osbi toxicology lab current technologies for blood testing robert g. weston forensic toxicologist

OSBI Toxicology Lab

Current Technologies

for Blood Testing

Robert G. WestonForensic 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 2010 – 3951 cases 2009 – 3854 cases 2008 – 3768 cases 2007 – 3217 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

ELISAEnzyme-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 testingin toxicology involves…

CHROMATOGRAPHY

The separation process

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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

• Mix

• 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

AM-2201

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…

oxazepam

temazepam

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 Opiates More synthetic cannabinoids “Bath salts” Cocaine

Quantitations