FORENSIC TOXICOLOGY - Bio Medical Forensics

temperature, thirty-seven degrees, for twenty minutes prior to purging.
The ability to detect explosives metabolites in biological matrices is time
limited because the body metabolizes substances at various rates.
Having the capability to preconcentrate using PT GC/MS gives a wider
range of time and concentrations to analyze trace metabolites in real
biological samples. The advantage of detecting low concentrations with
IMS will assist in rapid screening of explosives metabolites in
headspace. In the future, this research may aid in the development in a
method which would detect explosives metabolites in breath.
Methods for detecting explosives metabolites in headspace of
biological matrices can be useful to the investigation of bombers and
bomb-makers. This study primarily focuses on TNT and its metabolites,
2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and
dinitrotoluene. Other explosives will also be discussed in this
presentation. This information will present headspace data obtained in
urine and blood by PT GC/MS and IMS.
IMS, PT GC/MS, Explosives Metabolites
K19 Extraction and Analysis of Warfarin
From Whole Blood Using a Long
Chain SPE Sorbent
Albert A. Elian, MS, Massachusetts State Police Crime Laboratory,
59 Horsepond Road, Sudbury, MA 01776; and Jeffery Hackett, MSc*,
Center for Forensic Sciences, 100 Elizabeth Blackwell Street,
Syracuse, NY 13210
The goal of this presentation is to present information on a solid
phase extraction method that will improve on existing procedures for the
analysis of warfarin in postmortem blood samples.
This presentation will impact the forensic community and/or
humanity by improving the analysis of this drug in post mortem samples
by utilizing a more efficient extraction system i.e., a long chain SPE
sorbent in conjunction with both liquid and gas chromatographic systems.
Warfarin (Coumadin) is a popular pharmaceutical used as a bloodthinning
agent. In therapeutic use, blood levels range from 1000 ng to
3100 ng per mL has been reported. 1 Several methods have been used for
the analysis of this drug using liquid-liquid extraction. 2,3 This project
was developed in order to study this drug at low levels in post mortem
samples using a novel (C30 ) solid phase sorbent.
In this method, Warfarin and the internal standard (pchlorowarfarin
(100 ng)) were spiked into whole blood samples (1mL)
over a concentration range 0 through to 200 ng per mL. The samples
were treated with an aqueous phosphate buffer (9 mL) and the drugs
extracted onto a C30 SPE columns (200 mg). The columns were washed
with the phosphate buffer and hexane (1x 3 mL each) and eluted with
14% methanol acid in ethyl acetate (2x 3mL). The eluents were
collected and evaporated for further chromatographic analysis. Using
GC-MS, the samples were derivatized prior to analysis using BSTFA,
for analysis with LC-PDA the samples were reconstituted in DI water.
GC-MS separation was carried out using an Agilent Technologies
6890 GC coupled to a 5975 MSD for SIM analysis. HPLC analysis was
carried isocratically out using both PDA and Fluorescence detection.
From this method LOQ’s of 25 ng per mL of sample is easily
achievable by either chromatographic system. By using GC-MS (SIM)
in EI mode, 10 ng per mL of sample can be detected.
Examples of chromatograms and calibration curves are presented to
show the simplicity and efficiency of this methodology.
References:
1 C.Winek et al, Forensic Sci. Int’l 122 (2001) 107-123
2 Locatelli et al, J.Chrom.B., 818 (2005) 191-8
3 Naidong et al., J.Pharm.Biomed. Anal. 25 (2001) 219-29
Warfarin, SPE, Toxicology
K20 Stability of Exogenous GHB in
Antemortem Blood and Urine
Under Various Temperature and
Storage Conditions
Albert A. Elian, MS*, Massachusetts State Police Crime Laboratory,
59 Horse Pond Road, Sudbury, MA 01776
After attending this presentation, attendees will learn how storage
conditions will effect GHB concentration in blood and urine.
This presentation will impact the forensic community and/or
humanity by demonstrating the effect of long storage on GHB levels in
blood and urine.
The stability of exogenous GHB in three blood and three urine
samples under a variety of storage conditions; room temperature, 4°C and
-20°C, was evaluated over a period of six months. GHB concentration
increased the most at room temperature, with almost no change at the lower
temperature.
Gamma-hydroxybutyric acid (GHB) is an endogenous substance
found in the body. This central nervous system depressant, which was first
synthesized in the 1960s, has been used for induction of anesthesia,
treatment of narcolepsy, and for alcohol and opiates withdrawal. Recently,
GHB has been used illicitly by bodybuilders to increase the release of
growth hormone, ravers attendees for its euphoric, sedation and muscle
relaxation after ecstasy use, and victims of drug-facilitated sexual assault
(DFSA) [8-10].
Due to the increased demands on forensic toxicologists to analyze
GHB in cases such as DFSA and operating motor vehicles under the
influence, there are often variable time intervals between collection of the
specimen and analysis. A literature review has revealed no stability study
on antemortem blood or urine exogenous GHB levels. However, one study
reported the effect of storage on endogenous GHB antemortem urine levels,
and another study investigated the effect of storage conditions on GHB-free
and spiked urine antemortem concentration.
Quantitation of GHB was achieved by liquid-liquid extraction,
followed by concentration of the extracts and derivatization with BSTFA.
Analysis was performed on an Agilent 6890 gas chromatograph interfaced
with an Agilent 5975 mass selective detector. A 12m x 0.25mm (internal
diameter), 0.25mm (film thickness), HP-1MS column (100%
polydimethylsiloxane) was used with helium as the carrier gas at a flow rate
of 2.0 mL/min. An Agilent 7683 automatic sampler was used for injection
into the gas chromatograph. The splitless injection mode was used with the
valve closed for 0.25 min, and 2ml samples being injected. The operating
conditions for the analyses were injection port, 280°C; the detector, 300°C;
initial oven temperature, 60°C for 2 min increased at 30°C/min to 300°C,
holding for 1 min. The mass spectrometer was operated in the SIM mode.
The ions selected for monitoring were chosen from full scan mass spectral
analyses of the analytes that gave minimum interference. The following
ions were monitored: GHB: m/z 233,234,235 and GHB-d6 : m/z
239,240,241.
Three actual blood (20, 50, and 75 mg/L) were submitted to the
laboratory in test tubes containing sodium fluoride. Three actual urine
samples (33, 108, and 220 mg/L) were submitted in plastic jars with no
preservative added. The samples were chosen, from casework, to cover a
wide range of concentrations. The specimens were analyzed at the time of
arrival in the laboratory and then divided into three sets as described above.
For the blood stored at -20°C there was an increase in GHB
concentration of 1-12%, at 4°C 3.4-16%, and 20°C 9.6-28% (Fig. 1-3). For
the urine stored at -20°C there was an increase in GHB concentration of 1-
15%, at 4°C 1-27%, and at 20°C 3.6-44% (Fig. 4-6), with the highest
increase in GHB concentration in the lower concentrations (Fig. 1 and 4).
This could be attributed to the fact that a small increase in the GHB level
would be enough to significantly change to the measured level.
Storage, Gamma-Hydroxybutyric Acid (GHB), Exogenous
133 * Presenting Author