Analysis of Benzodiazepines

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 (t½) • 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 LCMS2) – 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 N2 • 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: NH3 • 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) • 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 • clonazepam (315/ 316) • nordiazepam (270/271) • flurazepam (387/388) • alprazolam (308/309) • flunitrazepam (313/314) • triazolam (343/343) • temazepam (300/301) • diazepam (284/285)

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

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