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

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

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

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

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

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

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!

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

Instrument Agilent Technologies 1100 LC Single Quadrupole –SL series

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

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

Atmospheric Pressure Ionization

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

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

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

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

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)

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

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

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

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

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

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

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.

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

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.

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

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.

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

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

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

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

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

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