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Drug Abuse Tests Mouth OffGC triple quadrupole mass spectrometry overcomes many of the challenges for confirming and quantifying THC in forensic toxicology analyses.by Eric Chi and Jason Cole, Thermo Fisher Scientific, Austin, TX
In forensic toxicology, the analysis of oral fluid is becoming a popular alternative to the use of urine or blood to test for abuse of drugs, such as marijuana. Unlike urine and blood, oral fluid samples can be taken from a subject without causing pain or embarrassment and without the need for extensive training.
The marijuana plant contains a pharmacologically active compound—∆9-tetrahydrocannabinol (THC)—known to have mind-altering properties. 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 overcomes many of these challenges. To demonstrate the capabilities of this technique, a method for the detection of THC in an oral fluid matrix was fully validated for linearity, precision and carryover. A Thermo Scientific TSQ Quantum GC analyzer was selected over a single quadrupole due to its ability to detect analytes to very low quantitation limits in complex matrices. Thermo Scientific ToxLabs Forms 2.5.1 software was used for sample analysis and quantitation, as well as method validation.
Experimental tests
Table 1. The sequence noted in this table summarizes the sample preparation, extraction, and derivatization steps used prior to loading the sample onto an autosampler for GCMS analysis. Click to enlarge. | Sample preparation plays a critical role in method validation since many certifying bodies recommend or require method validation performed in matrix. In this case, solid phase extraction was used due to its ease of use and the cleanliness of the resultant extracts. After extraction, the samples were derivatized with bis (trimethylsilyl) trifluoroacetamide (BSTFA). The silylated reaction products were analyzed using the TSQ Quantum GC triple stage quadrupole GC-MS-MS system using multiple reaction monitoring (MRM). Known negative oral fluid calibration standard was spiked and extracted at 0.2, 2, and 20 ng/mL for use as calibrators.
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 Scientific AS 3000 II autosampler for GC-MS analysis. Table 1 summarizes sample preparation, extraction and derivatization steps.
Instrument analysis
The Thermo Scientific TRACE GC Ultra was equipped with a standard split/splitless injector. A 5-mm ID Thermo Scientific deactivated glass liner (p/n 45350033) was used in the injector with a glass wool plug. The split/splitless injector temperature was set to 250 C. A 2-µL injection volume was programmed on the AS 3000 II autosampler, and a splitless injection was used. The analytical column was a TRACE TR-5MS 15-m x 0.25-mm ID x 0.25-µm film (p/n 260F130P), which was installed 64 mm into the injection port.
Carrier gas flow was set to a constant rate of 1.2 mL/min of helium. The initial temperature on the TRACE GC Ultra was set to 60 C. Upon injection of the sample, the oven temperature was immediately ramped at 35 C/min to a final temperature of 320 C with no final hold, for a total run time of 7.43 min and a THC retention time of 5.77 min. The TSQ Quantum GC source temperature was set to 200 C, and the mass spectrometer was tuned using default AutoTune parameters. These tune settings were used for acquisition, with the default detector gain for the MRM mode left at 2 x 106.
Table 2. This table reveals the set of multiple reaction monitoring (MRM) transitions, dwell times, collision energies and the collision cell pressure used to detect THC and its deuterated internal standard. Click to enlarge. | For initial mass spectrometer method development, high concentrations of derivatized THC and THC-D3 were injected and analyzed in electron ionization (EI) full scan to determine precursor masses for EI MRM. Methods were then created to measure each precursor ion’s product ion scan at various collision energies and collision pressures. From these product ion scans, the most intense ions were selected for each MRM transition at the optimum collision energy and collision cell pressure. The set of MRM transitions, dwell times, collision energies, and the collision cell pressure used to detect THC and its deuterated internal standard are shown in Table 2. The transition from m/z 386 to m/z 303 was used as the quantitative transition for THC, with the transition from m/z 371 to m/z 289 as the confirming transition. For THC-D3 the quantitative and confirming transitions were mass 389 to 306 and mass 374 to 292 respectively.
Sample processing
For sample acquisition, peak detection and quantitation, Thermo Scientific ToxLab Forms 2.5.1 software was utilized. Incorporating all of the vital components of analysis into a unified workflow oriented application, ToxLab Forms provides an integrated solution to THC GC-MS-MS confirmation.
To make use of ToxLab Forms for method validation, a Thermo Scientific Xcalibur instrument method was first created for the mass spectrometer, autosampler and GC. A Master Method was created within ToxLab Forms, including processing parameters for component identification and quantitation and QC criteria specific to the method. Batch creation was performed through the Batch Wizard function, which streamlined sample entry, particularly for the longer validation batches.
Concentration calculations were based on a three-point calibration at 0.2, 2 and 20 ng/mL, using THC-D3 as the internal standard. All validation batches had to conform to quality control (QC) criteria, including quantitative and qualitative bounds checking. Quantitative criteria for the batch included acceptable quantitation ranges for all samples in each batch. All calculated amounts for QC and calibration samples had to fall within ±20% of the expected concentration in order to accept the sample. Failure of a QC sample would mean the entire batch would need to be repeated. In addition to this quantitative window, negative controls were evaluated on two additional criteria. One means of assessing a negative control is a quantitative value for THC less than the method limit of detection (LOD), which in this case was 0.2 ng/mL.
Table 3. One batch of oral fluid samples was used to analyze THC for linearity and carryover using a Thermo Scientific TSQ Quantum GC system. Click to enlarge. | An alternate criterion for negative controls is that the calculated amount must be less than a pre-determined percentage of the method cutoff. For this method, a level of 5% of the cutoff, or 0.1 ng/mL, was used as a second criterion and all negative controls were evaluated for compliance to both criteria. Qualitative criteria included ion ratio and retention time target ranges based on an average of the calibrators, along with peak shape considerations. These criteria were applied to all sample types. Ratios were defined as follows: Ratios were calculated for THC-D3 (292:306) and THC (289:303), and for each ratio, an acceptable range of ±20% was established.
Similarly, the target retention time for THC and THC-D3 was set using a ±2% retention time window based on an average of the calibrators’ retention times. Each validation batch was reviewed for compliance with these criteria, and for a batch to be accepted, it had to comply with all of these QC criteria.
The analysis of THC in oral fluid using the TSQ Quantum GC system was validated through determination of linear range, carryover, and precision. Three separate batches were prepared and analyzed: one for linearity/carryover (Table 3) and two for precision (Table 4). The TSQ Quantum achieved coefficients of variation (CV) of 6% or less at 0.8 and 2.5 ng/mL as well as linearity and accuracy from 0.2 to 20 ng/mL with no significant carryover.
Table 4. Two batches of oral fluid samples were used to analyze THC for precision using the Thermo TSQ Quantum GC system. The TSQ achieved coefficients of variation of 6% or less at 0.8 and 2.5 ng/mL. Click to enlarge. |
In conclusion, the TSQ Quantum GC operated in selected reaction monitoring mode proved to be both selective and sensitive enough to routinely measure THC in oral fluid at a 2-ng/mL cutoff level. This was exemplified by the accuracy at the 0.2-ng/mL sample level analyzed during the linearity study, where all four injections at the level quantitated within 5% of the actual amount. The linearity study also demonstrated ample linear range for the assay, determined to be between 0.2 to 20 ng/mL.
Across this range, all samples also gave ion ratios which were within 20% of the ion ratios of the calibrator. Furthermore, the intra- and inter-day precision studies showed that the coefficients of variation for the assay at 0.8 and 2.5 ng/mL were well under the 10% value required by many regulatory bodies. Because instrument method development and validation were performed in an extracted oral fluid calibrating solution, the results demonstrate performance of the TSQ Quantum GC system for method validation as they would be performed within a working laboratory.
The TSQ Quantum GC was chosen for this assay not only because its performance exceeds that required for the analysis, but also because of its ease of use and speed of analysis relative to alternative approaches. Setup and daily use of this method was as easy as for a typical single quadrupole confirmation method, without requiring a complex multi-dimensional GC approach. Also, the ease of developing an MRM confirmation method on the TSQ Quantum GC allows the user more flexibility in expanding to other confirmation assays that prove difficult to analyze on a GC single quadrupole instrument. Finally, at a retention time of less than six minutes, the methodology described offers a productive means for high-throughput laboratories to confirm and quantitate the use of THC through oral fluid sampling.
For more information about the Thermo Scientific TSQ Quantum GC, please call +866-463-6522, E-mail analyze@thermofisher.com or visit www.thermo.com/gc.
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