Analysis of BenzodiazepinesTrevor D. Gillis, M.S., D-ABC
CriminalistSanta 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
http://web.lemoyne.edu
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
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 LCMS2) – 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 N2
•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 300L inserts
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
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: NH3
• Dry at 40°C• Reconstitute 300L 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+)AlprazolamMW=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 Analysis2 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 GradientsGroup Gradient (0.1% Formic Acid in
Acetonitrile)Total
Alprazolam 30% for 3 min. to 100% by 10 min. 16 min.
Clonazepam 20% for 3 min. to 100% by 10 min. 18 min.
Chlordiazepoxide
20% for 6 min. to 100% by 8 min. 16 min.
Diazepam 50% for 2 min. to 100% by 10 min. 12 min.
Flunitrazepam 30% for 3 min. to 100% by 10 min. 16 min.
Flurazepam 30% for 3 min. to 100% by 10 min. 16 min.
Lorazepam 30% for 3 min. to 100% by 10 min. 16 min.
Nitrazepam 30% for 3 min. to 100% by 10 min. 16 min.
Oxazepam 40% for 2 min. to 100% by 8 min. 14 min.
Triazolam 30% for 3 min. to 100% by 10 min. 16 min.
Confirmation Mass SpectrometryLorazepam
Channel 1SIM – 130V
Channel 2Scan – 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