Poster demonstrating the results from the development/verification project for the quantitation of synthetic cannabinoids in human urine.

1. Measurement of Synthetic Cannabinoids in Human Urine
by Liquid Chromatography-Tandem Mass Spectrometry
D. M. Garby, R. DelRosso, J. A. Edwards, L. A. Brunelle
Mayo Medical Laboratories, Andover, MA
© 2013 Mayo Foundation for Medical Education and Research
This method provides for the simultaneous and reliable analysis
of multiple synthetic cannabinoids in urine.
Conclusions
Figure 2: Linearity
Synthetic cannabinoids (i.e., K2, Spice) were originally
designed as research tools to aid in the investigation of the
endocannabinoid system due to their ability to bind to
cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2)
receptors. Often referred to as herbal incense or potpourri,
synthetic cannabinoids are gaining in popularity among
recreational drug users as an inexpensive and “legal”
alternative to marijuana. Synthetic cannabinoids do have
some effects common to that of marijuana and its primary
psychoactive compound, ∆9-tetrahydrocannabinol (THC),
with a higher degree of intoxication associated with their
consumption. Users of synthetic cannabinoids however may
demonstrate more serious side effects such as hypertension,
hallucinations, tachycardia, sinus bradycardia, chest pain,
dysrhythmias, seizures and even death. The metabolism of
synthetic cannabinoids occurs via cytochrome P450 enzymes
and generally includes either hydroxylation and/or
dehalogenation with excretion in the urine as glucuronide
conjugates.
Background
Precision: Precision was determined using drug-free human
urine fortified with certified standard material at various
concentrations. Intra-assay precision was determined from 20
serial measurements and inter-assay precision was
determined from 30 replicate measurements of each level of
material.
Accuracy: Comparisons were made to an external reference
laboratory in addition to the confirmation of traceability using
certified standards.
Linearity / Dynamic Range: Linearity and dynamic range
were assessed with serial dilutions of drug-free human urine
fortified with standard drug solutions and plotted versus the
expected concentrations. Dynamic range study data was
generated over 10 independent runs.
Specimen Stability: Specimen stability was assessed under
ambient, refrigerate and frozen storage conditions. Freeze-
thaw cycles were also evaluated.
Interference and Ion Suppression Studies: The potential
for method interference from common pharmaceuticals/illicit
drugs as well as hemoglobin and bilirubin was assessed by
spiking known concentrations of these interferences into
fortified drug-free human urine and comparing the data to the
results obtained in the absence of interferences. Ion
suppression was assessed by post-column infusion of drugs
and data was collected at the expected retention time.
Experimental Design
Table 1: Intra-assay Precision
Deuterated stable isotopes were added to 500µL of urine as
internal standards. Ammonium acetate buffer (0.5M, pH =
5.0) and beta-glucuronidase were then added and this mixture
was incubated at 50ºC for 30 minutes. The synthetic
cannabinoids and internal standard were extracted by solid
phase extraction using Bound Elut SPEC C18 3mL (15mg)
columns (Agilent Technologies, Santa Clara, CA.). The
samples underwent separation via liquid chromatography
using a Kinetex™ 5µm C18 50x4.6mm column (Phenomenex,
Torrance, CA) on a TLX4 high-throughput liquid
chromatography system (Thermo Fisher Scientific, Waltham,
MA), followed by analysis on a tandem mass spectrometer
(6500 QTRAP, AB SCIEX, Foster City, CA) equipped with an
electrospray ionization source in positive mode. Ion
transitions were monitored by multiple reaction monitoring
(MRM) mode.
With the exception of JWH-073, metabolites that are
structural isomers are not chromatographically separated.
Chromatographic separation was deemed unnecessary as the
clinical interpretation is identical and independent of which
isomer is detected. The above method includes detection of
JWH-018 N-(4/5-hydroxypentyl), JWH-073 N-(3-hydroxybutyl),
JWH-073 N-(4-hydroxybutyl), JWH-122 N-(4/5-hydroxypentyl),
JWH-210 N-(4/5-hydroxypentyl), JWH-250 N-(4/5-
hydroxypentyl), AM2201 N-(4-hydroxypentyl), RCS-4 N-(4/5-
hydroxypentyl), UR-144 N-(4/5-hydroxypentyl) and XLR11 N-
(4-hydroxypentyl).
Methods Table 3: Accuracy
Investigation into the effects of 80 different pharmaceuticals
and illict drugs as well as the presence of hemolysis and icterus
on the analysis demonstrated no interference with the assay.
Ion suppression was also demonstrated to be absent.
Interferences / Ion Suppression
Analyte
(N=20)
QC Level 1
(ng/mL)
Mean (SD, %CV)
QC Level 2
(ng/mL)
Mean (SD, %CV)
QC Level 3
(ng/mL)
Mean (SD, %CV)
AM2201 4-OH 1.1 (0.0, 4.1%) 9.8 (0.3, 3.0%) 39.8 (1.0, 2.6%)
JWH-018
4/5-OH
2.1 (0.1, 3.1%) 18.9 (0.5, 2.6%) 78.0 (1.6, 2.0%)
JWH-073 3-OH 1.0 (0.0, 4.6%) 8.9 (0.3, 3.0%) 37.4 (1.4, 3.9%)
JWH-073 4-OH 1.1 (0.1, 4.7%) 9.4 (0.2, 2.4%) 37.7 (0.8, 2.1%)
JWH-122
4/5-OH
2.0 (0.1, 3.0%) 18.5 (0.4, 2.0%) 77.3 (1.8, 2.3%)
JWH-210
4/5-OH
1.7 (0.0, 2.9%) 15.1 (0.3, 2.3%) 64.0 (1.3, 2.1%)
JWH-250
4/5-OH
2.2 (0.1, 4.2%) 20.4 (0.7, 3.2%) 80.5 (2.4, 2.9%)
RCS-4 4/5-OH 2.3 (0.1, 3.4%) 20.2 (0.8, 4.0%) 79.7 (2.3, 2.8%)
UR-144 4/5-OH 2.1 (0.1, 2.8%) 19.8 (0.4, 2.1%) 81.0 (2.1, 2.5%)
XLR11 4-OH 1.2 (0.0, 3.8%) 10.6 (0.3, 3.2%) 42.5 (1.5, 3.4%)
Table 2: Inter-assay Precision
Analyte
(N=20)
QC Level 1
(ng/mL)
Mean (SD, %CV)
QC Level 2
(ng/mL)
Mean (SD, %CV)
QC Level 3
(ng/mL)
Mean (SD, %CV)
AM2201 4-OH 1.1 (0.1, 7.0%) 9.8 (0.6, 5.8%) 39.5 (2.4, 6.1%)
JWH-018
4/5-OH
2.1 (0.2, 7.5%) 19.2 (1.3, 6.9%) 77.4 (6.4, 8.3%)
JWH-073 3-OH 1.1 (0.1, 7.1%) 9.6 (0.7, 7.1%) 39.4 (3.1, 7.8%)
JWH-073 4-OH 1.1 (0.1, 5.3%) 9.8 (0.7, 6.6%) 39.1 (2.3, 5.8%)
JWH-122
4/5-OH
2.1 (0.2, 7.6%) 18.4 (1.4, 7.5%) 75.8 (5.3, 7.0%)
JWH-210
4/5-OH
1.8 (0.2, 11.1%) 16.0 (1.6, 10.0%) 68.0 (5.6, 8.3%)
JWH-250
4/5-OH
2.3 (0.1, 5.9%) 20.3 (1.8, 8.9%) 79.8 (5.7, 7.1%)
RCS-4 4/5-OH 2.2 (0.2, 6.9%) 20.7 (3.4, 16.2%) 79.0 (6.7, 8.5%)
UR-144 4/5-OH 2.1 (0.2, 10.7%) 19.2 (2.3, 11.8%) 79.8 (8.1, 10.1%)
XLR11 4-OH 1.2 (0.1, 11.2%) 10.1 (1.1, 11.2%) 41.0 (4.6, 11.1%)
Chimalakonda KC, Seely KA, Bratton SM, et al: Cytochrome P450-mediated oxidative
metabolism of abused synthetic cannabinoids found in K2/Spice: identification of novel
cannabinoid receptor ligands. Drug Metab Dispos 2012;40:2174-2184
Crews BO: Synthetic Cannabinoids The Challenge of Testing for Designer Drugs. Clinical
Laboratory News, February 2013, pp 8-10
DEA Moves to Emergency Control Synthetic Marijuana. Available at
http:www.justice.gov/dea/pubs/pressrel/pr112410.html
Scheidweiler KB, Huestis MA: Simultaneous quantification of 20 synthetic cannabinoids and 21
metabolites, and semi-quantification of 12 alkyl hydroxy metabolites in human urine by liquid
chromatography-tandem mass spectrometry. J Chromatogr A 2014;1327:105-117
Synthetic Drug Abuse Prevention Act of 2012. 112th Congress 2D Session, S. 3190
Wohlfarth A, Scheidweiler KB, Chen X, Liu H, et al: Qualitative confirmation of 9 synthetic
cannabinoids and 20 metabolites in human urine using LC-MS/MS and library search. Anal
Chem 2013;85:3730-3738
References
Specimens are stable when stored under ambient, refrigerate
or frozen/ultra-frozen conditions for up to 35 days. Multiple
freeze-thaw cycles did not have an effect on stability.
Stability
Figure 1: Chromatography
Table 4: Dynamic Range Study
Analyte Slope Y-Intercept R2 Value
AM2201 4-OH 0.9883 0.0871 0.9999
JWH-018
4/5-OH
0.9796 0.0783 0.9993
JWH-073 3-OH 0.9870 0.0526 0.9998
JWH-073 4-OH 0.9883 0.0814 0.9998
JWH-122
4/5-OH
0.9727 0.1180 0.9990
JWH-210
4/5-OH
0.9433 -0.2244 0.9906
JWH-250
4/5-OH
0.9925 0.5649 0.9998
RCS-4 4/5-OH 0.9898 0.8399 0.9997
UR-144 4/5-OH 0.9951 1.0650 0.9993
XLR11 4-OH 1.0248 0.0669 0.9970
**External testing of RCS-4 4/5-OH and XLR11 4-OH was not available.
External Reference Lab
Negative Positive
Mayo
Method
Negative 16 0
Positive 1 79
% Agreement 94% 99%