1. Analysis of Benzodiazepines Trevor D. Gillis, M.S., D-ABC Criminalist Santa Clara County District Attorney ’ s Crime Laboratory

2. Medical Indications Anxiety (Anxiolytics) –associated with social/medical/personal problems Insomnia (Sedative) –as a result of anxiety/age Chronic pain –muscular, spasm, headaches, menopause/menses Skin conditions Dementia Anesthesia Muscle relaxant Withdrawal treatment Anticonvulsant

3. Pharmacological Action GABA receptor complex –Major inhibitory pathway –Composed of various subunits (  /  /  /  /  /  ) –Different brain regions have different subunit structures Drug actions differ based on subunit affinity http://web.lemoyne.edu

4. Medical Classification (t ½ ) Ultra-Short Acting (<6 hrs – Sedatives) –E.g. midazolam, triazolam Short Acting (<12 hrs – Sedatives) –E.g. oxazepam, temazepam, lorazepam Intermediate Acting (12-24 hrs - Anxiolytics) –E.g. clonazepam, flunitrazepam, alprazolam Long Acting (>24 hrs - Anxiolytics) –E.g. chlordiazepoxide, diazepam, flurazepam, nitrazepam, medazepam

5. Effects & Side Effects sedation anterograde amnesia ataxia low blood pressure poor balance cognitive impairment respiratory problems dependency drug interactions withdrawal

6. Common Forensic Encounters Implicated in drug facilitated sexual assaults Can impair performance and behavior Abuse is increasing Additive/Synergistic with many sedatives

7. Possible Analytical Schemes EIA – Not sensitive to every benzodiazepine GC or LC – Possible option (qualitative issues) GCMS – Possible option (sensitivity issues) LCMS (or LCMS 2 ) – of course!

8. Analytical Choice: LC/MSD Easy sample prep. Great selectivity –Screening –Confirmation Great sensitivity –Low LODs –Small sample volume (1 mL)

9. Instrument Agilent Technologies 1100 LC Single Quadrupole –SL series

10. Instrument Design LC – In-line solvent degasser Binary Pump with solvent selection 96-wellplate autosampler with needlewash Thermostated column compartment with column selection In-line DAD

11. Instrument Design MSD API (ESI) or APCI 2 modes (positive & negative) Single quadrupole 4 data channels Chemstation Software

12. Atmospheric Pressure Ionization

13. Spray Chamber Design API (ESI) Nebulizing Needle Hot N 2 Ionization Aid Instrument Potential

14. The Analytical Approach SPE Extraction Screening (Slow Gradient) - SIM –Low fragmentation voltage Confirmation – (Fast Gradient) – SIM and Full Scan –High fragmentation voltage

15. Specifications 2mm SB-C8 guard 150 x 2.1m Zorbax SB-C18 Column Varian Certify SPE Cartridges glass vials with 300  L inserts

16. Static Instrument Settings 2 sec. Needlewash Pump flow 0.200 mL/min. Isothermal 50°C column Spray chamber settings (API) –Drying gas 350°C @ 10.0 L/min. –Nebulizer pressure 25 psig –Capillary Voltage 2500 V MS in Positive Mode

17. Sample Preparation 1 mL blood or urine sample 30 ng Prazepam (300  L of 1.0  g/mL) 2 hour, 37°C urine hydrolysis (2000 units  - glucuronidase Type L-II e. coli pH 6.8) 4 mL of 0.1 M Phosphate buffer pH 6.0 Sonicate 15 min. Centrifuge 10 min. (5000 rpm)

18. SPE Extraction Bond Elut Certify –130 mg mixed-mode sorbent bed: octyl & benzene sulfonic acid Column Prep (Methanol then pH 6 buffer) Sample Added Wash and dry column Elution with 98:2 Ethyl Acetate: NH 3 Dry at 40°C Reconstitute 300  L 1:2 Acetonitrile

19. Screening Analysis 10  l injected Gradual Gradient (0.200 mL/min.) –30% Acetonitrile (0.1% formic acid) for 14 min. to 100% at 19 min. –Total time 27 min. QC procedures: –Standard mix first & last in run –Cutoff mix first & last in run –Blanks first and last in run

20. Screening – Single Ion (M+H + ) Alprazolam MW=308 SIM windows Optimal Ionization Settings Greatest Signal Extremely Sensitive

21. Compounds in the Procedure (MW/SIM Signal) clonazepam (315/ 316) nordiazepam (270/271) flurazepam (387/388) alprazolam (308/309) flunitrazepam (313/314) triazolam (343/343) temazepam (300/301) diazepam (284/285) 7-aminoclonazepam (285/286) norchlordiazepoxide (285/286) 7-aminoflunitrazepam (283/284) chlordiazepoxide (299/300) desalkylflurazepam* (288/289) nitrazepam (281/282) oxazepam (286/287) lorazepam (321/321) * not tested in urine

22. Screening - Analytical Requirements Integration is optimized for each compound based on cut-off standards Screening is positive if: –Peak shape is similar to the standards –Integration is acceptable –Retention Time match (  0.1 min.) –All blanks are negative –Cutoff standards contain results

23. Why Confirm at all? SIM M+H + ion is not enough character, especially at low levels The potential for co- eluting compounds Provides a greater level of certainty

24. Confirmation Options Targeted Analysis 2 Options: –Fragmentation – SIM –Fragmentation – SCAN Each drug group has its own method –Clonazepam/7-Aminoclonazepam –Diazepam/Nordiazepam/Oxazepam/ Temazepam –Etc.

25. Confirmation Analysis 20  l injected Standard: –Only 1 drug class per standard –Concentration similar to sample (based on screening result) Example: –Screening: 89 ng/mL 7-aminoclonazepam 85 ng/mL clonazepam 25 ng/mL lorazepam –Confirmation standards used: 100 ng/mL Clonazepam Mix 20 ng/mL Lorazepam

26. Confirmation Analysis Gradients GroupGradient (0.1% Formic Acid in Acetonitrile)Total Alprazolam30% for 3 min. to 100% by 10 min.16 min. Clonazepam20% for 3 min. to 100% by 10 min.18 min. Chlordiazepoxide20% for 6 min. to 100% by 8 min.16 min. Diazepam50% for 2 min. to 100% by 10 min.12 min. Flunitrazepam30% for 3 min. to 100% by 10 min.16 min. Flurazepam30% for 3 min. to 100% by 10 min.16 min. Lorazepam30% for 3 min. to 100% by 10 min.16 min. Nitrazepam30% for 3 min. to 100% by 10 min.16 min. Oxazepam40% for 2 min. to 100% by 8 min.14 min. Triazolam30% for 3 min. to 100% by 10 min.16 min.

27. Confirmation Mass Spectrometry Lorazepam Channel 1 SIM – 130V Channel 2 Scan – 250V

28. Confirmation Analytical Requirements Detected if (SIM): –All peaks are present –Peak shape is similar to standard –Retention times within ± 0.1 min. for all peaks –Ion ratios for all qualifiers within ± 20% of standard –Acceptable integration Detected if (Scan): –Spectral Match is clear –Retention times within ± 0.1 min.

29. Detection Limits (Blood) 1 ng/mL –flurazepam, nitrazepam, oxazepam, lorazepam, clonazepam, nordiazepam, desalkylflurazepam, alprazolam, flunitrazepam, triazolam, temazepam, diazepam 5 ng/mL –7-aminoclonazepam, norchlordiazepoxide, chlordiazepoxide, 7-aminoflunitrazepam

30. Detection Limits (Urine) 5 ng/mL –chlordiazepoxide, norchlordiazepoxide, flunitrazepam, 7-aminoflunitrazepam, flurazepam, alprazolam, triazolam 10 ng/mL –nitrazepam, lorazepam, diazepam, nordiazepam 20 ng/mL –7-aminoclonazepam, clonazepam, oxazepam, temazepam

31. Interferences Used NIST Compound Search Search compounds with the same MW Tested all compounds where a standard could be obtained Tested 29 different compounds No interferences detected

32. Carry-Over Carry-over exists in all methods where the same instrument is used multiple times 0.025% was detected for flurazepam None detected after 100  g/mL injection for remainder

33. Extract Stability stable for at least 1 week (instrument) most are stable up to 4 weeks chlordiazepoxide and clonazepam are known to be light sensitive 80-95% loss of norchlordiazepoxide and 7-aminoflunitrazepam by 4 weeks 30-65% loss of nitrazepam, oxazepam, nordiazepam, alprazolam, and temazepam by 4 weeks

34. Summary LCMSD Powerful analytical tool Easy to maintain Meets the analytical requirements for a forensic toxicology laboratory