Forensic Toxicol DOI 10.1007/s11419-013-0187-4 ORIGINAL ARTICLE Characterization of the designer benzodiazepine pyrazolam and its detectability in human serum and urine Bjoern Moosmann • Melanie Hutter • Laura M. Huppertz • Sascha Ferlaino • ¨ fer • Volker Auwa ¨ rter Lisa Redlingsho Received: 6 March 2013 / Accepted: 1 April 2013 Ó Japanese Association of Forensic Toxicology and Springer Japan 2013 Abstract In 2012, online shops selling so-called research chemicals started offering pyrazolam, a new benzodiazepine that differs from phenazepam and etizolam, which have also recently appeared on the ‘‘gray market’’, in that it is not marketed by pharmaceutical companies anywhere in the world. This article describes the characterization of pyrazolam (8-bromo-1-methyl-6-pyridin-2-yl-4H-[1,2,4] triazolo[4,3–a][1, 4]benzodiazepine) using gas chromatography-mass spectrometry, liquid chromatographytandem mass spectrometry (LC–MS–MS), liquid chromatography quadrupole time-of-flight mass spectrometry (LC–Q–TOF–MS), and nuclear magnetic resonance spectroscopy. In addition, a study was carried out in which one of the authors ingested two 0.5-mg pyrazolam tablets. Serum and urine samples were then obtained to investigate the metabolism of pyrazolam and to obtain preliminary results for the elimination half-life and the detectability of a 1-mg dose in serum and urine using a highly sensitive LC–MS–MS method and immunoassays. The results showed an elimination half-life of about 17 h and no detectable metabolism. The parent compound was detected with the described LC–MS–MS method in serum for more B. Moosmann Á M. Hutter Á L. M. Huppertz Á ¨ fer Á V. Auwa ¨ rter (&) L. Redlingsho Institute of Forensic Medicine, Forensic Toxicology Department, University Medical Center Freiburg, Albertstr. 9, 79104 Freiburg, Germany e-mail:
[email protected] B. Moosmann Á M. Hutter Hermann Staudinger Graduate School, University of Freiburg, Hebelstraße 27, 79104 Freiburg, Germany S. Ferlaino Institute of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany than 50 h and in urine for approximately 6 days. Immunoassays showed cross-reactivity, but poor detection in the study samples demonstrated that consumption or administration of this presumably potent drug could go undetected unless instrumental analytical techniques are also used. Keywords Pyrazolam Á Designer benzodiazepine Á LC–MS–MS Á NMR Á Serum Á Urine Introduction The nonmedical use of prescription drugs is a growing health problem, with benzodiazepines being among the main substances of concern [1]. In Germany alone, 39 million ‘‘defined daily doses’’ of benzodiazepines and 81 million defined daily doses of ‘‘Z-drugs’’ (zolpidem, zopiclone, and zaleplone) were prescribed in 2010 [2], while the number of people addicted to benzodiazepines is estimated to be 1.1–1.2 million [3]. However, in most countries benzodiazepines are available by prescription only and in Germany all benzodiazepines offered by pharmaceutical companies are controlled by the narcotics law, making it more difficult for addicted persons to obtain the drugs without visiting a physician or forging a prescription. In the past few years, ‘‘research chemicals’’ sold over the Internet as ‘‘legal highs’’ have become more and more popular because they enable drug users to circumvent narcotics laws. In the early stages of this development, the main substances available were synthetic cannabinoids sold as ‘‘herbal mixtures’’ [4–7], designer amphetamines [8] and cathinone derivatives [9] sold as ‘‘bathsalts’’. More recently the two benzodiazepines phenazepam and etizolam were offered as ‘‘legal’’ alternatives for benzodiazepines. Both substances are widely available over the Internet as they are 123 Forensic Toxicol registered drugs that are produced and sold in some countries (e.g., Russia, India) [10]; they are not yet scheduled in Germany and many other countries. However, with the synthetic routes to various structural classes of benzodiazepines being published in the literature, along with their relative potency, it comes as no surprise that producers of socalled research chemicals are beginning to use this knowledge. In August 2012, the first reported seizure of a new benzodiazepine in the European Union was made by Finnish Customs [11]. The seized compound pyrazolam (8-bromo-1methyl-6-pyridin-2-yl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine) combines structural elements of bromazepam and alprazolam (Fig. 1), and was first reported in 1979 [12]. This benzodiazepine is, to our knowledge, the first benzodiazepine on the ‘‘legal high’’ market that is not marketed anywhere in the world by a pharmaceutical company for medical purposes. This has led us to assume that it was produced solely for the drug market in a similar modus operandi as observed in the case of many synthetic cannabinoids and designer stimulants. It is distributed over the Internet as tablets that contain 0.5 mg of pyrazolam per tablet and the vendor stated a half-life of 6 h. In the present study, a volunteer took two 0.5-mg pyrazolam tablets orally. Afterward, serum and urine samples were obtained to investigate the metabolism of pyrazolam and to obtain preliminary results for the approximate elimination half-life and the detectability of a 1-mg dose in serum and urine samples using a highly sensitive liquid chromatography-tandem mass spectrometry (LC–MS–MS) method. Another point to consider was whether immunoassays that are commonly applied for screening of forensic samples for benzodiazepines would show cross-reactivity for pyrazolam. Due to the structural similarities to bromazepam and alprazolam, a positive test result seemed plausible. However, due to the low dosage of 1 mg, questions arose regarding the sensitivity and the window of detection for serum and urine samples. Materials and methods Chemicals, reagents, and blank serum Formic acid (HCOOH) (RotipuranÒ C98 %, p.a.), monopotassium phosphate, and potassium chloride (C99.5 %, p.a., ACS) were purchased from Carl Roth (Karlsruhe, Germany). 1-Chlorobutane (LiChrosolvÒ) and sodium carbonate were obtained from Merck (Darmstadt, Germany). Boric acid and methanol (MeOH) (HPLC grade) were purchased from J.T. Baker (Deventer, The Netherlands) and ammonium formate (99.995 %), ethanol (EtOH) (analytical grade), ethyl acetate (analytical grade), and potassium hydroxide were purchased from Sigma Aldrich (Steinheim, Germany). Deuterated chloroform (CDCl3) was obtained from Euriso–Top (Saint–Aubin, France) and acetic acid (AnalaR NORMAPUR 100 %) from VWR International (Darmstadt, Germany). Deionized water was prepared using a cartridge deionizer from Memtech (Moorenweis, Germany). Alprazolam-d5 (0.1 mg/ml) was obtained from Lipomed (Arlesheim, Switzerland) and b-glucuronidase/arylsulfatase (Helix pomatia, b-glucuronidase 5.5 U/ml, arylsulfatase 2.6 U/ml at 38 °C) from Roche Diagnostics (Mannheim, Germany). Pyrazolam tablets (declared amount: 0.5 mg per tablet) were ordered from an online retailer selling ‘‘research chemicals’’. Human blank serum was provided by volunteers after informed consent was obtained, and was stored at -20 °C prior to use. Borate buffer (pH 9) was prepared by mixing 630 ml of solution 1 (61.8 g/l H3BO3 and 74.6 g/l KCl in deionized water) with 370 ml of solution 2 (106 g/l Na2CO3 in deionized water). The pH was adjusted to 9 by addition of solution 2. Phosphate buffer (0.1 M, pH 6) was prepared by dissolving 13.61 g of KH2PO4 in 1 l of deionized water and adjusting the pH to 6 by addition of 1 M KOH. Fig. 1 Structural and molecular formulas of alprazolam, pyrazolam, and bromazepam 123 Forensic Toxicol Isolation and identification of pyrazolam No commercial standard was available for pyrazolam, so reference material for quantification in serum, urine, and tablet samples was isolated from tablets using thin-layer chromatography (TLC). For this purpose, six tablets were dissolved in 2 ml of borate buffer (pH 9). Afterward, 2 ml of 1-chlorobutane was added and the sample was vortexed for 1 min. Following centrifugation at 2,8609g for 5 min (Heraeus Megafuge 1.0, Thermo Scientific, Schwerte, Germany), the organic layer was transferred into a separate vial. The extract was loaded on a TLC plate (silica gel 60, 10 9 20 cm, F256, Merck) and separated using acetic acid (99 %), deionized water, MeOH, ethyl acetate (2:15:20:80 v/v/v/v) as mobile phase. The mobile phase was chosen based on the recommendation in the European Pharmacopoeia [13] for testing alprazolam. After separation, the band was scraped from the plate and was extracted with EtOH. Analyses by gas chromatography-mass spectrometry (GC–MS), LC–MS–MS, liquid chromatography quadrupole time-of-flight mass spectrometry (LC–Q–TOF– MS), and nuclear magnetic resonance (NMR) spectroscopy were conducted for identification and purity testing. For GC–MS analysis, 200 ll of a 1 mg/ml solution in MeOH was transferred into a glass vial, evaporated to dryness, and reconstituted in 1 ml of ethyl acetate prior to injection of 1 ll into the GC–MS system. A 6890 series GC system with a 5973 series mass selective detector, and 7683 B series injector were used with Chemstation G1701GA version D.03.00.611 software (Agilent, Waldbronn, Germany). The GC parameters and MS conditions were similar to the conditions used by Maurer et al. [14], using splitless injection; column, HP-5-MS capillary (30 9 0.25 mm i.d., 0.25 lm film thickness; Agilent); injection port temperature, 270 °C; carrier gas, helium; flow rate, 1 ml/min; oven temperature, 100 °C for 3 min, ramped to 310 °C at 30 °C/min, 310 °C for 10 min; transfer line heater, 280 °C; ion source temperature, 230 °C; electron impact ionization (EI) mode; ionization energy, 70 eV. Analysis was performed in scan mode from 50 to 550 amu at a speed of 1.5 scans/s. The solvent delay was set to 3.5 min. The obtained EI–GC–MS mass spectra were compared with those published in commonly used EI–GC–MS spectra libraries (Maurer Pfleger Weber 2007 Mass Spectral and GC Library, National Institute of Standards and Technology Mass Spectral Library 08, Wiley Registry of Mass Spectral Data sixth edn.). LC–MS–MS analysis was carried out on a Shimadzu Prominence HPLC system coupled with a QTRAP 4000 triple-quadropole linear ion trap fitted with a TurboIonSpray interface and AnalystÒ software version 1.5.2 for data acquisition (AB Sciex, Darmstadt, Germany). The HPLC system consisted of two LC-20AD SP isocratic pumps, a SIL-20AC autosampler, a CTO-20AC column oven, a DGU-20A3 degasser, and a CBM-20A controller (Shimadzu, Duisburg, Germany). For analysis, a 100 ng/ml sample solution was prepared in mobile phase (A/B 80:20 v/v), where mobile phase A contained 0.1 % HCOOH (v/v) and 1 mM ammonium formate in deionized water and mobile phase B was 0.1 % HCOOH (v/v) in MeOH. The injection volume was 20 ll. For separation of the compounds, gradient elution was applied on a Synergi 4u Polar RP column (150 9 2 mm, 4 lm) with a corresponding guard column (Polar RP 4 9 2 mm), both from Phenomenex (Aschaffenburg, Germany). The gradient elution started with 20 % mobile phase B and increased to 95 % mobile phase B in 10 min, followed by a 1.5-min hold at 95 % mobile phase B. Starting conditions were restored within 0.5 min and the system was equilibrated for 3 min. The flow rate was set at 0.4 ml/min. The samples were stored in the autosampler at 4 °C prior to analysis and the column oven was heated to 40 °C. For LC–Q–TOF–MS analysis, a maXis impact Q–TOF instrument (Bruker Daltonik, Bremen, Germany) coupled with a Dionex UltiMate 3000 RSLC HPLC system, consisting of a SRD-3600 solvent rack degasser, a HPG3400RS binary pump with solvent selection valve, a WPS3000TRS thermostated autosampler, and a TCC-3000RS thermostated column compartment (Thermo Fisher Scientific, Dreieich, Germany). Chromatographic separation was performed on a Dionex Acclaim RSLC 120 C18 col˚ pore diameter, umn (2.2 lm particle size, 120 A 2.1 9 100 mm; Thermo Fisher Scientific) using H2O/ MeOH 90/10 (v/v) with 5 mM ammonium formate and 0.01 % HCOOH (A) and MeOH with 5 mM ammonium formate and 0.01 % HCOOH (B). Gradient elution was as follows: 1 % mobile phase B at a flow rate of 0.2 ml/min for 1 min, increase to 39 % mobile phase B in 2 min, increase to 99.9 % mobile phase B and a flow rate of 0.4 ml/min in 9 min, hold at 99.9 % mobile phase B for 2 min and increase flow rate to 0.48 ml/min. The initial mobile phase composition was restored within 0.1 min and the flow rate decreased to 0.2 ml/min after 3 min. Starting conditions were held for 0.9 min. The temperature of the column compartment and the autosampler were set to 30 and 5 °C, respectively. HyStar and DataAnalysis (including the software tool SmartFormula) software (Bruker Daltonik) were used for data acquisition and evaluation, respectively. Full scan and broadband CID (bbCID) data were acquired in two individual runs. The collision energy applied for bbCID was 25 eV. NMR spectra were recorded at room temperature in CDCl3 using a DRX 400 (Bruker BioSpin, Rheinstetten, Germany). The chemical shifts are reported in ppm relative to CHCl3 (1H: d = 7.27) and CDCl3 (13C: d = 77.23) as internal standards. For full characterization of pyrazolam, 123 Forensic Toxicol one-dimensional (1D) 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively. Selective 1DTOCSY, 15N HMBC, 2D 1H/13C HSQC, COSY, and 1 H/13C HMBC spectra were also recorded. Sampling of serum and urine A male volunteer (42 years) (CYP1A2: *1F/*1F; CYP2C9: *2/WT; CYP2C19: WT/WT; CYP2D6:*4/WT; CYP3A4: WT/WT genotype and phenotype indices, measured according to Wohlfarth et al. [15], of CYP1A2: 0.884; CYP2C9: 0.880; CYP2C19: 2.018; CYP2D6: 247; CYP3A4: 1.470) took two pyrazolam tablets (stated content: 0.5 mg pyrazolam per tablet) (in Germany, approval by the ethics committee is not required for scientific selfexperiments). Serum and urine samples were obtained prior to ingestion. In addition, eight serum samples (1, 2, 3, 6, 18, 25, 42, 50 h after ingestion) and 31 urine samples (24 samples ad libitum up to 89 h after ingestion plus seven samples in the morning of days 4–10 post administration) were obtained. All the samples were stored at -20 °C prior to analysis. No physical or mental effects were observed after ingestion by the volunteer. Identification of the main metabolites For identification of the main metabolites, the urine samples obtained 5 h 20 min and 15 h 20 min after ingestion were analyzed using the LC–MS–MS system described above. All urine samples were screened for metabolites anticipated by analogy to the metabolites reported for midazolam, triazolam, and alprazolam [16–19]. Several enhanced product ion scan (EPI) experiments were performed after b-glucuronidase/arylsulfatase incubation and protein precipitation, and hypothetic masses of potential phase I and phase II metabolites were selected as precursor masses. The following phase I reactions were included: monohydroxylation, dihydroxylation, carboxylation, dealkylation, dehalogenation, and combinations of these reactions. In addition, precursor ion scans with characteristic fragments of pyrazolam were carried out and samples were screened for potential metabolites using LC–Q–TOF–MS in full scan and bbCID mode, respectively. The metabolism was also investigated by incubation of pooled human liver microsomes (HLM) (1 mg/ml) with 20 lM pyrazolam. The incubation time was 30 min at 37 °C. As a positive control, the assay was also performed with alprazolam. Serum sample preparation For sample preparation, 100 ll of serum was fortified with 20 ll of the internal standard solution (1 lg/ml alprazolam-d5) (IS). Afterward, 900 ll of borate buffer (pH 9) was added, followed by 1 ml of 1-chlorobutane. The sample was then placed in an overhead shaker for 5 min and centrifuged at 2,8509g for 5 min. Then the organic supernatant was transferred into an HPLC vial and evaporated to dryness at 40 °C under a gentle stream of nitrogen. Prior to LC–MS–MS analysis, the sample was reconstituted in 100 ll of the mobile phase. Urine sample preparation Twenty microliters of the IS solution and 0.5 ml of phosphate buffer (pH 6) were added to 100 ll of urine. Afterward, 50 ll of b-glucuronidase/arylsulfatase was added, followed by 2-h incubation at 45 °C for enzymatic hydrolysis. Then 900 ll of borate buffer (pH 9) and 1 ml of 1-chlorobutane were added and further treatment followed the same procedure used for serum samples. Pyrazolam tablets For determination of the amount of pyrazolam per tablet, one tablet was dissolved in EtOH and after centrifugation at 2,8609g for 5 min the solution transferred into an HPLC vial. The tablet remnant was extracted two more times with EtOH and the solutions added to the HPLC vial before evaporation to dryness at 40 °C under a stream of nitrogen. The residue was reconstituted in 1 ml of MeOH and a 1:50,000 dilution in mobile phase (A/B 80:20) was analyzed using LC–MS–MS. Limit of detection The limit of detection (LOD) for pyrazolam was evaluated by analyzing serum and urine samples spiked with the purified compound in the expected concentration range (0.1–1 ng/ml). A signal-to-noise ratio of at least 3:1 was required for both the target and qualifier ions. Calibration curve Calibration curves (1–100 ng/ml) were prepared by spiking the purified compound to the corresponding matrix. A weighted calibration model (1/x2) was applied to account for heteroscedasticity. LC–MS–MS instrumentation The HPLC system for analysis of the serum, urine, and tablet samples consisted of two LC-10AD VP pumps, a SCL-10A VP controller, and a CTO-10AC column oven (Shimadzu, Duisburg, Germany); and an ERC–3415a degasser (ERC, Riemerling, Germany). The autosampler 123 Forensic Toxicol was an HTC PAL (CTC Analytics, Zwingen, Switzerland) equipped with a 100-ll syringe from Hamilton (Reno, NV, USA). The HPLC was coupled to an API 5000 triplequadrupole instrument equipped with a TurboIonSpray interface for detection, and AnalystÒ software version 1.5.2 was used for data acquisition (AB Sciex, Darmstadt, Germany). For separation of the analytes, the same column and guard column as described above were used. LC–MS–MS conditions Apart from storage of the samples in the autosampler at room temperature, the LC conditions were as described for the identification of the compound. Five transitions for pyrazolam and one transition for the deuterated internal standard were recorded in multiple reaction monitoring (MRM) mode. The declustering potentials (DP), collision energies (CE), and cell exit potentials (CXP) were optimized for pyrazolam and the internal standard. The MRM transitions and potentials are shown in Table 1. The mass spectrometer was operated using positive electrospray ionization (ESI). The ion source temperature was set at 400 °C, the ion source voltage at ?2,000 V, the curtain gas pressure was 40 psi, ion source gas 1 was 60 psi, ion source gas 2 was 70 psi, and the collision gas pressure was 2 psi. The total cycle time was 0.15 s with a minimum dwell time for each analyte of 20 ms. Immunoassay Urine samples were tested for benzodiazepines by fluorescence polarization immunoassay (FPIA) using an AxsymÒ 4602 instrument and benzodiazepine calibrators (Abbott Laboratories, Abbott Park, IL, USA). Selected urine samples were tested by cloned enzyme donor immunoassay (CEDIA) using a CobasÒ 8000 instrument and benzodiazepine calibrators supplied by Roche Diagnostics (Mannheim, Germany). All the serum samples were tested for benzodiazepines by CEDIA using a KonelabÒ 30 instrument (Thermo Fisher Scientific, Waltham, MA, USA) and benzodiazepine calibrators (Microgenics, Fremont, CA, USA). An in-house protocol allowing for enhanced sensitivity was applied in all three assays. Results and discussion Identification and characterization of pyrazolam 1 H and 13C NMR analysis confirmed the compound as 8-bromo-1-methyl-6-pyridin-2-yl-4H-[1,2,4]triazolo[4,3-a] [1,4]benzodiazepine (Table 2). The respective GC–EI–MS analysis (Fig. 2) shows the major ion signals at m/z (relative intensity %) 353 (100), 355 (99), 274 (85), and 205 (76). Based on the EPI spectrum (positive mode) of pyrazolam (Fig. 3), the DP, CE, and CXP of the five most abundant ions (m/z 206.1; 167.1; 247.1; 285.0, and 326.0) were optimized and the resulting MRM transitions were chosen for analysis of the serum, urine, and tablet samples. LC–Q–TOF–MS data affirming the proposed molecular formula and fragments are listed in Table 3. Isolation of pyrazolam Using liquid–liquid extraction and TLC, pyrazolam was extracted out of the tablet and isolated to a purity of greater Table 2 Nuclear magnetic resonance data for pyrazolam in CDCl3 (for numbering see Fig. 1) Carbon no. 1 3a 4 6 6a 7 8 9 10 10a 10 30 40 50 60 100 d13C (ppm) 150.4 154.6 46.3 166.8 129.8 135.3 120 134.4 124.6 132.7 155.6 148.5 125.0 136.9 123.7 12.4 d1H (ppm) – – 4.19; 5.57 – – 7.68 – 7.80 7.34 – – 8.60 7.40 7.89 8.17 2.66 Table 1 Multiple reaction monitoring transitions, optimized mass spectrometry parameters of pyrazolam and the internal standard alprazolam-d5 Analyte Pyrazolam Q1 mass (amu) 354 Q3 mass (amu) 326.1 285.2 247.3 206.3 167.1 Alprazolamd5 314 210.6 a DP (V) 25 25 25 25 25 40 a EP (V) 8 8 8 8 8 8 a CE (V) 39 36 38 46 40 62 a CXP (V) 45 25 30 25 30 26 a DP declustering potential; EP entrance potential; CE collision energy; CXP collision cell exit potential a Quantifier ion For numbering of compounds, see Fig. 1 123 Forensic Toxicol Fig. 2 Electron impact ionization gas chromatography-mass spectrometry and proposed fragmentation of pyrazolam Fig. 3 Enhanced product ion scan of pyrazolam and proposed fragmentation 80 %, as confirmed by NMR and GC–MS analysis. The purified compound was later used for the quantification of pyrazolam in serum, urine, and tablet samples. Metabolism In the EPI scan experiments, none of the postulated phase I and phase II metabolites could be detected. LC– Q–TOF–MS analysis with broadband CID scans also did not reveal any metabolites. While most of the benzodiazepines with chemical structures similar to pyrazolam (e.g., alprazolam and triazolam) are metabolized to ahydroxy or 4-hydroxy metabolites, mainly by CYP3A4 isoymes [19, 20], neither of these hydroxylation reactions was observed for pyrazolam after cleavage by b-glucuronidase/arylsulfatase. In addition, none of the glucuronidated or sulfated analogues was detected after protein precipitation. Based on the genotyping and phenotyping results of the subject, a poor metabolism regarding CYP3A4 can be ruled out. However, the poor metabolizing genotype and phenotype of the volunteer for the CYP2D6 isozyme may serve as an explanation. On the other hand, no metabolites could be detected in the pooled human liver microsome assay either, strengthening the hypothesis that pyrazolam is not metabolized extensively (the incubation of alprazolam with HLM led to detection of typical oxidative metabolites proving the suitability of the system). 123 Forensic Toxicol Table 3 Accurate masses and elemental composition of pyrazolam and its characteristic fragment ions Monoisotopic acurate mass Molecular ion [M?H]? (m/z = 354) Fragment (m/z = 326) Fragment (m/z = 285) Fragment (m/z = 247) Fragment (m/z = 206) Fragment (m/z = 167) 354.0352 326.0278 285.0020 247.1107 206.0838 167.0730 Elemental composition C16H13BrN5 C16H13BrN3 C14H10BrN2 C16H13N3 C14H10N2 C12H9N e- configuration Even Even Even Odd Odd Odd Error (ppm) 0.4 0.9 -0.1 0.3 0.0 0.1 Fig. 4 Pyrazolam concentrations in serum measured after the intake of two tablets (1 mg) and best fit curve resulting from the calculated Bateman function with ka and ke values of 0.399 and 0.041 and C0 of 62 ng/ml Analysis of serum and urine samples Figure 4 shows the pyrazolam concentrations in serum measured after the intake of two tablets. The highest pyrazolam concentration was measured in the sample obtained 3 h after ingestion. The sample obtained at 50 h still showed a pyrazolam concentration of ca. 8.8 ng/ml. To keep methods simple and to obtain a first impression of the pharmacokinetic properties of pyrazolam, a onecompartment model was used to fit the data from the experiment. The Excel software add-in ‘‘solver’’ (Microsoft, Redmond, WA, USA) was applied to find the best fit for ka, ke, and C0 using the Bateman function with the analytical results. The method of least squares was used as a measure of best fit, resulting in ka = 0.399 and ke = 0.041. Based on these results, the approximate elimination half-life of pyrazolam was estimated to be 17 h which is almost three times the half-life claimed on the website of the vendor. Taking into account the typical biphasic or polyphasic elimination kinetics of benzodiazepines [21], the terminal elimination half-life may be even longer, despite pyrazolam most probably being less lipophilic than other representatives of this class of drugs (given the 1,2,4-triazole moiety in combination with the pyridine ring). The concentrations of pyrazolam in the urine samples are shown in Fig. 5 with the highest concentration of ca. Fig. 5 Pyrazolam concentrations in urine measured after the intake of two tablets (1 mg), normalized to the creatinine concentrations 160 ng/mg measured in the sample obtained 5 h 20 min after intake. Good linearity over the applied range of 1–100 ng/ml (r(serum) = 0.9990; r(urine) = 0.9992) was obtained in both matrices. Quantification of tablet pyrazolam The amount of pyrazolam extracted from the tablet was 0.47 mg, which approximately amounts to the declared dose per tablet. However, larger numbers of tablets need to be analyzed to determine whether the dosing in all distributed tablets is similar. In addition, the extraction 123 Forensic Toxicol Table 4 Urine benzodiazepine immunoassay and liquid chromatography-tandem mass spectrometry (LC–MS–MS) results next to creatinine concentrations of the first 3 days of the study AxsymÒ (nordazepam equivalents, ng/ml) 32 75 91 120 140 110 82 81 84 119 84 107 71 79 63 85 CobasÒ 8000 (nordazepam equivalents, ng/ml) 5 n.t. n.t. 271 402 356 n.t. n.t. n.t. 179 n.t. 238 n.t. n.t. n.t. n.t. Time after ingestion (h) Creatinine concentration (mg/dl) 110 100 91 190 260 160 110 110 150 180 280 300 160 130 100 240 Pyrazolam concentration (LC–MS–MS, ng/ml) 0 44 140 200 200 120 88 74 88 200 93 94 64 52 40 58 0.00 2.10 4.00 5.20 15.20 20.15 23.50 26.10 30.50 31.40 33.10 39.25 45.25 Axsym : positive [200 units, ‘‘gray area’’ = 100–200 units; CobasÒ 8000: positive [200 units, n.t.: not tested Ò 48.25 51.25 56.20 efficiency may vary because of differences in adjuvant composition. Window of detection in serum and urine samples With an LOD of 1.0 ng/ml in serum and urine for analysis by LC–MS–MS, pyrazolam was detected in all the serum samples collected in our study. In urine samples, pyrazolam could be detected up to 6 days after ingestion. Therefore, despite the lack of detectable metabolites, it seems that consumption of even small doses can be detected for a relatively long period because the parent compound is excreted unchanged in urine. Using recommended cutoffs provided with the calibrators, none of the urine and serum samples tested positive for benzodiazepines by immunoassay using the Axsym or Konelab (serum assay) instrument. Applying our in-house cutoffs, five urine samples and two serum samples (6 and 18 h) showed results in the ‘‘gray area’’. These five urine samples were also analyzed using the CobasÒ 8000 instrument, resulting in four positive tests using the cutoff recommended by the manufacturer. The immunoassay results are listed in Table 4 along with the creatinine concentrations of the urine samples obtained in the first 3 days. The inability of the immunoassays to detect pyrazolam in most of the tested samples may lead to undetected consumption or administration, unless samples are collected shortly after the incidence or high doses were applied; otherwise, instrumental techniques such as LC– MS–MS are required. Conclusions After designer opiods (the first designer drugs were esters of morphine that were synthesized in the 1930s to circumvent the legal ban of heroin), designer amphetamines and cathinones, synthetic cannabinoids, cocaine analogs and fentanyl derivates, the emergence of pyrazolam as a designer benzodiazepine marks the next logical step on the market for ‘‘legal highs’’. Because phenazepam and etizolam are already controlled substances or about to be scheduled as such in many countries, producers are once again turning to poorly characterized drug candidates from pharmaceutical research. From the data obtained from one volunteer and from HLM experiments, pyrazolam showed no detectable metabolism. Nevertheless, the long window of detection of the parent compound seems sufficient to solve forensic cases. One critical aspect of this new drug is its ready availability over the Internet in combination with a likely high potency. Therefore, this drug might be used by benzodiazepine-addicted persons and possibly as a date-rape drug. As a consequence pyrazolam should be taken into consideration in cases of suspected drugfacilitated crimes and in cases where positive immunoassay findings cannot be confirmed with commonly applied MRM methods. 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