Development and validation of an automated liquid-liquid extraction GC/MS method for the determination of THC, 11-OH-THC, and free THC-carboxylic acid (THC-COOH) from blood serum

Abstract

The analysis of Δ9-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) from blood serum is a routine task in forensic toxicology laboratories. For examination of consumption habits, the concentration of the phase I metabolite THC-COOH is used. Recommendations for interpretation of analysis values in medical-psychological assessments (regranting of driver’s licenses, Germany) include threshold values for the free, unconjugated THC-COOH. Using a fully automated two-step liquid-liquid extraction, THC, 11-OH-THC, and free, unconjugated THC-COOH were extracted from blood serum, silylated with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), and analyzed by GC/MS. The automation was carried out by an x-y-z sample robot equipped with modules for shaking, centrifugation, and solvent evaporation. This method was based on a previously developed manual sample preparation method. Validation guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) were fulfilled for both methods, at which the focus of this article is the automated one. Limits of detection and quantification for THC were 0.3 and 0.6 μg/L, for 11-OH-THC were 0.1 and 0.8 μg/L, and for THC-COOH were 0.3 and 1.1 μg/L, when extracting only 0.5 mL of blood serum. Therefore, the required limit of quantification for THC of 1 μg/L in driving under the influence of cannabis cases in Germany (and other countries) can be reached and the method can be employed in that context. Real and external control samples were analyzed, and a round robin test was passed successfully. To date, the method is employed in the Institute of Legal Medicine in Giessen, Germany, in daily routine. Automation helps in avoiding errors during sample preparation and reduces the workload of the laboratory personnel. Due to its flexibility, the analysis system can be employed for other liquid-liquid extractions as well. To the best of our knowledge, this is the first publication on a comprehensively automated classical liquid-liquid extraction workflow in the field of forensic toxicological analysis.Graphical abstractGC/MS with MPS Dual Head at the Institute of Legal Medicine, Giessen, Germany. Modules from left to right: quickMix (for LLE), wash station, tray 1 (vials for extracts), solvent reservoir, mVAP (for extract evaporation), Solvent Filling Station (solvent supply), cooled tray 2 (vials for serum samples), and centrifuge (for phase separation)

Determination of cannabinoids in hemp nut products in Taiwan by HPLC‐MS/MS coupled with chemometric analysis: quality evaluation and a pilot human study

“…Subsequently, 11‐hydroxy‐THC is oxidized to the psychotropically inactive metabolite 11‐nor‐9‐carboxy‐THC. Then it is followed by phase II metabolism, the metabolite enzymatically catalyzed conjugation with glucuronic acid . Concerns have been raised regarding the consideration of hemp nut consumers as dope users.…”

“…Then it is followed by phase II metabolism, the metabolite enzymatically catalyzed conjugation with glucuronic acid. [44] Concerns have been raised regarding the consideration of hemp nut consumers as dope users. The cut-off of 15-50 ng/mL for 11nor-9-carboxy-THC in the urine is used in most countries; in Taiwan, 15 ng/mL has been established.…”

Assessing cannabis consumption frequency: Is the combined use of free and glucuronidated THCCOOH blood levels of diagnostic utility?

“…Another point in favor of using the newly proposed decision limits is the instability of THCCOOH‐glucuronide. Under certain sample transport, storage and processing conditions, substantial deconjugation of THCCOOH‐glucuronide can occur, leading to erroneously elevated blood concentrations of free THCCOOH, drivers being falsely classified as heavy users and drivers being classified differently by different laboratories since (pre‐)analytical procedures can vary among laboratories. Therefore, taking into account the concentrations of both free and glucuronidated THCCOOH, either by measuring free THCCOOH and THCCOOH‐glucuronide separately or together after hydrolysis, is highly advisable for a reliable assessment of the cannabis consumption frequency.…”

“…Therefore, the THCCOOH blood level is not only determined by the consumption frequency, but also depends on the glucuronidation efficiency and kinetics. Sample collection, storage, and processing conditions are other important factors influencing the amount of free THCCOOH since THCCOOH‐glucuronide has limited stability and is prone to deconjugation . As reported for THCCOOH, plasma, serum, and whole blood concentrations of THCCOOH‐glucuronide were higher in heavy cannabis users than in occasional smokers .…”

Gas chromatographic analysis of naturally occurring cannabinoids: A review of literature published during the past decade

“…Simple GC-MS methods have also been used extensively to determine cannabinoids in plasma samples using various extraction and sample preparation techniques. For example, Camargo and co-workers24 developed an efficient liquid-liquid extraction and GC-MS method for the determination of CBD (2) at a concentration range of 2.5-250 ng/mL of plasma.An automated liquid-liquid extraction coupled with a GC-MS method was reported for the analysis of THC (10), THCA (11) and 11-OH-THC (8) from human blood serum samples after derivatisation by N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) 25. As this method could detect and quantify THC (10) as low as 0.3 mu g/L of concentration, when extracting only 0.5 mL of blood serum, this method was deemed suitable for the limit of quantification for THC (10) of 1 mu g/L in driving under the influence of cannabis cases in Germany and other countries.…”