OSBI Toxicology Lab Current Technologies for Blood Testing Robert G. Weston Forensic Toxicologist

We are… Seven full-time toxicologists One part-time technician Combined 90 years of tox experience

What law enforcement officers do…

Recent DUI caseload DUI cases submitted: 2011 – 3987 cases

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.

Everyone knows how drugs get in…

…but how do we get them out???

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

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)

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

Enzyme-linked immunosorbent assay ELISA Enzyme-linked immunosorbent assay

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

At OSBI, all confirmatory testing in toxicology involves… CHROMATOGRAPHY

The separation process Injector Detector MP SP Column

The separation process Injector Detector MP SP Column

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Alkaline drug analysis 2 milliliters of blood +internal standards +borate buffer +n-butyl chloride +mixing +centrifuging DRUGS MOVE

Alkaline drug analysis Transfer top layer to new tube Add hydrochloric acid Mix Centrifuge DRUGS MOVE

Alkaline drug analysis Dispose of top layer Add sodium hydroxide Mix Add chloroform Centrifuge Transfer chloroform to vial

Gas chromatography mass spectrometry

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

New technology for OSBI

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

Blank blood

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!!

A case sample – AM-2201 found on driver and in blood Small amounts of other “spice” compounds.

What else can we do? 12 benzodiazepines Under 4 minutes Limit of detection: 5 ng/mL

An real case example.

ELISA positive for benzos, but none found Temazepam would be found here.

Now we can see… temazepam oxazepam

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.

High concentrations of other drugs Blank Opiates Mix

High concentrations of other drugs Blank THCs Mix

High concentrations of other drugs Blank Spice Mix

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

Upcoming projects Continue to expand capacities Quantitations Opiates More synthetic cannabinoids “Bath salts” Cocaine Quantitations