Download Acid-Base-Neutral Drug Screen

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Document related concepts

Diazepam wikipedia, lookup

Lorazepam wikipedia, lookup

Drug discovery wikipedia, lookup

Transcript
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
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
300L 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 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+)
• SIM windows
• Optimal Ionization
Settings
• Greatest Signal
• Extremely
Sensitive
Alprazolam
MW=308
Compounds in the Procedure
•
•
•
•
•
•
•
•
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
•
•
•
•
•
•
•
•
(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
•
•
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
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):
–
–
–
–
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