Rapid, Robust, and Sensitive Detection of 11-nor-Δ9-Tetrahydrocannabinol-9-Carboxylic Acid in Hair

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.

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