Download - OSBI Toxicology Lab Current Technologies for Blood Testing Robert G. Weston Forensic Toxicologist
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OSBI Toxicology Lab
Current Technologies
for Blood Testing
Robert G. WestonForensic Toxicologist
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We are…
Seven full-time toxicologists One part-time technician Combined 90 years of tox experience
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What law enforcement officers do…
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Recent DUI caseload
DUI cases submitted: 2011 – 3987 cases 2010 – 3951 cases 2009 – 3854 cases 2008 – 3768 cases 2007 – 3217 cases
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Unlike CSI, we… must separate the drugs from the blood through sample
preparation. set up the analysis and wait for the data to be collected. review and interpret the data. do not know everything about every drug seen in the blood. determine the identity of substances we haven’t seen before. do not have a universal test that is effective for all drugs.
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Everyone knows how drugs get in…
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…but how do we get them out???
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Composition of blood
Red blood cells (40-50% of blood volume) White blood cells (1 % of blood volume) Platelets Plasma
• Water (>92%)• Sugars
• Lipids• Vitamins• Minerals
• Hormones• Enzymes• Antibodies• Proteins
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Volatiles analysis
Blood placed into a vial Internal standard is added Vial is heated Only volatile components evaporate into the headspace
Alcohols (ethanol, methanol, isopropyl) Acetone Toluene Difluoroethane (from spray dusters)
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Volatiles analysis A portion of the headspace gas is introduced into a gas chromatograph
Comparison is made between the sample and known standards
Quantification of ethanol
Identification of other volatiles
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ELISAEnzyme-linked immunosorbent assay
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ELISA Presumptive for classes of compounds Eight classes currently tested for are
Barbiturates Benzodiazepines Carisoprodol/meprobamate Cocaine/benzoylecgonine Phencyclidine Phenethylamines (meth) Opiates Tetrahydrocannabinols (marijauna)
Results must be confirmed by a chromatography/mass spectrometry method
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At OSBI, all confirmatory testingin toxicology involves…
CHROMATOGRAPHY
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
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DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
Injector
Column
DetectorMP
SP
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The separation process
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The separation process
Injector
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DetectorMP
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The separation process
Injector
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DetectorMP
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The separation process
Injector
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The separation process
Injector
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DetectorMP
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The separation process
Injector
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DetectorMP
SP
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The separation process
Injector
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DetectorMP
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The separation process
Injector
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DetectorMP
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The separation process
Injector
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DetectorMP
SP
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The separation process
Injector
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DetectorMP
SP
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The separation process
Injector
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DetectorMP
SP
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The separation process
Injector
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DetectorMP
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The separation process
Injector
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DetectorMP
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The separation process
Injector
Column
DetectorMP
SP
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Alkaline drug analysis
2 milliliters of blood+internal standards+borate buffer+n-butyl chloride+mixing+centrifuging DRUGS
MOVE
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Alkaline drug analysis
• Transfer top layer to new tube
• Add hydrochloric acid
• Mix
• Centrifuge
DRUGS MOVE
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Alkaline drug analysis
• Dispose of top layer
• Add sodium hydroxide
• Mix
• Add chloroform
• Mix
• Centrifuge
• Transfer chloroform to vial
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Gas chromatography mass spectrometry
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Why negative results?
Not all drugs extract well Drug may be present in an amount less
than what our methods can detect
Some drugs metabolize very quickly We don’t have methods to test for every
drug
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New technology for OSBI
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LC/MS/MS Advantages
Less sample needed Less sample prep (generally) Lower limits of detection Now capable of detecting previously unseen drugs
• 2.0 milliliters of blood for GC/MS testing
• 0.2 milliliters of blood for LC/MS/MS testing
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Blank blood
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New analysis capability - SPICE
• Seven synthetic cannabinoids
• 0.5 ng/mL
• AM-2201
• JWH-018
• JWH-073
• JWH-081
• JWH-122
• JWH-210
• JWH-250
3.0 minutes!!
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A case sample – AM-2201 found on driver and in blood
AM-2201
Small amounts of other “spice” compounds.
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What else can we do?
• 12 benzodiazepines
• Under 4 minutes
• Limit of detection: 5 ng/mL
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An real case example.
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ELISA positive for benzos, but none found
Temazepam would be found here.
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Now we can see…
oxazepam
temazepam
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Why lower limits of detection?
Fewer steps in sample prep – fewer opportunities to “lose” drugs.
We can program the instrument to “see” only the drugs we are interested in.
Other drugs/chemicals present do not cause a response.
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High concentrations of other drugs
Blank Opiates Mix
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High concentrations of other drugs
Blank THCs Mix
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High concentrations of other drugs
Blank Spice Mix
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LC/MS/MS limitations
Limited to analyzing what we have validated Method validation time consuming Not currently set up for unknown screening Different methods for different compounds –
multiple analyses required for multiple compound types
We will be able to analyze for most drugs with LC/MS/MS – it is just a matter of time
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Upcoming projects
Continue to expand capacities Opiates More synthetic cannabinoids “Bath salts” Cocaine
Quantitations
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