Quantitation of Drugs in Dried Bloodstains. Thomas Meyer Anna Gomenyuk Nadiah Lester

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1 Quantitation of Drugs in Dried Bloodstains Thomas Meyer Anna Gomenyuk Nadiah Lester

2 Objective To find the quantitative value of drugs in dried bloodstains

3 Currently Cloth With Dried Bloodstain Containing a Drug or Drugs GC/MS Amount of drug But the volume of blood on cloth is Unknown

4 With our research Cloth With Dried Bloodstain Containing a Drug or Drugs We want to split the experiment into two parts

5 The goal of our research Cloth With Dried Bloodstain Containing a Drug or Drugs GC/MS ID the drug Unit weight of drug ICP-AES Determine amount of iron Unit volume of blood on cloth Unit weight of drug Unit volume of blood on cloth

6 Part I: Drug Analysis

7 PCP 1-(1-phenylcyclohexyl)piperidine Phencyclidine Central Nervous System Stimulant 1950 s - used as an anesthetic 1965 became illegal

8 Materials for Drug Analysis Pipette Syringe Sonicator Rotator Centrifuge Aspirator TurboVap Gas Chromatograph Mass Spectrometer Auto Sampler Vials Reagents PCP PCP-D5 (Internal Standard) DI H 2 O Methylene Chloride Ammonium Hydroxide Methanol

9 Bloodstain Preparation 1. Spike tubes of whole blood with PCP 2. Apply blood to fabric Leave a piece of fabric free of blood and use it as a blank 3. Allow stains to dry

10 Experimental Conditions PCP Concentration Variations 50 ng/ml 90 ng/ml 150 ng/ml Fabric Variations Denim Cotton Volume of Bloodstain Variations 0.5 ml 1 ml

11 Analysis Preparation 1. Cut out bloodstain and weigh 2. Cut stain in half and weigh each half 3. Set one half aside for an iron analysis 4. Cut other half into small strips and place into test tube

12 PCP Procedure 1. Add 7 ml of methanol to bloodstained strips 2. Sonicate for 1.5 hours 3. Let stand for two days 4. Liquid/Liquid Extraction 5. GC/MS analysis

13 Liquid/Liquid Extraction Ammonium Hydroxide Methylene Chloride PCP + + OH - = PCP PCPOH Methylene Water Chloride

14 Dry and Reconstitute im doing the speech at nms = PCP Methylene Concentrated Chloride PCP in methanol

15 Gas Chromatograph/Mass Spectrometer

16 Gas Chromatograph

17 Gas Chromatograph Injector Port Mass Spectrometer Helium Gas (Mobile Phase) - PCP - PCPD5 Column (Stationary Phase) -Stationary Phase Retention Times

18 Mass Spectrometry GC Impurities Analyte ELECTRON IMPACT IONIZER + + QUADRUPOLE ELECTRON MULTIPLIER TUBE MASS SPECTRUM DETECTOR

19 PCP Calibration Curve What a cc is Calibrators: 5, 10, 40, 60, 80, 100, 200 ng/ml Controls: 30, 150 ng/ml

20 Determination of PCP in Entire Stain Half Stain 10 ng PCP 0.1 g cloth Whole Stain x ng PCP 0.2 g cloth Total PCP in stain 20 ng *Values are hypothetical and are for demonstration purposes only

21 Results Initial Stained Volume (ml) Spiked Sample Concentration (ng/ml) Denim Expected Total PCP (ng) Experimental Total PCP (ng) N/A N/A Samples ran in duplicate

22 Cotton Results Initial Stained Volume (ml) Spiked Sample Concentration (ng/ml) Expected Total PCP (ng) Experimental Total PCP (ng) N/A

23 Part II: Iron Analysis

24 Iron 70% found in Hemoglobin, a protein in red blood cells used for oxygen transport 20% free 10% bound to other proteins

25 Materials for Iron Analysis Micro vessel vials Concentrated HNO 3 DI H 2 O ETHOS-TC Microwave unit ICP-AES

26 Iron Procedure 1. Acid Digestion Specimen preparation Wash Add digestion reagents Perform digestion using ETHOS-TC microwave Cool Transfer the sample for next step 2. Analyze using ICP-AES

27 Inductively Coupled Plasma - Atomic Emission Spectroscopy (ICP-AES)

28 Radio frequency (AC) Generation of ICP Argon Magnetic Field Electrical Spark Resistance High Temp. Inductively Coupled Plasma

29 Atomic Emission Spectroscopy Excited Digested Bloodstained Sample ICP 0 0 Light is emitted Prism Iron Emission Spectrum

30 Determination of the Volume of Blood in a Dried Bloodstain Volume = g of bloodstained 0.503cloth µg of Fe g of bloodstained cloth 440 µg of Fe ml of whole blood Average male has µg/ml of total iron in whole blood

31 Determination of the Volume of Blood in a Dried Bloodstain Volume = g of bloodstained 0.354cloth µg of Fe g of bloodstained cloth 625 µg of Fe ml of whole blood ml of whole blood Average male has µg/ml of total iron in whole blood

32 Denim Volume Results Initial Stained Volume (ml) Experimental Volume Range (ml) Average Average

33 Cotton Volume Results Initial Stained Volume (ml) Experimental Volume Range (ml) Average Initial Stained Volume (ml) Experimental Volume Range (ml) Average

34 OVERALL RESULTS

35 Determination of Concentration Bloodstained cloth contained From GC/MS drug analysis Weight of PCP From Iron analysis Volume range of whole blood on stain Weight of PCP Highest volume of blood in stain Weight of PCP Lowest volume of blood in stain Lowest Concentration of PCP (ng/ml) Highest Concentration of PCP (ng/ml)

36 Overall Results Denim Cotton Initial Stained Volume (ml) Spiked Sample Conc. (ng/ml) Experimental Conc. Range (ng/ml) Initial Stained Volume (ml) Spiked Experimental Sample Conc. Conc. Range (ng/ml) (ng/ml) N/A 50 N/A N/A

37 CONCLUSION

38 Conclusion

39 FUTURE GOALS

40 Future Research Possibilities Stain more cloth Using smaller/larger volumes of blood Using different drugs Using different fabrics Using female whole blood Perform solid phase extraction to possibly clear samples of interfering substances Attempt experimentation using the whole bloodstain instead of cutting it in two

41 References 1. Dunn, W.A., Labay, L.M., Lipman, J.J., A Method for the Estimation of Drug Concentrations in Dried Bloodstains, NMS (2007) 2. Geigy Scientific Tables. 8th ed. Vol. 3. West Caldwell, New Jersey: Medical Education Division, CIBA-GEIGY, Print 3. Baselt, Randall C. Disposition of Toxic Drugs and Chemicals in Man. 6th ed. Foster City, California: Biomedic Publications, Print 4. Clarke's Isolation and Identification of Drugs. 2nd ed. Vol. 1. The Pharmaceutical Society of Great Britain, Print 5. Cecil, Russell L. Cecil Textbook of Medicine. 2nd ed. Vol. 21. W.B. Saunders Company, Print.

42 QUESTIONS?

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