Quantitative determination of five cannabinoids in blood and urine by gas chromatography tandem mass spectrometry applying automated on‐line solid phase extraction

Abstract Cannabis is the most frequently consumed illegal substance worldwide. More recently, an increasing number of legal cannabis products low in psychoactive Δ9‐tetrahydrocannabinol (THC) but high in non‐intoxicating cannabidiol (CBD) are being more widely consumed. While the detection and quantification of THC and its metabolites in biological matrices is an important forensic‐toxicological task, additional detection of CBD is also important, for example, when examining the plausibility of consumer's statements. This report describes the method validation for the quantitative determination of THC and its two major metabolites, 11‐hydroxy‐THC (OH‐THC) and 11‐nor‐9‐carboxy‐THC (THC‐COOH), as well as CBD and cannabinol (CBN) in whole blood and urine. The method employs automated on‐line solid phase extraction coupled to gas chromatography tandem mass spectrometry (GC–MS/MS). The method was fully validated according to guidelines of the Swiss Society of Legal Medicine (SGRM) and the Society of Toxicological and Forensic Chemistry (GTFCh). The method fulfilled the validation criteria regarding analytical limits, accuracy and precision, extraction efficacy, and sample stability. The limits of detection (LODs) in whole blood and urine were 0.15 ng/mL for THC, OH‐THC and CBD, 0.1 ng/mL for CBN, and 1.0 ng/mL for THC‐COOH. The limits of quantification (LOQ) in whole blood and urine were 0.3 ng/mL for THC, OH‐THC and CBD, 0.2 ng/mL for CBN, and 3.0 ng/mL for THC‐COOH. The fully validated and automated method allows sensitive and robust measurement of cannabinoids in whole blood and urine. Detection of CBD provides additional information regarding consumed products.

Cannabis rich in THC is the most frequently consumed illicit substance worldwide 9 ; however, legalization efforts for medical and recreational use have been on the increase, particularly in recent years. [10][11][12][13] Often products low in THC can be legally purchased. 14,15 For instance, under current Swiss law, cannabis flowers containing less than 1% THC are not banned under the narcotics law. 16 Dried flower buds of these cannabis varieties are advertised as having high concentrations of CBD. 17 With regard to driving, Switzerland, among other countries, 18 pursues a zero tolerance for THC, meaning that detection of THC proves driving inability regardless of impairment. 19 A limit concentration of 1.5 ng/mL THC in whole blood is applied, above which THC is considered as detected. 20 In practice, the Swiss-wide measurement uncertainty of ±30% has to be applied, which means that the legal limit is only exceeded with sufficient certainty at a measured concentration of ≥2.2 ng/mL. However, not only consumption of drug-type cannabis high in THC but also legal CBD-cannabis (which contains less than 1% THC) can lead to blood concentrations exceeding this limit. 21,22 According to Swiss guidelines, 23 detection of THC and its two major metabolites 11-hydroxy-THC (OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) by accredited forensic-toxicological laboratories is mandatory for concluding driving inability. Nonetheless, the detection of further plant ingredients is of major interest, as it might allow one to distinguish the uptake of CBD-rich cannabis from drug-type, THC-rich products. This is of particular interest if drug abstinence has to be verified in countries where CBD-rich/THC-low cannabis products are legally available and consumed. Additionally, minor cannabinoids including CBN are discussed as potential markers to identify recent cannabis uptake, [24][25][26][27] to distinguish the use of different types of cannabis, 28 and play a role in anti-doing testing. 29 The major THC-metabolite THC-COOH is glucuronidated by several UDP-glucuronyltransferases. 30 The ratio of glucuronidated to free THC-COOH in blood is highly variable and ranges from approximately 0.5 to 5 and is dependent on consumption frequency and time difference between intake and blood sampling. [31][32][33] Cut-off concentrations of free THC-COOH and/or its glucuronide in blood have been proposed as a marker for frequent THC-consumption. 33,34 Based on the work by Fabritius et al., 34 the Swiss Society of Legal Medicine (SGRM) has implemented a cut-off concentration of 40 ng/mL for free THC-COOH in whole blood, above which a frequent consumption (i.e., twice or more per week) is presumed. In cases involving driving under the influence of drugs (DUID), a concentration of ≥40 ng/mL of THC-COOH can result in an evaluation of general driving fitness.
In urine samples, cleavage of phase-II-metabolites prior to analysis is standard practice. The ratio of free to glucuronidated THC-COOH in urine ranges from 1.3 and 4.5. 35 To measure total THC-COOH, cleavage of THC-COOH-glucuronide is often necessary.
Cleavage of the glucuronide ester bond can be achieved chemically, for instance by basic hydrolysis, or enzymatically, usually by addition of β-glucuronidase. 36,37 This paper describes an automated method for the detection and quantification of THC, OH-THC, THC-COOH, CBD, and CBN ( Figure 1) in whole blood and urine via on-line solid phase extraction (SPE), followed by derivatization with N-methyl-N-(trimethylsilyl) trifluoracetamid (MSTFA), coupled to gas chromatography tandem mass spectrometry (GC-MS/MS). The advantages of automated sample preparation lie mainly in a less labor-intensive sample preparation, increased sample throughput, and improved reproducibility. The method is based on a previously reported sample preparation; 22 however, a reduction of the sample volume from 0.5 to 0.25 mL and optimization of the extraction efficacy were key goals for this current method. The detection of CBD alongside THC and THC-metabolites provides additional information, especially with regard to potential differentiation between the consumption of drug-type cannabis from (legally available) products rich in CBD but low in THC. Compared with previously reported automated methods for the detection of THC, OH-THC, and THC-COOH, 38,39 this F I G U R E 1 Chemical structure overview of the target analytes incorporated into the method: Δ 9 tetrahydrocannabinol (THC), 11-hydroxy-THC (OH-THC), 11-nor-9-carboxy-THC (THC-COOH), cannabidiol (CBD), and cannabinol (CBN) method represents an extension, by two analytes, for the analysis of two different matrices (urine and whole blood). Furthermore, special attention was paid to THC-COOH-glucuronide stability during whole blood sample preparation, to ensure accuracy in the determination of the free THC-COOH fraction.     Urine was collected in 100 mL sample cups made from high density (HD) polyethylene (Sarstedt, Sevelen, Switzerland) and poured into 30 mL tubes made from polypropylene (also from Sarstedt) for storage. Blood and urine specimens were stored at À20 C until analysis.

| Sample pretreatment
As shown in Figure 2

| GC-MS/MS parameters
An injection volume of 1 μL was chosen. The programmed temperature vaporization (PTV) inlet was used in splitless mode. An initial temperature of 70 C was held for 0.5 min. The PTV inlet was heated with a ramp for 14.5 C/min up to 250 C, with a transfer time of 1 min.
After the transfer step, the PTV was cleaned at a temperature of 300 C for 10 min.
Helium (≥99.9999%; Carbagas, Gümligen, Switzerland) was used as the carrier gas at a flow rate of 1.5 mL/min. The oven temperature gradient program started at 70 C, which was held for 0.5 min. The temperature was then increased by 80 C/min until 200 C was reached. In a second ramp, the temperature was increased for 10 C/ min to a final temperature of 300 C, which was held for another 3 min. Including the cool-down period of 5 min, one GC-run required 18 min.
The transfer line and ion source were held at 250 C. MS was used in electron ionization mode (EI; emission current of 50 μA, electron energy of À70 eV). Argon (≥99.999%, Carbagas) was used as the collision gas at 1.2 mTorr. All analytes were measured in positive ion mode. In Table 1, the MS/MS detection parameters via single reaction monitoring (SRM) are summarized.
Due to the small sample volumes of fully prepared samples, that is, the tertiary extract and potential decomposition of analytes, a continuous workflow was chosen. Hereby, samples are prepared one at a time, whereby preparation of a subsequent sample occurs while the previous one is being analyzed by GC-MS/MS. Overall, the method enables immediate injection of a fully prepared sample every 46 min, including blank measurements in-between samples.

| Method development
To evaluate the influence of pH, mixing of the primary extract with

| Validation
The method was validated according to the guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) and the SGRM. 40,41 Validation parameters included selectivity and specificity, limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy with precision and trueness, extraction efficiency, testing for carry-over, stability of samples at various time points during sample preparation, F I G U R E 2 Schematic overview of the optimized sample preparation protocol (method A and B): (a,b) pretreatment of samples to generate primary extract; (c,d) solid phase extraction (SPE) parameters to generate secondary extract; (e) derivatization to generate tertiary extract stability of THC-COOH-glucuronide in whole blood samples, and hydrolysis of THC-COOH-glucuronide by enzymatic cleavage via β-glucuronidase in urine samples. In order to be used as a test sample, unless otherwise indicated, 0.25 mL drug-free whole blood or urine was spiked with ISTD and the required reference analyte(s) (which had first been diluted in MeOH as required).
Selectivity was tested by analysis of six blank samples (only MSTFA) and two drug free samples (containing only ISTD). LOD and LOQ were tested in whole blood and urine according to DIN 32645 by generation of 9-point calibration curves spanning the expected analytical limits. For THC, OH-THC, and CBD, the concentrations ranged from 0.1 to 1.0 ng/mL (interval of 0.1 ng/mL between levels), for CBN, the curve ranged from 0.06 ng/mL up to 0.6 ng/mL (interval of 0.06 ng/mL between levels), and for THC-COOH, the concentrations ranged from 1.0 ng/mL up to 10 ng/mL (interval of 1.0 ng/mL between each calibrator). Additionally, calculated LOD were verified by determination of signal-to-noise(S/N)-ratio using Xcalibur software, which needed to be greater than 3.
Linearity was tested in both, whole blood and urine, respectively, by measuring calibrations in six replicates, followed by Mandel F test for linearity, Grubb's test for outliers, and Cochran's C test for variance homogeneity (each at a 99% level of significance). Calibration  Extraction efficiency (EE) was determined over the following cal- Also for CBD determination in urine, enzymatic hydrolysis of phase-IImetabolites seems crucial. 44 However, as CBD-glucuronide was not available as a reference standard, no evaluation of the cleavage step could be performed for this method. Furthermore, also phase-I-metabolites of CBD and CBN were not commercially available at the start of the method validation and therefore not incorporated in the method.

| Whole blood samples
The method was successfully validated, in accordance with the guidelines of the GTFCh and SGRM. 40,41 No interfering signal was detected in any of the samples used to test selectivity and specificity. This indicates stability of analyte precursors THC-acid, CBD-acid, and CBN-acid, which upon decarboxylation would yield THC, CBD, and CBN. Decarboxylation is presumably prevented by protection of the carboxylic acid moiety via silylation. In Table 2 The preliminary test for carry-over in the 10 μL syringe revealed that washing of the syringe with n-hexane three times before and after injection was necessary to remove residual analytes (i.e., no signal in ensuing blank). Also, DCM and EtOAc were tested. DCM gave equal cleaning results but was dismissed due to its higher toxicity, whereas EtOAc did not fully remove residual analytes.
Additionally, washing of the 2.5 mL syringe and carry-over in the

| Urine samples
The method was also validated for urine samples and fulfilled all requirements. 40,41 In Table 3, a summary of the evaluation of LOD, LOQ, accuracy (given as RSD intra-day [RSD r ] and inter-day [RSD (T) ] and bias), EE, and linearity range after sample preparation of urine samples can be seen. Overall, LOD and LOQ as determined according T A B L E 2 Summary of validation of whole blood including limit of detection (LOD), limit of quantification (LOQ), relative standard deviation intra-day (RSD r ) and inter-day (RSD (T) ), bias, extraction efficiency (EE) T A B L E 3 Summary of validation results of urine including limit of detection (LOD), limit of quantification (LOQ), relative standard deviation intra-day (RSD r ) and inter-day (RSD (T) ), extraction efficiency (EE), and 95% β-tolerance interval 300 ng/mL for CBD; and 20 ng/mL CBN) did not indicate any carryover using the syringe cleaning steps as previously described for whole blood samples.
Regarding sample stability of analytes in the primary and tertiary extract of urine samples, which were tested over 3 days and 40 h, respectively, no significant loss of signal could be observed for any analyte. For the tertiary extract, an increase in peak area was observed for all analytes, as described for the blood samples.
Nonetheless, the derived analyte concentrations remained unchanged.
To elucidate if the method is fit for purpose, it was compared with the established method of analysis of cannabinoids as applied at the Institute of Forensic Medicine. Hereby, authentic blood and urine samples covering a range of concentrations were analyzed with both methods within a time frame of 1 week. No diverging results (i.e., difference >15%) were found (data not shown). Additionally, stored samples from previous ring trials (whole blood as matrix, because urine was not available) were re-measured and lay within acceptable ranges as provided by the ring trial provider. Chromatograms of negative and positive tested authentic whole blood and urine samples, especially at an analyte concentration at the analytical limits, are shown in Figures S13 and S14 in the supporting information.