Evaluation of the Impact of Expanding ELISA Screening in DUID ...€¦ · %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

Evaluation of the Impact of Expanding ELISA Screening in DUID Investigations

Aileen Lu*, Karen S. Scott, Aya Chan-Hosokawa, and Barry K. Logan

FSF Emerging Forensic Scientist Award

Oral Presentation

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

Outline

• Background

• Aims and Objectives

• ELISA Validation

• Testing on case samples

• Conclusions

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.

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)

Carisoprodol and Meprobamate

• Impairing effects:

– Slowed movements/reflexes

– Confusion/disorientation

– Impaired coordination

– Slurred speech

– Dazed/groggy appearance

– Horizontal gaze nystagmus

Zolpidem

• CNS Depressant

• Used therapeutically to treat insomnia

• Therapeutic range 60-230 µg/L

• Duration 4-6 h, impairing for additional 8-16 h

Zolpidem

• Impairing effects:

– Slowed reactions

– Slurred speech

– Poor coordination

– Lack of balance

– Flaccid muscle tone

– Horizontal/vertical gaze nystagmus

– Sleep driving

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)

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)

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



TOF Screening (n=1672)


Additional 300+ compounds




Carisoprodol/

Meprobamate

5.9% (n=99)

Zolpidem

5.3% (n=89)


Both (n=6)



? ?

Incidence of Drugs of Abuse in C/M/Z-positive Cases


0

10

20

30

40

50

60

% In

cid

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ce

Carisoprodol/Meprobamate (n=99) Zolpidem (n=89)

All DUID Cases (n=11621)

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

ELISA Validation

• Titertek-Berthold Crocodile miniWorkstation

– Dispenser

– Shaker

– Incubator

– Washer

– Reader

• Neogen® Carisoprodol, Meprobamate, and Zolpidem Forensic (RTU) ELISA kits

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


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


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


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


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


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

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


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


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%

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

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


• 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


• 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


• 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


• 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


• My results:

– 5/322 (1.6%) positive for zolpidem

– 4/322 (1.2%) positive for carisoprodol/meprobamate


Carisoprodol/

Meprobamate

5.9% (n=99)

Zolpidem

5.3% (n=89)

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)

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)


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%)

Conclusions

2007 NHTSA National

Roadside Survey

(Randomly selected drivers)

This Study

(DUID population)

NMS Labs

(Expanded drug screen)

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%

Acknowledgements

Arcadia University

Center for Forensic Science Research and Education

NMS Labs

Amanda Mohr

Warren Korn

Jaron Quinlan

Dr. Sherri Kacinko

FSF Emerging Forensic Scientist Award

Oral Presentation

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%

Documents

Evaluation of the Impact of Expanding ELISA Screening in DUID ...€¦ · %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