Analysis of oral fluids for illicit drugs is becoming increasingly popular in forensic toxicology. The testing of this type of biological fluid is being used as an alternative to blood and urine. Unlike urine, the collection of oral fluid can be monitored without embarrassment. Also, it has the advantages over blood in its ability to be collected without pain or the need for extensive training.

Marijuana represents the largest proportion of positive drug screens as reported by one laboratory system.¹ The marijuana plant contains a pharmacologically active compound-Δ9-tetrahydrocannabinol (THC)-known to have mind-altering properties. When THC enters the body, it is rapidly metabolized to, among others, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol, which is the metabolite typically analyzed in urine, blood and hair. However, in saliva there is still a large amount of parent THC remaining, so its presence in oral fluid can be measured as an indication of use. Since marijuana is one of the most commonly used illegal drugs, THC analysis in oral fluid is becoming increasingly important; however, this type of analysis can be particularly challenging due to the low concentration of THC typically found in oral fluid, as well as the low volumes of oral fluid that are generally available for analysis. Like many biological fluids, the chemical background from the oral fluid matrix can limit the low detection levels required for this analysis.

The use of gas chromatography coupled with a triple quadrupole mass spectrometer can overcome many of these challenges. To demonstrate the capabilities of this technique, a method was developed which highlights the use of a triple stage quadrupole mass spectrometer for rapid and reliable analysis of trace amounts of THC in oral fluid. A Thermo Scientific TSQ Quantum GC analyzer was selected over a single quadrupole due to its ability to detect analytes at very low concentrations in complex matrices. Thermo Scientific ToxLab Forms 2.5.1 software was used for sample analysis and quantitation, as well as method validation.

Methods

Sample preparation plays a critical role in method validation since many certifying bodies recommend or require method validation performed in matrix.

For this experiment, Negative Calibrator Oral Fluid (Orasure, Bethlehem, PA) was used for sample preparation. A sample size of 200 µL was selected. Calibrators, quality controls and linearity samples were spiked with appropriate amounts of THC (Cerilliant, Round Rock, TX). Three point calibration at 0.2, 2, 20 ng/mL was used for calculation of all quantitative amounts. A 40% (0.8 ng/mL) and a 125% control (2.5 ng/mL) were prepared from a 20 ng/mL THC working solution. Batches contained a negative control, the three calibrators, and a 40% control and 125% control. THC-D3 (Cerilliant) was used as the deuterated internal standard, and was added to each sample at a final concentration of 2 ng/mL.

Prior to extraction, the samples were brought to an approximate pH of 6 by adding  2mL of pH 6 phosphate buffer. Each sample was extracted by solid phase extraction on Thermo Scientific HyperSep Verify-AX 200mg 10mL columns (P/N 60108-764). The extraction columns were conditioned with sequential rinses of the following: 2 mL methanol, 2 mL DI water, and 1 mL 0.1 M HCl. Between each conditioning step, the columns were not allowed to dry. The pH-adjusted samples were then loaded onto the column and extracted under low vacuum. The columns were then washed sequentially with 2 mL of DI water and 2 mL of 0.1 M HCl: acetonitrile solution (70:30 v:v). The columns were dried under high vacuum for five minutes, and the sample eluents were collected in clean tubes under low vacuum with 3 mL of elution solvent (hexane:ethyl acetate, 75:25 v:v).

The extracts were evaporated to dryness at 40°C under nitrogen. Next, the dried samples were derivatized with 30 µL of BSTFA at 80°C for 20 minutes, after which the excess BSTFA was evaporated to dryness at 40°C under nitrogen. For analysis, 50 µL of toluene was added to the derivatized extracts, and the resulting samples were transferred to autosampler vials with glass inserts and loaded onto the Thermo Scientifc AS 3000 II autosampler for GC/MS analysis.


Table 1: Sample Preparation Steps

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