evaluation of the impact of expanding elisa screening in duid ...€¦ · %cv 2.4% meprobamate conc...
TRANSCRIPT
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Evaluation of the Impact of Expanding ELISA Screening in DUID Investigations
Aileen Lu*, Karen S. Scott, Aya Chan-Hosokawa, and Barry K. Logan
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FSF Emerging Forensic Scientist Award
Oral Presentation
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Disclosure
• This presentation will mention the following commercial products and companies:
– Neogen®
– Agilent®
– Titertek-Berthold
– NMS Labs®
– UCT® Clean Screen®
• No financial support was provided by any of the above mentioned entities
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Outline
• Background
• Aims and Objectives
• ELISA Validation
• Testing on case samples
• Conclusions
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Background
• In 2013, Logan et al. published a set of recommendations for screening in DUID cases.
– Added carisoprodol and zolpidem to screen due to prevalence in laboratories across the United States and their confirmed impairing effects on driving.
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Carisoprodol and Meprobamate
• CNS Depressant
• Centrally-acting muscle relaxant, treatment for anxiety
• Often prescribed with narcotic analgesics or benzodiazepines
• Therapeutic range 1-5 mg/L
• Duration 4-6 h (longer for meprobamate)
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Carisoprodol and Meprobamate
• Impairing effects:
– Slowed movements/reflexes
– Confusion/disorientation
– Impaired coordination
– Slurred speech
– Dazed/groggy appearance
– Horizontal gaze nystagmus
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Zolpidem
• CNS Depressant
• Used therapeutically to treat insomnia
• Therapeutic range 60-230 µg/L
• Duration 4-6 h, impairing for additional 8-16 h
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Zolpidem
• Impairing effects:
– Slowed reactions
– Slurred speech
– Poor coordination
– Lack of balance
– Flaccid muscle tone
– Horizontal/vertical gaze nystagmus
– Sleep driving
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2007 Top Drugs in DUID Labs
Drug In top 10
Cannabis 39
Benzodiazepines 37
Cocaine 37
Hydrocodone 30
Morphine/Codeine 28
Methamphetamine 26
Carisoprodol/Meprobamate 26
Oxycodone 16
Methadone 12
Antidepressants 11
Zolpidem 10
Survey of 40 toxicology labs, asked to list their top 10 most frequently-encountered drugs.
(Farrell et al., 2007)
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2012 Top Drugs in DUID Labs
Compound In Top 20 Drugs
THC and metabolites 13
Alprazolam/Met. 13
Diazepam/Nordiazepam 13
Cocaine and metabolites 13
Morphine 13
Oxycodone 12
Hydrocodone 12
Carisoprodol/Meprobamate 11
Zolpidem 11
Methamphetamine 9
Clonazepam/ Met. 9
Amphetamine 9
Methadone 9
Lorazepam 9
Codeine 7
Diphenhydramine 6
Tramadol 6
PCP 5
Hydromorphone 5
Compound In Top 20 Drugs
Citalopram 4
Temazepam 3
Oxazepam 2
Trazodone 2
Oxymorphone 2
Butalbital 2
Dihydrocodeine 2
Pseudoephedrine 2
6-acetylmorphine 2
Fentanyl 2
MDMA 2
Fluoxetine/ Met. 1
Venlafaxine/ Met. 1
Gabapentin 1
Cyclobenzaprine 1
Amitriptyline 1
Topiramate 1
(Logan et al., 2013)
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C/M/Z Positivity Rates
DUID Cases (n=11,621)
Data from NMS Labs (06/2013 – 06/2014)
ELISA Screen for Drugs of Abuse: -Amphetamines -Barbiturates -Benzodiazepines -Cannabinoids -Cocaine -Methadone -Opiates -Phencyclidine -Propoxyphene ±Ethanol
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C/M/Z Positivity Rates
DUID Cases (n=11,621)
TOF Screening (n=1672)
Data from NMS Labs (06/2013 – 06/2014)
Additional 300+ compounds
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C/M/Z Positivity Rates
DUID Cases (n=11,621)
TOF Screening (n=1672)
Carisoprodol/
Meprobamate
5.9% (n=99)
Zolpidem
5.3% (n=89)
Data from NMS Labs (06/2013 – 06/2014)
Both (n=6)
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C/M/Z Positivity Rates
DUID Cases (n=11,621)
? ?
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Incidence of Drugs of Abuse in C/M/Z-positive Cases
Data from NMS Labs (06/2013 – 06/2014)
0
10
20
30
40
50
60
% In
cid
en
ce
Carisoprodol/Meprobamate (n=99) Zolpidem (n=89)
All DUID Cases (n=11621)
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Aims and Objectives
• To evaluate the addition of carisoprodol/ meprobamate and zolpidem to the initial DUID screening procedure
• Validate an ELISA platform in order to gather data on the incidence of carisoprodol/ meprobamate and zolpidem in blood in DUID cases
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ELISA Validation
• Titertek-Berthold Crocodile miniWorkstation
– Dispenser
– Shaker
– Incubator
– Washer
– Reader
• Neogen® Carisoprodol, Meprobamate, and Zolpidem Forensic (RTU) ELISA kits
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ELISA Validation - Linearity
0
0.025 0.05
0.1
0.2
0.375
0.5 0.75 1 2
0
0.5
1
1.5
2
2.5
3
0 1 2
OD
Conc (mg/L)
Meprobamate
0 0.025
0.05
0.1 0.2
0.375
0.5 0.75 1 1.5
0
0.5
1
1.5
2
2.5
0 0.5 1 1.5
Conc (mg/L)
Carisoprodol
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ELISA Validation - Linearity
0
0.025 0.05
0.1
0.2
0.375
0.5 0.75 1 2
0
0.5
1
1.5
2
2.5
3
0 1 2
OD
Conc (mg/L)
Meprobamate
0 0.025
0.05
0.1 0.2
0.375
0.5 0.75 1 1.5
0
0.5
1
1.5
2
2.5
0 0.5 1 1.5
Conc (mg/L)
Carisoprodol
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ELISA Validation - Linearity
0
0.025 0.05
0.1
0.2
0.375
0.5 0.75 1 2
0
0.5
1
1.5
2
2.5
3
0 1 2
OD
Conc (mg/L)
Meprobamate
0 0.025
0.05
0.1 0.2
0.375
0.5 0.75 1 1.5
0
0.5
1
1.5
2
2.5
0 0.5 1 1.5
Conc (mg/L)
Carisoprodol
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ELISA Validation - Linearity
0 1 2 3
5
7.5
10 15
25 40
0
0.5
1
1.5
2
2.5
3
3.5
0 10 20 30 40 50
OD
Conc (µg/L)
Zolpidem
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ELISA Validation - Linearity
0 1 2 3
5
7.5
10 15
25 40
0
0.5
1
1.5
2
2.5
3
3.5
0 10 20 30 40 50
OD
Conc (µg/L)
Zolpidem
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ELISA Validation - Linearity
0 1 2 3
5
7.5
10 15
25 40
0
0.5
1
1.5
2
2.5
3
3.5
0 10 20 30 40 50
OD
Conc (µg/L)
Zolpidem
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ELISA Validation - Precision
Carisoprodol
Conc Mean SD
75 0.790 0.036
100 0.746 0.035
125 0.737 0.037
Intra-array %CV
4.2%
Inter-array %CV
2.4%
Meprobamate
Conc Mean SD
75 0.689 0.042
100 0.648 0.043
125 0.602 0.048
Intra-array %CV
6.4%
Inter-array %CV
2.6%
Zolpidem
Conc Mean SD
3.75 0.824 0.048
5 0.797 0.050
6.25 0.725 0.053
Intra-array %CV
6.4%
Inter-array %CV
1.5%
n = 12
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ELISA Validation - Precision
Carisoprodol
Conc Mean SD
75 0.790 0.036
100 0.746 0.035
125 0.737 0.037
Intra-array %CV
4.2%
Inter-array %CV
2.4%
Meprobamate
Conc Mean SD
75 0.689 0.042
100 0.648 0.043
125 0.602 0.048
Intra-array %CV
6.4%
Inter-array %CV
2.6%
Zolpidem
Conc Mean SD
3.75 0.824 0.048
5 0.797 0.050
6.25 0.725 0.053
Intra-array %CV
6.4%
Inter-array %CV
1.5%
n = 12
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ELISA Validation - Precision
Carisoprodol
Conc Mean SD
75 0.790 0.036
100 0.746 0.035
125 0.737 0.037
Intra-array %CV
4.2%
Inter-array %CV
2.4%
Meprobamate
Conc Mean SD
75 0.689 0.042
100 0.648 0.043
125 0.602 0.048
Intra-array %CV
6.4%
Inter-array %CV
2.6%
Zolpidem
Conc Mean SD
3.75 0.824 0.048
5 0.797 0.050
6.25 0.725 0.053
Intra-array %CV
6.4%
Inter-array %CV
1.5%
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ELISA Validation
• Carryover – Row of a high concentration, followed by 2 rows
of blanks • 10 & 50 mg/L for carisoprodol and meprobamate
• 200 & 1000 µg/L for zolpidem
– Determined no significant carryover
• Cross-reactivity – Kits came with extensive list of cross-reactivity
– Confirmed that meprobamate kit had some cross-reactivity with carisoprodol
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Testing on case samples
• 322 DUID samples from NMS Labs
– Whole blood
– Gray-top tubes except one lavender-top
– Time period: May-Aug 2014
– All screened for ELISA DUID panel (no C/M/Z)
• 48 TOF-screened (included C/M/Z)
– Representative subset of DUID population
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Testing on case samples
• ELISA Screen for M & Z SPE GC/MS
– Confirmatory testing on the 9 positives + 9 random negatives
• Neat and 1+9 dilution
– Calibration range
• C/M: 0.5-20 mg/L
• Z: 10-400 µg/L
– 3 low QCs, 3 high QCs
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Testing on case samples
• Solid Phase Extraction – 130 mg Clean Screen® mixed mode cartridges
– Conditioned with methanol, water, pH6 phosphate buffer
– Added sample, buffered to pH6
– Washed with water, acetic acid, hexane
– Eluted with 78/20/2 (DCM/IPA/NH4OH)
– Dried and reconstituted in 50 µL ethyl acetate
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Testing on case samples
• GC/MS confirmation
– Methadone-D9 as internal standard (250 µg/L)
– Agilent® 5975 GC/MS
– SIM/SCAN method
SIM Ions (m/z)
Methadone-D9 78, 232, 303
Carisoprodol 55, 104, 158
Meprobamate 83, 114, 144
Zolpidem 219, 235, 307
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Testing on case samples
• My results:
– 5/322 (1.6%) positive for zolpidem
– 4/322 (1.2%) positive for carisoprodol/meprobamate
– The 9 random samples were negative for both
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Testing on case samples
• My results:
– 5/322 (1.6%) positive for zolpidem
– 4/322 (1.2%) positive for carisoprodol/meprobamate
TOF Screening (n=1672)
Carisoprodol/
Meprobamate
5.9% (n=99)
Zolpidem
5.3% (n=89)
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Comparison with NMS Results
Other drugs found in the 48 TOF-screened cases
Drug Occurrence Drug Occurrence
Ethanol 48% Lorazepam 6%
THC 27% Nordiazepam 6%
Alprazolam 19% Quetlapine 6%
Clonazepam 17% Topiramate 6%
Oxycodone 13% Trazodone 6%
Diazepam 10% Zolpidem 6%
Morphine 10% Cocaine 4%
Amphetamine 8% Cyclobenzaprine 4%
Buprenorphine 8% Diphenhydramine 4%
Tramadol 8% Lamotrigine 4%
Bupropion 6% Paroxetine 4%
Fentanyl 6% Venlafaxine 4%
Hydrocodone 6% Other 2% Data from NMS Labs (05/2014 – 08/2014)
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Other Drugs found in 9 Positives
0
1
2
3
4
# o
f O
ccu
rre
nce
s
Carisoprodol/Meprobamate (n=4) Zolpidem (n=5)
Data from NMS Labs (05/2014 – 08/2014)
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Conclusions
• Of 322 DUID cases that were initially not screened for zolpidem or meprobamate:
– 1.6% subsequently tested positive for zolpidem
– 1.2% subsequently tested positive for carisoprodol/meprobamate
• 274 of the cases received only ELISA screening
– When zolpidem and carisoprodol were added to the ELISA scope:
• Zolpidem was found in 1 case (0.4%)
• Carisoprodol/meprobamate was found in 4 cases (1.5%)
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Conclusions
2007 NHTSA National
Roadside Survey
(Randomly selected drivers)
This Study
(DUID population)
NMS Labs
(Expanded drug screen)
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Conclusions
2007 National Roadside Survey
(Randomly selected drivers)
This study
(DUID population)
NMS Labs (Expanded screen requested)
• C/M: 0.05%
• Z: 0.12%
• C/M: 1.2%
• Z: 1.6%
• C/M: 5.9%
• Z: 5.4%
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Acknowledgements
Arcadia University
Center for Forensic Science Research and Education
NMS Labs
Amanda Mohr
Warren Korn
Jaron Quinlan
Dr. Sherri Kacinko
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FSF Emerging Forensic Scientist Award
Oral Presentation
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Comparison with NMS Results
NMS Labs TOF-screened DUID
samples
(June 2013 – June 2014)
n = 1672
C/M: 5.9%
Z: 5.3%
This study’s 322 DUID samples
(Feb 2014 – Aug 2014)
n = 322
C/M: 1.2%
Z: 1.6%
NMS Labs TOF-screened DUID
samples
(Feb 2014 – Aug 2014)
n = 1158
C: 4.3%
M: 4.6%
Z: 4.7%