1. A Preliminary Investigation into Retrospective Calculation of In-Vivo Drug Concentrations in Dried Crime Scene Blood. Dwain C. Fuller*, Patricia Pisana, Veterans Affairs North Texas Health Care System, Dallas, TX, U.S.A.

2. Why? Evaluating injured drivers who may have fled the scene of an accident. Evaluating whether a wounded aggressor who has fled the scene was possibly drug-impaired. Because law enforcement asks.

3. Analytical Obstacles What was the original blood volume? Are drugs stable in dried blood? Can one adequately recover drugs from dried blood?

4. Approach Phase 1 – Determine if the original volume of blood can be calculated from the weight of its dried residue. Phase 2 – Determine the stability and recovery of some representative drugs from a dried blood matrix.

5. Phase 1 – Weight/Volume Ratio Selected 50 random blood specimens submitted for CBC analysis. – CBC’s selected to avoid any particular disease state – CBC’s are submitted in 5 mL purple-top tubes containing an average of 7.4 mg EDTA as an anticoagulant, but no preservative – Each tube contained 3.5 - 4 mL blood

6. Phase 1 – Weight/Volume Ratio Demographics – Gender: Males – 48, Females – 2 – Age: Mean – 63 (33-90) – Race: Caucasian – 28 African American – 13 Hispanic – 3 Unknown - 6

7. Phase 1 – Weight/Volume Ratio Specimens were mixed for at least 15 min. Weighing boat weighed and weight recorded. 500 µL of blood was pipetted into weighing boat. Total weight was immediately recorded. Boat was dried for at least 72 hours at room conditions (71 ±1⁰F, Relative humidity: 42 ±8%)

8. Phase 1 – Weight/Volume Ratio Boat with dried blood was re-weighed and weight recorded. Weight of dried blood per milliliter of liquid blood was calculated.

9. Other considerations Weight of dried blood was corrected for weight of EDTA anticoagulant (2 mg/mL). One analyst performed all pipetting to avoid interindividual differences in technique. A few specimens were subjected to a 60⁰C oven for an additional 24 hours and re- weighed to determine if drying conditions were adequate.

10. Results Mean: g dried blood/mL liquid blood = 0.215 ± 0.025 g (0.161 - 0.259) CV = 11.6 % Prediction Uncertainty: approximately ± 23% with 95% confidence

11. Also calculated: Blood density % Water

12. Comparison to other studies Density: 1.052 ± 0.020 g/mL Present Study 1.055 g/mL (Karch, Forensic Issues in Alcohol Testing, CRC Press, 2007) 1.06 g/mL (Cutnell, et al. Physics, 4 th edition, Wiley, 1998) 1.025 – 1.125 g/mL (Benson, Katherine, MCAT Review, Emory University,1999) 1.043 – 1.057 g/mL (Hinghofer-Szalkay, et al., Continous Monitoring of blood volume changes in Humans, Journal of Applied Physiology, Vol 63, 1987)

13. Comparison to other studies Percent water: 79.6 ± 2.4 % g/g Present Study 76.9 – 82.0 % g/g 80.5 % g/g – Females 78.9 % g/g – Males Lijnema, et al., Gravimetric determination of the water concentration in whole blood, plasma and erythrocytes and correlations with hematological and clinicochemical parameters. Clinica Chimica Acta, 214, 1993

14. Limitations of study Sample size Age Gender Race Disease states (diabetes, etc.) EDTA blood may not be representative of crime scene blood

15. Phase 2 – Drug Stability and Recovery Pooled EDTA blood and screened for drugs of interest Spiked blood with amphetamine, methamphetamine, MDA, MDMA, codeine, morphine, hydrocodone, hydromorphone, cocaine, ecgonine methyl ester, and benzoylecgonine Determined original concentration

16. Phase 2 – Drug Stability and Recovery Prepared 1 mL aliquots in weighing boats and dried as previously described Analyzed for target drugs as convenient over a period of weeks and months Compared results of dried specimens converted the concentration to the original liquid blood concentration

17. Brief Analytical Method Dried blood specimen was transferred into a disposable 16 x 100 mm culture tube. Dried blood specimen was ground to a powder using a 10 mm diameter glass stir rod. Powdered dried blood was transferred to a tared tube for analysis and weight was recorded.

18. Brief Analytical Method Reconstitute dried blood with buffer used for extraction either liquid:liquid or SPE. Add appropriate internal standard(s). Rotate on laboratory rotator for 10 minutes. Shake vigorously, (centrifuge if proceeding with SPE extraction.) Proceed with extraction as normal.

19. Recovery (Compared to original spiked blood) Amphetamine95 % Methamphetamine93 % MDA94 % MDMA85 % Codeine96 % Morphine99 % Hydrocodone95 % Hydromorphone94 % Cocaine84 % * Ecgonine methyl ester103 % * Benzoylecgonine98% *These values may reflect some breakdown of cocaine to EME

20. Amphetamine

21. Methamphetamine

22. MDA

23. MDMA

24. Codeine

25. Morphine

26. Hydrocodone

27. Hydromorphone

28. Cocaine studies Because of expected lability of cocaine and its metabolites, their stability was determined by comparison to a freshly spiked liquid blood specimen using the original spiking solution stored at -80⁰C, rather than the original liquid blood specimen itself.

29. Cocaine

30. Benzoylecgonine

31. Ecgonine Methyl Ester

32. Observations on cocaine stability in dried blood Benzoylecgonine is considerably more stable in dried blood at room temperature than is EME. Isenschmid, Levine, Caplan. JAT, 13:250; 1989

33. Observations on cocaine stability in dried blood Benzoylecgonine probably breaks down to ecgonine. Thus ecgonine would probably be an important target analyte to demonstrate cocaine use when analyzing dried blood. Logan. JAT, 25:219; 2001 Skopp, Klingman, et al. Ther. Drug. Monit. 23:174; 2001

34. A special note about cocaine stability in dried blood. Present study – Loss of 44% of cocaine in dried blood in 14 days Alfazil and Anderson, JAT, 32:511; 2008 – Loss of 19.9% of cocaine in dried blood spots on filter paper in one month at room temperature – blood matrix was prepared by diluting packed cells with isotonic saline

35. A special note about cocaine stability in dried blood. (cont.) Baselt, Journal of Chromatography, 268, 1983, 502-505 – Cocaine loss in unpreserved plasma was more rapid than that of unpreserved whole blood – This may reflective of a higher pseudocholinesterase concentration in plasma compared to whole blood. Thus Alfazil and Anderson’s results may be skewed due to their choice of matrix.

36. Cocaine Stability – Liquid vs. Dried Blood

37. BE Stability – Liquid vs. Dried Blood

38. EME – Liquid vs. Dried Blood

39. Observations of Cocaine stability in dried blood vs. liquid blood Both cocaine and BE appear to be more stable in unpreserved dried blood than liquid blood. Cocaine appears to degrade more to BE rather than EME in unpreserved dried blood as compared to liquid blood.

40. Limitations of Study Spiked EDTA blood may not be representative of crime scene blood – Clotting – Protein binding – Issues regarding removal dried blood from differing substrates Actual crime scene blood may be subjected to a wide array of temperatures and weather conditions

41. Future studies Evaluate larger more diverse population Evaluate cocaine and metabolite concentration changes in the short term Evaluate more drugs Evaluate authentic blood specimens – Logistically difficult – Postmortem specimens at autopsy? ??????

42. Conclusions It may be possible to quantitate drugs in unpreserved dried blood obtained from a crime scene, convert the drug concentrations to their previous liquid blood equivalent, and accurately assess the uncertainty of this process.

43. Questions