Comparison of Drug Concentrations in Postmortem Blood and Skeletal Tissue (Bone)

Eric S. Lavins1*, BS , John F. Wyman1, PhD, James H. Watterson2, PhD, Kathleen E. Toomey3,

Claire K. Naso-Kaspar1, BS, Kimberly D. Snell1, BS, Szabolcs Sofalvi1, MS, Laura. D. Wilkins1, BS, Carrie D. Mazzola1, BS, Rindi N. Norris1, BS, Jason M. Stibley1, BS, Thomas P. Gilson1, MD

1Cuyahoga County Medical Examiner’s Office, Toxicology Department, Cleveland, Ohio,

2Forensic Toxicology Research Laboratory, Laurentian University, Ontario, Canada, 3Hofstra University, Hempstead, New York

The authors would like to acknowledge the cooperation of the Cuyahoga County Medical Examiner’s Office and Cuyahoga County Regional Forensic Crime Lab for providing the facilities for the work, and thanks to Drs., Erica Armstrong, Joseph Felo, Dan Galita, Krista Pekarski-Timm, Andrea Weins, and autopsy assistants, Paul Weaton, Tony Arunski, Jim Mullaly and Michael Vitovich for providing the bone samples. Finally, thanks to Mr. James Wentzel and the Photography Department at CCMEO.

Abstract After attending this presentation, attendees will have a better understanding of the utilization and interpretive value of the detection of drugs in postmortem skeletal tissue (bone).

This presentation will impact the forensic community by informing forensic professionals to utilize alternate matrices such as bone when traditional specimens such as blood, body fluids and tissues are not available for toxicological analysis.

A recent multiple homicide case where only skeletonized and extremely putrefied fluids were available prompted the laboratory to establish a standardized approach for the analysis of bone in future cases. To do this, bones from non-decomposed specimens were evaluated. Cardiac and femoral blood (FB), along with routine postmortem specimens, iliac crest or vertebrae were collected in 20 cases. Specimens were analyzed using an enzyme-linked immunosorbent assay (ELISA-13-panel), base and acidic/neutral, EMIT, acetaminophen and salicylate analyses. Confirmations & quantitations were performed using gas chromatography/mass spectrometry.

Bones were stored (-200C) for three years prior to analysis. Cleaned bones were rinsed with DI water, dried (48 hours), manually crushed, and ground in a commercial grinder. Bone (3 g) was added to 10 mL methanol, vortexed 1 minute, incubated 48 hrs (room temperature), and rotary extracted for 8 hrs. Methanol supernatants were recovered by centrifugation, evaporated to near dryness and combined with 100 µl of 0.1% methanolic HCl. Phosphate buffer (PB: 2 mL, 100 mM, pH 6) and 4mL acetonitrile: methanol (2:1 v/v ratio) were added to evaporated extracts. Vortexed extracts were stored (-200C) for 12 hours, centrifuged 5 minutes. Supernatants were reduced to 2 mL by evaporation, diluted to 5 mL with PB. Bone extracts were analyzed in the same manner as blood. Drug free bone from postmortem blood screening served as negative controls, (n=3).

Opioids were detected in 8/9 cases which screened positive by ELISA. In 2 heroin cases, only codeine was detected, 6.6 ng/g and 15 ng/mL, in bone and FB, respectively, no opiates were present in the other. Two cases contained hydrocodone, 36 and 8.3 ng/g bone; 76 and 44 ng/mL FB. Dihydrocodeine was detected in both FB samples but not in bone. Oxycodone was detected in 3 cases at 161, 25 and 83.5 ng/g, bone and 520, 65 and 920 ng/mL FB. Oxymorphone was present in one bone specimen compared to three cases in FB. Fentanyl, methadone and propoxyphene were detected in one case each: (4.17 ng/g, bone, 8.7 ng/mL, FB), (217 ng/g, bone, 530 ng/mL, FB), (1.35 ng/g bone, and 2.9 mg/L FB), respectively. EDDP was present in FB, 220 ng/mL, but not in bone. Norpropoxyphene was present in both matrices.

Methylenedioxypyrovalerone (MDPV) and N-benzylpiperazine (BZP) were present in individual cases in FB, urine and bone. Synthetic Cannabinoids: JWH-18 and JWH-250 were detected in one case, bone, 0.33 and 1.4 ng/mL and, FB, 0.48 and 4.6 ng/mL, respectively. AM-2201 and RCS-4 were found in bone only, (synthetic cannabinoid and stimulant analysis performed by NMS Labs.)

Cocaine/metabolites were present in 4 cases: Benzoylecgonine, < 20, 62, 258 and 512 ng/g for bone, and 88, 96, 830 and >2000 ng/mL for FB. EME was in two bone samples, anhydroecgonine methylester (AEME) was present in one bone sample and not detected in FB. Cocaine was present in one case at 58.4 ng/g, bone, 278 ng/ml, FB. Nordiazepam was detected at 67.8 ng/g, bone, and was, qualitatively present in FB. Clonazepam, detected in 2 FB samples, but not in the corresponding bone.

Cyclobenzaprine was present in one specimen, 183 ng/g, bone, 14 ng/mL FB. Venlafaxine was present in one case, 768 ng/g, bone, 0.69 mg/L, blood. Norvenlafaxine and lidocaine, present in both matrices.

Dextromethorphan was present in bone, 968 ng/g and >2000 ng/L FB, in an overdose case. Diphenhydramine was present in 2 FB cases, 1.45 and 0.075 mg/L, but was only seen in the first case at 300 ng/g, bone. Cotinine correlated in 6/7 cases with FB and urine. Nicotine and caffeine were not found in bone, but both were detected in FB. Doxylamine, promethazine and ibuprofen were also found in both bone and FB.

In these cases, most drug findings in blood were also detected in bone. In most cases drug concentrations were higher in blood than in bone. These findings demonstrate that bone may be a useful matrix to document drug usage in postmortem cases when traditional specimens are unavailable.

Keywords: Bone, Skeletal Tissue, Alternate Specimen

Introduction Conclusions

References

Acknowledgements

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The bones were then weighed and placed into 50 mL polypropylene tubes prior to further treatment. Processing/Treatment of bones:

3 g of bone was transferred to 50 mL screw capped centrifuged tubes and 10 mLs of methanol was added.

Samples were then vortexed for 1 minute and allowed to sit for 48 hours and then rotated for 8 hours.

Bone extract was centrifuged and the methanol was recovered. To the extract, 100 µl of 0.1% methanolic HCl was added to each tube and was evaporated to near dryness under a stream of air at 500C. 2 mLs of sodium phosphate buffer 0.1 M (100 mM), pH 6 was added, to the samples. Acetonitrile: methanol (2:1 v/v ratio) solution was added to each specimen. To precipitate lipids, samples were vortexed for 1 minute and placed in a freezer -200C for 12 hours, and then centrifuged for 5 minutes. The supernatant was transferred to a 15 mL centrifuge tubes and reduced under a stream of air at 500C to 2 mLs. Samples were then adjusted with the sodium phosphate buffer 0.1 M (100 mM), pH 6 for a total volume of 5 mLs. Extraction and analysis: Drug free bone established from post- mortem blood screening and case history served as negative controls, (n=3). The routine specimens and bone extracts were subjected to general toxicological screening for illicit and non-illicit drugs using previously validated extraction protocols used by CCMEO. Screening was performed using enzyme -linked immunosorbent assay (ELISA). Specific classes of drugs include opiates, oxycodone, methamphetamine, amphetamine, tricyclic antidepressants, carisoprodol, fentanyl, cannabinoids, cocaine and metabolites, benzodiazepines, phencyclidine, barbiturates and methadone. The screen also included a base and acidic neutral analysis, an EMIT Screen, acetaminophen and salicylate analysis. Confirmations and quantitations were performed using typical gas chromatography- mass spectrometry (GC/MS) analysis. Analysis and confirmation of synthetic stimulants and synthetic cannabinoids were performed by NMS Labs, Willow Grove, PA.

Bone sample preparation: Bones were extracted using a modified, previously published 1 isolation protocol for skeletal tissue. Bones were cleaned of surrounding tissue and rinsed with DI water. Bones were then dried in weighing boats in the hood for 48 hours. Bones were then manually crushed with a steel pipe with a treaded end cap. The specimens were then ground in a mechanical grinder.

ELISA bone and blood results generally correlated and were either positive or elevated. Drug concentrations in bone were less than the blood levels except for JWH-18, cyclobenzaprine, doxylamine and venlafaxine. A number of drug classes were detected in the bone (iliac crest and vertebral) tissue, including sympathomimetic amines, antidepressants, benzodiazepines, opiates, opioids, cocaine and metabolites, and anticonvulsants. Methylenedioxypyrovalerone (MDPV), benzylpiperazine (BZP) and the synthetic cannabinoids, JWH-18, JWH-250, RCS-4 and AM-2201 were detected in bone samples. To our knowledge, this is the first reported documentation of cathinones and synthetic stimulants and synthetic cannabinoids being reported in skeletal (iliac crest and vertebral bone) tissue.

The primary matrices of choice in post-mortem forensic toxicological analysis have focused on utilizing blood, urine, bile, vitreous humor, liver and other tissues. Many times, only skeletal remains, extreme putrefied fluids or multiple trauma cases are provided to the laboratory for toxicological analysis. Bone is a highly complex and organized porous vascular connective tissue which may be utilized after skeletonization. The bone marrow consists of a lipid matrix that may act as a drug storage site. The goal of the study was to: Improve and standardize procedures for the post-mortem analysis of alternate specimens such as skeletal tissue (bone, iliac crest, vertebra, pelvic bone, femur, sternum, and ribs) when traditional specimens were unavailable. Assess the respective forensic interpretive value in correlating blood and skeletal tissue drug relationships.

Forensic autopsy sample collection: A complete autopsy was performed at the Cuyahoga County Medical Examiner’s Office (CCMEO), Cleveland, Ohio and samples were collected from cases involving drug intoxications and random cases. Bones, (iliac crest or vertebral) were collected from non-decomposed cases, n=20), along with routine postmortem specimens, cardiac and femoral blood (FB), liver, urine, bile, vitreous humor and gastric. The subjects’ ages ranged from 21-89 years, including 5 female, and 15 males. Iliac crest and vertebrae from autopsy were stored at -200C, for three years prior to analysis.

Methods Results

The method proved to be an efficient means of examining the bone tissue iliac crest and vertebral bone for the presence of drugs. The majority of drugs and some metabolites that were detected in the post-mortem blood were detected in the skeletal tissue (bone) specimen and qualitatively correlated to the blood results. Drug parent and metabolite were stable in bone matrix stored at -200C, for three years prior to analysis. Other drugs found to be present in bone: dextromethorphan, diphenhydramine, doxylamine, promethazine, ibuprofen, cotinine, lidocaine, and beta-Phenethylamine. However, morphine, 6-AM and caffeine were absent in the bone. Additional studies in this area are needed to determine linear correlations, ratios of drug and metabolite levels between blood, bone and bone marrow and optimal tissue type for analysis. These findings demonstrate that bone may be a useful matrix to document qualitative drug usage in forensic postmortem cases when traditional specimens are not present

GC/MS and LC/MS/MS results

Drugs Detected in Femoral Bone

Amphetamines 1 case Amphetamine: present Amphetamine: not detected beta-Phenethylamine: 15 of 20 cases positive

Synthetic Stimulants:

Cathinones 2 cases

Methylenedioxypyrovalerone (MDPV): 520 ng/mL Heart, 530 ng/mL Femoral,

Benzylpiperazine (BZP): present

*MDPV: present > 10 ng/mL, BZP: present > 10 ng/mL

*Vertebral bone

Cannabinoids 3 cases THCOOH: 1-urine positive, 2-bloods: Not detected Testing not performed

Synthetic Cannabinoids 1 case

JWH-18: 0.24, 0.48 ng/mL Heart and Femoral, JWH-250: 2.8, 4.6 ng/mL, Heart and Femoral,

RCS-4 and AM2201: not tested-Blood or Femoral

*Vertebral bone. *JWH-18: 0.33 ng/mL, *JWH-250: 1.4 ng/mL,

*RCS-4: 0.26 ng/mL, *AM2201: 0.49 ng/mL

Cocaine and metabolites 4 cases

BE: 88, 96, 830 and > 2000 ng/mL, EME: present in all blood specimens, Cocaine: (1) 278 ng/mL,

AEME: Not detected (ND)

BE: < 20, 62, 258, 512 ng/g, EME: present in 2 of 4 specimens, Cocaine: 58.4 ng/g,

AEME: present in 1 specimen

Benzodiazepines 3 cases Diazepam, Desmethyldiazepam, Temazepam, Clonazepam, 7-amino-clonazepam: present

Desmethyldiazepam 67.8 ng/g, Clonazepam, 7-amino-clonazepam: ND

Opioids: 6-Acetyl-

morphine (6-AM) 2 cases 6-AM: Not detected in Femoral, 6-AM: present in Vitreous Humor - 2 cases 6-AM: not detected

Codeine 1 case Codeine: 15 ng/mL Codeine: 6.6 ng/g Dihydrocodeine 2 cases Dihydrocodeine: present Dihydrocodeine: 4 ng/mL Hydrocodone 2 cases Hydrocodone: 76 and 44 ng/mL Hydrocodone: 36 and 8.3 ng/g

Morphine 2 cases Morphine: 62 & 233 ng/mL Morphine: not detected Oxycodone 3 cases Oxycodone: 520, 65.0, 920 ng/mL Oxycodone:161.0, 25.2 and 83.5 ng/g

Oxymorphone 1 case Oxymorphone: present Oxymorphone: present in 1 specimen Fentanyl/ Norfentanyl 1 case Fentanyl: 8.7 ng/mL, Norfentanyl: 4.3 ng/mL Fentanyl: 3.34 ng/g

Methadone 1 case Methadone: 530 ng/mL , EDDP: 220 ng/mL Methadone: 217 ng/g, EDDP: not detected Propoxyphene 1 case Propoxyphene: 2.9 mg/L & mtb: present Propoxyphene: 1.35 ng/g , NorPropox: present

Other Drugs: Caffeine 5 cases Caffeine: present in 5 cases Caffeine: not detected

Carisoprodol 1 case Carisoprodol ELISA: negative Carisoprodol: not detected in bone, ELISA Carisoprodol: positive

Cyclobenzaprine 1 case Cyclobenzaprine:14 ng/mL Cyclobenzaprine: 183 ng/g Dextromethorphan 1 case Dextromethorphan: > 2.0 mg/L femoral Dextromethorphan: 968 ng/g Diphenhydramine 2 cases Diphenhydramine: 1.45 and 0.75 mg/L Diphenhydramine: 300 ng/g 1st case

Doxylamine 1 case Doxylamine: 0.24 mg/L Doxylamine: 267 ng/g Gabapentin 1 case Gabapentin: not detected Gabapentin: present Ibuprofen 1 case Ibuprofen: present Ibuprofen: present Lidocaine 1 case Lidocaine: present Lidocaine: present

Lamotrigine 1 case Lamotrigine: 0.41 mg/L Lamotrigine: not detected Mirtazapine 1 case Mirtazapine: 26 ng/mL Mirtazapine: not detected

Nicotine & Cotinine 7 cases Nicotine/Cotinine: present in 7 cases Cotinine: Present in 6 of 7 cases, Nicotine: not detected

Promethazine 1 case Promethazine: 81 ng/mL Promethazine: present Quetiapine 2 cases Quetiapine: 220 ng/mL Quetiapine: not detected

Venlafaxine & mtb. 1 case Venlafaxine: 0.69 ng/mL & Norvenlafaxine: present Venlafaxine: 768 ng/g & Norvenlafaxine: present

Figure 4: Processed Bone Samples

Figure 1: Bone Drying Process

Figure 2: Mechanical Grinder

Figure 3: Ground bone