Synthesis of possible BZP metabolites by online electrochemical oxidation

Transcription

1 Synthesis of possible BZP metabolites by online electrochemical oxidation Lars Ambach, Stefan König, Wolfgang Weinmann Institute of Forensic Medicine Department of Forensic Toxicology University of Bern, Switzerland

2 Electrochemistry mimics Cytochrome P 450 (CYP) Typical CYP-catalysed reactions Allylic and alliphatic hydroxylation Benzylic hydroxylation Hydroxylation of aromatics Desalkylation of amines Desalkylation of ethers and thioethers CH 3 CH 3 H H H + CH H + CH Simulation by EC Possible at high potentials Possible Possible Possible Ethers: possible Thioethers: low yield, acidic conditions Epoxide formation xidation of heteroatoms (,S) H H H - C + or ot mimicked Possible Dehydrogenation H Possible

3 bjectives» Find possible oxidation products of benzylpiperazine (BZP) by electrochemical oxidation» Analyse BZP-positive urine specimen for these products

4 adapted from: Staack F, Fritschi G, Maurer HH. J Chromatogr B. 773 (2002) Known Metabolism of BZP Glucuronides or Sulfates H H» m/z [M+H]: 193 H H» m/z [M+H]: 209 H 3 C H» m/z [M+H]: 223 H» m/z [M+H]: 87» m/z [M+H]: 177 2» m/z [M+H]: 108 2» m/z [M+H]: 151

5 Electrochemical eactor Cell (XY, Antec)» Flow: max. 10 µl/min» Electrodes: Glassy Carbon Magic Diamond Pt Au

6 System Configuration» Syringe pump» Potentiostat and EC cell» AB Sciex QTrap 5500

7 Platinum Electrode, ph 2.9, 1 µm BZP XIC of +Q1: 318 Da from Sample 1 ( _MSV_C_Pt_0-2500mV_BZP_04) of _MSV_C_Pt_0-2500mV_BZP_04.wiff (Turbo S e6 3.0e6 Max. 1.0e6 cps In te n s ity, c p s 2.5e6 2.0e6 1.5e6 1.0e6 5.0e Time, min

8 200 mv 400 mv 600 mv 800 mv 1000 mv 1200 mv 1400 mv 1600 mv 1800 mv 2000 mv 2200 mv 2400 mv 2500 mv Platinum Electrode XIC of +Q1: 318 Da from Sample 1 ( _MSV_C_Pt_0-2500mV_BZP_04) of _MSV_C_Pt_0-2500mV_BZP_04.wiff (Turbo S e6 3.0e6 Max. 1.0e6 cps In te n s ity, c p s 2.5e6 2.0e6 1.5e6 1.0e e Time, min

9 HPLC-MS/MS analysis» Create MM method (2-3 MMs per product)» Keep XY at constant potential (1400, 1600 and 1800 mv) and collect eluate offline» Analyse XY samples and urine sample with MM method

10 HPLC-MS/MS esults Q1 XY Urine 159 Yes (1) o 161 Yes (1) o

11 HPLC-MS/MS esults Q1 XY Urine 159 Yes (1) o 161 Yes (1) o 177 Yes, 2 products, rearrangement? 1 product? In te n s ity, c p s XIC of +MM (31 pairs): Exp 1, / Da ID: BZP MM 1 from Sample 13 (BZP 1800mV 2) of BZP-x.wiff (Turbo Spray) Max. 7.9e5 cps.» XY e5 7.5e5 7.0e5 6.5e mv 6.0e5 5.5e5 5.0e5 4.5e5 4.0e5 3.5e5 3.0e5 2.5e e5 1.5e5 1.0e5 5.41» 4.5 min 5.4 min» min 5.0e4 In te n s ity, c p s XIC of +MM (31 pairs): Exp 1, / Da ID: BZP MM 1 from Sample 10 ( Urin ohne Gluc Methode 03) of BZP-x.wif... Max. 1.3e6 cps.» Urine e6 1.20e6 1.15e6 1.10e6 1.05e6 1.00e6 9.50e5 9.00e5 8.50e5 8.00e5 7.50e5 7.00e5 6.50e5 6.00e5 5.50e5 5.00e5 4.50e5 4.00e e5 3.00e5 2.50e5 2.00e In te n s ity, c p s Time, min XIC of +MM (31 pairs): Exp 1, / Da ID: BZP MM 1 from Sample 6 ( Urin +Gluc Methode 03) of BZP-x.wiff (Tur... Max. 3.4e6 cps.» Urine e6 3.2e6 3.0e6 2.8e6 Glucuronidase 2.6e6 2.4e6 2.2e6 2.0e e6 1.6e6 1.4e6 1.2e6» min» min 1.50e5 4.0e e5 2.0e5 5.00e Time, min Time, min 1.0e6 8.0e5 6.0e5 4.01

12 HPLC-MS/MS esults Q1 XY Urine 159 Yes (1) o 161 Yes (1) o 177 Yes, 2 products, rearrangement? 191 Yes (3) o 1 product? H

13 HPLC-MS/MS esults Q1 XY Urine 159 Yes (1) o 161 Yes (1) o 177 Yes, 2 products, rearrangement? 191 Yes (3) o 1 product? 193 o Yes (2) In te n s ity, c p s XIC of +MM (31 pairs): Exp 1, / Da ID: BZP-193 MM 1 from Sample 5 (BZP 1400mV 2) of BZP-x.wiff (Turbo Spray) 1.00e5 9.50e4 9.00e4 8.50e4 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 1.50e4 1.00e » XY 1400 mv Time, min XIC of +MM (31 pairs): Exp 1, / Da ID: BZP-193 MM 1 from Sample 10 ( Urin ohne Gluc Methode 03) of BZP-...» Urine e5 2.8e5 2.6e e Max. 1.0e5 cps. Max. 2.9e5 cps. 2.2e5 H In te n s ity, c p s 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4» 2.9 min 3.2 min e4 4.0e4 2.0e Time, min

14 HPLC-MS/MS esults Q1 XY Urine 159 Yes (1) o 161 Yes (1) o 177 Yes, 2 products, rearrangement? 191 Yes (3) o 1 product? 193 o Yes (2) 207 Yes (2) o In te n s ity, c p s XIC of +MM (31 pairs): Exp 1, / Da ID: BZP-207 MM 1 from Sample 9 (BZP 1600mV 2) of BZP-x.wiff (Turbo Spray) 4.8e5 4.6e5 4.4e5 4.2e5 4.0e5 3.8e5 3.6e5 3.4e5 3.2e5 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e Time, min XIC of +MM (31 pairs): Exp 1, / Da ID: BZP-207 MM 1 from Sample 10 ( Urin ohne Gluc Methode 03) of BZP-...» Urine 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5» XY 1600 mv Max. 4.9e5 cps.» 4.6 min 10.8 min Max. 4.6e4 cps. In te n s ity, c p s 9.0e4 8.0e4 7.0e4 6.0e4 5.0e e e e e Time, min

15 HPLC-MS/MS esults Q1 XY Urine 159 Yes (1) o 161 Yes (1) o 177 Yes, 2 products, rearrangement? 191 Yes (3) o 1 product? 193 o Yes (2) 207 Yes (2) o» 199, 229, 258, 272 and 318 showed no HPLC results for XY and urine» Possible artefacts or reactive intermediates

16 Why is there no H-metabolite in the XY?» m/z 177» m/z 193» m/z 209» m/z 207 H H H» inactivated aromatic system» activated aromatic system» second hydroxylation requires less energy than the first» observed EC product

17 What we learned:» Some metabolites can be produced easily in the EC system, but others can not» Variation of parameters may be laborious (electrodes, ph etc.)» nline setup to observe reactive intermediates ffline setup for micro-preparative synthesis» Underlying mechanisms of product formation differ for EC and enzymes, artefact formation possible in EC» Complex interpretation

18 Acknowledgements» Herbert beracher and obert Erb, Institute of Legal Medicine, Innsbruck» Daniel Vetter, EC GmbH» GTFCh for providing the generous stipend for presenting at TIAFT 2012