FORENSIC TOXICOLOGY - Bio Medical Forensics

important of quickly identifying the metabolites as markers of use.
Urine was collected from participants who smoked incense containing
JWH-018 and JWH-073. These specimens were used to identify urine
metabolites of these two compounds based on literature reports and LC-
TOF analysis. Based on the literature and in-house analysis, JWH-018
and JWH-073 undergo mono-, di- and tri-hydroxylation followed by
glucuronidation. Qualitative screen and confirmation methods for
identifying exposure to JWH-018 and JWH-073 were developed and
validated based on the presence of these urinary metabolites.
Specimens were screened for the monohydroxy glucuronide
metabolites. Solid phase extraction was used to clean and concentrate
unhydrolyzed urine specimens and extracts were analyzed on an
LC/MS/MS for the detection of monohydroxy-glucuronide metabolites.
The instrument was operated in positive ionization mode employing
atmospheric pressure chemical ionization. Separation was achieved
using gradient elution on a C18 HPLC analytical column. Source
fragmentation of JWH-073-monohydroxy-glucuronide and JWH-018monohydroxy-glucuronide
was employed and the transitions resulting
from the loss of the glucuronide moiety were monitored. Further
fragmentation was then induced in the collision cell and two transitions
monitored for identification purposes. The confirmation method
employed is based on the presence of multiple urinary metabolites.
Urine specimens underwent enzymatic hydrolysis and a liquid-liquid
extraction prior to analysis. LC-MS/MS with electrospray ionization
was performed on an Applied Biosystems API5000 system. Multiple
transitions were monitored for each analyte. The following table
summarizes the monitored transitions for the screening and confirmation
methods:
Synthetic Cannabinoids, JWH-018, JWH-073
K30 Toxicological Analysis of Synthetic
Cannabinomimetic Spice Drugs
Francisco I. Ortiz, BS*, 1013 21st Street, #15, Huntsville, TX 77340;
Jeffrey P. Walterscheid, PhD, Harris County Institute of Forensic
Science, 1885 Old Spanish Trail, Houston, TX 77054; Michael Chen,
PhD, 1885 Old Spanish Trail, Houston, TX 77054; and Ashraf
Mozayani, PhD, PharmD, Harris County Institute of Forensic Science,
1885 Old Spanish Trail, Houston, TX 77054
After attending this presentation, attendees will be informed of an
LC/MS/MS method for determining the concentration of “spice” drugs
in forensic blood and urine specimens.
This presentation will benefit the forensic science community by
providing a method for qualitatively and quantitatively detecting seven
emerging indole cannabinoid drugs of abuse using liquid
chromatography-tandem mass spectrometry (LC/MS/MS). The lack of
methods available to analyze these drugs makes detection difficult in
suspected cases of “spice.’’ Therefore, as the prevalence of use
increases, the need for validated detection methods becomes important.
Synthetic cannabinomimetic drugs have been studied primarily for
their activity as CB 1 and CB 2 cannabinoid receptor agonists.
Additionally, their strong binding affinity for CB 1 receptors has made
* Presenting Author
these synthetic drugs potent marijuana alternatives which have become
increasingly popular in recent years. Many of these drugs are sold as
herbal incense under the name “spice” or more commonly “K2” in the
United States. JWH-018 is the most commonly found drug in these
herbal blends. The drugs analyzed in this study include JWH-015, JWH-
018, JWH-019, JWH-073, JWH-200, JWH-250, and WIN55212-2.
“Spice” toxicity can present itself in conflicting psychological
states such as nausea, excitability, sedation, and panic. Physiological
changes can include sweating, tachycardia, dyspnea, and xerostomia.
Numerous hospitalizations as well as a suicide have been following
reported acute doses of “spice.” Furthermore, long term effects include
panic attacks, blurred vision, muscle spasms, and a case of diagnosed
dependence syndrome.
Liquid-liquid extractions were used to extract the drugs from blood
and urine. Drug standards were spiked into negative blood and urine
specimens. Various extraction conditions were compared in order to
optimize extraction efficiencies of the drugs in both blood and urine. In
the end, pH10 sodium borate buffer and ethyl acetate provided the best
extraction efficiency. LC/MS/MS was used to analyze the extracted
drugs and develop a method to qualitatively and quantitatively identify
the drugs. Prior to LC/MS/MS analysis, drug optimization on the
instrument was performed in order to select the appropriate qualifier and
quantifier ions for each drug as well as the fragmentor and collision
voltages. To ensure optimal chromatography, diazepam-D5 was chosen
as the internal standard after comparison with hydrocodone-D3 and
fentanyl-D5. Methanol with 0.1% formic acid was chosen as the mobile
phase as it allowed for adequate separation of the compounds of interest.
The method was validated using the laboratory’s validation
guidelines. A five-point calibration curve was developed from 1-250
ng/mL. Linear ranges were from 1-100 ng/mL for all drugs except JWH-
200 and WIN55212-2 which maintained linearity from 1-250 ng/mL
with R 2 greater than 0.995 for all drugs. To validate the method, two
extractions were performed on separate days. Accuracy and precision
were calculated at 10 ng/mL and 100 ng/mL using three replicates for
each concentration. LOQ for all drugs was 1ng/mL.
This presentation provides a rapid, sensitive method for
determining the presence and concentration of several indole-based
cannabinomimetic drugs in blood. The combination of chromatographic
separation and ion monitoring with LC/MS/MS allows for multiple
drugs to be accurately detected. This method can prove useful with the
increasing rate of synthetic cannabinomimetic drug use in
the population.
Spice, Cannabinoid, LC/MS/MS
K31 Analysis and Stability Determination of
Salvinorin A and B in Human Blood,
Plasma, and Urine by Liquid
Chromatography Tandem Mass Spectrometry
Barry K. Logan, PhD*, Allan Xu, PhD, and Matthew M. McMullin, MS,
NMS Labs, 3701 Welsh Road, Willow Grove, PA 19090
After attending this presentation, attendees will be able to describe
the origins of and effects associated with abuse of Salvia divinorum,
optimum methods for its analysis by liquid chromatography/tandem
mass spectrometry, and limitations on its analysis based on
analyte stability.
This presentation will impact the forensic science community by
identifying a novel analytical approach to detection of an emerging
hallucinogenic drug of abuse.
Salvinorin-A is an hallucinogenic compound that has no approved
medical use in the United States. It is a naturally occurring, nonnitrogenous
kappa opioid receptor agonist, and is the active component
of the plant, Salvia divinorum, belonging to the mint family. The leaves
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