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