Method development and validation for the identification and quantitation of gamma-hydroxybutyrate in urine, blood, and oral fluid using gas chromatography-mass spectrometry
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Gamma-hydroxybutyrate (GHB) is an endogenous compound in the human body, found in regions of the mammalian brain and believed to be a cerebral neurotransmitter.1,2 GHB also acts as a powerful central nervous system depressant commonly used as a “date rape” drug due to its hypnotic and sedative properties.1 The drug has also been used medicinally to treat alcohol withdrawal, opiate-withdrawal syndrome, and narcolepsy.3–5 Toxicological analysis of GHB in drug facilitated sexual assault (DFSA) cases is typically performed using blood and urine specimens.1 However, due to the endogenous nature of GHB, toxicological interpretation of these biological specimens can be complex and challenging.1,4 Additionally, urine and blood analysis of GHB can be impacted by sample collection, sample analysis times, and sample storage conditions.6,7 Due to the challenges and limitations associated with blood and urine analysis of GHB along with the prominence of GHB in DFSA cases, it would be beneficial to determine the possibility of GHB analysis using alternative biological matrices. The primary goal of this research was to develop a sample preparation method that could accurately and reliably identify and quantify GHB in oral fluid, as an alternative biological matrix. Additionally, this research was carried out to compare the identification and quantitation capabilities of GHB in oral fluid to that of traditional biological matrices, specifically urine and blood. The methods employed in this study utilized gas chromatography – mass spectrometry (GC-MS) instrumentation in order to correctly identify GHB. A deuterated internal standard, GHB-d6, was used to quantify all samples. The methods were assessed using the parameters set forth by the Scientific Working Group of Forensic Toxicologists (SWGTOX) for quantitative analysis methods. The following factors were considered: calibration model, bias, precision, limit of detection and quantitation, carryover, and interferences. Urine and blood samples were prepared using 200 uL of urine (UTAK Laboratories, Inc., Valencia, CA, U.S.A.) or blood (Equitech-Bio Inc., Kerrville, TX, U.S.A.), varying amounts of the 200 mg/L working calibrator and control solution prepared using certified reference standards (Cerilliant, Round Rock, TX, U.S.A.), and 50 uL of 100 mg/L working internal standard solution resulting in an internal standard concentration of 25 mg/L in each sample. Solid phase extraction (SPE) was performed using United Chemical Technologies (UCT), Inc. (Bristol, PA, U.S.A) Clean Screen GHB columns (ZSGHB020) on all samples.1 Samples were reconstituted, derivatized, and analyzed using GC-MS. Oral fluid samples were prepared using 1.0 uL of drug-free oral fluid, and 1.0 mg/mL (as salt) in methanol GHB received from Cerilliant. The samples were spiked with 1 uL of 1.0 mg/mL (as salt) in methanol GHB-d6 received from Cerilliant. Each sample had an internal standard concentration of 10 mg/L. Samples were fortified with 100 uL of ethyl acetate, and derivatized with 100 uL of bis(trimethyl)trifluoroacetamide (BSTFA) with 1% Trimethylchlorosilane (TMCS) received from Cerilliant. The samples were incubated, and analyzed using GC-MS.8 All analyses were conducted using an Agilent 7890A GC system, Agilent 5975C Mass Detector System (MSD), and an Agilent 7683B Autosampler (Agilent Technologies Inc. Santa Clara, CA). The chromatographic component was carried out using an Agilent HP5-MS 30m x 250um x 0.25um capillary column and an Agilent HP- 5MS 15m x 250um x 0.25um capillary column. All data was analyzed using Agilent MSD ChemStation software (version E.02.02.1431). The method has a total length of 12.75 minutes. Selective ion monitoring (SIM) was used to monitor the ions of interest for each analyte. GHB-d6 was monitored using the ions 239, 240, and 241. GHB was monitored using the ions 233, 234, 235.1 Results revealed that GHB and GHB-d6 could be identified and differentiated due to their fragmentation patterns. All calibration curves for the three matrices exhibited R2 values > 0.98 using a linear dynamic range of 5-100 mg/L with a minimum of four calibration points. The limit of detection for the three matrices was determined to be 1 mg/L, and the limit of quantitation for the three matrices was determined to be 5 mg/L. Bias and precision were analyzed at concentrations of 8 mg/L, 45 mg/L, and 90 mg/L for each matrix. All urine and blood samples were calculated to be within the acceptance range of +20% bias and +20% coefficient of variation. Oral fluid samples were outside of the +20% acceptance range for both bias and coefficient of variation. The highest concentration analyzed that did not produce carryover into subsequent matrix blanks was found to be 350 mg/L for each matrix. Significant interferences were found to be present in urine and blood samples, but negligible for all oral fluid samples. This research illustrates that the developed sample preparation method can be used to accurately and reliably identify GHB in oral fluid. Additionally, this research suggests that the quantitation capabilities of GHB in oral fluid are not as accurate and precise as those of urine and blood. Therefore, the developed method has better qualitative analysis capabilities, while the urine and blood methods have better quantitative analysis capabilities for forensic toxicology casework.