TY - JOUR
AU1 - DeGregorio, Michael, W
AU2 - Audino, Susan, A
AU3 - Montoya,, Edward
AU4 - Phong,, Aron
AU5 - Kao,, Chiao-Jung
AU6 - Wurz, Gregory, T
AB - Abstract Background Cannabis legalization is expanding rapidly throughout the United States, but there is no reliable means of establishing recent use. Objective To develop and validate a bioanalytical method for determination of Δ9-tetrahydrocannabinol (Δ9-THC), cannabinol, 11-hydroxy-Δ9-THC, 11-nor-9-carboxy-Δ9-THC, and 8β,11-dihydroxy-Δ9-THC in whole blood microsamples by liquid chromatography high-resolution mass spectrometry (LC-HRMS). Methods Cannabinoid extraction from whole blood was performed using a mixture of n-hexane/ethyl acetate (90:10, v/v). Chromatographic separation was performed with a C18 column using a binary mobile phase gradient of water and acetonitrile, each with 0.1% formic acid. Detection was performed by positive ion mode heated electrospray ionization with full scan MS on an Orbitrap mass spectrometer. A clinical study was performed in 30 subjects to identify recent cannabis use based on analysis of cannabinoids in blood samples up to 200 min post-smoking. Results Acceptable linearity of all calibration curves was observed (r2>0.99) for all analytes over a 1–100 ng/mL concentration range, with acceptable accuracy. Limit of detection (LOD) was 0.5 ng/mL. Accuracy and precision met acceptance criteria for all analytes. Repeatability (CV) was <5% at low (3 ng/mL) and high (90 ng/mL) concentrations. In the clinical study, the ratios between 11-nor-9-carboxy-Δ9-THC and Δ9-THC fell immediately after smoking and returned to near baseline levels by 200 min post-smoking, which is consistent with recent use. Conclusions and Highlights The developed LC-HRMS bioanalytical method is suitable for quantification of five key cannabinoids in whole capillary blood microsamples and can be used in conjunction with a test for determining recent cannabis use. Cannabis (Cannabis spp.) remains the most commonly used illicit drug in the United States and throughout the world (1). This complex plant, which has been used by human beings for thousands of years, contains over 100 different cannabinoids, also known as phytocannabinoids, as well as hundreds of other chemical compounds such as terpenes and flavonoids (2). The primary psychoactive constituent of cannabis is Δ9-tetrahydrocannabinol (Δ9-THC), which exerts its activity by binding to endogenous cannabinoid receptors known as CB1 and CB2 (3). Other common cannabinoids found in cannabis include cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), cannabichromene (CBC), and Δ9-tetrahydrocannabivarin (Δ9-THCV). In cannabis, these compounds occur largely in their carboxylic acid forms (e.g., Δ9-tetrahydrocannabinolic acid or THCA, which requires decarboxylation to Δ9-THC to become psychoactive). In the United States, cannabis has been approved for medicinal use in a total of 34 states, with recreational use having been legalized in 11 states and the District of Columbia as of June 2019. Canada became only the second country to legalize cannabis nationwide in October 2018. Driving under the influence (DUI) of drugs has become a critical public safety issue, and will become only more so with the expanding legalization of cannabis for medicinal and recreational use. According to data from the National Highway Traffic Safety Administration, the number of drivers testing positive for cannabis who were killed in crashes in the United States doubled between the years 2007 and 2015 (4). In May 2016, the AAA Foundation for Traffic Safety published a study titled “Prevalence of Marijuana Involvement in Fatal Crashes: Washington, 2010–2014,” the results of which showed that the number of drivers involved in fatal crashes in the state of Washington who tested positive for Δ9-THC more than doubled, from approximately 8.3% in 2013 to 17.0% in 2014 (5). However, these results do not show whether these drivers were actually impaired by Δ9-THC at the time of the crash. The state of Washington legalized recreational cannabis in November 2012. In the state of Colorado, where recreational cannabis was also legalized following the November 2012 election, a sharp increase of approximately 153% (from 55 to 139) was observed between 2013 and 2017 in the number of drivers involved in fatal crashes who tested positive for marijuana use (6). Cannabis is the most commonly detected illicit drug in drivers (4, 7); unfortunately, there is still no reliable method of proving recent cannabis use in DUI investigations. Recent use is defined as use within a window of 3 h for inhalation, e.g., smoking and vaping, and within 8 h for edibles. Cannabis impairment has been associated with use within these time windows. Although some states, e.g., Colorado and Washington, have enacted per se limits for cannabis DUI, no definitive correlation between the degree of impairment and specific blood levels of Δ9-THC has been established (8–10). The state of California and other U.S. states where cannabis has been legalized presently rely on specially-trained police officers known as drug recognition experts (DRE) to make a determination of DUI due to cannabis or other drugs. Law enforcement agencies in the United States and abroad desperately need an objective means of assessing cannabis impairment to support their initial determinations of driver intoxication. Employers likewise need a means of distinguishing between legal, responsible use of cannabis by employees and recent use during work hours, which poses a safety risk in the workplace. To address this pressing public and workplace safety issue, a liquid chromatography high-resolution mass spectrometry (LC-HRMS) bioanalytical method for the determination of five key cannabinoids [Δ9-THC; CBN; 11-hydroxy-Δ9-THC (11-OH-Δ9-THC); 11-nor-9-carboxy-Δ9-THC; 8β,11-dihydroxy-Δ9-THC (8β,11-diOH-Δ9-THC)] in microsamples of human whole capillary blood (50 µL) was developed and validated as part of a test for determining recent cannabis use (the RCU Test). These particular cannabinoids were chosen because Δ9-THC is the primary psychoactive compound, and 11-OH-Δ9-THC, also psychoactive, and 11-nor-9-carboxy-Δ9-THC are its two main metabolites. Cannabinol and 8β,11-diOH-Δ9-THC were selected because these compounds have been associated with recent cannabis use (11, 12). This report describes the method validation with respect to the assay parameters of limit of detection, lower limit of quantification (LLOQ), intra-run, inter-run, and pooled inter-run accuracy and precision over a period of 5 consecutive days, reinjection reproducibility (assay repeatability), and extraction efficiency. Deuterated Δ9-THC (Δ9-THC-D3) was employed as the internal standard. The validation study was based on the U.S. Food and Drug Administration’s industry guidance for bioanalytical methods (13). Finally, the described bioanalytical method was used in a clinical study to evaluate blood cannabinoid concentrations over a period of 200 min after smoking in 30 human subjects who participated in a study designed to assess recent cannabis use. Materials and Methods Chemicals and Reagents Two of the five cannabinoid analytes (Δ9-THC and CBN) were obtained as certified reference materials (CRMs) manufactured by Cerilliant (Round Rock, TX). The Cerilliant CRMs were supplied as sealed, 1.0 mL glass ampules containing 1000 µg/mL of analyte in methanol. The three cannabinoid metabolites (11-OH-Δ9-THC, 11-nor-9-carboxy-Δ9-THC, 8β,11-diOH-Δ9-THC) were obtained as reference standards from ElSohly Laboratories, Inc. (Oxford, MS). The ElSohly Laboratories standards were supplied as sealed, individual 1.0 mL ampules containing 100 µg/mL of each respective analyte in methanol. The internal standard (IS), deuterated Δ9-THC (Δ9-THC-D3), was manufactured by Cerilliant as a CRM. The IS was supplied in a sealed, 1.0 mL glass ampule containing 100 µg/mL of Δ9-THC-D3 in methanol. When not in use, concentrated stock solutions of these agents and working solutions made therefrom were stored at –20°C. Acetonitrile, formic acid, methanol, and n-hexane were purchased from Thermo Fisher Scientific (Waltham, MA) and were of LC/MS grade. Ethyl acetate (Acros Organics) was purchased from Thermo Fisher Scientific and was of spectroscopy grade (>99.5%). High purity water (18.2 MΩ) required for preparing the mobile phase and for sample extraction was produced using an EMD Millipore Simplicity water purification system. When not in use, these agents were stored at room temperature (20–25°C). Nitrogen (N2), supplied as a cryogenic liquid in a 230 L dewar at a purity of 99.998%, or as compressed nitrogen gas at a purity of 99.999% in T-type cylinders, was obtained from Praxair (Danbury, CT). Analytical Conditions The LC-HRMS system consisted of a Thermo Scientific Vanquish ultra-high-performance liquid chromatography (UHPLC) system and a Thermo Scientific Q Exactive Orbitrap mass spectrometer. All analytical data were collected and processed using TraceFinder version 4.1 software (Thermo Fisher Scientific). At least once each week, the Q Exactive Orbitrap mass spectrometer was calibrated using the positive ion calibration solution (Thermo Scientific ESI positive ion calibration solution; product 88323). For analyzing cannabinoids and metabolites, the mass spectrometer had the following settings: runtime 14 min; polarity positive; scan range 150–550 m/z; resolution 70 000 (full MS); automatic gain control (AGC) target 1.0 × 106; maximum inject time (IT) 250 ms. The following tune settings were saved and loaded prior to analyzing each sample batch: sheath gas 12; auxiliary gas 6; sweep gas 1; spray voltage 3.5 kV; capillary temperature 320°C; auxiliary gas heater 300°C; all other settings default. Detection was performed by positive ion mode heated electrospray ionization (HESI). Analyte verification was based on the presence of molecular ions, at least two isotopic ions, and intensity ratios between the isotopic ions and the molecular ions. The LC system was configured as follows: flow rate 0.300 mL/min; column temperature 37 ± 1.0°C; sample compartment 8 ± 1.0°C. The injection volume was set to 5 µL. The LC system was equipped with the following column: Restek (Bellefonte, PA) Raptor ARC-18, 1.8 µm, 2.1 × 150 mm (We would prefer the use of Cat. No. 9314262). Samples were held at 8 ± 1°C in the autosampler racks until analyzed. The mobile phase was composed of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. Samples were eluted according to the following mobile phase gradient: initial composition 75% B; increase to 100% B by 6 min; hold at 100% B for 3.5 min; decrease to 75% B by 10 min; hold at 75% B for 4 min; end run at 14 min. Preparation, Extraction, and Analysis of Calibration Standards and Validation Samples Before validation samples and calibration standards could be prepared, sufficient quantities of the matrix (whole blood) were obtained from a reliable, cannabis-free donor. All supplies of whole blood were tested prior to use and did not have any detectable amounts of the analytes of interest. Supplies of whole blood were kept refrigerated (2–8°C) for up to six weeks. Standard calibration working solutions were prepared daily in methanol at 15, 75, 150, 375, 750, and 1500 ng/mL of all five cannabinoids combined by serial dilution of the 100 µg/mL and 1000 µg/mL concentrated standard stock solutions. Following extraction from whole blood, the final standard concentrations were 1.0, 5.0, 10, 25, 50, and 100 ng/mL, determined based on the outcome of the LLOQ study. Quality control validation samples were prepared separately from the calibration standards using standard stock solutions dedicated for QC sample preparation. The internal standard (IS: Δ9-THC-D3) working solution was prepared in methanol at a concentration of 75 ng/mL using the 100 µg/mL concentrated stock solution. To prepare for extraction, 5.0 µL of each calibration standard (or QC validation) working solution and 5.0 µL IS working solution were spiked into appropriately labeled tubes containing 50 µL whole blood matrix mixed with 100 µL high-purity water. The final concentration of the IS was 5 ng/mL following extraction (final volume: 75 µL). After all samples had been spiked with IS and calibration standard or QC solutions, they were gently vortexed for 30 s to ensure thorough mixing of compounds with the matrix. Matrix blanks were spiked with 10 µL of methanol, and matrix blanks with IS were spiked with 5.0 µL methanol and 5.0 µL of the 75 µg/mL IS working solution. To extract cannabinoids from whole blood, 500 µL of a solution containing 90% n-hexane and 10% ethyl acetate (v/v) was added to each sample. After adding the extraction solvent, samples were vortexed vigorously for 30 s. After vortexing, samples were centrifuged at 0°C at a speed of 10 000 rpm [9300 rcf (relative centrifugal force)] for 10 min using an Eppendorf 5415R microcentrifuge. Following centrifugation, the supernatant (organic layer) was transferred to a 16 × 125 mm borosilicate glass culture tube. The samples were then placed in an N-Evap Model 112 analytical nitrogen evaporator (Organomation Associates, Berlin, MA) and evaporated to dryness under a gentle stream of nitrogen gas, with the water bath temperature set to 50°C. Once evaporation was complete (approximately 30 s), the samples were allowed to cool to room temperature (20–25°C; approximately 2 min) and reconstituted by adding 75 µL of a solution containing 65% acetonitrile and 35% water with 0.1% formic acid. After vigorously vortexing for 30 s, the samples were transferred into individual glass microinsert-equipped autosampler vials and placed in the autosampler compartment for analysis. Experimental Design Limit of detection To determine the LOD, a range of five different concentrations of the cannabinoid analytes and IS combined were prepared in methanol: 0.5, 0.75, 1.0, 2.5, and 5.0 ng/mL. Each concentration was analyzed in triplicate. Any sample with a signal to noise ratio of five or greater for any analyte compared to the blank sample was considered to be “detected.” To verify the LOD in blood, matrix-matched samples were prepared in the same concentration range. Lower limit of quantification The LLOQ is the lowest concentration level on the standard calibration curve that gives a signal to noise ratio of at least five compared to the matrix blank and has a coefficient of variation (CV) of 20% or less (13). Assay LLOQ was determined by preparing six replicates at each of five concentration levels (0.5, 1.0, 2.5, 5.0 and 10 ng/mL) in whole blood, extracting, and analyzing according to the described method. The CV was calculated using the average signal intensity ratio (peak area of analyte to IS) at each concentration level. Each sample was spiked to contain 5 ng/mL IS. The matrix blank was prepared by extracting a sample of the whole blood used to prepare the calibration standards and QC validation samples, which contained no added compounds other than the IS. Accuracy and precision The within run, or intra-run, accuracy and precision of the bioanalytical method following extraction from whole blood were calculated using five replicates at each level of the standard curve. These replicates were made using QC validation samples prepared separately from the calibration standards. Accuracy was calculated by subtracting the theoretical concentration from the average determined concentration and then dividing by the theoretical concentration. Precision, also known as the CV or relative standard deviation (RSD), was calculated by dividing the standard deviation (SD) by the mean. Both values are expressed as percentages. The accuracy and precision at each concentration level on the standard curve was subjected to the acceptance criteria given below. Between run, or inter-run, accuracy and precision were determined at low (3 ng/mL), intermediate (45 ng/mL) and high (90 ng/mL) controls prepared in whole blood. This was accomplished by performing accuracy and precision determinations at each of these concentration levels (n = 5) on 5 consecutive days. The determined concentration values for each day were averaged, and the accuracy and precision were calculated as described and subjected to the acceptance criteria given below. On each of the 5 consecutive days of inter-run accuracy and precision, a matrix blank and a matrix blank containing internal standard were extracted and analyzed. Pooled inter-run accuracy and precision were determined by combining the data from all 5 days for each cannabinoid analyte at each concentration level and subjected to the given acceptance criteria. Reinjection reproducibility (assay repeatability) The reinjection reproducibility or assay repeatability was determined by analyzing in succession six repetitions of the same low (3 ng/mL) and high concentration (90 ng/mL) prepared in whole blood and extracted according to the described method. The CV was calculated from the signal intensity ratios (analyte peak area to IS peak area). The CV must be 5% or less to be acceptable. Extraction efficiency The extraction efficiency of the analytes was determined by LC-HRMS. A low concentration (3 ng/mL) and a high concentration (90 ng/mL) of each cannabinoid analyte was added to whole blood and extracted according to the described method. To determine the theoretical maximum (100%) values, the same concentrations of cannabinoids were prepared in sample tubes containing no matrix. After samples were extracted, they were transferred to autosampler vials after being reconstituted and then analyzed according to the method. Average signal intensities obtained from stock solutions (not extracted) versus those obtained after extraction from whole blood were compared to obtain extraction efficiency values (n = 6). Method specificity To assess analytical method specificity, whole blood was used to prepare matrix blanks with and without IS each day of the 5-day inter-run accuracy and precision evaluation. These samples were analyzed along with the calibration standards and validation samples each day. Statistical Methods The statistical relationship between the standard calibration points was determined by using linear regression analysis to derive the equation of the line of best fit in the form y = mx + b, where y = peak area ratio (analyte to IS), m = slope of the regression line, and b = y-axis intercept. Concentrations of each cannabinoid analyte were automatically determined by the TraceFinder software. TraceFinder was configured to perform a linear curve fit with 1/Y weighting using Δ9-THC-D3 as the internal standard. Acceptance Criteria Acceptance criteria were based on the FDA’s industry guidance for bioanalytical method validation (13). Standard Calibration Curves For a standard curve to be accepted, the back-calculated values of at least 75%, or a minimum of five of the points should be within 15% of the theoretical concentration, except at the LLOQ where the value should be within 20% of the theoretical concentration. Back-calculated results were generated by fitting the signal intensity ratios of each calibration point to the standard curve. Accuracy and Precision For accuracy and precision determinations, average determined concentrations should be within 15% of the theoretical concentrations and the CV should be no more than 15%, except at the LLOQ, where average determined concentrations should be within 20% of the theoretical concentrations, with CVs not exceeding 20%. Clinical Study As part of the development of a test for determining recent use of cannabis (the RCU Test), an open-label clinical trial was conducted in 30 healthy human subjects between the ages of 21 and 29 who were self-reported frequent cannabis smokers. This study was conducted under a clinical protocol approved by the Institutional Review Board of the Cancer Immunotherapy Research Institute (CIRI). Subjects were instructed to abstain from using cannabis products for at least 12 h prior to their participation. After providing written informed consent, subjects were each given a cannabis cigarette to smoke. Blood samples were collected prior to smoking, immediately after smoking, and then at 20, 60, 80, 120, 140, 180, and 200 min after smoking. Capillary blood (approximately 50 µL) was collected into heparinized microsample tubes (BD, Franklin Lakes, NJ) by finger prick using 17-gauge lancets. All subjects were compensated for their participation in the study. The described analytical method was used to assess the blood concentrations of five cannabinoids and metabolites (Δ9-THC, CBN, 11-OH-Δ9-THC, 11-nor-9-carboxy-Δ9-THC, and 8β,11-diOH-Δ9-THC). Results Table 1 summarizes the analytical method target masses, retention times (RT), and confirmation ion settings for the five cannabinoid analytes and internal standard. The mass and RT tolerances employed in the method are included. Analyte verification further considered ion intensity ratios between isotopic ions and the molecular ions. Throughout the study, all RTs for the target analytes and IS fell within a target range of ± 2 s. Results for each phase of the validation study are detailed below. Figure 1 shows representative chromatograms at each level of the standard calibration curve. Figure 1. Open in new tabDownload slide Representative chromatograms of Δ9−THC, CBN, 11-OH-Δ9-THC, 11-nor-9-carboxy-Δ9-THC, 8β,11-diOH-Δ9-THC, Δ9-THC-D3. Figure 1. Open in new tabDownload slide Representative chromatograms of Δ9−THC, CBN, 11-OH-Δ9-THC, 11-nor-9-carboxy-Δ9-THC, 8β,11-diOH-Δ9-THC, Δ9-THC-D3. Table 1. Target masses, retention times (RTs), and confirming ions Compound . Target m/za . RT (min)b . Confirming ion 1 . Confirming ion 2 . Δ9-THC 315.2318 4.55 316.2345 317.2381 CBN 311.2005 3.99 312.2042 313.2077 11-nor-9-carboxy-Δ9-THC 345.2060 2.30 346.2099 347.2127 11-OH-Δ9-THC 331.2268 2.35 332.2304 333.2334 8β,11-diOH-Δ9-THC 329.2116 1.48 347.2216 348.2261 Δ9-THC-D3 318.2506 4.53 319.2353 315.2324 Compound . Target m/za . RT (min)b . Confirming ion 1 . Confirming ion 2 . Δ9-THC 315.2318 4.55 316.2345 317.2381 CBN 311.2005 3.99 312.2042 313.2077 11-nor-9-carboxy-Δ9-THC 345.2060 2.30 346.2099 347.2127 11-OH-Δ9-THC 331.2268 2.35 332.2304 333.2334 8β,11-diOH-Δ9-THC 329.2116 1.48 347.2216 348.2261 Δ9-THC-D3 318.2506 4.53 319.2353 315.2324 a Mass tolerance ± 5 ppm (parts per million). b RT tolerance ± 0.25 min. Open in new tab Table 1. Target masses, retention times (RTs), and confirming ions Compound . Target m/za . RT (min)b . Confirming ion 1 . Confirming ion 2 . Δ9-THC 315.2318 4.55 316.2345 317.2381 CBN 311.2005 3.99 312.2042 313.2077 11-nor-9-carboxy-Δ9-THC 345.2060 2.30 346.2099 347.2127 11-OH-Δ9-THC 331.2268 2.35 332.2304 333.2334 8β,11-diOH-Δ9-THC 329.2116 1.48 347.2216 348.2261 Δ9-THC-D3 318.2506 4.53 319.2353 315.2324 Compound . Target m/za . RT (min)b . Confirming ion 1 . Confirming ion 2 . Δ9-THC 315.2318 4.55 316.2345 317.2381 CBN 311.2005 3.99 312.2042 313.2077 11-nor-9-carboxy-Δ9-THC 345.2060 2.30 346.2099 347.2127 11-OH-Δ9-THC 331.2268 2.35 332.2304 333.2334 8β,11-diOH-Δ9-THC 329.2116 1.48 347.2216 348.2261 Δ9-THC-D3 318.2506 4.53 319.2353 315.2324 a Mass tolerance ± 5 ppm (parts per million). b RT tolerance ± 0.25 min. Open in new tab Limit of Detection The limits of detection for the five cannabinoids and the IS were determined by analyzing each of five different concentration levels in triplicate and comparing the average peak areas to the background noise level for each analyte in the blank sample. Table 2 shows the average peak areas for each of the five analytes and the IS at each concentration level. The signal intensities for all of the cannabinoid analytes at the 0.5 ng/mL concentration level exceeded five times the background (matrix blank) response, thus meeting the established detection criterion. Analysis of matrix-matched samples prepared in the same concentration range verified a 0.5 ng/mL LOD. Table 2. Limit of detection . Average peak area (n = 3) . Concentration (ng/mL) . Δ9-THC-D3 (IS) . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 0 (Matrix blank) Nfa Nf Nf Nf Nf Nf 0.5 26 135 36 688 6040 20 651 6550 511 0.75 55 932 59 221 19 370 32 554 12 355 935 1.0 72 932 83 190 28 202 47 680 20 365 538 2.5 202 606 192 479 82 703 125 794 60 671 5971 5.0 417 041 404 839 178 023 239 452 118 264 19 349 . Average peak area (n = 3) . Concentration (ng/mL) . Δ9-THC-D3 (IS) . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 0 (Matrix blank) Nfa Nf Nf Nf Nf Nf 0.5 26 135 36 688 6040 20 651 6550 511 0.75 55 932 59 221 19 370 32 554 12 355 935 1.0 72 932 83 190 28 202 47 680 20 365 538 2.5 202 606 192 479 82 703 125 794 60 671 5971 5.0 417 041 404 839 178 023 239 452 118 264 19 349 a Not found. Open in new tab Table 2. Limit of detection . Average peak area (n = 3) . Concentration (ng/mL) . Δ9-THC-D3 (IS) . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 0 (Matrix blank) Nfa Nf Nf Nf Nf Nf 0.5 26 135 36 688 6040 20 651 6550 511 0.75 55 932 59 221 19 370 32 554 12 355 935 1.0 72 932 83 190 28 202 47 680 20 365 538 2.5 202 606 192 479 82 703 125 794 60 671 5971 5.0 417 041 404 839 178 023 239 452 118 264 19 349 . Average peak area (n = 3) . Concentration (ng/mL) . Δ9-THC-D3 (IS) . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 0 (Matrix blank) Nfa Nf Nf Nf Nf Nf 0.5 26 135 36 688 6040 20 651 6550 511 0.75 55 932 59 221 19 370 32 554 12 355 935 1.0 72 932 83 190 28 202 47 680 20 365 538 2.5 202 606 192 479 82 703 125 794 60 671 5971 5.0 417 041 404 839 178 023 239 452 118 264 19 349 a Not found. Open in new tab Lower Limit of Quantification The LLOQ for the five cannabinoids was determined by analyzing six replicates at each of five different concentration levels. Table 3 shows the average peak area ratio, SD and CV for each of the cannabinoid analytes at the 1.0 ng/mL concentration level. The acceptance criteria for LLOQ were met at the 1.0 ng/mL concentration level for all of the cannabinoids, and thus the 1.0 ng/mL concentration was accepted as the LLOQ for the validation study (13). The remaining concentration levels chosen for the validation study were: 5, 10, 25, 50, and 100 ng/mL. Open in new tab Open in new tab Intra-run accuracy and precision The intra-run accuracy and precision of the LC-HRMS bioanalytical method in the chosen concentration range were assessed by analyzing five replicates of each of the 1.0, 5.0, 10, 25, 50, and 100 ng/mL cannabinoid calibration standards as unknowns. A separately prepared standard calibration curve was analyzed to determine the calculated concentrations. Table 4 summarizes the intra-run accuracy and precision of the method at the six different concentration levels. Table 4. Summary of intra-run accuracy and precision Theoretical (ng/mL) . Parameter . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 1.0 Average (ng/mL) determined 0.98 0.94 0.90 0.87 0.97 SD 0.18 0.06 0.14 0.10 0.10 Accuracy (%) −1.90 −6.36 −10.26 −12.76 −3.04 Precision (%) 17.89 6.12 15.32 11.20 10.02 5.0 Average (ng/mL) determined 4.51 4.56 4.81 4.58 4.92 SD 0.47 0.29 0.47 0.23 0.51 Accuracy (%) −9.78 −8.76 −3.83 −8.35 −1.67 Precision (%) 10.43 6.26 9.68 4.98 10.30 10 Average (ng/mL) determined 8.53 8.57 8.93 8.52 8.81 SD 0.39 0.32 0.48 0.47 0.41 Accuracy (%) −14.74 −14.27 −10.66 −14.82 −11.86 Precision (%) 4.61 3.79 5.43 5.46 4.69 25 Average (ng/mL) determined 21.23 21.08 22.96 21.38 22.11 SD 0.90 1.43 1.33 1.06 2.11 Accuracy (%) −15.09 −15.70 −8.16 −14.49 −11.57 Precision (%) 4.24 6.79 5.77 4.95 9.55 50 Average (ng/mL) determined 43.63 43.77 47.00 45.66 46.29 SD 1.20 1.54 2.36 2.00 2.04 Accuracy (%) −12.74 −12.46 −6.00 −8.68 −7.42 Precision (%) 2.76 3.52 5.03 4.38 4.42 100 Average (ng/mL) determined 85.50 85.63 90.38 88.35 88.21 SD 5.60 7.05 4.94 4.90 5.90 Accuracy (%) −14.50 −14.37 −9.62 −11.65 −11.79 Precision (%) 6.55 8.23 5.46 5.55 6.69 Theoretical (ng/mL) . Parameter . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 1.0 Average (ng/mL) determined 0.98 0.94 0.90 0.87 0.97 SD 0.18 0.06 0.14 0.10 0.10 Accuracy (%) −1.90 −6.36 −10.26 −12.76 −3.04 Precision (%) 17.89 6.12 15.32 11.20 10.02 5.0 Average (ng/mL) determined 4.51 4.56 4.81 4.58 4.92 SD 0.47 0.29 0.47 0.23 0.51 Accuracy (%) −9.78 −8.76 −3.83 −8.35 −1.67 Precision (%) 10.43 6.26 9.68 4.98 10.30 10 Average (ng/mL) determined 8.53 8.57 8.93 8.52 8.81 SD 0.39 0.32 0.48 0.47 0.41 Accuracy (%) −14.74 −14.27 −10.66 −14.82 −11.86 Precision (%) 4.61 3.79 5.43 5.46 4.69 25 Average (ng/mL) determined 21.23 21.08 22.96 21.38 22.11 SD 0.90 1.43 1.33 1.06 2.11 Accuracy (%) −15.09 −15.70 −8.16 −14.49 −11.57 Precision (%) 4.24 6.79 5.77 4.95 9.55 50 Average (ng/mL) determined 43.63 43.77 47.00 45.66 46.29 SD 1.20 1.54 2.36 2.00 2.04 Accuracy (%) −12.74 −12.46 −6.00 −8.68 −7.42 Precision (%) 2.76 3.52 5.03 4.38 4.42 100 Average (ng/mL) determined 85.50 85.63 90.38 88.35 88.21 SD 5.60 7.05 4.94 4.90 5.90 Accuracy (%) −14.50 −14.37 −9.62 −11.65 −11.79 Precision (%) 6.55 8.23 5.46 5.55 6.69 Open in new tab Table 4. Summary of intra-run accuracy and precision Theoretical (ng/mL) . Parameter . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 1.0 Average (ng/mL) determined 0.98 0.94 0.90 0.87 0.97 SD 0.18 0.06 0.14 0.10 0.10 Accuracy (%) −1.90 −6.36 −10.26 −12.76 −3.04 Precision (%) 17.89 6.12 15.32 11.20 10.02 5.0 Average (ng/mL) determined 4.51 4.56 4.81 4.58 4.92 SD 0.47 0.29 0.47 0.23 0.51 Accuracy (%) −9.78 −8.76 −3.83 −8.35 −1.67 Precision (%) 10.43 6.26 9.68 4.98 10.30 10 Average (ng/mL) determined 8.53 8.57 8.93 8.52 8.81 SD 0.39 0.32 0.48 0.47 0.41 Accuracy (%) −14.74 −14.27 −10.66 −14.82 −11.86 Precision (%) 4.61 3.79 5.43 5.46 4.69 25 Average (ng/mL) determined 21.23 21.08 22.96 21.38 22.11 SD 0.90 1.43 1.33 1.06 2.11 Accuracy (%) −15.09 −15.70 −8.16 −14.49 −11.57 Precision (%) 4.24 6.79 5.77 4.95 9.55 50 Average (ng/mL) determined 43.63 43.77 47.00 45.66 46.29 SD 1.20 1.54 2.36 2.00 2.04 Accuracy (%) −12.74 −12.46 −6.00 −8.68 −7.42 Precision (%) 2.76 3.52 5.03 4.38 4.42 100 Average (ng/mL) determined 85.50 85.63 90.38 88.35 88.21 SD 5.60 7.05 4.94 4.90 5.90 Accuracy (%) −14.50 −14.37 −9.62 −11.65 −11.79 Precision (%) 6.55 8.23 5.46 5.55 6.69 Theoretical (ng/mL) . Parameter . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 1.0 Average (ng/mL) determined 0.98 0.94 0.90 0.87 0.97 SD 0.18 0.06 0.14 0.10 0.10 Accuracy (%) −1.90 −6.36 −10.26 −12.76 −3.04 Precision (%) 17.89 6.12 15.32 11.20 10.02 5.0 Average (ng/mL) determined 4.51 4.56 4.81 4.58 4.92 SD 0.47 0.29 0.47 0.23 0.51 Accuracy (%) −9.78 −8.76 −3.83 −8.35 −1.67 Precision (%) 10.43 6.26 9.68 4.98 10.30 10 Average (ng/mL) determined 8.53 8.57 8.93 8.52 8.81 SD 0.39 0.32 0.48 0.47 0.41 Accuracy (%) −14.74 −14.27 −10.66 −14.82 −11.86 Precision (%) 4.61 3.79 5.43 5.46 4.69 25 Average (ng/mL) determined 21.23 21.08 22.96 21.38 22.11 SD 0.90 1.43 1.33 1.06 2.11 Accuracy (%) −15.09 −15.70 −8.16 −14.49 −11.57 Precision (%) 4.24 6.79 5.77 4.95 9.55 50 Average (ng/mL) determined 43.63 43.77 47.00 45.66 46.29 SD 1.20 1.54 2.36 2.00 2.04 Accuracy (%) −12.74 −12.46 −6.00 −8.68 −7.42 Precision (%) 2.76 3.52 5.03 4.38 4.42 100 Average (ng/mL) determined 85.50 85.63 90.38 88.35 88.21 SD 5.60 7.05 4.94 4.90 5.90 Accuracy (%) −14.50 −14.37 −9.62 −11.65 −11.79 Precision (%) 6.55 8.23 5.46 5.55 6.69 Open in new tab As shown in Table 4, the accuracy and precision of the method in the range between 1.0 ng/mL and 100 ng/mL met the described acceptance criteria for all compounds at all concentration levels (13). Although the average determined concentrations of Δ9-THC and CBN were slightly low at the 25 ng/mL level, all other analytes at this level, including Δ9-THC and CBN at all other concentration levels, met the acceptance criteria. The standard calibration curves used to determine the calculated concentrations in Table 4 also met the acceptance criteria for all compounds at all concentration levels (see Table 5). Table 5. Summary of intra-run accuracy and precision calibration curves . Determined concentration, ng/mL [Accuracy (%)] . Theoretical concentration (ng/mL) . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 1.0 1.03 [3.20] 1.02 [2.00] 1.09 [9.20] 1.06 [5.50] 1.09 [8.90] 5.0 4.78 [−4.44] 4.94 [−1.18] 5.01 [0.16] 5.15 [2.92] 5.18 [3.68] 10 10.07 [0.66] 9.69 [−3.14] 9.59 [−4.06] 9.58 [−4.25] 9.54 [−4.56] 25 26.42 [5.67] 26.96 [7.84] 25.65 [2.58] 25.18 [0.72] 24.79 [−0.82] 50 47.21 [−5.57] 46.72 [−6.56] 43.76 [−12.47] 44.78 [−10.44] 42.90 [−14.20] 100 101.78 [1.78] 102.10 [2.10] 107.33 [7.33] 106.28 [6.28] 109.54 [9.54] r2 0.9983 0.9974 0.9913 0.9940 0.9878 . Determined concentration, ng/mL [Accuracy (%)] . Theoretical concentration (ng/mL) . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 1.0 1.03 [3.20] 1.02 [2.00] 1.09 [9.20] 1.06 [5.50] 1.09 [8.90] 5.0 4.78 [−4.44] 4.94 [−1.18] 5.01 [0.16] 5.15 [2.92] 5.18 [3.68] 10 10.07 [0.66] 9.69 [−3.14] 9.59 [−4.06] 9.58 [−4.25] 9.54 [−4.56] 25 26.42 [5.67] 26.96 [7.84] 25.65 [2.58] 25.18 [0.72] 24.79 [−0.82] 50 47.21 [−5.57] 46.72 [−6.56] 43.76 [−12.47] 44.78 [−10.44] 42.90 [−14.20] 100 101.78 [1.78] 102.10 [2.10] 107.33 [7.33] 106.28 [6.28] 109.54 [9.54] r2 0.9983 0.9974 0.9913 0.9940 0.9878 Open in new tab Table 5. Summary of intra-run accuracy and precision calibration curves . Determined concentration, ng/mL [Accuracy (%)] . Theoretical concentration (ng/mL) . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 1.0 1.03 [3.20] 1.02 [2.00] 1.09 [9.20] 1.06 [5.50] 1.09 [8.90] 5.0 4.78 [−4.44] 4.94 [−1.18] 5.01 [0.16] 5.15 [2.92] 5.18 [3.68] 10 10.07 [0.66] 9.69 [−3.14] 9.59 [−4.06] 9.58 [−4.25] 9.54 [−4.56] 25 26.42 [5.67] 26.96 [7.84] 25.65 [2.58] 25.18 [0.72] 24.79 [−0.82] 50 47.21 [−5.57] 46.72 [−6.56] 43.76 [−12.47] 44.78 [−10.44] 42.90 [−14.20] 100 101.78 [1.78] 102.10 [2.10] 107.33 [7.33] 106.28 [6.28] 109.54 [9.54] r2 0.9983 0.9974 0.9913 0.9940 0.9878 . Determined concentration, ng/mL [Accuracy (%)] . Theoretical concentration (ng/mL) . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 1.0 1.03 [3.20] 1.02 [2.00] 1.09 [9.20] 1.06 [5.50] 1.09 [8.90] 5.0 4.78 [−4.44] 4.94 [−1.18] 5.01 [0.16] 5.15 [2.92] 5.18 [3.68] 10 10.07 [0.66] 9.69 [−3.14] 9.59 [−4.06] 9.58 [−4.25] 9.54 [−4.56] 25 26.42 [5.67] 26.96 [7.84] 25.65 [2.58] 25.18 [0.72] 24.79 [−0.82] 50 47.21 [−5.57] 46.72 [−6.56] 43.76 [−12.47] 44.78 [−10.44] 42.90 [−14.20] 100 101.78 [1.78] 102.10 [2.10] 107.33 [7.33] 106.28 [6.28] 109.54 [9.54] r2 0.9983 0.9974 0.9913 0.9940 0.9878 Open in new tab Inter-run accuracy and precision Inter-run accuracy and precision were determined using a low (3 ng/mL), intermediate (45 ng/mL) and high (90 ng/mL) control. This was accomplished by performing accuracy and precision determinations at each concentration level (n = 5) on 5 consecutive days. Tables 6–8 show the pooled inter-run accuracy and precision data at these concentration levels. As shown in the tables, the pooled inter-run accuracy and precision data for all five cannabinoids were within the stated acceptance criteria at all three concentration levels. As with the intra-run validation data, all standard calibration curves for all components during the 5-day inter-run validation met the established acceptance criteria for accuracy (13), and all r2 values were >0.99. Representative calibration curves for each of the five analytes are shown in Figure 2. Figure 2. Open in new tabDownload slide Representative calibration curves for Δ9−THC, CBN, 11-OH-Δ9-THC, 11-nor-9-carboxy-Δ9-THC, and 8β,11-diOH-Δ9-THC. Figure 2. Open in new tabDownload slide Representative calibration curves for Δ9−THC, CBN, 11-OH-Δ9-THC, 11-nor-9-carboxy-Δ9-THC, and 8β,11-diOH-Δ9-THC. Table 6. Pooled inter-run accuracy and precision: theoretical concentration 3 ng/mL Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 3 at Day 1 2.78 2.70 3.03 3.10 3.01 3 at Day 2 2.75 2.73 2.92 2.89 2.87 3 at Day 3 2.99 2.84 3.04 2.98 3.05 3 at Day 4 3.12 2.87 3.02 3.08 3.04 3 at Day 5 2.88 2.68 2.71 2.79 2.76 Average 2.90 2.76 2.94 2.97 2.95 SD 0.16 0.09 0.14 0.13 0.13 Accuracy, % −3.18 −7.87 −1.87 −0.99 −1.79 Precision, % 5.34 3.18 4.75 4.46 4.29 Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 3 at Day 1 2.78 2.70 3.03 3.10 3.01 3 at Day 2 2.75 2.73 2.92 2.89 2.87 3 at Day 3 2.99 2.84 3.04 2.98 3.05 3 at Day 4 3.12 2.87 3.02 3.08 3.04 3 at Day 5 2.88 2.68 2.71 2.79 2.76 Average 2.90 2.76 2.94 2.97 2.95 SD 0.16 0.09 0.14 0.13 0.13 Accuracy, % −3.18 −7.87 −1.87 −0.99 −1.79 Precision, % 5.34 3.18 4.75 4.46 4.29 Open in new tab Table 6. Pooled inter-run accuracy and precision: theoretical concentration 3 ng/mL Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 3 at Day 1 2.78 2.70 3.03 3.10 3.01 3 at Day 2 2.75 2.73 2.92 2.89 2.87 3 at Day 3 2.99 2.84 3.04 2.98 3.05 3 at Day 4 3.12 2.87 3.02 3.08 3.04 3 at Day 5 2.88 2.68 2.71 2.79 2.76 Average 2.90 2.76 2.94 2.97 2.95 SD 0.16 0.09 0.14 0.13 0.13 Accuracy, % −3.18 −7.87 −1.87 −0.99 −1.79 Precision, % 5.34 3.18 4.75 4.46 4.29 Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 3 at Day 1 2.78 2.70 3.03 3.10 3.01 3 at Day 2 2.75 2.73 2.92 2.89 2.87 3 at Day 3 2.99 2.84 3.04 2.98 3.05 3 at Day 4 3.12 2.87 3.02 3.08 3.04 3 at Day 5 2.88 2.68 2.71 2.79 2.76 Average 2.90 2.76 2.94 2.97 2.95 SD 0.16 0.09 0.14 0.13 0.13 Accuracy, % −3.18 −7.87 −1.87 −0.99 −1.79 Precision, % 5.34 3.18 4.75 4.46 4.29 Open in new tab Table 7. Pooled inter-run accuracy and precision: theoretical concentration 45 ng/mL Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 45 at Day 1 41.41 39.50 42.36 42.77 43.82 45 at Day 2 43.67 40.56 46.45 43.32 44.06 45 at Day 3 41.95 40.48 41.37 41.15 39.26 45 at Day 4 41.17 39.64 41.70 42.15 42.30 45 at Day 5 38.76 37.12 41.99 39.75 39.46 Average 41.39 39.46 42.78 41.83 41.78 SD 1.77 1.39 2.09 1.41 2.31 Accuracy, % −8.02 −12.31 −4.94 −7.05 −7.15 Precision, % 4.27 3.53 4.88 3.38 5.54 Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 45 at Day 1 41.41 39.50 42.36 42.77 43.82 45 at Day 2 43.67 40.56 46.45 43.32 44.06 45 at Day 3 41.95 40.48 41.37 41.15 39.26 45 at Day 4 41.17 39.64 41.70 42.15 42.30 45 at Day 5 38.76 37.12 41.99 39.75 39.46 Average 41.39 39.46 42.78 41.83 41.78 SD 1.77 1.39 2.09 1.41 2.31 Accuracy, % −8.02 −12.31 −4.94 −7.05 −7.15 Precision, % 4.27 3.53 4.88 3.38 5.54 Open in new tab Table 7. Pooled inter-run accuracy and precision: theoretical concentration 45 ng/mL Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 45 at Day 1 41.41 39.50 42.36 42.77 43.82 45 at Day 2 43.67 40.56 46.45 43.32 44.06 45 at Day 3 41.95 40.48 41.37 41.15 39.26 45 at Day 4 41.17 39.64 41.70 42.15 42.30 45 at Day 5 38.76 37.12 41.99 39.75 39.46 Average 41.39 39.46 42.78 41.83 41.78 SD 1.77 1.39 2.09 1.41 2.31 Accuracy, % −8.02 −12.31 −4.94 −7.05 −7.15 Precision, % 4.27 3.53 4.88 3.38 5.54 Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 45 at Day 1 41.41 39.50 42.36 42.77 43.82 45 at Day 2 43.67 40.56 46.45 43.32 44.06 45 at Day 3 41.95 40.48 41.37 41.15 39.26 45 at Day 4 41.17 39.64 41.70 42.15 42.30 45 at Day 5 38.76 37.12 41.99 39.75 39.46 Average 41.39 39.46 42.78 41.83 41.78 SD 1.77 1.39 2.09 1.41 2.31 Accuracy, % −8.02 −12.31 −4.94 −7.05 −7.15 Precision, % 4.27 3.53 4.88 3.38 5.54 Open in new tab Table 8. Pooled inter-run accuracy and precision: theoretical concentration 90 ng/mL Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 90 at Day 1 82.92 79.07 87.65 88.22 88.83 90 at Day 2 82.62 76.46 87.19 82.25 81.72 90 at Day 3 84.44 81.05 93.20 90.98 87.27 90 at Day 4 82.67 78.96 82.11 86.36 83.85 90 at Day 5 78.74 76.56 85.33 83.42 79.27 Average 82.28 78.42 87.09 86.25 84.19 SD 2.12 1.93 4.05 3.55 3.92 Accuracy, % −8.58 −12.87 −3.23 −4.17 −6.46 Precision, % 2.57 2.46 4.65 4.11 4.66 Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 90 at Day 1 82.92 79.07 87.65 88.22 88.83 90 at Day 2 82.62 76.46 87.19 82.25 81.72 90 at Day 3 84.44 81.05 93.20 90.98 87.27 90 at Day 4 82.67 78.96 82.11 86.36 83.85 90 at Day 5 78.74 76.56 85.33 83.42 79.27 Average 82.28 78.42 87.09 86.25 84.19 SD 2.12 1.93 4.05 3.55 3.92 Accuracy, % −8.58 −12.87 −3.23 −4.17 −6.46 Precision, % 2.57 2.46 4.65 4.11 4.66 Open in new tab Table 8. Pooled inter-run accuracy and precision: theoretical concentration 90 ng/mL Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 90 at Day 1 82.92 79.07 87.65 88.22 88.83 90 at Day 2 82.62 76.46 87.19 82.25 81.72 90 at Day 3 84.44 81.05 93.20 90.98 87.27 90 at Day 4 82.67 78.96 82.11 86.36 83.85 90 at Day 5 78.74 76.56 85.33 83.42 79.27 Average 82.28 78.42 87.09 86.25 84.19 SD 2.12 1.93 4.05 3.55 3.92 Accuracy, % −8.58 −12.87 −3.23 −4.17 −6.46 Precision, % 2.57 2.46 4.65 4.11 4.66 Theoretical concentration, ng/mL . Average determined concentrations, ng/mL . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 90 at Day 1 82.92 79.07 87.65 88.22 88.83 90 at Day 2 82.62 76.46 87.19 82.25 81.72 90 at Day 3 84.44 81.05 93.20 90.98 87.27 90 at Day 4 82.67 78.96 82.11 86.36 83.85 90 at Day 5 78.74 76.56 85.33 83.42 79.27 Average 82.28 78.42 87.09 86.25 84.19 SD 2.12 1.93 4.05 3.55 3.92 Accuracy, % −8.58 −12.87 −3.23 −4.17 −6.46 Precision, % 2.57 2.46 4.65 4.11 4.66 Open in new tab Reinjection Reproducibility (Assay Repeatability) To assess the assay repeatability, six repetitions each of two different standard concentrations were analyzed. Table 9 shows the assay repeatability for the concentrations 3 and 90 ng/mL based on average peak area ratios. For each concentration level, the coefficients of variation for all cannabinoid components were found to be within 5%. Table 9. Assay repeatability . . Peak area ratios . Concentration, ng/mL . Parameter . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 3.0 Average 0.61220 0.39957 0.07800 0.18097 0.07319 SD 0.00925 0.01052 0.00335 0.00595 0.00245 CV, % 1.51 2.63 4.30 3.29 3.35 90 Average 18.59461 12.55071 2.47570 6.15605 2.46247 SD 0.32153 0.23843 0.06478 0.10779 0.05858 CV, % 1.73 1.90 2.62 1.75 2.38 . . Peak area ratios . Concentration, ng/mL . Parameter . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 3.0 Average 0.61220 0.39957 0.07800 0.18097 0.07319 SD 0.00925 0.01052 0.00335 0.00595 0.00245 CV, % 1.51 2.63 4.30 3.29 3.35 90 Average 18.59461 12.55071 2.47570 6.15605 2.46247 SD 0.32153 0.23843 0.06478 0.10779 0.05858 CV, % 1.73 1.90 2.62 1.75 2.38 Open in new tab Table 9. Assay repeatability . . Peak area ratios . Concentration, ng/mL . Parameter . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 3.0 Average 0.61220 0.39957 0.07800 0.18097 0.07319 SD 0.00925 0.01052 0.00335 0.00595 0.00245 CV, % 1.51 2.63 4.30 3.29 3.35 90 Average 18.59461 12.55071 2.47570 6.15605 2.46247 SD 0.32153 0.23843 0.06478 0.10779 0.05858 CV, % 1.73 1.90 2.62 1.75 2.38 . . Peak area ratios . Concentration, ng/mL . Parameter . Δ9-THC . CBN . 11-nor-9-carboxy-Δ9-THC . 11-OH-Δ9-THC . 8β,11-diOH-Δ9-THC . 3.0 Average 0.61220 0.39957 0.07800 0.18097 0.07319 SD 0.00925 0.01052 0.00335 0.00595 0.00245 CV, % 1.51 2.63 4.30 3.29 3.35 90 Average 18.59461 12.55071 2.47570 6.15605 2.46247 SD 0.32153 0.23843 0.06478 0.10779 0.05858 CV, % 1.73 1.90 2.62 1.75 2.38 Open in new tab Extraction Efficiency The extraction efficiency of the five cannabinoid analytes and the IS from the whole blood matrix was assessed by analyzing six replicates of a low (3 ng/mL) and a high (90 ng/mL) control added to whole blood and extracted according to the stated method. Average peak areas obtained from stock solutions (not extracted) were compared to those obtained following extraction from the matrix for each cannabinoid component and the IS to obtain extraction efficiency values (seeTable 10). Recovery of the five cannabinoid analytes and the IS from the whole blood matrix using the described extraction method was acceptable except for 11-nor-9-carboxy-Δ9-THC. Although the recovery of 11-nor-9-carboxy-Δ9-THC was relatively low, this did not present a problem, as the method is highly sensitive for this metabolite as well as for the other analytes. Table 10. Extraction efficiency Concentration, ng/mL . Δ9-THC-D3 (IS), % . Δ9-THC, % . CBN, % . 11-nor-9-carboxy-Δ9-THC, % . 11-OH-Δ9-THC, % . 8β,11-diOH-Δ9-THC, % . 3.0 39.8 46.9 43.6 18.6 60.0 38.6 90 41.7 39.5 39.2 18.0 57.8 34.3 Concentration, ng/mL . Δ9-THC-D3 (IS), % . Δ9-THC, % . CBN, % . 11-nor-9-carboxy-Δ9-THC, % . 11-OH-Δ9-THC, % . 8β,11-diOH-Δ9-THC, % . 3.0 39.8 46.9 43.6 18.6 60.0 38.6 90 41.7 39.5 39.2 18.0 57.8 34.3 Open in new tab Table 10. Extraction efficiency Concentration, ng/mL . Δ9-THC-D3 (IS), % . Δ9-THC, % . CBN, % . 11-nor-9-carboxy-Δ9-THC, % . 11-OH-Δ9-THC, % . 8β,11-diOH-Δ9-THC, % . 3.0 39.8 46.9 43.6 18.6 60.0 38.6 90 41.7 39.5 39.2 18.0 57.8 34.3 Concentration, ng/mL . Δ9-THC-D3 (IS), % . Δ9-THC, % . CBN, % . 11-nor-9-carboxy-Δ9-THC, % . 11-OH-Δ9-THC, % . 8β,11-diOH-Δ9-THC, % . 3.0 39.8 46.9 43.6 18.6 60.0 38.6 90 41.7 39.5 39.2 18.0 57.8 34.3 Open in new tab Method Specificity In the LOD study, no analytes were detected in the solvent blanks. At the assay LLOQ (1 ng/mL), calibration standards for all analytes showed an average peak area ratio of at least nine times the background noise seen in the whole blood matrix blanks that were prepared with and without internal standard and analyzed during the 5-day inter-run accuracy and precision evaluation. Clinical Study In the clinical trial conducted as part of the development of the RCU Test, blood collected from 30 human subjects at various time points before and after smoking was analyzed using the described method. Table 11 shows the concentration ratios between 11-nor-9-carboxy-Δ9-THC and Δ9-THC at baseline through 200 min post-smoking. As seen from the median values, the ratio falls substantially between baseline and the time immediately after smoking, gradually returning to near baseline levels by 200 min post-smoking, a pattern typically seen with recent cannabis use. The RCU Test employs this ratio, in conjunction with other indicators, to assess the probability of recent cannabis use in a subject. Table 11. 11-nor-9-carboxy-Δ9-THC/Δ9-THC concentration ratios before and after smoking cannabis in human subjects Subject . 11-nor-9-carboxy-Δ9-THC/Δ9-THC concentration ratios (min after smoking) . Baseline . Immediately after smoking . 20 . 60 . 80 . 120 . 140 . 180 . 200 . 1 4.01 0.17 0.65 0.78 1.75 0.69 0.92 0.50 1.52 2 1.59 0.03 0.16 0.47 0.45 0.22 0.23 0.05 0.63 3 0.24 <0.01 0.05 0.06 0.02 0.04 0.09 0.19 0.30 4 2.12 0.22 1.93 2.34 0.65 1.63 2.05 2.63 0.86 5 0.13 0.08 0.11 0.65 0.72 0.60 0.28 0.34 0.54 6 2.90 0.24 0.30 0.77 0.94 1.60 2.28 1.82 2.08 7 10.67 0.70 5.12 10.01 9.41 9.25 14.59 20.79 23.80 8 1.06 0.37 0.69 2.23 1.39 1.14 1.43 1.86 2.07 9 2.35 0.26 0.76 1.37 1.37 1.03 0.49 1.45 1.56 10 8.50 0.79 3.38 4.81 5.16 7.25 7.61 6.96 8.44 11 23.95 0.87 4.27 11.53 11.30 9.93 10.60 11.62 9.77 12 33.67 1.47 16.53 13.12 9.92 12.96 11.39 20.34 12.66 13 8.50 0.58 3.01 3.71 3.75 3.01 7.65 2.46 5.48 14 21.39 1.39 5.83 17.84 12.29 14.87 21.47 15.29 19.08 15 17.81 0.40 2.88 5.01 1.32 15.06 13.45 4.42 17.70 16 15.50 2.66 16.90 6.95 11.19 6.64 15.33 19.30 14.97 17 34.60 10.78 34.57 72.26 55.36 78.40 42.50 44.54 28.24 18 3.00 0.23 1.45 1.38 1.96 3.28 2.35 2.92 2.72 19 13.23 0.43 3.30 3.58 5.73 6.50 5.59 5.65 4.52 20 0.36 0.07 0.46 1.67 1.53 0.56 0.77 0.64 0.50 21 8.14 0.30 1.49 3.06 3.14 5.27 5.57 6.23 6.67 22 0.56 0.28 0.60 1.74 0.86 2.49 0.97 3.63 3.29 23 2.36 0.22 0.63 0.98 0.90 1.75 1.91 2.36 2.56 24 9.08 2.26 2.84 5.82 8.06 8.15 7.04 8.63 9.10 25 2.58 0.23 2.76 3.15 3.65 3.72 1.37 3.23 4.30 26 5.34 0.32 1.48 2.55 1.45 1.75 2.93 —a 3.34 27 23.71 0.15 1.25 0.40 2.33 2.98 3.75 1.50 3.55 28 1.56 0.39 6.18 3.10 3.79 9.84 7.33 7.95 6.78 29 0.87 0.79 1.68 1.87 2.32 0.97 1.58 3.35 1.49 30 6.12 0.69 1.51 1.18 0.96 0.69 0.98 0.70 1.71 Median 4.68 0.34 1.60 2.45 2.14 3.00 2.64 3.23 3.44 Range 0.13–34.60 0.004-–0.78 0.05–34.57 0.06–72.26 0.02–55.36 0.04–78.40 0.09–42.50 0.05–44.54 0.30–28.24 Subject . 11-nor-9-carboxy-Δ9-THC/Δ9-THC concentration ratios (min after smoking) . Baseline . Immediately after smoking . 20 . 60 . 80 . 120 . 140 . 180 . 200 . 1 4.01 0.17 0.65 0.78 1.75 0.69 0.92 0.50 1.52 2 1.59 0.03 0.16 0.47 0.45 0.22 0.23 0.05 0.63 3 0.24 <0.01 0.05 0.06 0.02 0.04 0.09 0.19 0.30 4 2.12 0.22 1.93 2.34 0.65 1.63 2.05 2.63 0.86 5 0.13 0.08 0.11 0.65 0.72 0.60 0.28 0.34 0.54 6 2.90 0.24 0.30 0.77 0.94 1.60 2.28 1.82 2.08 7 10.67 0.70 5.12 10.01 9.41 9.25 14.59 20.79 23.80 8 1.06 0.37 0.69 2.23 1.39 1.14 1.43 1.86 2.07 9 2.35 0.26 0.76 1.37 1.37 1.03 0.49 1.45 1.56 10 8.50 0.79 3.38 4.81 5.16 7.25 7.61 6.96 8.44 11 23.95 0.87 4.27 11.53 11.30 9.93 10.60 11.62 9.77 12 33.67 1.47 16.53 13.12 9.92 12.96 11.39 20.34 12.66 13 8.50 0.58 3.01 3.71 3.75 3.01 7.65 2.46 5.48 14 21.39 1.39 5.83 17.84 12.29 14.87 21.47 15.29 19.08 15 17.81 0.40 2.88 5.01 1.32 15.06 13.45 4.42 17.70 16 15.50 2.66 16.90 6.95 11.19 6.64 15.33 19.30 14.97 17 34.60 10.78 34.57 72.26 55.36 78.40 42.50 44.54 28.24 18 3.00 0.23 1.45 1.38 1.96 3.28 2.35 2.92 2.72 19 13.23 0.43 3.30 3.58 5.73 6.50 5.59 5.65 4.52 20 0.36 0.07 0.46 1.67 1.53 0.56 0.77 0.64 0.50 21 8.14 0.30 1.49 3.06 3.14 5.27 5.57 6.23 6.67 22 0.56 0.28 0.60 1.74 0.86 2.49 0.97 3.63 3.29 23 2.36 0.22 0.63 0.98 0.90 1.75 1.91 2.36 2.56 24 9.08 2.26 2.84 5.82 8.06 8.15 7.04 8.63 9.10 25 2.58 0.23 2.76 3.15 3.65 3.72 1.37 3.23 4.30 26 5.34 0.32 1.48 2.55 1.45 1.75 2.93 —a 3.34 27 23.71 0.15 1.25 0.40 2.33 2.98 3.75 1.50 3.55 28 1.56 0.39 6.18 3.10 3.79 9.84 7.33 7.95 6.78 29 0.87 0.79 1.68 1.87 2.32 0.97 1.58 3.35 1.49 30 6.12 0.69 1.51 1.18 0.96 0.69 0.98 0.70 1.71 Median 4.68 0.34 1.60 2.45 2.14 3.00 2.64 3.23 3.44 Range 0.13–34.60 0.004-–0.78 0.05–34.57 0.06–72.26 0.02–55.36 0.04–78.40 0.09–42.50 0.05–44.54 0.30–28.24 a Δ9-THC undetectable in this sample. Open in new tab Table 11. 11-nor-9-carboxy-Δ9-THC/Δ9-THC concentration ratios before and after smoking cannabis in human subjects Subject . 11-nor-9-carboxy-Δ9-THC/Δ9-THC concentration ratios (min after smoking) . Baseline . Immediately after smoking . 20 . 60 . 80 . 120 . 140 . 180 . 200 . 1 4.01 0.17 0.65 0.78 1.75 0.69 0.92 0.50 1.52 2 1.59 0.03 0.16 0.47 0.45 0.22 0.23 0.05 0.63 3 0.24 <0.01 0.05 0.06 0.02 0.04 0.09 0.19 0.30 4 2.12 0.22 1.93 2.34 0.65 1.63 2.05 2.63 0.86 5 0.13 0.08 0.11 0.65 0.72 0.60 0.28 0.34 0.54 6 2.90 0.24 0.30 0.77 0.94 1.60 2.28 1.82 2.08 7 10.67 0.70 5.12 10.01 9.41 9.25 14.59 20.79 23.80 8 1.06 0.37 0.69 2.23 1.39 1.14 1.43 1.86 2.07 9 2.35 0.26 0.76 1.37 1.37 1.03 0.49 1.45 1.56 10 8.50 0.79 3.38 4.81 5.16 7.25 7.61 6.96 8.44 11 23.95 0.87 4.27 11.53 11.30 9.93 10.60 11.62 9.77 12 33.67 1.47 16.53 13.12 9.92 12.96 11.39 20.34 12.66 13 8.50 0.58 3.01 3.71 3.75 3.01 7.65 2.46 5.48 14 21.39 1.39 5.83 17.84 12.29 14.87 21.47 15.29 19.08 15 17.81 0.40 2.88 5.01 1.32 15.06 13.45 4.42 17.70 16 15.50 2.66 16.90 6.95 11.19 6.64 15.33 19.30 14.97 17 34.60 10.78 34.57 72.26 55.36 78.40 42.50 44.54 28.24 18 3.00 0.23 1.45 1.38 1.96 3.28 2.35 2.92 2.72 19 13.23 0.43 3.30 3.58 5.73 6.50 5.59 5.65 4.52 20 0.36 0.07 0.46 1.67 1.53 0.56 0.77 0.64 0.50 21 8.14 0.30 1.49 3.06 3.14 5.27 5.57 6.23 6.67 22 0.56 0.28 0.60 1.74 0.86 2.49 0.97 3.63 3.29 23 2.36 0.22 0.63 0.98 0.90 1.75 1.91 2.36 2.56 24 9.08 2.26 2.84 5.82 8.06 8.15 7.04 8.63 9.10 25 2.58 0.23 2.76 3.15 3.65 3.72 1.37 3.23 4.30 26 5.34 0.32 1.48 2.55 1.45 1.75 2.93 —a 3.34 27 23.71 0.15 1.25 0.40 2.33 2.98 3.75 1.50 3.55 28 1.56 0.39 6.18 3.10 3.79 9.84 7.33 7.95 6.78 29 0.87 0.79 1.68 1.87 2.32 0.97 1.58 3.35 1.49 30 6.12 0.69 1.51 1.18 0.96 0.69 0.98 0.70 1.71 Median 4.68 0.34 1.60 2.45 2.14 3.00 2.64 3.23 3.44 Range 0.13–34.60 0.004-–0.78 0.05–34.57 0.06–72.26 0.02–55.36 0.04–78.40 0.09–42.50 0.05–44.54 0.30–28.24 Subject . 11-nor-9-carboxy-Δ9-THC/Δ9-THC concentration ratios (min after smoking) . Baseline . Immediately after smoking . 20 . 60 . 80 . 120 . 140 . 180 . 200 . 1 4.01 0.17 0.65 0.78 1.75 0.69 0.92 0.50 1.52 2 1.59 0.03 0.16 0.47 0.45 0.22 0.23 0.05 0.63 3 0.24 <0.01 0.05 0.06 0.02 0.04 0.09 0.19 0.30 4 2.12 0.22 1.93 2.34 0.65 1.63 2.05 2.63 0.86 5 0.13 0.08 0.11 0.65 0.72 0.60 0.28 0.34 0.54 6 2.90 0.24 0.30 0.77 0.94 1.60 2.28 1.82 2.08 7 10.67 0.70 5.12 10.01 9.41 9.25 14.59 20.79 23.80 8 1.06 0.37 0.69 2.23 1.39 1.14 1.43 1.86 2.07 9 2.35 0.26 0.76 1.37 1.37 1.03 0.49 1.45 1.56 10 8.50 0.79 3.38 4.81 5.16 7.25 7.61 6.96 8.44 11 23.95 0.87 4.27 11.53 11.30 9.93 10.60 11.62 9.77 12 33.67 1.47 16.53 13.12 9.92 12.96 11.39 20.34 12.66 13 8.50 0.58 3.01 3.71 3.75 3.01 7.65 2.46 5.48 14 21.39 1.39 5.83 17.84 12.29 14.87 21.47 15.29 19.08 15 17.81 0.40 2.88 5.01 1.32 15.06 13.45 4.42 17.70 16 15.50 2.66 16.90 6.95 11.19 6.64 15.33 19.30 14.97 17 34.60 10.78 34.57 72.26 55.36 78.40 42.50 44.54 28.24 18 3.00 0.23 1.45 1.38 1.96 3.28 2.35 2.92 2.72 19 13.23 0.43 3.30 3.58 5.73 6.50 5.59 5.65 4.52 20 0.36 0.07 0.46 1.67 1.53 0.56 0.77 0.64 0.50 21 8.14 0.30 1.49 3.06 3.14 5.27 5.57 6.23 6.67 22 0.56 0.28 0.60 1.74 0.86 2.49 0.97 3.63 3.29 23 2.36 0.22 0.63 0.98 0.90 1.75 1.91 2.36 2.56 24 9.08 2.26 2.84 5.82 8.06 8.15 7.04 8.63 9.10 25 2.58 0.23 2.76 3.15 3.65 3.72 1.37 3.23 4.30 26 5.34 0.32 1.48 2.55 1.45 1.75 2.93 —a 3.34 27 23.71 0.15 1.25 0.40 2.33 2.98 3.75 1.50 3.55 28 1.56 0.39 6.18 3.10 3.79 9.84 7.33 7.95 6.78 29 0.87 0.79 1.68 1.87 2.32 0.97 1.58 3.35 1.49 30 6.12 0.69 1.51 1.18 0.96 0.69 0.98 0.70 1.71 Median 4.68 0.34 1.60 2.45 2.14 3.00 2.64 3.23 3.44 Range 0.13–34.60 0.004-–0.78 0.05–34.57 0.06–72.26 0.02–55.36 0.04–78.40 0.09–42.50 0.05–44.54 0.30–28.24 a Δ9-THC undetectable in this sample. Open in new tab Discussion An LC-HRMS bioanalytical method for the quantification of Δ9-THC, CBN, 11-nor-9-carboxy-Δ9-THC, 11-OH-Δ9-THC, and 8β,11-diOH-Δ9-THC in human whole blood microsamples using Δ9-THC-D3 as internal standard was developed and validated according to current industry guidance documents (13, 14) for use as part of a test for determining recent cannabis use. The assay parameters of LOD, LLOQ, intra- and inter-run accuracy and precision, assay repeatability and extraction efficiency were examined. The rapidly evolving regulatory environment regarding the legalization of medicinal and recreational cannabis both in the U.S. and internationally has created a pressing need for an accurate means of assessing recent cannabis use that can be utilized by law enforcement and employers to protect public safety on the highways and in the workplace. Current methods of testing, which rely on determining Δ9-THC and metabolite concentrations in blood, urine or saliva, are inadequate because cannabis impairment has never been correlated with specific concentrations of Δ9-THC or its metabolites (8–10). Overall, the described LC-HRMS Orbitrap-based bioanalytical method performed well within the pre-established specifications for each assay parameter in a concentration range of 1–100 ng/mL, with acceptable LOD, LLOQ, and intra-run and inter-run accuracies and precisions over a period of 5 consecutive days at low, intermediate, and high concentrations. Assay repeatability for the five cannabinoid analytes and internal standard was excellent, with coefficients of variations well within the 5% limit. Calibration curves displayed good linearity and met the acceptance criteria for accuracy. Although the extraction efficiency for 11-nor-9-carboxy-Δ9-THC was relatively low, this was not considered to be a major issue because of the high sensitivity of the assay for this metabolite. Although this validation study evaluated five cannabinoid analytes, additional analytes such as Δ9-THCV, CBG, and CBC (11, 15, 16) can easily be added to this assay method. Our laboratory is already ISO/IEC 17025-accredited for analyzing Δ9-THC, Δ9-THCA, Δ9-THCV, CBN, CBC, CBG, cannabigerolic acid (CBGA), CBD, and cannabidiolic acid (CBDA) by standard HPLC, and the described bioanalytical method will be added to our testing scope in the near future. The Q Exactive Orbitrap mass spectrometer used in this study is advantageous for its high-resolution accurate mass (HRAM) MS data for targeted quantification and non-targeted screening of cannabinoids of interest and unknowns. HRAM MS allows for the detection and quantification of all precursor ions in a sample without the need for individual specific analyte tuning and optimization as would be required for analytical MS methods that employ triple quadrupole mass spectrometers. The HRAM and full-scan capabilities of the Orbitrap mass spectrometer capture all sample data, enabling retrospective and informative data analysis in complex matrices (e.g., whole blood). Using the validated LC-HRMS bioanalytical method to quantify cannabinoid and metabolite levels in blood, a clinical study was performed in 30 human subjects for the purpose of identifying parameters that can be used to determine recent use of cannabis following both inhalation and oral routes of administration. After quantifying the five target analytes, specific recent use parameters, including the ratio of 11-nor-9-carboxy-Δ9-THC/Δ9-THC presented here, were identified based on the pharmacology of Δ9-THC. This parameter, in combination with others that were identified in this study, were able to identify recent use with at least 95% confidence within 3 h following smoking (unpublished data). The complete results of this clinical study are currently being prepared for publication. In summary, the LC-HRMS Orbitrap-based bioanalytical method was identified to be suitable for the quantification of five cannabinoids (Δ9-THC, CBN, 11-nor-9-carboxy-Δ9-THC, 11-OH-Δ9-THC, and 8β,11-diOH-Δ9-THC) with acceptable accuracy and precision, according to accepted industry practices, in the concentration range of 1–100 ng/mL. This bioanalytical method, designed to quantify cannabinoids in microsamples of whole capillary blood, was successfully employed in a clinical study used to develop a pharmacological parameter-based test for recent cannabis use (the RCU Test). Acknowledgments The authors would like to acknowledge the Cancer Immunotherapy Research Institute (CIRI) for supporting the clinical studies. Funding This work was funded by RCU Labs, Inc., the Cancer Immunotherapy Research Institute, and ImmunoTess, Inc. References 1 United Nations Office on Drugs and Crime ( 2017 ) World Drug Report (United Nations Publication, Sales No. E.17.XI.7). United Nations Office on Drugs and Crime, Vienna, Austria 2 ElSohly M.A. , Radwan M.M., Gul W., Chandra S., Galal A. ( 2017 ) Prog. Chem. Org. Nat. Prod. 103 , 1 – 36 . doi:10.1007/978-3-319-45541-9_1 PubMed 3 Maccarrone M. , Bab I., Biro T., Cabral G.A., Dey S.K., Di Marzo V., Konje J.C., Kunos G., Mechoulam R., Pacher P., Sharkey K.A., Zimmer A. ( 2015 ) Trends Pharmacol. Sci. 36( 5), 277 – 296 . doi:10.1016/j.tips.2015.02.008 Crossref Search ADS PubMed 4 Berning A. , Compton R., Wochinger K. 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TI - Development and Validation of a Liquid Chromatography High-Resolution Mass Spectrometry (LC-HRMS) Bioanalytical Method for Quantifying Cannabinoids in Whole Blood: Application for Determining Recent Cannabis Use
JF - Journal of AOAC International
DO - 10.1093/jaocint/qsz011
DA - 2020-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/development-and-validation-of-a-liquid-chromatography-high-resolution-DONxuMxLrs
SP - 725
EP - 735
VL - 103
IS - 3
DP - DeepDyve
ER -