A Preliminary Investigation into Retrospective Calculation of In

Report
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.
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.
Analytical Obstacles
• What was the original blood volume?
• Are drugs stable in dried blood?
• Can one adequately recover drugs from dried
blood?
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.
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
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
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%)
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.
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 reweighed to determine if drying conditions
were adequate.
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
Also calculated:
• Blood density
• % Water
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, 4th 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)
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
Limitations of study
•
•
•
•
•
•
Sample size
Age
Gender
Race
Disease states (diabetes, etc.)
EDTA blood may not be representative of
crime scene blood
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
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
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.
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.
Recovery
(Compared to original spiked blood)
Amphetamine
95 %
Methamphetamine
93 %
MDA
94 %
MDMA
85 %
Codeine
96 %
Morphine
99 %
Hydrocodone
95 %
Hydromorphone
94 %
Cocaine
84 % *
Ecgonine methyl ester
103 % *
Benzoylecgonine
98%
*These values may reflect some breakdown of cocaine to EME
Amphetamine
% [amphetamine] vs. Original Liquid Specimen
40%
30%
Percent
20%
10%
0%
-10%
-20%
-30%
-40%
0
50
100
Day
150
200
Methamphetamine
% [Methamphetamine ] vs. Original Liquid Specimen
40%
30%
Percent
20%
10%
0%
-10%
-20%
-30%
-40%
0
50
100
Day
150
200
MDA
% [MDA] vs. Original Liquid Specimen
40%
30%
Percent
20%
10%
0%
-10%
-20%
-30%
-40%
0
50
100
Day
150
200
MDMA
% [MDMA] vs. Original Liquid Specimen
40%
30%
Percent
20%
10%
0%
-10%
-20%
-30%
-40%
0
50
100
Day
150
200
Codeine
% [Codeine] vs. Original Liquid Specimen
40%
30%
20%
Percent
10%
0%
-10%
-20%
-30%
-40%
0
50
100
Day
150
200
Morphine
% [Morphine] vs. Original Liquid Specimen
40%
30%
20%
Percent
10%
0%
-10%
-20%
-30%
-40%
0
50
100
Day
150
200
Hydrocodone
% [Hydrocodone] vs. Original Liquid Specimen
40%
30%
Percent
20%
10%
0%
-10%
-20%
-30%
-40%
0
50
100
Day
150
200
Hydromorphone
% [Hydromorphone] vs. Original Liquid Specimen
40%
30%
Percent
20%
10%
0%
-10%
-20%
-30%
-40%
0
50
100
Day
150
200
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.
Cocaine
% [Cocaine] vs. Spiked Standard
80%
60%
40%
Percent
20%
0%
-20%
-40%
-60%
-80%
0
50
100
150
Day
200
250
Benzoylecgonine
% [Benzoylecgonine] vs. Spiked Standard
40%
30%
Percent
20%
10%
0%
-10%
-20%
-30%
-40%
0
50
100
150
Day
200
250
Ecgonine Methyl Ester
% [Ecgonine Methyl Ester] vs Spiked Standard
Percent
20%
10%
0%
-10%
-20%
-30%
-40%
-50%
-60%
-70%
-80%
0
50
100
150
Day
200
250
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
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
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
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.
Cocaine Stability – Liquid vs. Dried Blood
20%
0%
-20%
-40%
% Diff Dried blood
% Diff liquid Blood
-60%
-80%
-100%
0
50
100
Day
150
200
250
BE Stability – Liquid vs. Dried Blood
10%
0%
-10%
-20%
-30%
-40%
% Diff Dried Blood
-50%
% Diff Liquid Blood
-60%
-70%
-80%
0
50
100
150
Day
200
250
EME – Liquid vs. Dried Blood
60%
40%
20%
0%
-20%
% Diff Dried blood
-40%
% Diff liquid Blood
-60%
-80%
-100%
0
50
100
150
Day
200
250
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.
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
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?
• ??????
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.
Questions

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