Evaluation of the Impact of Expanding ELISA Screening in DUID Investigations. Aileen Lu*, Karen S. Scott, Aya Chan-Hosokawa, and Barry K.

Transcription

1 Evaluation of the Impact of Expanding ELISA Screening in DUID Investigations Aileen Lu*, Karen S. Scott, Aya Chan-Hosokawa, and Barry K. Logan

2 FSF Emerging Forensic Scientist Award Oral Presentation

3 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

4 Outline Background Aims and Objectives ELISA Validation Testing on case samples Conclusions

5 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.

6 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)

7 Carisoprodol and Meprobamate Impairing effects: Slowed movements/reflexes Confusion/disorientation Impaired coordination Slurred speech Dazed/groggy appearance Horizontal gaze nystagmus

8 Zolpidem CNS Depressant Used therapeutically to treat insomnia Therapeutic range µg/l Duration 4-6 h, impairing for additional 8-16 h

9 Zolpidem Impairing effects: Slowed reactions Slurred speech Poor coordination Lack of balance Flaccid muscle tone Horizontal/vertical gaze nystagmus Sleep driving

10 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 frequentlyencountered drugs. (Farrell et al., 2007)

11 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)

12 C/M/Z Positivity Rates DUID Cases (n=11,621) ELISA Screen for Drugs of Abuse: -Amphetamines -Barbiturates -Benzodiazepines -Cannabinoids -Cocaine -Methadone -Opiates -Phencyclidine -Propoxyphene ±Ethanol Data from NMS Labs (06/ /2014)

13 C/M/Z Positivity Rates DUID Cases (n=11,621) TOF Screening (n=1672) Additional 300+ compounds Data from NMS Labs (06/ /2014)

14 C/M/Z Positivity Rates DUID Cases (n=11,621) TOF Screening (n=1672) Carisoprodol/ Meprobamate 5.9% (n=99) Both (n=6) Zolpidem 5.3% (n=89) Data from NMS Labs (06/ /2014)

15 C/M/Z Positivity Rates DUID Cases (n=11,621)??

16 % Incidence Incidence of Drugs of Abuse in C/M/Z-positive Cases Carisoprodol/Meprobamate (n=99) All DUID Cases (n=11621) Zolpidem (n=89) Data from NMS Labs (06/ /2014)

17 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

18 ELISA Validation Titertek-Berthold Crocodile miniworkstation Dispenser Shaker Incubator Washer Reader Neogen Carisoprodol, Meprobamate, and Zolpidem Forensic (RTU) ELISA kits

19 OD ELISA Validation - Linearity 3 Meprobamate Carisoprodol Conc (mg/l) Conc (mg/l)

20 OD ELISA Validation - Linearity 3 Meprobamate Carisoprodol Conc (mg/l) Conc (mg/l)

21 OD ELISA Validation - Linearity 3 Meprobamate Carisoprodol Conc (mg/l) Conc (mg/l)

22 OD ELISA Validation - Linearity Zolpidem Conc (µg/l)

23 OD ELISA Validation - Linearity Zolpidem Conc (µg/l)

24 OD ELISA Validation - Linearity Zolpidem Conc (µg/l)

25 ELISA Validation - Precision Carisoprodol Conc Mean SD Meprobamate Conc Mean SD Zolpidem Conc Mean SD Intra-array %CV 4.2% Intra-array %CV 6.4% Intra-array %CV 6.4% Inter-array %CV 2.4% Inter-array %CV 2.6% Inter-array %CV 1.5% n = 12

26 ELISA Validation - Precision Carisoprodol Conc Mean SD Meprobamate Conc Mean SD Zolpidem Conc Mean SD Intra-array %CV 4.2% Intra-array %CV 6.4% Intra-array %CV 6.4% Inter-array %CV 2.4% Inter-array %CV 2.6% Inter-array %CV 1.5% n = 12

27 ELISA Validation - Precision Carisoprodol Conc Mean SD Meprobamate Conc Mean SD Zolpidem Conc Mean SD Intra-array %CV 4.2% Intra-array %CV 6.4% Intra-array %CV 6.4% Inter-array %CV 2.4% Inter-array %CV 2.6% Inter-array %CV 1.5%

28 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 crossreactivity with carisoprodol

29 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

30 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: mg/l Z: µg/l 3 low QCs, 3 high QCs

31 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/NH 4 OH) Dried and reconstituted in 50 µl ethyl acetate

32 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

33 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

34 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)

35 Comparison with NMS Results 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% Other drugs found in the 48 TOFscreened cases Data from NMS Labs (05/ /2014)

36 # of Occurrences Other Drugs found in 9 Positives 4 Carisoprodol/Meprobamate (n=4) Zolpidem (n=5) Data from NMS Labs (05/ /2014)

37 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%)

38 Conclusions 2007 NHTSA National Roadside Survey (Randomly selected drivers) This Study (DUID population) NMS Labs (Expanded drug screen)

39 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%

40 Acknowledgements Arcadia University Center for Forensic Science Research and Education NMS Labs Amanda Mohr Warren Korn Jaron Quinlan Dr. Sherri Kacinko

41 FSF Emerging Forensic Scientist Award Oral Presentation

42 Comparison with NMS Results NMS Labs TOFscreened DUID samples (June 2013 June 2014) n = 1672 This study s 322 DUID samples (Feb 2014 Aug 2014) n = 322 NMS Labs TOFscreened DUID samples (Feb 2014 Aug 2014) n = 1158 C/M: 5.9% Z: 5.3% C/M: 1.2% Z: 1.6% C: 4.3% M: 4.6% Z: 4.7%