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THC-COOH Quantification in Urine Using
Dilute and Shoot LC-MS/MS Method for
Forensic Toxicology
Samuele Scurati, Thermo Fisher Scientific, Rodano, Italy
Maura Brambilla, Ludovico D’Amato, Paolo Brambilla, University Department of Laboratory Medicine, Hospital of Desio, Desio, Italy
Application
Note: 548
Key Words
• TSQ Quantum
Access MAX
• Accela Pump
• Cannabinoids
• Forensic
Toxicology
Introduction
Cannabis sativa
is a widely used drug of abuse. Tetrahy-
drocannabinol (THC) is the major psychoactive chemical
compound in the cannabis plant. After smoke inhalation,
THC is absorbed and distributed in blood. Subsequently,
it is rapidly metabolized to THC-COOH, conjugated
with glucuronic acid, and excreted through urine. Liquid
chromatography-tandem mass spectrometry (LC-MS/MS)
is considered a useful tool to establish the consumption of
cannabis by the assessment of THC-COOH in urine for
forensic toxicology purposes.
Goal
To develop a reliable and fast analytical method for the
quantitative determination of THC-COOH in urine using
a Thermo Scientific TSQ Quantum Access MAX triple
stage quadrupole mass spectrometer.
Experimental
Sample Preparation
A urine sample was hydrolyzed with 10M NaOH and
heated at 60 °C for 15 minutes. The pH was restored with
Fisher Chemical acetic acid. Hydrolyzed samples as well as
calibrators were diluted 1:10 in Fisher Chemical water/ace-
tonitrile (1:1). Then, 10 µL were directly injected. Quan-
titative analysis was performed on the basis of calibration
curves prepared in urine, ranging from 7.8 to 1000 ng/mL.
Calibrators were injected in duplicate.
UHPLC conditions
Liquid chromatography separation was performed using
a Thermo Scientific Accela autosampler and pump. The
sample was injected directly on a Thermo Scientific Hyper-
sil GOLD column (50 × 2.1 mm, 1.9 μm). A gradient LC
method used mobile phases A (0.1% aqueous formic acid)
and B (Fisher Chemical Optima LC/MS acetonitrile) at a
flow rate of 300 μL/min. The run time was 6 minutes.
Mass Spectrometry
MS analysis was carried out on a TSQ Quantum Access
MAX™ triple stage quadrupole mass spectrometer
equipped with a Thermo Scientific Ion Max source with
a heated electrospray ionization (HESI) probe. The MS
conditions were as follows:
Scan type:
SRM
Divert valve:
2 - 4 min to source
Selected ions for quantification:
m/z
343
→
299 + 245 for THC-COOH in
negative mode
Results and Discussion
Figures 1 and 2 show the ion chromatograms of the lowest
and highest calibration points. Excellent linearity
(r
2
= 0.99) fits for the calibration curve were observed over
the range of 7.8-1000 ng/mL urine, with a Coefficient of
Variation (%CV) at the lower end of 6.5%. The limit of
quantitation (LOQ) was established as 7.8 ng/mL in urine.
Figure 4 reports an ion chromatogram of a real urine
sample positive for cannabinoids (225 ng/mL urine), ana-
lyzed as described.
To examine the difference between hydrolyzed and
non-hydrolyzed urine, we analyzed the same urine sample
without the hydrolysis step. When urines were not hy-
drolyzed, the portion excreted as free THC-COOH was
detected at 3.06 minutes, while THC-COOH-glucuronide
was detected at 2.58 minutes (Figure 5). The precursor ion
m/z
343 was generated as result of an in-source fragmenta-
tion and a consequent loss of glucuronic acid.
Because THC-COOH is mainly excreted as glucuronic
acid conjugate, it is always necessary to perform urine
hydrolysis before the LC-MS analysis to obtain an accurate
quantification of THC-COOH.