Transcript Acid-Base-Neutral Drug Screen

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
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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
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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
300L inserts
Static Instrument Settings
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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
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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
Screening Analysis
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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.
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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+)
• SIM windows
• Optimal Ionization
Settings
• Greatest Signal
• Extremely
Sensitive
Alprazolam
MW=308
Compounds in the Procedure
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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
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(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)
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 coeluting 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
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20 l injected
Standard:
– Only 1 drug class per standard
– Concentration similar to sample (based on
screening result)
Example:
– Screening:
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89 ng/mL 7-aminoclonazepam
85 ng/mL clonazepam
25 ng/mL lorazepam
Confirmation standards used:
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100 ng/mL Clonazepam Mix
20 ng/mL Lorazepam
Confirmation Analysis Gradients
Group
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 Spectrometry
Lorazepam
Channel 1
SIM – 130V
Channel 2
Scan – 250V
Confirmation Analytical Requirements
Detected if (SIM):
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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