Testing hair for drugs of abuse has been practiced for
over 50 years, due in large part to the ability to detect
drug use over a longer period of time, as compared to
other biological matrices, because many drugs are well-preserved
in hair. Hair testing is widely used in criminal investigations
such as monitoring abstinence of parolees, verifying drug
use history, and identifying drug-facilitated sexual assault. It is
also commonly used to screen and monitor drug use in employees,
drug treatment participants, and parties involved in child
custody cases. Workplace programs include hair testing due
to the ease of collection, difficulty of adulteration, and longer
detection times.
Marijuana is one of the drugs tested most
often in forensic and drug screening
applications. The parent compound, tetrahydrocannabinol
(THC), is found in
higher concentration in hair samples, but
detection of the acid metabolite THCA
(11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid) is preferred, in order
to eliminate the possibility of potential
environmental contamination from
marijuana smoke. While guidelines
for workplace hair testing have not yet
been adopted by the Substance Abuse
Mental Health Services Administration
(SAMHSA) in the United States, a cut-off
concentration for THCA as low as
0.05 pg/mg of hair has been suggested,
and such guidelines are a topic of additional
study and analysis by this regulatory
body. The Society of Hair Testing
recommends a limit of quantification
(LOQ) of ≤0.2 pg/mg for THCA.
This article describes a method developed
on the Agilent 7890A GC system (Agilent Technologies, Inc., Santa Clara, CA) coupled with an Agilent 7000B triple quadrupole GC-MS system that provides rapid
and sensitive detection of a THC metabolite in hair using 2-D GC
and negative ion chemical ionization (CI) MS-MS in the multiple
reaction monitoring (MRM) mode (also called SRM, selected reaction
monitoring). The method is modified from a previous GC-MSD
method1 to take advantage of the lower chemical background and
higher sensitivity provided by triple quadrupole MS-MS analysis.
Backflushing is used to increase robustness, and low thermal mass
(LTM) column modules speed the chromatography process, enabling
a run time of 7 min and a cycle time of 9 min. MRM MS-MS analysis
on the triple quadrupole GC-MS system delivers very high sensitivity,
with a limit of detection (LOD) of 0.002 pg/mg and a limit of
quantification (LOQ) of 0.01 pg/mg.
Experimental
Standards and reagents
Figure 1 - Schematic representation of the system used to
develop the THCA method.
Tri-deuterated THCA, which was used as the internal standard
(100 μg/mL in methanol), and unlabeled THCA (100 μg/mL in
methanol) were obtained from Cerilliant (Round Rock, TX).
The internal standard concentration in the method was 0.05 pg/mg of hair. Methanol, acetonitrile,
toluene, ethyl acetate, hexane, glacial
acetic acid, and methylene chloride
were obtained from Spectrum
Chemicals (Gardena, CA). All solvents
were HPLC grade or better, and
all chemicals were ACS grade. Bond
Elut Certify I solid-phase extraction (SPE) columns (130 mg) from Agilent,
or Clean Screen ZSTHC020 extraction
columns (200 mg) from United
Chemical Technologies, Inc. (Bristol,
PA) were interchangeable for the
assay. The derivatizing agents, pentafluoropropionic
anhydride (PFPA)
and 1,1,1,3,3,3-hexafluoro-2-propanol
(HFIP), were purchased from Sigma-Aldrich (St. Louis, MO) and Campbell
Science (Rockton, IL), respectively.
Instruments
The experiments were performed on an
Agilent 7890N GC system equipped with
a multimode inlet (MMI) and an LTM
system, coupled to an Agilent 7000B triple
quadrupole GC-MS system. Two-dimensional chromatography was
performed using a pre-column for backflushing, two LTM columns connected
by a Deans switch, and a Purged Ultimate Union (Figure 1). The
instrument conditions are listed in Table 1.
Table 1 - Agilent 7890N/7000B gas chromatograph and triple quadrupole GC-MS conditions
Sample preparation
Samples were prepared as previously described.2 Calibrators, controls,
or hair specimens (20 mg) were weighed into silanized glass tubes and
washed with methylene chloride (1.5 mL). The solvent was decanted
and the hair samples were allowed to dry. The internal standard, THCA-d3 (0.05 pg/mg), was added to each hair
specimen. For the calibration curve, unlabeled
THCA was added to the hair at concentrations of
0.002, 0.01, 0.02, 0.05, 0.1, and 0.5 pg/mg of hair.
Deionized water (0.5 mL) and 2N sodium hydroxide
(0.5 mL) were added, and the hair was heated
at 75 °C for 15 min. The sample was allowed to
cool and then centrifuged (2500 rpm, 15 min).
The supernatant was poured into glass tubes
already containing acetic acid (1 mL), 1 M acetic
acid (3 mL), and 0.1 M sodium acetate buffer (pH
4, 2 mL). The tubes were capped and mixed. SPE
columns were conditioned with hexane/ethyl acetate
(75:25, v/v; 2 mL), methanol (3 mL), deionized
water (3 mL), and 0.1 M hydrochloric acid
(1 mL). The acidified samples were loaded onto
the SPE columns and allowed to dry. The SPE columns were washed with deionized water
(2–3 mL) and allowed to dry for 5 min. The
SPE columns were washed with 0.1 M hydrochloric
acid/acetonitrile (70:30 v/v; 3 mL) and
allowed to dry at 30 psi for 10 min. The SPE
columns were finally rinsed with hexane/ethyl
acetate (75:25 v/v; 3 mL) in order to elute the
THCA into silanized glass tubes. The eluent
was evaporated to dryness under nitrogen at
40 °C and reconstituted in PFPA (70 μL) and
HFIP (30 μL) for derivatization. The mixture
was transferred to autosampler vials with glass
inserts and capped. The vials were heated at 80
°C for 20 min, and then left at room temperature
for 10 min. The extracts were evaporated
to dryness in a vacuum oven. The samples were
finally reconstituted in toluene (50 μL) for
injection into the GC-MS system.
Analysis parameters
The Agilent triple quadrupole GC-MS system parameters used are
shown in Table 2.
Table 2 - Agilent 7000B triple quadrupole GC-MS system analysis parameters
Results
Two-dimensional GC with heartcutting
The use of two serial GC columns to separate background from the
required peak is a well-established technology that is widely used to provide
excellent separation of the analyte from matrix interferences. Once
the analyte retention time on the first column has been determined, the
pneumatic switch (Deans switch) is turned on at that time to divert the
flow to the second column, and turned off a short time later. This diverts
a narrow, heartcut “window” of the effluent from the first column that
contains the analyte and minimal background for further separation
on the second column (Figure 1). The two columns function optimally
when the stationary phases are as different as possible.