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Characterization of the Designer Benzodiazepine Pyrazolam and Its Detectability in Human Serum and Urine

Characterization of the Designer Benzodiazepine Pyrazolam and Its Detectability in Human Serum and Urine

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Interesting that the pyridine ring means that there is NO metabolism so it won't interact with other drugs nor stress liver or kidneys.....
Interesting that the pyridine ring means that there is NO metabolism so it won't interact with other drugs nor stress liver or kidneys.....

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10/16/2013

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ORIGINAL ARTICLE
Characterization of the designer benzodiazepine pyrazolamand its detectability in human serum and urine
Bjoern Moosmann
Melanie Hutter
Laura M. Huppertz
Sascha Ferlaino
Lisa Redlingsho ¨fer
Volker Auwa ¨rter
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 researchchemicals started offering pyrazolam, a new benzodiaze-pine that differs from phenazepam and etizolam, whichhave also recently appeared on the ‘‘gray market’’, in that itis not marketed by pharmaceutical companies anywherein the world. This article describes the characterizationof pyrazolam (8-bromo-1-methyl-6-pyridin-2-yl-4
 H 
-[1,2,4]triazolo[4,3–a][1, 4]benzodiazepine) using gas chroma-tography-mass spectrometry, liquid chromatography-tandem mass spectrometry (LC–MS–MS), liquid chroma-tography quadrupole time-of-flight mass spectrometry(LC–Q–TOF–MS), and nuclear magnetic resonance spec-troscopy. In addition, a study was carried out in which oneof the authors ingested two 0.5-mg pyrazolam tablets.Serum and urine samples were then obtained to investigatethe metabolism of pyrazolam and to obtain preliminaryresults for the elimination half-life and the detectability of a 1-mg dose in serum and urine using a highly sensitiveLC–MS–MS method and immunoassays. The resultsshowed an elimination half-life of about 17 h and nodetectable metabolism. The parent compound was detectedwith the described LC–MS–MS method in serum for morethan 50 h and in urine for approximately 6 days. Immu-noassays showed cross-reactivity, but poor detection in thestudy samples demonstrated that consumption or adminis-tration of this presumably potent drug could go undetectedunless 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 growinghealth problem, with benzodiazepines being among themain substances of concern [1]. In Germany alone,39 million ‘‘defined daily doses’’ of benzodiazepines and81 million defined daily doses of ‘Z-drugs’(zolpidem,zopiclone, and zaleplone) were prescribed in 2010 [2],while the number of people addicted to benzodiazepines isestimated to be 1.1–1.2 million [3]. However, in mostcountries benzodiazepines are available by prescriptiononly and in Germany all benzodiazepines offered by phar-maceutical companies are controlled by the narcotics law,making it more difficult for addicted persons to obtain thedrugs without visiting a physician or forging a prescription.In the past few years, ‘‘research chemicals’’ sold over theInternet as ‘legal highshave become more and morepopular because they enable drug users to circumvent nar-cotics laws. In the early stages of this development, the mainsubstances available were synthetic cannabinoids sold as‘herbal mixtures[47], designer amphetamines [8] and cathinone derivatives [9] sold as ‘‘bathsalts’’. More recentlythe two benzodiazepines phenazepam and etizolam wereoffered as ‘legal’alternatives for benzodiazepines. Bothsubstances are widely available over the Internet as they are
B. Moosmann
Á
M. Hutter
Á
L. M. Huppertz
Á
L. Redlingsho¨fer
Á
V. Auwa¨rter (
&
)Institute of Forensic Medicine, Forensic Toxicology Department,University Medical Center Freiburg, Albertstr. 9,79104 Freiburg, Germanye-mail: volker.auwaerter@uniklinik-freiburg.deB. Moosmann
Á
M. HutterHermann Staudinger Graduate School, University of Freiburg,Hebelstraße 27, 79104 Freiburg, GermanyS. FerlainoInstitute of Pharmaceutical Sciences, University of Freiburg,Albertstr. 25, 79104 Freiburg, Germany
 123
Forensic ToxicolDOI 10.1007/s11419-013-0187-4
 
registered drugs that are produced and sold in some coun-tries (e.g., Russia, India) [10]; they are not yet scheduled inGermany and many other countries. However, with thesynthetic routes to various structural classes of benzodiaze-pines being published in the literature, along with their rel-ative potency, it comes as no surprise that producers of so-called research chemicals are beginning to use this knowl-edge. In August 2012, the first reported seizure of a newbenzodiazepine in the European Union was made by FinnishCustoms [11]. The seized compound pyrazolam (8-bromo-1-methyl-6-pyridin-2-yl-4
 H 
-[1,2,4]triazolo[4,3-a][1,4]benzo-diazepine) combines structural elements of bromazepam andalprazolam (Fig.1), and was first reported in 1979 [12]. This benzodiazepine is, to our knowledge, the first benzodiaze-pine on the ‘‘legal high’’ market that is not marketed any-where in the world by a pharmaceutical company formedical purposes. This has led us to assume that it wasproduced solely for the drug market in a similar modusoperandi as observed in the case of many synthetic can-nabinoids and designer stimulants. It is distributed over theInternet as tablets that contain 0.5 mg of pyrazolam pertablet and the vendor stated a half-life of 6 h.In the present study, a volunteer took two 0.5-mg py-razolam tablets orally. Afterward, serum and urine sampleswere obtained to investigate the metabolism of pyrazolamand to obtain preliminary results for the approximate elimi-nation half-life and the detectability of a 1-mg dose in serumand urine samples using a highly sensitive liquid chroma-tography-tandem mass spectrometry (LC–MS–MS) method.Another point to consider was whether immunoassaysthat are commonly applied for screening of forensic sam-ples for benzodiazepines would show cross-reactivity forpyrazolam. Due to the structural similarities to bromaze-pam and alprazolam, a positive test result seemed plausi-ble. However, due to the low dosage of 1 mg, questionsarose regarding the sensitivity and the window of detectionfor serum and urine samples.
Materials and methods
Chemicals, reagents, and blank serumFormic acid (HCOOH) (Rotipuran
Ò
C
98 %, p.a.), mo-nopotassium phosphate, and potassium chloride (
C
99.5 %,p.a., ACS) were purchased from Carl Roth (Karlsruhe,Germany). 1-Chlorobutane (LiChrosolv
Ò
) and sodiumcarbonate were obtained from Merck (Darmstadt, Ger-many). Boric acid and methanol (MeOH) (HPLC grade)were purchased from J.T. Baker (Deventer, The Nether-lands) and ammonium formate (99.995 %), ethanol (EtOH)(analytical grade), ethyl acetate (analytical grade), andpotassium hydroxide were purchased from Sigma Aldrich(Steinheim, Germany). Deuterated chloroform (CDCl
3
)was obtained from Euriso–Top (Saint–Aubin, France) andacetic acid (AnalaR NORMAPUR 100 %) from VWRInternational (Darmstadt, Germany). Deionized water wasprepared using a cartridge deionizer from Memtech(Moorenweis, Germany). Alprazolam-
5
(0.1 mg/ml) wasobtained from Lipomed (Arlesheim, Switzerland) and
b
-glucuronidase/arylsulfatase (
 Helix pomatia
,
b
-glucuron-idase 5.5 U/ml, arylsulfatase 2.6 U/ml at 38
°
C) fromRoche Diagnostics (Mannheim, Germany). Pyrazolamtablets (declared amount: 0.5 mg per tablet) were orderedfrom an online retailer selling ‘research chemicals’.Human blank serum was provided by volunteers afterinformed consent was obtained, and was stored at
-
20
°
Cprior to use.Borate buffer (pH 9) was prepared by mixing 630 mlof solution 1 (61.8 g/l H
3
BO
3
and 74.6 g/l KCl indeionized water) with 370 ml of solution 2 (106 g/lNa
2
CO
3
in deionized water). The pH was adjusted to 9by addition of solution 2. Phosphate buffer (0.1 M, pH 6)was prepared by dissolving 13.61 g of KH
2
PO
4
in 1 l of deionized water and adjusting the pH to 6 by addition of 1 M KOH.
Fig. 1
Structural and molecularformulas of alprazolam,pyrazolam, and bromazepamForensic Toxicol
 123
 
Isolation and identification of pyrazolamNo commercial standard was available for pyrazolam, soreference material for quantification in serum, urine, andtablet samples was isolated from tablets using thin-layerchromatography (TLC). For this purpose, six tablets weredissolved in 2 ml of borate buffer (pH 9). Afterward, 2 mlof 1-chlorobutane was added and the sample was vortexedfor 1 min. Following centrifugation at 2,860
9
g
for 5 min(Heraeus Megafuge 1.0, Thermo Scientific, Schwerte,Germany), the organic layer was transferred into a separatevial. The extract was loaded on a TLC plate (silica gel 60,10
9
20 cm, F256, Merck) and separated using aceticacid (99 %), deionized water, MeOH, ethyl acetate(2:15:20:80 v/v/v/v) as mobile phase. The mobile phasewas chosen based on the recommendation in the EuropeanPharmacopoeia [13] for testing alprazolam. After separa-tion, the band was scraped from the plate and was extractedwith EtOH. Analyses by gas chromatography-mass spec-trometry (GC–MS), LC–MS–MS, liquid chromatographyquadrupole time-of-flight mass spectrometry (LC–Q–TOF–MS), and nuclear magnetic resonance (NMR) spectroscopywere conducted for identification and purity testing.For GC–MS analysis, 200
l
l of a 1 mg/ml solution inMeOH was transferred into a glass vial, evaporated todryness, and reconstituted in 1 ml of ethyl acetate prior toinjection of 1
l
l into the GC–MS system. A 6890 seriesGC system with a 5973 series mass selective detector, and7683 B series injector were used with ChemstationG1701GA version D.03.00.611 software (Agilent, Wald-bronn, Germany). The GC parameters and MS conditionswere 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
l
m 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; ionizationenergy, 70 eV. Analysis was performed in scan mode from50 to 550 amu at a speed of 1.5 scans/s. The solvent delaywas set to 3.5 min. The obtained EI–GC–MS mass spectrawere compared with those published in commonly usedEI–GC–MS spectra libraries (Maurer Pfleger Weber 2007Mass Spectral and GC Library, National Institute of Stan-dards and Technology Mass Spectral Library 08,Wiley Registry of Mass Spectral Data sixth edn.).LC–MS–MS analysis was carried out on a ShimadzuProminence HPLC system coupled with a QTRAP 4000triple-quadropole linear ion trap fitted with a TurboIon-Spray interface and Analyst
Ò
software version 1.5.2 fordata acquisition (AB Sciex, Darmstadt, Germany). TheHPLC system consisted of two LC-20AD SP isocraticpumps, a SIL-20AC autosampler, a CTO-20AC columnoven, a DGU-20A3 degasser, and a CBM-20A controller(Shimadzu, Duisburg, Germany).For analysis, a 100 ng/ml sample solution was preparedin mobile phase (A/B 80:20 v/v), where mobile phase Acontained 0.1 % HCOOH (v/v) and 1 mM ammonium for-mate in deionized water and mobile phase B was 0.1 %HCOOH (v/v) in MeOH. The injection volume was 20
l
l.For separation of the compounds, gradient elution wasapplied on a Synergi 4u Polar RP column (150
9
2 mm,4
l
m) with a corresponding guard column (Polar RP4
9
2 mm), both from Phenomenex (Aschaffenburg, Ger-many). The gradient elution started with 20 % mobile phaseB and increased to 95 % mobile phase B in 10 min, fol-lowed by a 1.5-min hold at 95 % mobile phase B. Startingconditions were restored within 0.5 min and the system wasequilibrated for 3 min. The flow rate was set at 0.4 ml/min.The samples were stored in the autosampler at 4
°
C prior toanalysis and the column oven was heated to 40
°
C.For LC–Q–TOF–MS analysis, a maXis impact Q–TOFinstrument (Bruker Daltonik, Bremen, Germany) coupledwith a Dionex UltiMate 3000 RSLC HPLC system, con-sisting of a SRD-3600 solvent rack degasser, a HPG-3400RS binary pump with solvent selection valve, a WPS-3000TRS thermostated autosampler, and a TCC-3000RSthermostated column compartment (Thermo Fisher Scien-tific, Dreieich, Germany). Chromatographic separationwas performed on a Dionex Acclaim RSLC 120 C18 col-umn (2.2
l
m particle size, 120 A˚pore diameter,2.1
9
100 mm; Thermo Fisher Scientific) using H
2
O/ MeOH 90/10 (v/v) with 5 mM ammonium formate and0.01 % HCOOH (A) and MeOH with 5 mM ammoniumformate and 0.01 % HCOOH (B). Gradient elution was asfollows: 1 % mobile phase B at a flow rate of 0.2 ml/minfor 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 for2 min and increase flow rate to 0.48 ml/min. The initialmobile phase composition was restored within 0.1 min andthe flow rate decreased to 0.2 ml/min after 3 min. Startingconditions were held for 0.9 min. The temperature of thecolumn compartment and the autosampler were set to 30and 5
°
C, respectively. HyStar and DataAnalysis (includ-ing the software tool SmartFormula) software (BrukerDaltonik) were used for data acquisition and evaluation,respectively. Full scan and broadband CID (bbCID) datawere acquired in two individual runs. The collision energyapplied for bbCID was 25 eV.NMR spectra were recorded at room temperature inCDCl
3
using a DRX 400 (Bruker BioSpin, Rheinstetten,Germany). The chemical shifts are reported in ppm relativeto CHCl
3
(
1
H:
d
=
7.27) and CDCl
3
(
13
C:
d
=
77.23) asinternal standards. For full characterization of pyrazolam,
Forensic Toxicol
 123

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