazepam, lorazepam, lormetazepam and of the internal standards used. Analyte
Q1 mass [amu]
Diclazepam
319
Delorazepam
305
Lorazepam
321
Lormetazepam
335
Diazepam-D5 Lorazepam-D4 Nordazepam-D5 Temazepam-D5
290 327 276 306
Q3 mass [amu] 227 154 256 140 241 275 303 289 177 154 281 140 198
DP [V] 50 50 50 70 70 40 40 80 80 20 50 40 60
CE [V] 42 42 38 45 43 32 23 33 60 50 32 20 53
CXP [V] 32 22 37 18 35 38 40 42 21 38 41 30 27
Entrance potential was set to 8 V Data in bold are ion transitions used for quantification DP: Declustering potential CE: Collision energy CXP: Collision cell exit potential
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Drug Testing and Analysis
B. Moosmann, P. Bisel and V. Auwärter
and a cut-off at 200 ng/mL nordazepam equivalents (both Abbott Laboratories, Wiesbaden, Germany) [fluorescence polarization immunoassay (FPIA)]. Cross-reactivities of all three immunochemical assays were assessed by spiking blank serum and urine with diclazepam at 200 ng/mL, respectively. In the case of the FPIA assay, the determined cross-reactivity was 75% and for the CEDIA assay 72% on the Konelab® 30 instrument and 136 % on the Olympus AU400 instrument.
Results and discussion Identification of diclazepam Applying NMR techniques the compound extracted from the tablet could be confirmed as 7-chloro-5-(2-chlorophenyl)-1-methyl-1,3dihydro-2H-1,4-benzodiazepin-2-one. Besides the N-methyl group
and the methylene moiety of the diazepinon ring, two spin systems have been identified in the aromatic region, one of them consisting of three coupling protons with a 1,2,4-relationship. The second aromatic spin system is more complex but consists of two protons with only one ortho-coupling each and two protons with two orthocouplings each, strongly indicating an ortho-disubstituted phenyl moiety. The 1H- and 13C-data are shown in Table 2. The three most abundant ions in the LC-MS/MS EPI spectrum (Figure 2) (m/z: 227, 154 and 256) were included as MRM transitions in the quantitative LC-MS/MS method. The most intense peaks in the GC-EI-MS spectrum were the signal for the molecular ion (m/z: 318; relative intensity 95 %) and signals at m/z 282.9 (100 %), 292.0 (68 %), 255.0 (53 %) and 176.9 (30 %) (Figure 3).
Quantification of diclazepam tablets Table 2. NMR data for diclazepam in CDCl3 (for numbering, refer to Figure 1). Carbon number
δ
13
C NMR (ppm)
2 3
169.3 56.8
5 5a 6 7 8 9 9a 1’ 2’ 3’ 4’ 5’ 6’ 1’’
168.6 129.6 128.0 131.2 131.4 122.7 141.8 133.0 137.9 130.1 127.1 131.0 131.1 34.9
1
δ H NMR (ppm)
3.8 (d, 10.28, 1H) / 4.8 (d, 10.28, 1H)
The amount of diclazepam per tablet ranged from 0.59 to 1.39 mg (median: 0.95 mg, mean: 0.94 mg, SD: 0.23) (n = 13), which does not comply with pharmaceutical guidelines.[14] Furthermore it should be noted that extraction efficiency could not be determined and the actual amount per tablet might be higher. The wide concentration range bears the risk for consumer to overdose since it is not possible to predict the amount of active compound per tablet.
7.04 (d, 2.58, 1H) 7.5 (dd, 2.58, 8.84, 1H) 7.3 (d, 8.87, 1H)
7.35-7.42 (m, 1H) 7.40-7.46 (m, 1H) 7.38-7.44 (m, 1H) 7.57-7.63 (m, 1H) 3.4 (s, 3H)
Identification of the main metabolites Using EPI scan experiments, signals at m/z corresponding to a demethylated, a monohydroxylated as well as to a demethylated monohydroxylated metabolite could be detected in serum and urine samples. Using commercially available standards of delorazepam, lorazepam and lormetazepam the spectra could clearly be assigned to these three benzodiazepines. However, none of these metabolites was excreted unconjugated in the obtained urine samples. In the serum samples no glucuronidated metabolites could be detected. The main metabolic pathway of diclazepam is shown in Figure 4.
Figure 2. LC-MS/MS EPI spectrum of diclazepam and proposed fragmentation. (Collision energy spread: Collection of data at 20, 35 and 50 eV in one EPI spectrum).
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Drug Testing and Analysis
Diclazepam: a new designer benzodiazepine
Figure 3. GC-EI-MS spectrum and diclazepam.
proposed fragmentation of
Limit of detection and long-term stability A signal-to-noise ratio of at least 3:1 could be obtained for all the monitored MRM transitions of the four analytes at the concentration of the lowest calibrator (0.25 ng/mL). Therefore, 0.25 ng/mL was set as the limit of detection. Long-term stability could be shown for all four analytes over the storage period of 14 days. The mean concentration ranged from 91 to 101% and the 90% confidence interval fell within 85–104% of the mean concentration of the control samples, respectively.
Analysis of the serum samples The serum concentrations over time of diclazepam and its metabolites delorazepam, lorazepam and lormetazepam after the intake of one tablet (declared amount: 1 mg) are shown in Figure 5 (no noticeable effects were reported by the volunteer at any time during the days after uptake). The highest concentration of diclazepam (3.4 ng/mL) was detected in the sample obtained 3 h after the intake and maximum serum levels of the
Figure 5. Concentration time profile of diclazepam and its metabolites delorazepam, lorazepam and lormetazepam in serum after the consumption of one tablet diclazepam (declared amount: 1 mg) obtained by LCMS/MS analysi.
metabolites were reached after 36 h for delorazepam (2.0 ng/mL), 99 h for lorazepam (0.4 ng/mL) and 6 h for lormetazepam (0.34 ng/mL), respectively. For calculation of basic pharmacokinetic parameters of diclazepam a demo version of the software Kinetica 5.1 (Thermo Fisher Scientific, Waltham, MA, USA) was utilized, fitting the results into a two-compartment model. Based on the data obtained from one volunteer, diclazepam has an approximate elimination half-life in the distribution phase of 1.9 h and in the terminal phase of 42 h and can therefore be regarded as a long-acting benzodiazepine. Volume of distribution and clearance were 8 l/kg and 165 mL/min. Both of these values have to be treated with caution as the exact dose ingested by the volunteer is not known, although it was most probably close to 1 mg (0.94 ± 0.23 mg). Applying the above described LC-MS/MS method, intake of one tablet could in our case be detected in serum for 99 h targeting the parent compound and for 10 days analyzing the metabolite delorazepam.
Figure 4. Metabolism of diclazepam in a human volunteer.
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Drug Testing and Analysis
B. Moosmann, P. Bisel and V. Auwärter
Figure 6. Concentration time profile of the diclazepam metabolites delorazepam, lorazepam and lormetazepam in urine, after the consumption of one tablet diclazepam (declared amount: 1 mg) obtained by LC-MS/MS analysis after enzymatic hydrolysis using β-glucuronidase (Escherichia coli). The dotted line shows the response of the applied immunoassay (Olympus AU400 instrument) normalized to the creatinine concentration.
Applying an immunochemical assay, all serum samples tested negative, demonstrating that these kind of analytical tests are not suitable for comprehensive benzodiazepine screening in this matrix despite a sufficient cross-reactivity of 72%, a problem shared with other highly potent benzodiazepines like flunitrazepam or triazolam. Analysis of the urine samples Unmetabolized diclazepam could only be detected in some of the urine samples collected between 3 and 56 h post consumption and only in very low concentrations (0.1–0.5 ng/mg; normalized to a creatinine of 100 mg/dL for easier comparison). The concentration profiles of the three main metabolites after hydrolysis with β-glucuronidase are shown in Figure 6. The main metabolite detected in urine is delorazepam, which is already present in the urine sample obtained 3 h post ingestion and detectable up to day 6 post ingestion. Both lorazepam as well as lormetazepam allow for a longer window of detection of 19 days and 11 days post intake, respectively. However, as all three metabolites are marketed as pharmaceutical drugs in various countries worldwide, identification of the compound consumed may be hampered when based solely on urine results. As already seen with the serum samples, the sensitivity of one of the applied immunochemical assays was not sufficient for detection of this low-dosed drug, as no urine samples tested positive using the FPIA technique. However, utilizing the CEDIA on the Olympus AU400 instrument, seven urine samples tested positive (obtained: 14, 21, 25, 45, 50, 62, and 84 h post consumption) enabling detection even after low dosed uptake (Figure 6). Nevertheless, it has to be noted that all the positive urine samples (cut-off 50 ng/mL, calibrated for nitrazepam) showed rather high creatinine concentrations greater than 151 mg/dL.
Conclusion The typical approach of clandestine designer drug distributors particularly observed in the early stages of the ‘spice phenomenon’,[15] comprising of exploiting published literature from pharmaceutical
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drug candidate research, online marketing and rapid adaptation to changing narcotics laws, reached out to the drug class of benzodiazepines. Diclazepam (7-chloro-5-(2-chlorophenyl)-1-methyl-1,3dihydro-2H-1,4-benzodiazepin-2-one) represents the third designer benzodiazepine available from online vendors within less than one year. Similar to the preceding two, pyrazolam and flubromazepam, detectability in serum and urine samples utilizing immunochemical assays is limited due to the low doses applied as suggested by the low amounts of active compound per tablet. As a consequence, it has to be carefully assessed whether these types of assays are sufficient when analyzing for example samples from drug rehabilitation clinics where clients are more likely to consume ‘legal highs’ or research chemicals. However, applying a sensitive LC-MS/MS method covering the three commercially available benzodiazepines delorazepam, lorazepam and lormetazepam seems sufficient, as presence of all three analytes in the same sample may indicate an uptake of diclazepam. Nevertheless, for a definite proof serum samples should be analyzed since the parent drug was detectable for up to 99 h after the intake of one tablet in this matrix and only small amounts of unmetabolized diclazepam could be detected in the urine samples. The overall window of detection was 10 days in serum targeting the metabolite delorazepam and 19 days in urine targeting lorazepam, with the limitation that no statement regarding the actual drug consumed can be made as all three main metabolites are marketed by pharmaceutical companies in many countries worldwide. Diclazepam shows a rather long terminal elimination half-life of approximately 42 h and its pharmacokinetic profile seems to follow a multi-compartment model showing at least biphasic elimination. All three main metabolites of diclazepam are pharmacologically active with elimination half-lives of 78 h (delorazepam),[11] 12 h (lorazepam)[16] and 13 h (lormetazepam),[17] respectively. As a consequence, long-lasting sedative effects seem likely when applying higher or repeated doses. The latter could also lead to severe intoxication by accumulation. Some additional risk for consumers of this specific product comes from the considerable variation of drug content per tablet which is not at all predictable for the user and may lead to imprecise dosing.
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Diclazepam: a new designer benzodiazepine Acknowledgements This publication has been produced with the financial support of the Drug Prevention and Information Programme of the European Union (JUST/2011/DPIP/AG/3597), the German Federal Ministry of Health, and the City of Frankfurt/Main.
References [1] B. Moosmann, M. Hutter, L. Huppertz, S. Ferlaino, L. Redlingshöfer, V. Auwärter. Characterization of the designer benzodiazepine pyrazolam and its detectability in human serum and urine. Forensic Toxicol. 2013, 31, 263. [2] EMCDDA. Reporting form on the new drug Pyrazolam. REITOX, Finland, 2012. [3] B. Moosmann, L. Huppertz, M. Hutter, A. Buchwald, S. Ferlaino, V. Auwärter. Detection and identification of the designer benzodiazepine flubromazepam and preliminary data on its metabolism and pharmacokinetics. J. Mass Spectrom. 2013, 48, 1150. [4] L.H. Sternbach, R.I. Fryer, W. Metlesics, E. Reeder, G. Sach, G. Saucy, A. Stempel. Quinazolines and 1,4-Benzodiazepines. VI.1a Halo-, Methyl-, and Methoxy-substituted 1,3-Dihydro-5-phenyl-2H-1,4benzodiazepin-2-ones1b,c. J. Org. Chem. 1962, 27, 3788. [5] M. Babbini, M. Gaiardi, M. Bartoletti. Anxiolytic versus sedative properties in the benzodiazepines series: Differencies in structure activity relationships. Life Sci. 1979, 25, 15. [6] L.H. Sternbach, L.O. Randall, R. Banziger, H. Lehr. Chapter 6: Structure-activity relationships in the 1,3-benzodiazepine series, in Drugs Affecting the Central Nervous System, (Ed: A. Burger), Edward Arnold, London, 1968. [7] E. Sigel, M.T. Schaerer, A. Buhr, R. Baur. The benzodiazepine binding pocket of recombinant α1ß2γ2 γ-Aminobutyric AcidA receptors: Relative orientation of ligands and amino acid side chains. Mol. Pharmacol. 1998, 54, 1097.
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[8] C.M. Bradley, A.N. Nicholson. Activity of the chloro- and triazolobenzodiazepines: Behavioural studies in the monkey (Macaca mulatta). Neuropharmacology 1984, 23, 327. [9] UNODC. Recommended Methods for the Detection and Assay of Barbiturates and Benzodiazepines in Biological Specimens, manual for use by National laboratories. United Nations Office on Drugs and Crime, New York, 1997. [10] L.A. Berrueta, B. Gallo, F. Vicente. Biopharmacological data and high-performance liquid chromatographic analysis of 1,4benzodiazepines in biological fluids: A review. J. Pharmaceut. Biomed. 1992, 10, 109. [11] L.D. Bo, F. Marcucci, E. Mussini, D. Perbellini, A. Castellani, P. Fresia. Plasma levels of chlorodesmethyldiazepam in humans, Biopharm. Drug Disposition 1980, 1, 123. [12] T.H. Williams, G.J. Sasso, J.J. Ryan, M.A. Schwartz. Novel application of proton nuclear magnetic resonance spectroscopy in the identification of 2’-chloronordiazepam metabolites in the dog. J. Med. Chem. 1979, 22, 436. [13] F.T. Peters, M. Hartung, M. Herbold, G. Schmitt, T. Daldrup, F. Mußhoff. Anhang B zu den Richtlinien der GTFCh zur Qualitätssicherung bei forensisch-toxikologischen Untersuchungen; Anforderungen an die Validierung von Analysenmethoden. Toxichem. Krimtech. 2009, 76, 185. [14] Uniformity of content of single-dose preparations, European Pharmacopoeia, 8th edn. Council of Europe. Available at: http://online. edqm.eu/EN/entry.htm. [20 February 2014] [15] P. Griffiths, R. Sedefov, A.N.A. Gallegos, D. Lopez. How globalization and market innovation challenge how we think about and respond to drug use: ‘Spice’ a case study. Addiction 2010, 105, 951. [16] D.J. Greenblatt, R.T. Schillings, A.A. Kyriakopoulos, R.I. Shader, S.F. Sisenwine, J.A. Knowles, H.W. Ruelius. Clinical pharmacokinetics of lorazepam. I. Absorption and disposition of oral 14C-lorazepam. Clin. Pharmacol. Ther. 1976, 20, 329. [17] M. Hümpel, V. Illi, W. Milius, H. Wendt, M. Kurowski. The pharmacokinetics and biotransformation of the new benzodiazepine lormetazepam in humans I. Absorption, distribution, elimination and metabolism of 14 lormetazepam-5- C. Eur. J. Drug Metab. Ph. 1979, 4, 237.
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Research article Received: 18 November 2013
Revised: 24 January 2014
Accepted: 27 January 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1628
Characterization of the designer benzodiazepine diclazepam and preliminary data on its metabolism and pharmacokinetics Bjoern Moosmann,a,b Philippe Biselc and Volker Auwärtera* 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)-1methyl-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 selfexperiment 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. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: LC-MS/MS; benzodiazepines; designer drugs; diclazepam; metabolism
Introduction Recently, online retailers selling ‘research chemicals’ started offering diclazepam (other names: 2-chlorodiazepam, Ro 5-3448 or 7-chloro5-(2-chlorophenyl)-1-methyl-1,3-dihydro-2H-1,4-benzodiazepin-2one) (Figure 1) in addition to the two designer benzodiazepines pyrazolam[1,2] and flubromazepam,[3] as alternatives to prescriptiononly benzodiazepines. Similar to its predecessors, the synthesis of this compound was published in an article on pharmaceutical drug candidate research in the 1960s,[4] and the compound is not approved for medical use anywhere in the world. Regarding the potency of this new drug, only limited data can be found in the literature. One study conducted by Babbini et al. showed that diclazepam is approximately 4–8 times more potent than diazepam in terms of reducing motor activity and conflict behavior in rats[5] and studies carried out by Sternbach et al. found diclazepam to be approximately equally potent as diazepam with regard to muscle relaxant and sedative effects in mice and twice as potent than diazepam investigating the same effects in cats.[6] The Ki value at recombinant wild-type α1ß2γ2 γ-aminobutyric acid (GABAA) receptors demonstrated a 30 times higher binding affinity for diclazepam than for diazepam.[7] However, Bradley et al. found that diclazepam given in the same doses as diazepam showed no differences in effects on the behavioral activity of monkeys.[8]
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Due to the structural similarity to diazepam, demethylation, and hydroxylation can be anticipated as the main metabolic reactions which would lead to the benzodiazepines delorazepam, lorazepam, and lormetazepam, all of them being pharmacologically active. In the present study, preliminary data regarding pharmacokinetic properties and metabolism in humans as well as its window of detection in serum and urine are presented. Furthermore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the quantification of diclazepam and its three main metabolites in serum and urine samples. Additionally, different immunochemical assays were assessed for their capability of detecting uptake of a low dose of diclazepam.
* Correspondence to: V. Auwärter, Institute of Forensic Medicine, Forensic Toxicology Department, University Medical Center Freiburg, Albertstr. 9, 79104 Freiburg, Germany. E-mail: [email protected] a Institute of Forensic Medicine, Forensic Toxicology Department, University Medical Center Freiburg, Albertstr. 9, 79104 Freiburg, Germany b Hermann Staudinger Graduate School, University of Freiburg, Hebelstr. 27, 79104 Freiburg, Germany c Institute for Pharmaceutical Sciences, University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany
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B. Moosmann, P. Bisel and V. Auwärter
Figure 1. Molecular structures of diclazepam, diazepam and the two ‘designer-benzodiazepines’ flubromazepam and pyrazolam.
Experimental Materials and chemicals Ammonium formate (99.995%), ethanol (analytical grade), as well as ethyl acetate (analytical grade) were purchased from Sigma Aldrich (Steinheim, Germany). Methanol (HPLC grade) was obtained from Th. Geyer (Renningen, Germany) and acetonitrile (HPLC grade) from J.T. Baker (Deventer, the Netherlands). Formic acid (Rotipuran® ≥ 98%, p.a.) was purchased from Carl Roth (Karlsruhe, Germany) and 1-chlorobutane (LiChrosolv®) from Merck (Darmstadt, Germany). Acetic acid (AnalaR NORMAPUR 100%) was from VWR International (Darmstadt, Germany) and deuterated chloroform (CDCl3) from Euriso-Top (Saint-Aubin, France). Deionized water was prepared using a cartridge deionizer from Memtech (Moorenweis, Germany). Lorazepam-D4, Delorazepam (both 100 μg/mL), Diazepam-D5, Nordazepam-D5, Temazepam-D5, Lorazepam and Lormetazepam (all 1.0 mg/mL) were obtained from LGC Standards GmbH (Wesel, Germany). Diclazepam was extracted from tablets and the filtrate was evaporated to dryness (purity: 96 %, as determined by GC-MS and nuclear magnetic resonance (NMR)). The pure substance was exactly weighed in and dissolved in methanol leading to a 1 mg/mL stock solution. β-Glucuronidase (Escherichia coli, 140 U/mg at 37 °C) and βglucuronidase/arylsulfatase (Helix pomatia, β-glucuronidase 5.5 U/mL and arylsulfatase 2.6 U/mL at 38 °C) were from Roche Diagnostics (Mannheim, Germany). Diclazepam tablets (declared amount: 1 mg per tablet) were ordered from an online shop selling research chemicals. Human blank serum and urine samples were provided by volunteers and stored at -20 °C prior to use. Borate buffer (pH 9) was prepared as previously described.[1,3]
SP isocratic pumps, SIL-20 AC autosampler, CTO-20 AC column oven, DGU-20A3 degasser and CBM-20A controller (Shimadzu, Duisburg, Germany). For data acquisition Analyst® software version 1.5.2 was used (ABSciex, Darmstadt, Germany). Separation was performed as described elsewhere,[1] In brief, gradient elution was applied on a Synergi 4u Polar RP column (150 x 2 mm, 4 μm) with a Polar RP guard column (4 x 2 mm) (Phenomenex, Aschaffenburg, Germany). Mobile phase A consisted of 0.1% HCOOH and 1 mM ammonium formate in deionized water and mobile phase B of 0.1% HCOOH in methanol. The gradient increased from 20% mobile phase B to 95% mobile phase B in 10 min, was held for 1.5 min at 95% mobile phase B, decreased to 20% mobile phase B within 0.5 min and held for 3 min prior to the injection of the next sample. The flow rate was set to 0.4 mL/min and the injection volume was 20 μL. Column oven temperature and autosampler temperature were 40 °C and 4 °C, respectively. A GC-MS mass spectrum was recorded in electron impact ionization (EI) mode using a 6890 series GC system with a 5973 series mass selective detector and 7683 B series injector as well as Chemstation G1701GA version E.02.00.493 software (Agilent, Waldbronn, Germany). GC parameters and MS conditions were as follows and described elsewhere.[1] Splitless injection with an injection port temperature of 270 °C, separation on an HP-5-MS capillary (30 x 0.25 mm i.d., 0.25 μm film thickness (Agilent, Waldbronn, Germany)) with helium as carrier gas and a flow rate of 1 mL/min. Oven temperature was set to 100 °C for 3 min, increased to 310 °C at 30 °C/min and held at 310 °C for 10 min. Transfer line heater and ion source temperature were 280 °C and 230 °C, respectively, and the ionization energy was set to 70 eV. Self-administration study
Identification of diclazepam For identification and further characterization of diclazepem, NMR, LC-MS/MS and gas chromatography mass-spectrometry (GC-MS) techniques were applied after extraction of the compound from the tablets with ethanol. 1D-1H-NMR at 400 MHz, 13C-NMR at 100 MHz as well as selective 2D 1H/13C HSQC, 1H/1H COSY and 1H/13C HMBC spectra were recorded in CDCl3 utilizing a DRX 400 instrument (Bruker BioSpin GmbH, Rheinstetten, Germany). For LC-MS/MS characterization, an enhanced product ion (EPI) spectrum of the ions with m/z 319.0 Da was recorded applying collision energy spread (CES) (collection of data at 20, 35 and 50 eV in one EPI spectrum) and an ion trap fill time of 20 ms and a scan rate of 4000 Da/s on a QTRAP 4000 triple-quadrupole linear ion trap instrument (ABSciex, Darmstadt, Germany) equipped with a TurboIonSpray interface and coupled to a Shimadzu Prominence HPLC system consisting of two LC-20 AD
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To gain preliminary data on pharmacokinetic parameters and investigate the metabolism of diclazepam, one of the authors (43 years, 73 kg) ingested one diclazepam tablet (declared amount: 1 mg diclazepam). Next to a serum and urine sample obtained prior to the intake, 13 serum samples over 14 days and 46 urine samples over 21 days were collected and stored at -20 °C until analysis. In Germany, approval by an ethics committee is not required for self experiments. Identification of the main metabolites For identification of the main metabolites various serum samples (0; 1; 2; 3; 6; 12, 25, 36, 50, and 72 h post ingestion) and urine samples (0; 3; 6; 8; 10; 14; 21, 25, 45, 54, 58, 69, and 93 h post ingestion) were screened for potential metabolites applying LC-MS/MS EPI scan and precursor ion scan experiments utilizing the instrument and conditions described above. In case of the EPI experiments hypothetic masses of potential phase I
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Diclazepam: a new designer benzodiazepine metabolites including mono-, di- and trihydroxylation, demethylation, dehalogenation, methoxylation, formation of an aminobenzophenone or quinazoline-2-one as well as combinations and potential phase II metabolites (glucuronidation or sulfation) of the above, reflecting common metabolic steps for benzodiazepines,[9–12] were implied. For precursor ion experiments characteristic fragments of delorazepm, diclazepam, lorazepam and lormetazepam were included as precursor masses. All samples were analyzed after alkaline liquid-liquid extraction with 1-clorobutane and pretreatment with ß-glucuronidase/ arylsulfatase as well as after precipitation with ice-cold acetonitrile. Analysis of serum, urine and tablet samples Serum and urine sample preparation
A 100 μL aliquot of the sample was spiked with 20 μL of internal standard (IS) solution (2 μg/mL Diazepam-D5, Lorazepam-D4, Nordazepam-D5, Temazepam-D5). Subsequent, 500 μL of phosphate buffer (pH 6) and 50 μL of ß-glucuronidase for enzymatic hydrolysis were added to the urine samples and these samples were incubated for 2 h at 45 °C. Afterwards, liquid-liquid extraction under alkaline conditions, using 1 mL of 1-chlorobutane and 900 μL of borate buffer (pH 9), was performed for both the urine and serum samples. Finally, the supernatant was, after transfer into an HPLC vial and evaporation to dryness under a stream of nitrogen, reconstituted in 100 μL of mobile phase (A/B 80:20) and analyzed using LC-MS/MS. Quantification of diclazepam tablets
For quantification of diclazepam in the purchased tablets, 13 tablets were dissolved separately in 1 mL ethanol, centrifuged and the supernatant transferred into an HPLC vial. Afterwards, the remnant was extracted twice more and the respective supernatants combined. After evaporation to dryness under a stream of nitrogen the samples were reconstituted in 1 mL of methanol and 1:20 000 dilutions in mobile phase (A/B 80:20) were quantified using LC-MS/MS and a five point calibration curve (10–100 ng/mL) in mobile phase. Calibration range, limit of detection and long-term stability
Calibration curves were prepared by spiking blank urine and serum with delorazepam, diclazepam, lorazepam, and lormetazepam in the concentration range of 0.25–100 ng/mL. To account for heteroscedasticity a weighted calibration model (1/x2) was applied. Long-term stability was assessed as recommended by the Society of Toxicological and Forensic Chemistry (GTFCh),[13] by fortifying two pooled blank serum samples and dividing into 12 aliquots each at low (2 ng/mL) and high concentrations (8 ng/mL) of diclazepam, delorazepam, lorazepam, as well as lormetazepam. One set of six samples per concentration was analyzed on the day of preparation (control). The second set was stored at -20 °C for 14 days prior to analysis. Stability was assumed if the mean concentration of the stored samples were within 90–110% of the mean concentration of the control samples. Furthermore, the 90% confidence interval of the stored samples had to fall within 80–120% of the mean concentration of the control samples. As semi-quantitative results were sufficient for gaining preliminary pharmacokinetic data, a full method validation was not carried out. LC-MS/MS analysis
LC-MS/MS instrumentation consisted of an API 5000 triple-quadrupole instrument equipped with a TurboIonSpray interface (ABSciex,
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Darmstadt, Germany) coupled to a Shimadzu HPLC system (two LC-10 AD VP pumps, SCL-10A controller, CTO-10 AC column oven (Shimadzu, Duisburg, Germany), ERC-3415a degasser (ERC, Rimerling, Germany), HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland) equipped with a 100 μL syringe (Hamilton, Reno, NV, USA)). For data acquisition Analyst® software version 1.5.2 was used (ABSciex, Darmstadt, Germany). LC-MS/MS conditions and the applied column were identical, except from storage of the samples at room temperature in the autosampler, to the ones described for the identification of the compound. The MS was operated in multiple reaction monitoring (MRM) mode, including the MRM transitions listed in Table 1 with a minimum dwell time of 20 ms for each transition. Declustering potentials, collision energies and cell exit potentials were optimized for all internal standards, as well as for delorazepam, diclazepam, lorazepam, and lormetazepam. Ion source temperature and ion source voltage were set at 400 °C and +2000 V, respectively. Curtain gas (N2) pressure was 40 psi, ion source gas 1 and 2 (compressed air) pressure were 60 and 70 psi and collision gas (N2) pressure was set to 2 psi. Immunochemical assays
All the serum samples obtained within the first 50 h of the study were tested on a Konelab® 30 instrument (Thermo Fisher Scientific, Dreieich, Germany) with nitrazepam calibrators at 0, 300, and 800 ng/mL and a cut-off at 0 ng/mL nitrazepam equivalents (Microgenics GmbH, Passau, Germany) [cloned enzyme donor immunoassay (CEDIA)]. Furthermore, all urine sample obtained during the study were tested on an Olympus AU400 instrument (Beckman Coulter GmbH, Krefeld, Germany) with the same antibody kit as described above and calibrators at 0, 50, and 200 ng/mL and a cut-off at 50 ng/mL. Additionally the same urine samples were retested on an Axsym® 4602 instrument with nordazepam calibrators at 0, 200, 400, 800, 1200, and 2400 ng/mL
Table 1. Multiple reaction monitoring transitions and mass spectrometry parameters of diclazepam, delorazepam, lorazepam, lormetazepam and of the internal standards used. Analyte
Q1 mass [amu]
Diclazepam
319
Delorazepam
305
Lorazepam
321
Lormetazepam
335
Diazepam-D5 Lorazepam-D4 Nordazepam-D5 Temazepam-D5
290 327 276 306
Q3 mass [amu] 227 154 256 140 241 275 303 289 177 154 281 140 198
DP [V] 50 50 50 70 70 40 40 80 80 20 50 40 60
CE [V] 42 42 38 45 43 32 23 33 60 50 32 20 53
CXP [V] 32 22 37 18 35 38 40 42 21 38 41 30 27
Entrance potential was set to 8 V Data in bold are ion transitions used for quantification DP: Declustering potential CE: Collision energy CXP: Collision cell exit potential
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and a cut-off at 200 ng/mL nordazepam equivalents (both Abbott Laboratories, Wiesbaden, Germany) [fluorescence polarization immunoassay (FPIA)]. Cross-reactivities of all three immunochemical assays were assessed by spiking blank serum and urine with diclazepam at 200 ng/mL, respectively. In the case of the FPIA assay, the determined cross-reactivity was 75% and for the CEDIA assay 72% on the Konelab® 30 instrument and 136 % on the Olympus AU400 instrument.
Results and discussion Identification of diclazepam Applying NMR techniques the compound extracted from the tablet could be confirmed as 7-chloro-5-(2-chlorophenyl)-1-methyl-1,3dihydro-2H-1,4-benzodiazepin-2-one. Besides the N-methyl group
and the methylene moiety of the diazepinon ring, two spin systems have been identified in the aromatic region, one of them consisting of three coupling protons with a 1,2,4-relationship. The second aromatic spin system is more complex but consists of two protons with only one ortho-coupling each and two protons with two orthocouplings each, strongly indicating an ortho-disubstituted phenyl moiety. The 1H- and 13C-data are shown in Table 2. The three most abundant ions in the LC-MS/MS EPI spectrum (Figure 2) (m/z: 227, 154 and 256) were included as MRM transitions in the quantitative LC-MS/MS method. The most intense peaks in the GC-EI-MS spectrum were the signal for the molecular ion (m/z: 318; relative intensity 95 %) and signals at m/z 282.9 (100 %), 292.0 (68 %), 255.0 (53 %) and 176.9 (30 %) (Figure 3).
Quantification of diclazepam tablets Table 2. NMR data for diclazepam in CDCl3 (for numbering, refer to Figure 1). Carbon number
δ
13
C NMR (ppm)
2 3
169.3 56.8
5 5a 6 7 8 9 9a 1’ 2’ 3’ 4’ 5’ 6’ 1’’
168.6 129.6 128.0 131.2 131.4 122.7 141.8 133.0 137.9 130.1 127.1 131.0 131.1 34.9
1
δ H NMR (ppm)
3.8 (d, 10.28, 1H) / 4.8 (d, 10.28, 1H)
The amount of diclazepam per tablet ranged from 0.59 to 1.39 mg (median: 0.95 mg, mean: 0.94 mg, SD: 0.23) (n = 13), which does not comply with pharmaceutical guidelines.[14] Furthermore it should be noted that extraction efficiency could not be determined and the actual amount per tablet might be higher. The wide concentration range bears the risk for consumer to overdose since it is not possible to predict the amount of active compound per tablet.
7.04 (d, 2.58, 1H) 7.5 (dd, 2.58, 8.84, 1H) 7.3 (d, 8.87, 1H)
7.35-7.42 (m, 1H) 7.40-7.46 (m, 1H) 7.38-7.44 (m, 1H) 7.57-7.63 (m, 1H) 3.4 (s, 3H)
Identification of the main metabolites Using EPI scan experiments, signals at m/z corresponding to a demethylated, a monohydroxylated as well as to a demethylated monohydroxylated metabolite could be detected in serum and urine samples. Using commercially available standards of delorazepam, lorazepam and lormetazepam the spectra could clearly be assigned to these three benzodiazepines. However, none of these metabolites was excreted unconjugated in the obtained urine samples. In the serum samples no glucuronidated metabolites could be detected. The main metabolic pathway of diclazepam is shown in Figure 4.
Figure 2. LC-MS/MS EPI spectrum of diclazepam and proposed fragmentation. (Collision energy spread: Collection of data at 20, 35 and 50 eV in one EPI spectrum).
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Drug Testing and Analysis
Diclazepam: a new designer benzodiazepine
Figure 3. GC-EI-MS spectrum and diclazepam.
proposed fragmentation of
Limit of detection and long-term stability A signal-to-noise ratio of at least 3:1 could be obtained for all the monitored MRM transitions of the four analytes at the concentration of the lowest calibrator (0.25 ng/mL). Therefore, 0.25 ng/mL was set as the limit of detection. Long-term stability could be shown for all four analytes over the storage period of 14 days. The mean concentration ranged from 91 to 101% and the 90% confidence interval fell within 85–104% of the mean concentration of the control samples, respectively.
Analysis of the serum samples The serum concentrations over time of diclazepam and its metabolites delorazepam, lorazepam and lormetazepam after the intake of one tablet (declared amount: 1 mg) are shown in Figure 5 (no noticeable effects were reported by the volunteer at any time during the days after uptake). The highest concentration of diclazepam (3.4 ng/mL) was detected in the sample obtained 3 h after the intake and maximum serum levels of the
Figure 5. Concentration time profile of diclazepam and its metabolites delorazepam, lorazepam and lormetazepam in serum after the consumption of one tablet diclazepam (declared amount: 1 mg) obtained by LCMS/MS analysi.
metabolites were reached after 36 h for delorazepam (2.0 ng/mL), 99 h for lorazepam (0.4 ng/mL) and 6 h for lormetazepam (0.34 ng/mL), respectively. For calculation of basic pharmacokinetic parameters of diclazepam a demo version of the software Kinetica 5.1 (Thermo Fisher Scientific, Waltham, MA, USA) was utilized, fitting the results into a two-compartment model. Based on the data obtained from one volunteer, diclazepam has an approximate elimination half-life in the distribution phase of 1.9 h and in the terminal phase of 42 h and can therefore be regarded as a long-acting benzodiazepine. Volume of distribution and clearance were 8 l/kg and 165 mL/min. Both of these values have to be treated with caution as the exact dose ingested by the volunteer is not known, although it was most probably close to 1 mg (0.94 ± 0.23 mg). Applying the above described LC-MS/MS method, intake of one tablet could in our case be detected in serum for 99 h targeting the parent compound and for 10 days analyzing the metabolite delorazepam.
Figure 4. Metabolism of diclazepam in a human volunteer.
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Figure 6. Concentration time profile of the diclazepam metabolites delorazepam, lorazepam and lormetazepam in urine, after the consumption of one tablet diclazepam (declared amount: 1 mg) obtained by LC-MS/MS analysis after enzymatic hydrolysis using β-glucuronidase (Escherichia coli). The dotted line shows the response of the applied immunoassay (Olympus AU400 instrument) normalized to the creatinine concentration.
Applying an immunochemical assay, all serum samples tested negative, demonstrating that these kind of analytical tests are not suitable for comprehensive benzodiazepine screening in this matrix despite a sufficient cross-reactivity of 72%, a problem shared with other highly potent benzodiazepines like flunitrazepam or triazolam. Analysis of the urine samples Unmetabolized diclazepam could only be detected in some of the urine samples collected between 3 and 56 h post consumption and only in very low concentrations (0.1–0.5 ng/mg; normalized to a creatinine of 100 mg/dL for easier comparison). The concentration profiles of the three main metabolites after hydrolysis with β-glucuronidase are shown in Figure 6. The main metabolite detected in urine is delorazepam, which is already present in the urine sample obtained 3 h post ingestion and detectable up to day 6 post ingestion. Both lorazepam as well as lormetazepam allow for a longer window of detection of 19 days and 11 days post intake, respectively. However, as all three metabolites are marketed as pharmaceutical drugs in various countries worldwide, identification of the compound consumed may be hampered when based solely on urine results. As already seen with the serum samples, the sensitivity of one of the applied immunochemical assays was not sufficient for detection of this low-dosed drug, as no urine samples tested positive using the FPIA technique. However, utilizing the CEDIA on the Olympus AU400 instrument, seven urine samples tested positive (obtained: 14, 21, 25, 45, 50, 62, and 84 h post consumption) enabling detection even after low dosed uptake (Figure 6). Nevertheless, it has to be noted that all the positive urine samples (cut-off 50 ng/mL, calibrated for nitrazepam) showed rather high creatinine concentrations greater than 151 mg/dL.
Conclusion The typical approach of clandestine designer drug distributors particularly observed in the early stages of the ‘spice phenomenon’,[15] comprising of exploiting published literature from pharmaceutical
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drug candidate research, online marketing and rapid adaptation to changing narcotics laws, reached out to the drug class of benzodiazepines. Diclazepam (7-chloro-5-(2-chlorophenyl)-1-methyl-1,3dihydro-2H-1,4-benzodiazepin-2-one) represents the third designer benzodiazepine available from online vendors within less than one year. Similar to the preceding two, pyrazolam and flubromazepam, detectability in serum and urine samples utilizing immunochemical assays is limited due to the low doses applied as suggested by the low amounts of active compound per tablet. As a consequence, it has to be carefully assessed whether these types of assays are sufficient when analyzing for example samples from drug rehabilitation clinics where clients are more likely to consume ‘legal highs’ or research chemicals. However, applying a sensitive LC-MS/MS method covering the three commercially available benzodiazepines delorazepam, lorazepam and lormetazepam seems sufficient, as presence of all three analytes in the same sample may indicate an uptake of diclazepam. Nevertheless, for a definite proof serum samples should be analyzed since the parent drug was detectable for up to 99 h after the intake of one tablet in this matrix and only small amounts of unmetabolized diclazepam could be detected in the urine samples. The overall window of detection was 10 days in serum targeting the metabolite delorazepam and 19 days in urine targeting lorazepam, with the limitation that no statement regarding the actual drug consumed can be made as all three main metabolites are marketed by pharmaceutical companies in many countries worldwide. Diclazepam shows a rather long terminal elimination half-life of approximately 42 h and its pharmacokinetic profile seems to follow a multi-compartment model showing at least biphasic elimination. All three main metabolites of diclazepam are pharmacologically active with elimination half-lives of 78 h (delorazepam),[11] 12 h (lorazepam)[16] and 13 h (lormetazepam),[17] respectively. As a consequence, long-lasting sedative effects seem likely when applying higher or repeated doses. The latter could also lead to severe intoxication by accumulation. Some additional risk for consumers of this specific product comes from the considerable variation of drug content per tablet which is not at all predictable for the user and may lead to imprecise dosing.
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Diclazepam: a new designer benzodiazepine Acknowledgements This publication has been produced with the financial support of the Drug Prevention and Information Programme of the European Union (JUST/2011/DPIP/AG/3597), the German Federal Ministry of Health, and the City of Frankfurt/Main.
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