FORENSIC TOXICOLOGY - Bio Medical Forensics

Fast gas chromatography (GC) has the potential to be a very useful
tool in toxicological analysis by shortening retention times and
increasing the overall rate of analysis. The most common techniques for
achieving fast GC analysis include shortening of the GC column, raising
the GC oven temperature, and increasing the GC oven ramping parameters.
With these simple techniques in place, drugs can be analyzed
more efficiently.
In the present study, a fast GC method was developed for the
analysis of amobarbital, butalbital, pentobarbital, phenobarbital and secobarbital.
The barbiturates were isolated from 1.0 mL of whole blood
using CleanScreen ® solid-phase extraction (SPE) cartridges
(ZSDAU020) manufactured by United Chemical Technologies, Inc.
Following elution from the SPE cartridge, the extracts were dried under
a gentle stream of nitrogen at 40°C and reconstituted in a dilute
methanolic solution (0.02 M) of trimethylanilinium hydroxide.
The extracts were analyzed using a Hewlett-Packard 6890 Series
gas chromatograph equipped with a nitrogen-phosphorus detector. The
inlet and detector temperatures were set at 250°C and 330°C, respectively.
Helium was used as the carrier gas at a flow rate of 0.1 mL/min.
Automated injections of 0.5 mL, at a split ratio of 31:1, were made onto
an Agilent Technologies DB-5 (10 m x 100 mm x 0.1 mm) GC column.
The initial oven temperature of 120°C was held for 0.25 min., then
ramped 30°C/min. to a final temperature of 320°C for 0.75 min. The
total run time was 7.67 min. These oven parameters were achieved by
reducing the internal GC oven volume with the aid of an oven insert, as
well increasing the GC power supply voltage to 220 V, from the standard
120 V.
A five-point calibration curve was prepared in drug-free whole
blood in a range of 2.5 mg/L to 25 mg/L. Quantification was performed
with barbital as the internal standard fortified at a concentration of 10
mg/L. In order to assess the intra- and inter-run accuracy and precision
of the assay, control samples were prepared at 7.5 mg/L and 12.5 mg/L
and assayed five-times each in three separate experiments. Finally, a
correlative study utilizing specimens previously assayed by a conventional
GC method was conducted.
Under the fast GC conditions described, all barbiturates eluted from
the GC column within 5 min.; the total GC cycle time was 9-10 min.
This increase in throughput had no effect on chromatographic performance
and analyte resolution. The results of the validation studies
demonstrated excellent accuracy and precision with %CV values in the
range of 15% or less and % accuracy values in the range of 90% or
greater. Further, correlation was good between the conventional GC
and fast GC methods.
While the fast GC method has distinct advantages, mainly
improved efficiency, some limitations do exist. First, poor resolution
between some analytes was evident. For example, under the conditions
described, butalbital and butabarbital, and hexobarbital and caffeine,
_co-eluted. Similarly, high concentrations of caffeine interfered with the
quantitation of phenobarbital. Another potential limitation of the fast
GC method is the decreased capacity of a narrow bore GC column which
may lead to column overload and reduced range of analyte linearity.
In conclusion, fast GC has great potential to become an efficient
method for routine toxicological procedures in forensic toxicology laboratories.
Because this method reduces the GC cycle time by nearly twofold,
it significantly increases laboratory throughput.
Fast Gas Chromatography, Barbiturates, Solid-Phase Extraction
* Presenting Author
K20 A Tale of Two Drugs in Southwestern
Virginia: Oxycodone and Methadone
George S. Behonick, PhD, Virginia Division of Forensic Science,
Western Laboratory; William Massello III, MD, Office of The Chief
Medical Examiner, Western District, and James J. Kuhlman, Jr., PhD,
Virginia Division of Forensic Science, Western Laboratory, 6600
Northside High School Road, Roanoke, VA; and Joseph J.
Saady, PhD*, Virginia Division of Forensic Science, Central
Laboratory, 700 North Fifth Street, Richmond, VA
The objective of this presentation is to provide forensic toxicologists
and pathologists with statistical drug-related case data for a five
year period from 1997 to 2001 for the drugs oxycodone and methadone.
To understand patterns of oxycodone and methadone misuse and abuse
and become familiar with factors responsible for absolute and relative
changes in case statistical profiles over time.
The problem of prescription drug misuse and abuse contributes to
significant morbidity and mortality in Southwestern Virginia. Opiate
and opioid drugs are in great demand by misusers and abusers.
Beginning in 1999 the Toxicology Section of the Virginia Division of
Forensic Science (DFS) Western Laboratory together with the Office of
the Chief Medical Examiner (OCME) for the Western Region noted a
dramatic rise in drug-related fatalities involving oxycodone. The trend
continued through 2000 and 2001. Investigative information, drug paraphernalia
recovered from death scenes, decedent history and witness
statements chronicled in a substantial number of cases implicated
involvement of the sustained release formulation of oxycodone
(OxyContin ® ). OxyContin ® , a single-drug entity designed for sustained
release over a 12 hour period, is easily compromised by abusers
to achieve a powerful morphine-like high. The drug is diverted and supplied
to abusers by a number of means: Illegal prescriptions by
unscrupulous physicians (“pill mills”), illicit black market sales,
pharmacy thefts, fraudulent prescriptions, “doctor shopping,” and
diversion from sources in Mexico and Canada.
Methadone, a drug traditionally used as a heroin substitute for
treating addiction, exhibited a similar increase in frequency in Western
District postmortem cases over the same time period. Methadone is also
prescribed in the treatment of chronic pain syndromes. The data suggests
a hypothesis of a classic “supply and demand” scenario. Intense
interdictive efforts by law enforcement, attention by legislative officials
and widespread media attention curbed “supply” of oxycodone, but not
“demand.” Additionally, physicians cognizant of the controversy substitute
methadone for the treatment of chronic pain syndromes formerly
managed with oxycodone, more specifically, OxyContin ® .
The data presented includes: the total number of Western Region
drug-related deaths, cases in which oxycodone and methadone were
determined to be significant in terms of cause of death and statistics in
which oxycodone and methadone were determined to be present in the
blood and postmortem tissues of decedents. Retrospective review of
information in the DFS database and information derived from the
database of the OCME for the Commonwealth of Virginia constituted
the methodology of the study. OCME, Western Region certified 519
drug deaths from 1997 to 2001. Thirty-four percent (n=175) of the certifications
identified oxycodone (n=82) or methadone (n=93) as being
significant to the cause of death. The three-year period 1999 to 2001
illustrated increases in the total number of drug deaths and deaths
attributed to oxycodone and methadone. Deaths attributable to oxycodone
or methadone represented the following proportions for the
period 1999-2001: Thirty-one per cent, thirty per cent and fifty-eight per
cent, respectively. OCME, Western Region conducted 676 autopsies in
the most recent calendar year (2001), certifying 23 per cent as drugrelated
(n=155). Fifty-eight per cent (n=90) identified oxycodone or
methadone as primary agents in establishing cause of death. All cases
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