DOT/FAA/AM-17/12

Office of Aerospace Medicine

Washington, DC 20591

Postmortem Fluid and Tissue 

Concentrations of THC, 

11-OH-THC, and THC-COOH

Sunday R. Saenz1

Russell J. Lewis1

Michael K. Angier1

Jarrad R. Wagner2

1

Civil Aerospace Medical Institute 

Federal Aviation Administration 

Oklahoma City, OK 73125

2

School of Forensic Sciences

Oklahoma State University

Center for Health Sciences

Tulsa, OK 74107

April 2017

Final Report
NOTICE

This document is disseminated under the sponsorship of 

the U.S. Department of Transportation in the interest of 

information exchange. The United States Government 

assumes no liability for the contents thereof.

___________

This publication and all Office of Aerospace Medicine 

technical reports are available in full-text from the 

Civil Aerospace Medical Institute’s publications website: 

http://www.faa.gov/go/oamtechreports
i

Technical Report Documentation Page

1. Report No. 2. Government Accession No. 3. Recipient's Catalog No.

DOT/FAA/AM-17/12

4. Title and Subtitle 5. Report Date

Postmortem Fluid and Tissue Concentrations of THC, 11-OH-THC, and 

THC-COOH

April 2017

6. Performing Organization Code

7. Author(s) 8. Performing Organization Report No.

Saenz SR,1 Lewis RL,1 Angier MK1 and Wagner JR2

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS)

1

FAA Civil Aerospace Medical Institute

P.O. Box 25082, Oklahoma City, OK 73125

2

School of Forensic Sciences, Oklahoma State University

Center for Health Sciences, Tulsa, OK 74107

11. Contract or Grant No.

12. Sponsoring Agency name and Address 13. Type of Report and Period Covered

Office of Aerospace Medicine

Federal Aviation Administration

800 Independence Ave., S.W.

Washington, DC 20591 14. Sponsoring Agency Code

15. Supplemental Notes

This work was accomplished under the approved task AM-B-16-TOX-204

16. Abstract

Marijuana is the most commonly abused illicit drug worldwide. Marijuana is used for its euphoric and 

relaxing properties. However, marijuana use has been shown to result in impaired memory, cognitive skills, 

and psychomotor function. The Federal Aviation Administration’s Civil Aerospace Medical Institute 

conducts toxicological analysis on aviation fatalities. Due to severe trauma associated with aviation 

accidents, blood is not always available; therefore, the laboratory must rely on specimens other than blood 

for toxicological analysis in approximately 30-40% of cases. However, the postmortem distribution of 

cannabinoids has not been well characterized. The purpose of this research is to evaluate the distribution of 

THC, and its metabolites 11-OH-THC, and THC-COOH in postmortem fluid and tissue specimens from 11 

fatal aviation accident cases (2014-2015) previously found positive for cannabinoids. Specimens evaluated, 

when available, included: blood, urine, vitreous humor, liver, lung, kidney, spleen, muscle, brain, heart, and 

bile. We developed and validated (following SWGTOX guidelines) a sensitive and robust method using 

solid phase extraction and liquid chromatography-tandem mass spectrometry to identify and quantify THC, 

11-OH-THC and THC-COOH in postmortem fluids and tissues. The method readily identified and 

quantified these cannabinoids in postmortem fluids and tissues below 1 ng/mL. Qualitative cannabinoid 

results within each case were comparable between blood and non-blood specimens. However, there was no 

consistent distribution of the cannabinoids between blood and any other fluids or tissues. Therefore, while 

quantitative interpretation of non-blood specimens is not prudent, a majority of the non-blood specimens 

tested could be suitable alternative/supplemental choices for qualitative cannabinoid purposes.

17. Key Words 18. Distribution Statement

Forensic Toxicology, Postmortem Distribution, LC/MS, 

THC, 11-OH-THC, THC-COOH, Canabiniods

Document is available to the public through the Internet:

http://www.faa.gov/go/oamtechreports/

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price

Unclassified Unclassified 20

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized
ii

Contents

Introduction ............................................................................................................................... 1

Materials and Methods .............................................................................................................. 2

Chemicals and Reagents ....................................................................................................... 2

Sample selection and storage ................................................................................................ 2

Ultra-performance liquid-chromatography-tandem mass spectrometric conditions ............ 2

Calibration and control preparation ...................................................................................... 3

Sample preparation and extraction method ........................................................................... 4

Results and Discussion ............................................................................................................. 5

Analytical Method ................................................................................................................ 5

Postmortem specimen analysis ........................................................................................... 10

Conclusion .............................................................................................................................. 15

References ............................................................................................................................... 16
1 

POSTMORTEM FLUID AND TISSUE CONCENTRATIONS OF

THC,11-OH-THC, AND THC-COOH

INTRODUCTION

Approximately 147 million individuals worldwide currently use marijuana, making it the 

most widely used drug of abuse.(1) A survey conducted in 2010 showed that 6.9% of individuals 

12 and older in the United States used marijuana in the preceding month.(2) With an increasing 

number of states now legalizing marijuana, both recreationally and medicinally, marijuana use is 

on the rise.(3) 

The primary psychoactive compound in marijuana is Δ9-tetrahydrocannabinol (THC). Once 

introduced to the body, THC is rapidly metabolized to an equipotent psychoactive short-term 

metabolite, 11-hydroxy-tetrahydrocannabinol (11-OH-THC), which is then metabolized to the 

inactive metabolite 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH).(4) THC is typically 

used for its euphoric effects.(4) Non-desirable side effects include dizziness, dry mouth, 

increased heart rate, and impaired memory, concentration, and reaction time.(4) Numerous 

studies have shown that THC has detrimental effects on cognitive and psychomotor function and 

skills.(5-11) Moreover, operation of a motor vehicle under the influence of marijuana has been 

found to have similar effects as in individual with a blood alcohol content of 0.08%.(12) Another 

study compared ethanol ingestion with marijuana smoking; they found a similar degree of both 

perceived impairment and actual impairment.(5) Aircraft simulator studies have shown that 

following the use of marijuana, pilots deviated in altitude and had trouble following flight 

patterns compared to when they were “sober.”(13-16) 

Following toxicological evaluation of a fatality, toxicologists are expected to form educated 

opinions about the postmortem findings. However, there is minimal postmortem fluid and tissue 

data in the literature relating to THC, 11-OH-THC, and THC-COOH. Furthermore, the analysis 

of postmortem tissues for THC, 11-OH-THC, and THC-COOH is not common in the forensic 

community. Our laboratory currently utilizes solid-phase extraction (SPE) and gas 

chromatography with negative chemical ionization mass spectrometry (GC/MS-NCI) for the 

analysis of THC and THC-COOH in postmortem fluids and tissues.(17) While our current 

method is suitable for the analysis of postmortem fluids and tissues for THC and THC-COOH, 

there are a few limitations with this method: our extraction requires 3 mL of fluid specimen, 

GC/MS analysis requires multiple time-consuming derivatizations, GC/MS-NCI relies on 

retention time and the M-H ion only for identification, and it does not detect 11-OH-THC.

In order to have a method that uses a smaller sample size, eliminates time consuming 

derivatizations, and identifies/quantitates 11-OH-THC (a short-lasting metabolite of THC), we 

determined that we needed to utilize liquid chromatography-tandem mass spectrometry 

(LC/MS/MS). There are numerous LC/MS/MS methods in the literature for the identification 

and quantitation of THC and its metabolites; however, few analyzed THC, 11-OH-THC, and 
2 

THC-COOH in postmortem fluids and tissues. Furthermore, there have been a limited number of 

studies published on the distribution of these cannabinoids in postmortem fluids and tissues.(17-

19) This paper details the validation and application of SPE coupled with LC/MS/MS for the 

identification and quantitation of THC, 11-OH-THC, and THC-COOH in postmortem fluid and 

tissue specimen from 11 aviation accident fatalities.

MATERIALS AND METHODS

Chemicals and Reagents 

THC, 11-OH-THC, and THC-COOH methanolic standards were purchased from Cerilliant 

(Cerilliant Corp., Round Rock, TX) at 1.00 mg/mL. THC-d3, 11-OH-THC-d3, THC-COOH-d3, 

THC-glucuronide, and THC-COOH-glucuronide methanolic standards were purchased from 

Cerilliant at 100 μg/mL. Selectrazyme (abalone derived β-glucuronidase) was obtained from 

UCT (UCT, Inc., Bristol, PA). Sodium acetate and potassium oxalate were purchased from 

Sigma-Aldrich (Sigma-Aldrich; St. Louis, MO). Formic acid, sodium fluoride, hydrochloric acid, 

hexanes, ethyl acetate, and LC/MS grade acetonitrile, isopropanol, and methanol were purchased 

from Fisher (Fisher Scientific, Pittsburgh, PA). Double deionized (DI) water was obtained from a 

Millipore Direct Q-3 UV (Millipore, Continental Water Systems, El Paso, TX). Bovine blood 

was obtained from Country Home Meat Co. (Country Home Meat Co., Edmond, OK). 

Immediately upon collection, sodium fluoride and potassium oxalate were added to the blood 

and mixed to produce a final sodium fluoride/potassium oxalate concentration of 1.0% and 0.2% 

(w/v), respectively. 

Mobile phase A (MPA) was made with DI water with formic acid (999:1 v/v). Mobile phase 

B (MPB) was made with LC/MS grade acetonitrile with formic acid (999:1 v/v). A seal wash 

solution at a 25:25:25:25 (v/v) ratio was prepared with DI water, and LC/MS grade acetonitrile, 

methanol, and isopropanol. 

Sample selection and storage 

The toxicology laboratory’s database (ToxFLO, DiscoverSoft Development, LLC; Oklahoma 

City, OK) was searched for cases that were previously found positive for cannabinoids within the 

last 2 years (2014 – 2015) that had blood and a majority of the desired specimens available for 

analysis (urine, vitreous humor, liver, lung, kidney, spleen, muscle, brain, heart, and bile). Blood 

samples were stored in gray top tubes (1.0% and 0.2% (w/v) sodium fluoride/potassium oxalate, 

respectively). All specimens were stored at -20°C until analysis. 

Ultra-performance liquid-chromatography-tandem mass spectrometric conditions

Analysis was performed on a Waters® Xevo TQ-S Acquity Ultra Performance Liquid 

Chromatography (UPLC) (Waters Corporation; Milford, MA). The column manager temperature 

was set at 30°C. Acquity UPLC BEH C18 column (2.1 x 100 mm, 1.7 μm) was used for 

chromatographic separation. Prior to analysis, the column was equilibrated for 30 min. The 

UPLC was operated with a gradient of 35:65 MPA:MPB (MPA - water with 0.1% formic acid; 
3 

MPB - acetonitrile with 0.1% formic acid) to 10:90 MPA:MPB at 2.0 min, held until 2.5 min, 

and then returned to 35:65 MPA:MPB at 3.8 min and held until 4.0 min for a total run time of 

4.0 min. The UPLC flow rate was 0.60 mL/min and a sample injection volume was 2 μL. The 

autosampler was set at 10°C. UPLC pressures observed for these conditions were around 9,000 

psi. Following use, the column was washed and stored in acetonitrile.

All compounds were optimized on the mass spectrometer by direct infusion. Four different 

modes of compound ionization were evaluated (positive and negative atmospheric pressure 

chemical ionization - ±APCI, and positive and negative electrospray ionization - ±ESI) and the 

+ESI mode provided maximum ionization for the compounds. The source temperature was set at 

150°C, desolvation temperature at 550°C, and desolvation gas flow at 1,100 L/hr. Precursor 

(parent) and product (daughter) ions, quantitation ions, cone voltage, collision energy, and ion 

mode are listed in Table 1.

Table 1. MS parameters for THC, 11-OH-THC, and THC-COOH. 

Compound Retention 

Time

Cone 

Voltage 

(V)

Precursor 

Ion (m/z)

Product Ion 

(m/z) Collision Energy 

(V)

THC 2.43 38.0 315.26 123.12

193.19*

32.0

22.0

THC-d3 2.41 19.0 318.30 123.11

196.24*

33.0

21.0

11-OH-THC 1.47 20.0 331.25 193.19*

313.32

25.0

13.0

11-OH-THC-d3 1.46 20.0 334.30 196.24*

316.30

23.0

13.0

THC-COOH 1.51 20.0 345.23 299.29

327.31*

19.0

15.0

THC-COOH-d3 1.49 20.0 348.30 302.29

330.31*

19.0

15.0

 *Transition ion used for quantification.

There are three criteria set by our laboratory that must be met before an analyte can be 

reported as positive by LC/MS/MS. An analyte’s product ions must have a minimum signal-tonoise ratio of 10 (quant ion) and 3 (confirmation ion), a retention time +/- 5% of the average 

calibrator retention time, and product ion ratio +/- 20% of the average calibrator product ion 

ratio.

Calibration and control preparation

All calibrators and controls were prepared using bovine whole blood. Calibrators and 

controls were prepared using separate 1.00 mg/mL methanolic drug standards. Calibration curves 

consisted of eight calibrators and were prepared by serial dilution to produce concentrations 

ranging from 0.78 to 100 ng/mL. A 100 ng/mL internal standard solution was prepared in DI 

water using 100 μg/mL THC-d3, 11-OH-THC-d3, and THC-COOH-d3 methanolic standards. 

Controls were prepared at concentrations of 2, 20, and 80 ng/mL, covering low, medium, and 
4 

high portions of the calibration curve. Controls were used to determine the accuracy and 

precision of the method, both within-day and between-day, and analyte stability. Controls were 

analyzed with all case specimens to ensure the calibration curve accuracy.

THC, 11-OH-THC, and THC-COOH are present in urine, to some degree, as glucuronide 

conjugates. Therefore, we hydrolyzed each postmortem urine specimen using the enzyme 

β-glucuronidase (Selectrazyme, 100,000 units/mL, UCT; Bristol, PA). This solution was stored 

in a refrigerator at 4°C. THC and THC-COOH glucuronide controls, prepared as 25 ng/mL free 

drug, were made from a 100 μg/mL methanolic standards of THC-glucuronide and THC-COOHglucuronide. The 11-OH-THC-glucuronide standard was unavailable. 

Quantification was achieved via an internal standard calibration procedure. Response factors 

for each compound were determined for every sample analyzed. The response factor was 

calculated by dividing the area of the analyte quant ion peak by the area of the internal standard 

quant ion peak. Calibration curves were derived by plotting the analyte/internal standard 

response factor versus the analyte concentration for each respective calibrator and determining 

the mathematical model that best fit the calibration data. These calibration curves were then used 

to determine the concentrations of each cannabinoid compound in the prepared controls and 

biological specimens.

Sample preparation and extraction method 

All calibrators, controls, and specimens were prepared using the following procedure. Tissue 

samples were diluted 2:1 w:w (1% NaF solution:tissue) and then homogenized using an OMNI 

homogenizer (Omni International; Kennesaw, GA). One-half mL of liquid specimen and 1.5 g of 

tissue homogenate (0.5 g of wet tissue) were added to individual 16 x 100 mm screw-top tubes. 

Each specimen was spiked with 100 μL of the 100 ng/mL internal standard solution (10 ng total). 

Samples were vortexed briefly and allowed to stand at room temperature for 10 min. Fifty μL of 

stock Selectrazyme (10,000 units), followed by 2.00 mL of 1.0 M pH 5.00 sodium acetate buffer 

was added to a 16x150 screw top tube containing 0.5 mL urine sample. The urine samples were 

then vortexed briefly and incubated at 65°C for 3 h to facilitate hydrolysis of all glucuronide 

conjugates. In our initial investigations, complete hydrolysis of both THC and THC-COOHglucuronide conjugates was achieved after incubation with Selectrazyme for 2.5 h at 65°C 

(unpublished data). However, an incubation time of 3 h was chosen to ensure that specimens 

with elevated cannabinoid concentrations were also completely hydrolyzed. Following 

hydrolysis, samples were allowed to cool to room temperature. Three mL of ice-cold acetonitrile 

was added to all specimens (except urine specimens) and the specimens were immediately 

vortexed for 30 sec. Samples were capped, rotated for 5 min, and centrifuged for 10 min at 2300 

rpm in a Thermo Jouan C4i Centrifuge (Thermo Electron Corp.; Chateau-Gontier, France). The 

supernatant was transferred to a clean 16x100 mm round bottom tube and 5 mL of 0.1 M Sodium 

Acetate Buffer pH 3 was added.
5 

Buffered samples were transferred to the SPE Clean Screen THC columns that were 

preconditioned with 2 mL of methanol, 2 mL of DI water, and 1 mL of 0.1 M Sodium Acetate 

Buffer pH 3. The Clean Screen THC columns, 10 mL barrel, 200 mg sorbent bed, were obtained 

from UCT (UCT; Bristol, PA). After the sample passed through the column, the columns were 

washed with 2 mL of DI water, 2 mL of 95:5 100mm HCl:ACN, and 200 μL of hexanes. 

Samples were then eluted into a 16x100 mm conical tube. The first elution was 2 mL of hexanes 

(for THC). The second elution was 2 mL 50:50 hexanes:ethyl acetate (for 11-OH-THC and 

THC-COOH). Elutions were taken to dryness in a TurboVap LV nitrogen evaporator (Caliper 

Life Sciences; Hopkinton, MA) set at 40°C, reconstituted in 100 μL of LC/MS grade methanol, 

and transferred to 2 mL LC vials with inserts. The vials were capped and transferred to the auto 

sampler.

RESULTS AND DISCUSSION

Analytical Method 

This LC/MS/MS method proved to be sensitive, robust, and reproducible for the detection 

and quantitation of THC, 11-OH-THC, and THC-COOH. The UPLC provided sharp, narrow 

analyte peaks which were far superior to traditional HPLC. A typical chromatogram can be seen 

in Figure 1. This method was validated following SWGTOX guidelines.(20) These guidelines 

recommend the evaluation of calibration curve best fit model, carryover, limit of detection 

(LOD), limit of quantitation (LOQ), accuracy, precision, dilution integrity, stability, ion 

suppression/enhancement, and drug interference for the validation of a new analytical method. 

Figure 1. An 80 ng/mL control with 11-OH-THC, THC-COOH, and THC (left to right).

The calibration curve best fit model was determined by preparing and analyzing a calibration 

curve on 5 separate, but consecutive days. The THC, 11-OH-THC, and THC-COOH calibration 

curves were constructed of concentrations ranging from 0.78– 100 ng/mL. All curves best fit a 

linear regression model with a 1/x weighting. The linear dynamic range (LDR) for THC, 

11-OH-THC, and THC-COOH was found to be 0.78 – 100 ng/mL. Correlation coefficients were 

all greater than 0.99. 

LOD is the lowest analyte concentration meeting all identification criteria described in the 

materials and methods section, i.e., minimum signal-to-noise ratio of 10 for quantitation ion and 
6 

3 for all other ions, retention time +/- 5% of the average calibrator retention time, and product 

ion ratio +/- 20% of the average calibrator product ion ratio. The LOQ has the same 

identification requirements, plus it must have a measured concentration +/- 20% of target or 

expected concentration. The lowest calibrator tested (0.78 ng/mL) met the qualitative and 

quantitative criteria for THC, 11-OH-THC, and THC-COOH. Since concentrations below this 

were not tested, the LOD and LOQ were administratively determined to be 0.78 ng/mL. This 

method could readily determine concentrations far below the lowest calibrator (0.78 ng/mL), but 

we determined this was a low enough concentration for our laboratory’s needs and mission. All 

case specimens falling below the LOD/LOQ were considered negative.

Carryover was evaluated for all analytes; no carryover was observed. Carryover was 

evaluated by running a methanol blank after the highest calibrator during the linearity study 

described above. The blanks showed no trace of THC, 11-OH-THC, or THC-COOH. Carryover 

was also monitored throughout the study by including blanks between specimens to verify that 

no carryover occurred. 

Method accuracy was determined by calculating the difference between the target 

concentration and the determined concentration and is expressed as bias or relative error (%E). 

Precision is expressed as the coefficient of variation (CV) of multiple analyses at a given control 

level. Controls at 2, 20, and 80 ng/mL of each analyte were prepared on Day 1 of the accuracy, 

precision, and stability study. Controls were prepared in sufficient quantities (100 mL bovine 

blood) for use in all studies and were aliquoted into 16x100 mm screw-top tubes. Freshly 

prepared controls were extracted on Day 1 of the study. The remainder of the controls were 

stored either in the refrigerator (4°C) or in the freezer (-20°C) until use. 

The Day 1 accuracy and precision data demonstrate that this method is very accurate and 

precise. The largest Day 1 %E for all THC, 11-OH-THC, and THC-COOH control levels 

were -9, -8, and -14, respectively. The largest CV for all THC, 11-OH-THC, and THC-COOH 

control levels were 3, 8, and 2, respectively. On Days 2, 3, 4, and 5, controls were removed from 

the refrigerator and analyzed with a freshly prepared calibration curve. The within-day %E for 

all controls were less than +/- 20% and the CV was less than 10%, a SWGTOX recommendation. 

The grand mean, or between-day, accuracy and precision also demonstrated small %E and CV. 

The accuracy and precision data is summarized in Tables 2, 3, and 4. 

All three cannabinoids examined here exist to some degree in urine as a glucuronide 

conjugate. In order to determine total THC, 11-OH-THC and THC-COOH, the glucuronide bond 

must be cleaved. This is best achieved enzymatically, using a beta-glucuronidase hydrolysis step. 

To ensure this step provided a quantitatively reliable hydrolysis, a THC and THC-COOH 

glucuronide control was analyzed with all urine specimens. No 11-OH-THC-glucuronide was 

commercially available, so no control for this analyte was used. The target concentration for each 

glucuronide control was 25 ng/mL free-drug. The control values were within 20% of the target 

concentration. 
7 

Actual specimens may have cannabinoid concentrations that exceed the highest calibrator in 

the calibration curve. Since measured values must be within the LDR, concentrations outside the 

LDR cannot be reported and, thus, a specimen dilution may be necessary. Therefore, we 

evaluated the dilution integrity of the cannabinoids. Five 80 ng/mL controls were diluted 1:10 

and five 1 μg/mL controls were diluted 1:100. All dilutions were within 20% of the expected 

dilution concentration of 8 ng/mL and 100 ng/mL. 

Stability was evaluated in three ways: on-instrument stability (post-extraction stability), 

refrigerated stability, and freeze/thaw stability. On-instrument stability or post-extraction 

stability was determined by reanalyzing (re-injecting) all three levels of the Day 1 controls on 

Days 2, 3, 4, and 5. Specimens were left on the instrument in the autosampler that was set at 

10°C. All three compounds were found to be stable for five days post-extraction. Refrigeration 

stability was evaluated by extracting controls stored at refrigerator temperatures on Days 2, 3, 4, 

and 5. This data is the same as the accuracy and precision data described above. It was found that 

all three analytes were stable for five days in the refrigerator at 4°C. We also evaluated freezethaw stability. In order to determine if THC, 11-OH-THC and THC-COOH are stable after 

multiple freeze/thaw cycles, controls were placed in a freezer at -20°C on Day 1. On Day 2, all 

frozen controls were removed from the freezer and allowed to thaw for one hour at room 

temperature. Five specimens at each control concentration were analyzed and the remainders of 

the controls were placed back into the freezer. Three freeze/thaw cycles were completed. It was 

found that all three analytes were stable after three freeze/thaw cycles.

Ion suppression/enhancement was determined by analyzing multiple analyte-spiked solvent 

samples and spiked post-extraction fluid and tissue specimens and comparing their response. If 

the post-extraction specimen had a lower signal than the solvent control sample, it is ion 

suppression. If it has a higher signal, then it is ion enhancement. Ion suppression/enhancement 

was evaluated in blood, urine, serum, liver, lung, brain, and muscle. Ion suppression was not a 

factor for THC, 11-OH-THC, or THC-COOH in blood or urine specimens (<20%). THC and 

11-OH-THC also suffered no suppression in serum (<20%). THC-COOH did experience 

approximately 40% ion suppression in serum. Additionally, significant ion suppression was 

observed in tissue specimens for all 3 analytes. However, similar ion suppression also affected 

the deuterated internal standards, thus the quantification of these analytes in these specimens was 

not affected.

It is common to encounter multiple drugs in a typical case. Therefore, drug interference 

needed to be evaluated to determine if common drugs can alter the detection and/or quantitation 

of THC, 11-OH-THC, or THC-COOH. Drugs commonly encountered in our laboratory were 

prepared at final concentration of 5 μg/mL with 80 ng/mL cannabinoids. These drugs included 

acetaminophen, atenolol, atorvastatin, citalopram, dextromethorphan, diphenhydramine, 

hydrocodone, methamphetamine, naproxen, and sertraline. Five of these controls were analyzed 

and none suffered qualitative or quantitative interference. 
8 

Table 2. Accuracy and Precision for THC.

DAY 1 DAY 2 DAY 3 DAY 4 DAY 5

Target 

(ng/mL)

Mean 

(ng/mL) CV% E% Mean (ng/mL) CV% E% Mean (ng/mL) CV% E% Mean (ng/mL) CV% E% Mean (ng/mL) CV% E%

Grand 

Mean 

(ng/mL)

CV% E%

2 1.83 ± 0.02 1 -9 1.69 ± 0.02 1 -16 1.73 ± 0.04 2 -13 1.73 ± 0.05 3 -14 1.69 ± 0.01 1 -15 1.73 ± 0.06 3 -14

20 21.4 ± 0.2 1 7 20.4 ± 0.2 1 1 19.5 ± 0.3 1 -3 22.7 ± 0.2 1 13 23.8 ± 0.5 2 19 22 ± 2 9 10

80 81 ± 2 3 1 80 ± 1 2 1 76.5 ± 0.5 1 -5 88.6 ± 0.9 1 11 95 ± 1 1 19 84 ± 7 8 5 

n=5 for all measurements

Accuracy measured as relative error (%E) from target concentration.

Precision measured as CV in replicate measurements.

Table 3. Accuracy and Precision for 11-OH-THC.

DAY 1 DAY 2 DAY 3 DAY 4 DAY 5

Target 

(ng/mL)

Mean 

(ng/mL) CV% E% Mean (ng/mL) CV% E% Mean (ng/mL) CV% E% Mean (ng/mL) CV% E% Mean (ng/mL) CV% E%

Grand 

Mean 

(ng/mL)

CV% E%

2 1.8 ± 0.2 8 -8 1.72 ± 0.02 1 -14 1.68 ± 0.09 6 -16 1.9 ± 0.1 6 -3 1.79 ± 0.06 3 -11 1.8 ± 0.2 11 -10

20 20.5 ± 0.4 2 3 19.4 ± 0.6 3 -3 17.8 ± 0.5 3 -11 20.3 ± 0.9 4 1 19.2 ± 0.2 3 -4 20.5 ± 0.4 2 3 

80 80 ± 2 3 0 82 ± 2 2 3 73 ± 2 3 -8 81.5 ± 1 1 2 84 ± 1 1 5 80 ± 2 3 0 

n=5 for all measurements

Accuracy measured as relative error (%E) from target concentration.

Precision measured as CV in replicate measurements.
9 

Table 4. Accuracy and Precision for THC-COOH.

DAY 1 DAY 2 DAY 3 DAY 4 DAY 5

Target 

(ng/mL)

Mean 

(ng/mL) CV% E% Mean (ng/mL) CV% E% Mean (ng/mL) CV% E% Mean (ng/mL) CV% E% Mean (ng/mL) CV% E%

Grand 

Mean 

(ng/mL)

CV% E%

2 1.72 ± 0.04 2 -14 1.79 ± 0.08 4 -11 1.83 ± 0.07 4 -8 1.72 ± 0.04 2 -14 1.86 ± 0.08 4 -7 1.78 ± 0.08 4 -11

20 19.8 ± 0.3 2 -1 21.5 ± 0.6 3 7 20.8 ± 0.3 1 4 21.0 ± 0.3 2 5 22 ± 1 5 11 21 ± 1 5 5 

80 79.9 ± 0.7 1 0 85 ± 1 1 7 85 ± 1 2 6 88 ± 2 2 10 87 ± 2 2 9 85 ± 3 4 6 

n=5 for all measurements

Accuracy measured as relative error (%E) from target concentration.

Precision measured as CV in replicate measurements.
10 

Postmortem specimen analysis 

Postmortem toxicology evaluation is an essential part of aviation accident investigation. The 

most desired specimen for postmortem toxicology is blood. However, due to the nature of 

aviation accidents, blood is not always available for analysis. In fact, our laboratory relies 

exclusively on tissues in approximately 30 – 40% of our cases. Therefore, having the ability to 

reliably analyze postmortem tissues is important. Approximately 4% of aviation fatalities in 

2015-2016 were positive for marijuana use. Understanding the postmortem distribution of 

cannabinoids could potentially aid in the interpretation of postmortem cannabinoid results.

Eleven aviation fatalities (22 – 69 years of age, median 47 years old; all male) were analyzed 

with this newly validated method. These cases were previously confirmed and quantitated using 

GC/MS-NCI for THC and THC-COOH.

The distribution of THC, 11-OH-THC, and THC-COOH throughout various postmortem 

specimens is presented in Tables 5, 6, and 7. Blood specimens from all 11 cases were positive for 

THC-COOH, with concentrations ranging from 2.37 – 70.2 ng/mL. Eight of the 11 blood 

specimens were positive for THC, with concentrations ranging from 0.78 – 8.39 ng/mL. Only 

four of these THC-positive bloods had measurable concentrations of 11-OH-THC (1.03 – 4.59 

ng/mL). The analysis of 11-OH-THC is important as it is a short-lasting metabolite of THC, 

which has been suggested to be an indicator of recent THC exposure. 

Multiple non-blood specimens were analyzed to evaluate their possible usefulness as 

supplemental or alternative specimens for the identification and quantification of THC, 

11-OH-THC, and THC-COOH including urine, vitreous humor, liver, lung, kidney, spleen, 

muscle, brain, heart, and bile. Results can be seen in tables 5, 6, and 7. 

Ten of the 11 cases tested had non-blood fluids available for analysis (either urine, vitreous, 

and/or bile). Of the 8 cases with available urine, 7 were positive for THC (1.25 – 32.2 ng/mL). 

11-OH-THC and THC-COOH were detected in all of the cases where urine specimens were 

available for analysis. The concentration of 11-OH-THC ranged from 11.7 – 620 ng/mL, and the 

THC-COOH ranged from 24.2 – 970 ng/mL. The case with the highest THC concentration also 

had the highest 11-OH-THC and THC-COOH concentrations. THC was detected in bile in 5 of 

the 9 cases containing bile (0.78 – 50.4 ng/mL). 11-OH-THC (0.98 – 227 ng/mL) and 

THC-COOH (201 – 3,307 ng/mL) were detected in all bile specimens. Bile yielded the highest 

THC-COOH concentrations of all the specimens tested. Due to excretory nature of bile, these 

elevated concentrations were not surprising. There were only two cases that had vitreous humor 

available for analysis. Neither case was positive THC or 11-OH-THC, while 1 case showed 

positive for THC-COOH.
11 

Table 5. THC fluid and tissue concentrations obtained from 11 pilot fatalities.*

Case Blood Urine VH Liver Lung Kidney Spleen Muscle Brain Heart Bile

1 Neg 1.25 — Neg Neg Neg 0.78 5.21 Neg Neg 0.78

2 4.03 — — Neg 14.1 22.6 1.31 7.37 3.71 7.40 14.2

3 0.78 1.26 — Neg 1.82 1.91 Neg 2.43 Neg Neg Neg

4 1.78 1.43 Neg 52.2 143 450 16.9 81.4 43.6 472 50.4

5 3.45 Neg — Neg 42.6 32.3 5.14 11.9 — 1.70 6.35

6 Neg 1.35 Neg Neg Neg Neg Neg 5.31 Neg 6.58 Neg

7 4.67 32.2 — 22.3 151 16.4 20.0 377 30.6 158 —

8 1.50 — — Neg 48.1 9.08 0.96 1.19 1.34 5.35 Neg

9 5.00 — — Neg 62.6 5.21 5.58 3.24 16.4 54.7 —

10 Neg 3.04 — Neg Neg Neg Neg Neg Neg Neg Neg

11 8.39 3.62 — Neg Unsuit 0.99 6.39 3.10 8.80 Neg 5.60

* All concentrations shown in units of ng/mL or ng/g

— Specimen type not available for analysis

Table 6. 11-OH-THC fluid and tissue concentrations obtained from 11 pilot fatalities.*

Case Blood Urine VH Liver Lung Kidney Spleen Muscle Brain Heart Bile

1 Neg 13.5 — Neg Neg Neg Neg Neg Neg Neg 5.13

2 Neg — — Neg Neg Neg Neg Neg 3.31 Neg 21.2

3 Neg 13.4 — Neg Neg Neg Neg Neg Neg Neg 16.8

4 2.02 60.6 Neg 66.1 12.7 17.9 19.8 10.9 37.4 29.9 227

5 Neg 43.3 — 4.04 1.15 1.34 Neg Neg — Neg 20.6

6 Neg 11.7 Neg 1.27 Neg Neg Neg Neg Neg Neg 0.98

7 4.59 620 — 38.6 6.95 10.5 13.8 12.0 30.8 8.6 —

8 Neg — — Neg Neg Neg Neg Neg 0.99 Neg 1.93

9 1.03 — — Neg 2.18 1.77 Neg Neg 8.97 Neg —

10 Neg 53.4 — Neg Neg Neg Neg Neg Neg Neg 32.4

11 1.74 25.9 — 8.17 Neg Neg Neg Neg 3.01 Neg 61.7

* All concentrations shown in units of ng/mL or ng/g

— Specimen type not available for analysis
12 

Table 7. THC-COOH fluid and tissue concentrations obtained from 11 pilot fatalities.*

Case Blood Urine VH Liver Lung Kidney Spleen Muscle Brain Heart Bile

1 2.37 24.2 — 28.5 1.81 12.1 2.06 0.83 Neg Neg 291

2 52.3 — — 59.9 7.93 47.4 7.54 Neg 4.66 Neg 1000

3 3.13 109 — 29.1 3.11 10.1 Neg 0.80 0.98 Neg 565

4 68.0 102 2.7 3894 205 1774 284 66.8 73.4 346 3307

5 32.0 219 — 352 30.7 226 25.8 7.23 — Neg 2238

6 2.62 87.6 Neg 57.3 1.31 33.6 2.83 1.17 2.52 3.32 201

7 70.2 970 — 2238 31.3 1482 62.9 27.0 28.8 59.6 —

8 4.60 — — 77.0 2.66 45.6 4.63 1.76 2.54 3.16 310

9 9.32 — — 46.3 5.94 107 10.5 0.87 7.51 Neg —

10 3.17 200 — 114 14.3 28.0 16.9 Neg 3.39 Neg 485

11 65.7 189 — 529 50.0 222 15.7 10.2 12.3 Neg 301

* All concentrations shown in units of ng/mL or ng/g

— Specimen type not available for analysis
13 

All 11 cases had liver available for analysis. THC-COOH was detected in all liver samples 

(28.5 – 3,894 ng/g). THC was only detected in two cases (22.3 and 52.2 ng/g). Five liver 

specimens were positive for 11-OH-THC (1.27 – 66.1 ng/g). Two cases with the highest 

concentrations of 11-OH-THC were the same two cases where THC was detected. The liver 

contains the enzyme cytochrome P450, which is responsible for the metabolism of THC to 

11-OH-THC and further to THC-COOH.(21) This could explain why THC was identified in only 

2 liver specimens and 11-OH-THC was detected in 5 of the 11 livers.(22) While liver tissue does 

not show a large percentage of THC positives relative to other tissues, it may be a good 

alternative or supplemental specimen for cannabinoid testing because of the percentage of 

specimens positive for 11-OH-THC.

Lung was found to contain some of the highest concentrations of THC. Seven of the 11 lungs 

tested were found positive for THC (1.82 – 151 ng/g), while one was found to be unsuitable for 

analysis. 11-OH-THC was detected in four lung specimens (1.15 – 12.7 ng/g), primarily in cases 

with the higher THC values. THC-COOH was detected in all lung specimens (1.31 – 205 ng/g). 

The lung specimens with the highest THC concentration also had the highest concentration of 

THC-COOH. The high THC concentrations in this tissue may be due to the fact that the most 

common route of administration of THC is through the smoking of marijuana. Therefore, the 

lung is exposed to very high amounts of THC through the inhalation of marijuana smoke. Lung 

tissue appears to be a good alternative matrix for cannabinoid testing since it has a high number 

of positives and the cannabinoids are at high concentrations. 

THC-COOH was positive in all kidney specimens (10.1 – 1,774 ng/g) at high concentration 

relative to most other tissue specimens. High concentrations of THC-COOH in the kidney were 

also found by Kemp et al. and Brunet et al.(17,23) THC was found in 8 of 11 kidneys studied 

(0.99 – 450 ng/g). Four cases were positive for 11-OH-THC (1.34 – 17.9 ng/g). Kidney tissue 

showed high concentrations of THC and THC-COOH, which may be due to the function of the 

kidney. Kidney tissue also appears to be a good alternative matrix for cannabinoid testing with 

its high cannabinoid concentrations. 

Spleen and muscle also proved to be viable matrices for cannabinoid testing. THC was 

detected in 8 of 11 spleen specimens and 10 out of 11 muscle specimens. THC-COOH was 

detected in 10 of 11 spleen cases and 9 of 11 available muscles. The two cases with the highest 

THC and THC-COOH spleen and muscle concentrations were the only cases positive for 

11-OH-THC.

The brain is a highly perfused organ and THC is quickly able to cross the blood-brain barrier. 

The detection of cannabinoids in the brain, even if they were absent in the blood agreed with a 

study conducted by Mura et al.(24) THC was detected in 6 of 10 brain tissues (1.34 – 43.6 ng/g). 

Each brain specimen that had THC also contained 11-OH-THC (0.99 – 37.4 ng/g). Nine of 10

brain specimens were positive for THC-COOH (0.98 – 73.4 ng/g). While brain did not have the 

highest concentration or largest percentage of THC positives, it did have the highest percentage 
14 

of 11-OH-THC positives of any specimen. Therefore, brain could also be a suitable supplemental 

specimen for cannabinoid analysis if 11-OH-THC is of interest.

Seven of the 11 heart specimens were positive for THC (1.70 – 472 ng/g) and four for 

THC-COOH (3.16 – 346 ng/g). Only two heart tissues showed 11-OH-THC (8.60 and 29.9 

ng/g). These two heart specimens were the ones that had the highest concentration of THC and 

THC-COOH. With the low number of cannabinoid positive in this specimen type, heart does not 

appear to be as good of choice for an alternative matrix for cannabinoid analysis as are some of 

the other tissues. 

We also examined the postmortem distribution of THC, 11-OH-THC, and THC-COOH for 

each specimen type to determine if there was a consistent distribution between non-blood 

specimens and blood (non-blood specimen concentrations divided by the blood concentration). 

No consistent distribution was identified for any of the cannabinoids tested. There are numerous 

reasons why there could be a lack of consistent distribution within a given tissue type. When the 

body dies, postmortem changes begin instantly.(25-29) Cells begin degrading and postmortem 

redistribution can occur. The degree of postmortem redistribution may vary based on sampling 

site and the postmortem interval.(30) A study that examined postmortem redistribution of 

cannabinoids between central and peripheral blood observed a trend between postmortem 

interval and increased postmortem redistribution.(31) The postmortem intervals for the cases in 

this study are unknown. 

The blood collection site can also impact the apparent distribution when attempting to 

evaluate the distribution of a drug relative to its blood concentration. In a majority of the cases 

our laboratory receives for analysis, the blood collection site is not noted. When the collection 

site is reported, the blood typically is noted as having been collected from the chest cavity. Chest 

cavity blood is likely not representative of “true” blood, as it may be contaminated with fluids 

from the surrounding tissues and/or the stomach. Further, cannabinoid concentrations in cavity 

fluid could be affected by redistribution from surrounding tissues of higher cannabinoid 

concentrations, i.e., lung. In addition, cannabinoid concentrations in tissue specimens could vary 

based on the portion of tissue that is provided to our laboratory for analysis.

Individual characteristics such as gender, body composition, hepatic, and renal function 

could influence how the analytes distribute through the body. Since THC is lipophilic, 

individuals with high BMI, could have more THC stored in adipose tissue relative to other 

organs. There are many factors concerning the cannabis itself that could affect the distribution. 

Since these are postmortem cases, much information regarding the individual’s cannabis intake 

is unknown, including time of last use, how often they used cannabis, the potency, and if they 

smoked marijuana or consumed the THC orally by pill or in an edible. 

Cannabinoid concentrations in the body will vary based on how often the individual used 

cannabis. Studies show that chronic users could have a concentration of THC and THC-COOH 

in their blood after 7 days of not using cannabis.(32) This could mean that the person smoked 
15 

marijuana several days before death and it was just still able to be detected in the fluid and tissue 

specimens.

Route of administration and the concentration of THC introduced to the body could affect the 

concentration in the body and where analytes are detected at higher levels. For example, if an 

individual was to eat “pot brownies,” the concentration of THC may not be as high in various 

tissues as it would if the individual would have smoked marijuana. This is because of first-pass 

metabolism, where much of the THC is metabolized in the liver to 11-OH-THC prior to 

circulation throughout the body.(21) However, in this scenario more THC may actually be 

consumed than during smoking. This is because ingesting marijuana takes longer for effects to 

be felt, so an individual could consume more marijuana trying to obtain the desired euphoric 

effect.

CONCLUSION

A rapid, sensitive, and robust method for the confirmation and quantitation of THC, 

11-OH-THC, and THC-COOH in was developed using SPE and LC/MS/MS. This new method 

utilizes significantly less sample and is far less time consuming than our current GC/MS

procedure. The LDR for all three cannabinoids was 0.78 – 100 ng/mL, covering the expected 

concentrations of these analytes. This validated method was successfully applied to 11 aviation 

fatalities to determine the concentrations of THC, 11-OH-THC, and THC-COOH in postmortem 

blood, urine, vitreous humor, liver, lung, kidney, spleen, muscle, brain, heart, and bile. No 

consistent distribution within any specimen type was identified. Urine, bile and a majority of the 

tissue specimens tested appear to be good alternative specimens for qualitative cannabinoid 

testing. However, quantitatively the concentrations are relatively ambiguous, as little postmortem 

cannabinoids distribution data exists and the fact that postmortem specimens in general are not 

ideal due to postmortem changes. Postmortem concentrations should always be interpreted with 

a great deal of caution. This data should add to the limited information about cannabinoid 

distribution in postmortem fluids and tissues. 
16 

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