FORENSIC TOXICOLOGY - Bio Medical Forensics

same model. No significant variations in sensitivity or other aspects of
data quality were observed. The ability to transfer methods without
individual optimization of each instrument can save substantial time in
method set-up and implementation.
LC/MS/MS, Method Development, Toxicology
K8 Determination and Quantitation of
Noroxycodone in Human Urine Samples
Using High Performance Liquid
Chromatography - Electrospray Ionization-
Tandem Mass Spectrometry
Christopher Doctorman, BS*, University of Central Oklahoma, 100
North University, Edmond, OK 73034; and Chelsy L. Wingate, BS, 1517
Hawk Tree Drive, College Station, TX 77845
After attending this presentation, attendees will have a greater
understanding of opiate chemistry, metabolism, kinetics, and
pharmacology, as well as be familiar with and implement current
LC/MS/MS technology. Attendees will also gain information about an
analytical method for determination of noroxycodone, a metabolite of
oxycodone, and will understand the metabolic pattern for oxycodone.
This presentation will impact the forensic community by giving
greater insight into human metabolism of oxycodone. This information
can be utilized to perfect or improve current methods for detecting and
quantifying oxycodone and its metabolites in clinical and forensic
toxicological settings.
Oxycodone (4,5-epoxy-14-hydroxy-3-methoxy-17-methylmorphinan-6-one),
is an analgesic, semi synthetic opioid derived from thebaine.
Also known by its manufactured names OxyContinä, OxyNormä,
Roxicodone TM , and others, it comes in a variety of shapes and dosages.
Oxycodone is commonly prescribed for significant pain management
typically associated with cancer, and has been used clinically for this
purpose in the United States for the past eighty years. It has been a “drug
of abuse” for nearly 50 years.
Oxycodone is metabolized in the body by two isoenzymes
Cytochrome P450 (CYP) 3A4 and CYP2D6. CYP3A4-mediated
metabolism of the compound yields N-demethylated metabolites
noroxycodone, noroxymorphone, and a and b noroxycodol. CYP2D6mediated
metabolism produces O-demethylation of oxycodone to
oxymorphone and a and b noroxymorphol, and 6-keto-reduction to a and
b oxycodol.
Human urine samples, collected as part of another study to
determine the elimination rate of oxycodone, were used as test samples
for the detection and quantitation of noroxycodone. A method developed
for the simultaneous quantitation of several opiates, including codeine,
hydrocodone, hydromorphone, oxycodone, oxymorphone, and
morphine, was modified to also incorporate noroxycodone as one of the
compounds using selected ion monitoring (SIM). This method was
utilized on a 4-channel multiplexing HPLC system interfaced with triple
quadrupole mass spectrometer. Limit of quantitation, as well as between
day accuracy and precision (%deviation and %CV) of noroxycodone
was established at 100 ng/mL (3.9% and 24.9%).
Urine samples were collected over a period of a week from seven
individuals given one of three different concentrations of oxycodone,
along with a naltrexone blockade (50 mg per day). Concentrations of
noroxycodone, oxycodone, and oxymorphone resulting from the analysis
of an individual dosed with 80 mg tablets of oxycodone have shown
noroxycodone to be the primary metabolite (70.8%±4.7) followed by
oxycodone (18.5%±5.2) and oxymorphone (10.8%±2.1). Results for
samples from other individuals will be tabulated and presented. These
concentration results indicate that CYP3A4 mediation is the
predominant metabolic pathway of oxycodone in humans.
Noroxycodone, High Performance Liquid Chromatography,
Tandem Mass Spectrometry
K9 Development and Validation of a LC/MS
Method for the Determination of Guanfacine
in Urine
Sara J. Kester-Florin, BS*, 944 Wye Drive, Akron, OH 44303; and Carl
E. Wolf, PhD, and Alphonse Poklis, PhD, Medical College of Virginia,
Box 98-165, VCU/MCVH Station, Richmond, VA 23298-0165
After attending this presentation, attendees will become familiar
with a validated liquid chromatography/mass spectrometry (LC/MS)
method for detecting and quantifying guanfacine in urine specimens.
Guanfacine is a drug that was initially approved for the treatment of
hypertension in adults, but has been recently approved (2007) for the
treatment of attention deficit/hyperactivity disorder (ADHD) in
adolescents. Due to the new therapeutic use, an increase in both
availability and consumption of this drug required the development of an
analytical method to detect the use or abuse of guanfacine. A validation
of this LC/MS method will impact the forensic community by providing
the field of toxicology with a rapid, robust analytical method that
requires a small sample volume, and is also sensitive enough to detect
drug use at a therapeutic dose.
The validation of a LC/MS method for the detection and
quantification of guanfacine in urine is presented. Guanfacine was
extracted from alkaline buffered urine using a liquid-liquid extraction
scheme with ethyl acetate. Two hundred microliters of samples,
controls, and calibrators were prepared with the addition of 10µL of
protriptyline internal standard (2mg/L). Samples were buffered to a pH
of 9.5 with 200µL saturated carbonate:bicarbonate solution. Five
hundred microliters of ethyl acetate was added to the samples, followed
by two minutes of rotation and five minutes of centrifugation at
3000rpm. The organic layer was transferred to a clean test tube,
evaporated to dryness under a gentle stream of nitrogen, and
reconstituted in 200µL of mobile phase. Guanfacine and protriptyline
were separated and quantified on a reverse phase S-5 micron, 2.0 x
150mm column in a high performance liquid chromatography (HPLC)
separations module coupled to a mass spectrometer (MS) with
electrospray ionization operated in the positive ionization mode. The
mobile phase consisted of 40% 10mM ammonium formate in methanol,
and was delivered isocratically at a flow of 0.3 mL/min. Sample
injection volume was 10µL. The MS was operated in selected ion
resonance mode (SIR) using the following m/z ions: 246, 248, and 250
for guanfacine, and 264 and 265 for protriptyline. Under these
conditions the retention time for guanfacine and protriptyline were 2.1
min and 3.6 min, respectively.
The analytical measurement range for guanfacine ranged from
5ng/mL to 2000ng/mL with a 5ng/mL limit of detection (LOD) and a
20ng/mL limit of quantitation (LOQ). The method was shown to be both
precise and accurate. Precision for the assay was determined at
concentrations of 40ng/mL, 100ng/mL, and 500ng/mL, (n=6), the %CV
was