THC Toxic-Lab Test

1.0 DEFINITION OF THC
THC, or tetrahydrocannabinol, is the chemical responsible for most of marijuana's
psychological effects. THC changes behavior by binding fitting together like a lock and key
to receptors on nerve cells, which then respond with a change in activity.

Figure: Molecule of tetrahydrocannabinol
Cannabinoid receptors are concentrated in certain areas of the brain associated with
thinking, memory, pleasure, coordination and time perception. There are also cannabinoid
receptors on nerves in other parts of the body. THC relieves pain, but it doesn't bind to the
same receptors in the brain as opioids such as heroine, morphine and other drugs derived
from the poppy plant.
THC stimulates cells in the brain to release dopamine, creating euphoria. It also
interferes with how information is processed in the hippocampus, which is part of the brain
responsible for forming new memories. THC can induce hallucinations, change thinking and
cause delusions. These effects of marijuana make it a popular drug, but they also concern
mental health advocates. THC can trigger a relapse in schizophrenic symptoms, according to
the National Institute on Drug Abuse.
THC is one of many compounds found in the resin excreted by glands of the
marijuana plant. More of these glands are found around the reproductive organs of the plant

than on any other area of the plant. Other compounds unique to marijuana, called
cannabinoids, are present in this resin and may lessen the effect of THC. When THC is
exposed to air, it degrades into cannabinol, a cannabinoid which has its own psychological
effects. THC concentration also depends on the cultivation of the marijuana plant, known
scientifically as Cannabis sativa L.
Cannabis that has a minimal amount of THC up to 1 percent is cultivated as hemp.
Some strains of Cannabis can have as little as 0.3 percent THC by weight. In other strains,
THC makes up 20 percent of the weight in a sample. THC can be extracted from marijuana,
or synthesized as is the case for the FDA-approved drug dronabinol.

2.0 EFFECTS OF THC

2.1 Physiological Effects of Marijuana
The active ingredient in marijuana is THC. That's short for delta-9-
tetrahydrocannabinol. THC is rapidly absorbed after smoking pot. Within minutes, THC and
the other substances in marijuana smoke cause short-term medical effects.
Signs of using marijuana include:
 Rapid heart rate
 Increased blood pressure
 Increased rate of breathing
 Red eyes
 Dry mouth
 Increased appetite, or "the munchies"
 Slowed reaction time
These effects are reduced after three or four hours. However, marijuana hangs around
in your system for as long as a month after smoking. The lingering effects mean you're
impaired for several days to weeks after the high wears off.

2.2 Psychological Effects of Marijuana
According to the National Institute on Drug Abuse, the main effects of marijuana on
mood vary and may include euphoria, calmness, anxiety, or paranoia. Getting high or
"stoned" is the reason most pot smokers use marijuana.


2.3 Other short-term psychological effects of pot include:
 Distorted sense of time
 Paranoia
 Magical or "random" thinking
 Short-term memory loss
 Anxiety and depression
These psychological signs of using pot also generally ease after a few hours. But
residual effects can last for days.

2.4 Risks of Marijuana (THC) Use
The risks of smoking marijuana go up with heavy use. Although the link has never
been proven, many experts believe heavy pot smokers are at increased risk for lung cancer.
Heavy marijuana use lowers men's testosterone levels and sperm count and quality. Pot could
decrease libido and fertility in some heavy-smoking men.
Contrary to what many pot smokers may tell you, marijuana is addictive, at least
psychologically. Even among occasional users, one in 12 can feel withdrawal symptoms if
they can't get high when they want to. Among heavy pot smokers, the rates of dependence are
higher.
Many experts also believe that marijuana is physically addictive. Symptoms of
withdrawal from pot might include:
 Aggression
 Anxiety
 Depressed mood
 Decreased appetite

3.0 TYPES OF DRUGS THAT CONTAIN TETRAHYDRACANNABINOL (THC)
Pharmaceutical drugs have been developed which either contain or have similar
chemicals as those found in the marijuana (cannabis) plant. Some researchers have used their
understanding of how the brain processes cannabinoids to develop drugs which follow the
same pathways but work differently than marijuana. Pharmaceutical drugs based on
marijuana are divided into four categories and listed below with the names, trade names,
manufacturers, approval status, suggested medical use and cannabis-related properties. All
drugs referenced are in pill form unless otherwise noted (ProCog.org, 2013).

I. Drugs that contain chemicals taken directly from the marijuana plant
Name/Trade
Name
Manufacturer Suggested Medical
Use
Cannabis-Related
Properties
Sativex GW
Pharmaceuticals
Treatment of
neuropathic pain and
spasticity in patients
with Multiple Sclerosis
(MS); Analgesic
treatment in adult
patients with advanced
cancer who experience
moderate to severe
pain.
Mouth spray whose chemical
compound is derived from
natural extracts of the
cannabis plant. Sativex
contains two cannabinoids:
THC (delta-9-
tetrahydrocannabinol) and
CBD (cannabidiol).
II. Drugs that contain synthetic versions of chemicals naturally found in marijuana
Name/Trade
Name
Manufacturer Suggested Medical
Use
Cannabis-Related
Properties
Dronabinol/
Marinol
Unimed
Pharmaceuticals,
a subsidiary of
Solvay
Pharmaceuticals
Treatment of nausea
and vomiting for
patients in cancer
treatment; Appetite
stimulant for AIDS
patients; Analgesic to
ease neuropathic pain
in multiple sclerosis
patients
Synthetic Delta-9 THC
Dronabinol
Metered Dose
Inhaler (MDI)/
Marinol aerosol
Solvay
Pharmaceuticals
Treatment of nausea,
vomiting, migraines,
spasticity in MS
patients, and
neuropathic pain
Human-made THC
inhaler
III. Drugs that contain chemicals similar to those in marijuana but not found in the plant
Name/Trade
Name
Manufacturer Suggested Medical Use Cannabis-Related
Properties
Nabilone/
Cesamet
Valeant
Pharmaceuticals
International
Treatment of nausea and
vomiting in patients
undergoing cancer
treatment
Synthetic cannabinoid
similar to THC
Dexanabinol Pharmos Neuroprotective for use
after cardiac surgery
Regain memory and other
high-level function
following Traumatic
Brain Injury (TBI)
Synthetic non-psychotropic
cannabinoid which blocks
NMDA receptors and COX-
2 cytokines and chemokines

CT-3
(ajulemic
acid)
Atlantic
Technology
Ventures
Treatment of spasticity
and neuropathic pain in
MS patients
Synthetic, more potent
analog of THC metabolite
THC-11-oic acid
PRS-
211,375/
Cannabinor
and
Cannabinor
Oral
Pharmos Anti-inflammatory

Treatment of pain
Synthetic chemical that
specifically binds to the
brain's secondary
cannabinoid receptor (CB2)
HU 308 Pharmos
(licensed from
the Hebrew
University of
Jerusalem)
Treatment of
hypertension
Anti-inflammatory
Synthetic chemical that
specifically binds to the
brain's secondary
cannabinoid receptor (CB2)
HU 331 Cayman
Chemical
Treatment of memory,
weight loss, appetite,
neurodegeneration, tumor
surveillance, analgesia,
and inflammation
Synthetic chemical
compound composed of
central cannabinoid (CB1),
peripheral cannabinoid
(CB2), and non-CB
receptor-mediated
pharmacology
IV. Drugs that do not work like marijuana but use the same brain pathways
Name/Trade
Name
Manufacturer Suggested
Medical Use
Cannabis-Related Properties
Rimonabant/
Acomplia
Sanofi-Aventis Anti-obesity Synthetic chemical that blocks
endocannabinoids from being
received in the brain, and, as a
result, suppresses appetite.
Taranabant/
MK-0364
Merck Anti-obesity Targets receptors in the brain
linked to appetite. Acts as a
Cannabinoid receptor type 1
(CB1R) inverse agonist, blocking
cannabinoid receptors in the brain,
which suppresses appetite
URB597
or
KDS-4103
Cayman
Chemical
and
Kadmus
Pharmaceuticals
Inc
Treatment of pain
(acute - post-
surgical;
inflammatory -
arthritis;
neuropathic -
damaged nerve
cells from shingles,
H.I.V., diabetes),
anxiety, and
depression
Increases the amount of
endocannabinoids in the brain by
blocking the natural process of
deactivating them. The same
process that deactivates
endocannabinoids also blocks
chemicals which regulate mood
O-3246 Not available Treatment of
spasticity in MS
Increases the amount of
anandamide, an endocannabinoid,

patients by tricking the brain to produce
more instead of uptaking what is
already present
AM 281 Bachem
(formerly
Peninsula
Laboratories)
Neuroprotective
for use in
association with
septic shock
Synthetic chemical that blocks the
endocannabinoids from being
received in the brain, regulating
the flow of blood to the brain
during
septic shock

(ProCog.org, 2013).

4.0 PROCEDURE

TOXI-LAB PROCEDURE
In the TOXI-LAB procedure, urine samples were hydrolyzed at room temperature
with KOH and then extracted with a mixture of ethyl-acetate and hexane (1:9). The extracts
were concentrated onto discs, and those discs were inserted into a toxigram together with a
blank toxi disc and a standard disc containing 350ng of ∆
8
-THC-COOH. The plate was then
developed using a mixture of heptanes-acetone-glacial acetic acid (70:30:1) and visualized
with Fast Blue BB salt. The TOXI-LAB method allowed simultaneous extraction of 10
samples with one control and one standard, using a disposable applicator cartridge. In
general, the TOXI-LAB procedure was simple, easy to perform, and required minimal cost
and instrumentation. The system did not provide the high throughput capacity of automated
EMIT but was much better than conventional TLC. The TOXI-LAB assay was reported to be
successfully used as a screening method for urine samples or as a confirmatory technique to
the immunoassays to minimize the need and cost of the GC-MS confirmation (M.J. Bogusz,
2008).

THC II
THC II - A quick, easy, cost effective solution for screening or
confirmation of the Marijuana Metabolite, THC-COOH in urine.
Sensitivity of 15ng/mL for both screening and confirmation. Urine
samples are hydrolysed and then acidified, centrifuged and aspirated
through a SPEC-C18-1 column. The disc in the cartridge is then
washed and dried and inoculated (Microgen Bioproducts).
Figure 1 THC II

4.1 BACKGROUND
The TOXI-LAB THC TLC drug detection system provides for extraction, concentration,
inoculation, elution, and visualization steps for the detection of ∆
9
-THC-COOH in urine
specimens. The preliminary identification is based on matching the position of a drug (Rf)
and visualization color characteristics with that of corresponding reference material.

4.2 SCOPE
This method is an option to screen for the presence of ∆
9
-THC-COOH in urine. TOXI-
LAB THC II system provides a preliminary result that must be confirmed by GC-MSD.

4.3 SPECIMENS
4.3.1 The specimen volume is 2 mL for all specimen types except urine. The default
volume for urine is 1 mL.
4.3.2 Specimens include whole blood, serum, plasma, urine, and tissue homogenate.
4.3.3 Dilutions of specimens may be analyzed at the Forensic Scientist‟s discretion;
however, this should be done in addition to testing the standard specimen volume,
unless sample quantity dictates otherwise.
4.3.4 Analysis of larger specimen volumes must be approved and documented.

4.4 EQUIPMENT AND SUPPLIES
4.4.1 Tube rocker
4.4.2 Laboratory centrifuge
4.4.3 Solvent concentrator with appropriate concentration cups
4.4.4 Electric (plates) warmer
4.4.5 Fixed and adjustable volume single channel air displacement pipetters, and
appropriate tips, capable of accurate and precise dispensing of volume indicated.
4.4.6 Forceps
4.4.7 Disc handing pins
4.4.8 Index cards for use as disc press cards
4.4.9 TOXI-GRAMS Blank THC-II-PLUS
4.4.10 TOXI-GRAMS Blank THC-II
4.4.11 TOXI-DISCS THC
4.4.12 SPEC-C18-1 extraction cartridges
4.4.13 THC II wash reagent 1 bottle

4.4.14 THC II wash reagent 2 bottle
4.4.15 TOXI-DIP THC-1 reagent tank with Lid
4.4.16 TOXI-DIP THC-2 reagent tank with Lid
4.4.17 HCl reagent tank with Lid
4.4.18 Chromatography tank with Lid (THC-II-PLUS)
4.4.19 Chromatography jar with Lid (THC-II)
4.4.20 TOXI-LAB THC elution solvent bottle

4.5 REAGENTS
4.5.1 11.8N KOH
4.5.2 Methanol
4.5.3 Wash reagent 1: 20% acetic acid
4.5.4 Wash reagent 2: 20% methylene chloride in n-Heptane
4.5.5 TOXI-DIP THC-1 Fast Blue BB: prepare with approximately 1g Fast Blue BB salt
(purified grade). Add Fast Blue BB to reagent tank for TOXI-DIP THC-1. Add
approximately 700mL methylene chloride. Solution should be pale yellow in color.
Mix well. Store at room temperature. Solution stable for 2-3 months.
4.5.6 TOXI-DIP THC-2 Diethylamine (DEA) Fuming: pipet 40mL DEA through an
opening in the standoff to the bottom of the tank. Remove any DEA on standoff
surface. Store at room temperature. Replace DEA weekly.
4.5.7 HCl Fuming: pipet 40mL concentrated HCl through an opening in the standoff to the
bottom of the tank. Remove any HCl on standoff surface. Store at room temperature.
Replace HCl weekly.
4.5.8 THC II Stock Elution Solution: in the THC II Elution Solution bottle, mix 50mL n-
heptane, 50mL acetone and 1mL glacial acetic acid. Cap tightly and mix. Store at
room temperature.

4.6 REFERENCE MATERIAL

4.6.1 POSITIVE CONTROL
Positive control can be prepared by adding specified amount of Working Control
Solution to negative urine and/or obtained commercially. Use the same lot of negative
urine to prepare positive control as used to prepare negative control.


4.6.1.1 Stock Reference Solution
100µg/mL (+) 11-nor-9-carboxy-∆
9
-THC
4.6.1.2 Working Reference Solution (1800ng/mL)
Add 900µL Stock Solution to 49.1mL methanol. Solution is stable for six months
when stored at 4°C.
4.6.2 NEGATIVE CONTROL
Negative urine: may be either in-house verified or commercially obtained.

4.7 PROCEDURE
4.7.1 INITIAL SET-UP
Label extraction tubes and extraction cartridges for the negative contro, positive
control, and appropriate laboratory numbers.
4.7.2 60ng/mL CARBOXY-THC POSITIVE CONTROL
4.7.2.1 Transfer 6mL of negative urine to extraction tube.
Use the same lot of urine for negative control.
4.7.2.2 200µL of working reference solution. Vortex.
4.7.3 NEGATIVE CONTROL
Transfer 6mL of negative urine to extraction tube.
4.7.4 CASEWORK SAMPLE PREPARATION
Transfer 6mL of casework urine specimen to extraction tube.
4.7.5 SAMPLE HYDROLYSIS
4.7.5.1 To 6mL of urine, add 12 drops 11.8N KOH. Vortex.
4.7.5.2 Allow to hydrolyze for 10 minutes.
4.7.5.3 Add 1.5mL glacial acetic acid. Vortex.
4.7.6 EXTRACTION
4.7.6.1 Condition cartridge with 1mL methanol. Aspirate at approximately 5 in. HG. Do not
allow the disc to dry.
4.7.6.2 Add acidified samples to cartridge reservoirs. Aspirate such that the sample passes
through the column no faster than 2mL/min.
4.7.6.3 Once the sample is completely through the reservoir, remove filter.
4.7.6.4 Add 1mL 20% acetic acid. Aspirate ≥2 min at 10-12 in. Hg.
4.7.6.5 Ass 500µL wash reagent. Aspirate at 2 min at 10-12 in. Hg.
4.7.6.6 After solvent has past through, allow to aspirate ≥2 min.

4.7.6.7 Remove disc from cartridge and place into a pre heated concentrated cup to remove
all residual moisture.
4.7.7 TLC
4.7.7.1 Place disc into labeled 3 or 10-channel TOXI-GRAM for THC-II.
4.7.7.2 Add THC-DISC THC disc. (if not all channels are used, remove excess with razor
blade or scissors.)
4.7.7.3 Heat the GRAM, with the disc and slightly off the warmer edge, for 30-60 sec.
4.7.7.4 Add 12.5 mL of developing solution to chromatography tank (10 channel) or 3mL
solution to chromatography jar (3 channel).
4.7.7.5 Place GRAM into chromatography tank or jar. Allow dye marker to migrate to almost
4cm. (this only take 2-3 min)
4.7.7.6 Remove GRAM from tank/jar and place face down on warmer for 1-2 min.
4.7.7.7 Dip GRAM into TOXI-DIP THC-I, hold to dry until GRAM become speckled.
4.7.7.8 Place GRAM into TOXI-DIP THC-II until scarlet spots develop.
4.7.7.9 Place GRAM in hood so that the DEA can evaporate. If any DEA is present when the
HCl is added, fuming will occur.
4.7.7.10 For HCl fuming, either of the following options may be pursued. Option 2 will
produce a more intense color which photocopies better.
Option1: place GRAM into HCl fuming Tank until a purple spot develops. Place
GRAM into page protector, label and photocopy.
Option 2: place GRAM onto a page protector. With bulb pipet, add concentrated HCl
to just cover GRAM. Note desired color change to deep purple. Label and photocopy
GRAM.
4.7.7.11 Place a copy of GRAM into each associated casefile.
4.7.8 Detection and identification criteria
The position (Rf) and color characteristics at each stage of visualization of a spot
noted for a specimen must correspond to that of reference material (Quality Manager
of Idaho State Police, 2008).

4.8 Drug Test Detection Times
Drug tests detect not only drugs but metabolites as well. Metabolites are the
byproducts of a substance after it has run through system. To determine whether a person will
pass or not, it is important to know how much of the illicit metabolites are in urine, as well as
the test “cut off,” or Level of Detection (LOD). Usually marijuana (cannabis or THC)

tests will have a cutoff of 50ng/mL, but it can be as low as 25ng/mL. Home testing can show
whether a person level at the time of the test is above or below the 50ng/mL level, but it will
not show the exact level of THC metabolites in system.

4.8.1 Marijuana Detection Time and Half-Life of TetraHydraCannabinol (THC)
The half life of THC concentration is about 10 days. There is way too much variation
to even approximate how long THC will be detected in the urine of an individual. Infrequent
users with a fast metabolism will have the shortest detection time. Frequent or chronic users
with a slow metabolism will have the longest detection times. The only way to estimate a
detection time is to consider the lower and upper bounds (3-50 days), and estimate based on
the factors outlined here.
Marijuana Detection Time Based on Usage
Usage at 1 time only 5-8 days
Usage at 2-4 times per month 11-18 days
Usage at 2-4 times per week 23-35 days
Usage at 5-6 times per week 33-48 days
Daily Usage 49-63 days
Note: Detection times vary depending on many factors, including drug potency, tolerance,
patient‟s condition, fluid intake at time of test, method and frequency of marijuana use, body
type, metabolism, exercise frequency and many others. These are general guidelines only.
4.8.2 How is a “Positive Test” Defined?
“50 nanograms of THC metabolites per milliliter” defines a presumptive positive by
most laboratories and instant tests. This value was originally 20 ng/mL, but too many false
positives resulted. So the level was raised to 100 ng/mL to reduce false positives. As of
January 1995, the threshold was lowered back down to 50 ng/mL because it became known
that drinking excessive water could easily bring the level of metabolites in the urine below
100 ng/mL. Some employers may use a lower cutoff, but that is rare. If a specimen screens
non-negative for THC (anything other than negative or fails an integrity test), the specimen is
then sent through a Gas Chromatograph/Mass Spectrometer (GC/MS) for the specific
metabolite tripping the immunoassay screening. This level is set at 15 ng/m, and is used as
the ultimate confirmation for a positive on drug test.



4.8.3 Can Second hand Smoke Cause a Positive Drug Test?
In rare occurrences, second hand marijuana smoke can cause to fail drug test. It is
possible that second hand marijuana smoke will raise someone to the 50 ng/mL level.
However, extreme second hand exposure is required. For instance, being in a closed car full
of pot smokers for several hours might cause someone to test positive in a drug test the very
next day. Non-smokers are safe in a ventilated area such as an average living room or garage
where partygoers are smoking pot.

4.8.4 What about Other Drugs? How Long Do They Stay In System?
50% of all drug test positives are for marijuana. What about the other 50%? The table
below shows how long drugs other than marijuana typically stay in system:
Urine Blood Hair Saliva
Marijuana-
Single Use
1-7 + days 12-24 hours Doubtful
Not validated.
Estimated 0-24
hours
Marijuana –
Regular Use
7-100 days 2-7 days
Months
Amphetamines 1-3 days 24 hours
Cocaine 1-3 days 1-3 days
Heroin,
Opiates
1-4 days 1-3 days
PCP 3-7 days 1-3 days

The most popular kind of drug test is the urine test, which can detect marijuana for
days or weeks after use. Note that urine tests do not detect the psychoactive component in
marijuana, THC (delta-9-tetrahydrocannabinol), and therefore in no way measure
impairment; rather, they detect the non-psychoactive marijuana metabolite THC-COOH,
which can linger in the body for days and weeks with no impairing effects. Because of THC-
COOH's unusually long elimination time, urine tests are more sensitive to marijuana than
other commonly used drugs (California NORML Guide Interpreting Drug Test Results,
2012).
Blood tests are a better detector of recent use, since they measure the active presence
of THC in the system. Because they are invasive and difficult to administer, blood tests are
used less frequently. They are typically used in investigations of accidents, injuries and DUIs,

where they can give a useful indication of whether the subject was actually under the
influence (California NORML Guide Interpreting Drug Test Results, 2012).
Hair tests are the most objectionable form of drug testing, since they do not measure
current use, but rather non-psychoactive residues that remain in the hair for months
afterwards. These residues are absorbed internally and do not appear in the hair until 7-10
days after first use. Afterwards, they cannot be washed out by shampoos (though shampoos
may help remove external smoke particles that get stuck in the hair). Hair tests are more
likely to detect regular than occasional marijuana use. One study found that 85% of daily
users tested positive for marijuana, versus 52% of occasional smokers (1-5 times per week).
Ingested cannabis was less likely to be detected than smoked marijuana. It is doubtful
whether hair tests are sensitive to one-time use of marijuana (California NORML Guide
Interpreting Drug Test Results, 2012).
Saliva testing is a newer, less proven technology. The sensitivity of saliva tests is not
well established in the case of marijuana. In theory, they are supposed to detect recent use,
but this may range from several hours to over a day. They are supposed to detect secretions
from inside the oral tissues that cannot be washed out with mouthwash. Because they are less
intrusive than blood or urine tests, the industry has been eager to develop saliva tests. Due to
reliability problems, they have yet to gain acceptance in the U.S., but they have come into use
in some other countries, such as Australia. An international study of various onsite saliva
tests concluded that no device was reliable enough to be recommended for roadside screening
of drivers (California NORML Guide Interpreting Drug Test Results, 2012).

4.8.5 Why Choose Oral Fluid versus Urine Drug Testing?
Oral fluid testing can detect drugs anytime, from immediately after the most recent
use, up to three days after use. Conventional drug testing with urine cannot accurately detect
usage in the first four hours after use, thus making saliva superior for post-accident testing
(Forensic Fluids Laboratories, 2011).

4.8.6 Saliva THC VS Urine THC
In general, what is in the blood, is in the saliva. If a drug is smoked, like THC, you
may also find this in the mouth, thus giving a positive, saliva drug test. THC psychoactive
effects are seen for 3 to 5 hours after normal drug use, although further effects, for longer
periods, have been identified. Delta 9 THC, the parent, and 11 hydroxy 9 delta 9 THC, the
metabolite, are the psychoactive compounds (Forensic Fluids Laboratories, 2011).

Saliva THC
Delta 9 THC is the parent compound. When found in saliva, this means impairment,
because the drug has entered the blood stream and affected the brain, thus affecting motor
skills. Levels we have measured in saliva in the lab range from 0.5ng/mL to 150ng/mL (1
ng/mL = 1 part per billion). All of these mean the parent compound is available to the brain.
The higher the level, the more drug ingested or the more recently the drug was used. Delta 9
THC has been measured up to 72 hours after smoking, in saliva (Forensic Fluids Laboratories,
2011).
Urine THC
11 nor 9 carboxy THC is the major metabolite found in urine. A single dose of THC
may be detected in urine for an average of 24 to 120 hours. In most peer reviewed papers, the
highest doses produced positive screens for up to 120 hours, with most papers reporting an
average of 26 to 33 hours. (These data are taken from over 80 peer reviewed papers). Urine
excretion of THC does not decrease monotonously so urine screenings may fluctuate between
negative and positive for a while (Forensic Fluids Laboratories, 2011).

5.0 IDENTIFICATION OF THC

In general, immunoassays (IA) are adopted as a preliminary test method in drug
testing programs. However, false-positive (or false-negative) results occur from structurally
related drugs that are recognized by the antibodies or occasionally by artefacts, such as
adulterants affecting pH, detergents, and other surfactants. For this reason, any positive result
must be confirmed by a Chromatographic technique, preferably with mass spectral
identification. Gas chromatography and liquid chromatography coupled with mass
spectrometry (GC/MS or LC/MS) are the „„gold standard‟‟ procedures. In some previous
studies, the window of detection for THC-COOH or cannabinoids in urine samples was
demonstrated with ranges from several days in infrequent users to weeks or months in
frequent users (Table 1).




6.0 ANALYSIS OF THC

Example Study: THC detection time in occasional marijuana smoker
(using an immunochromatographic assay)

This document contains the results and specifics of a study designed to test THC
metabolite detection time in urine. A primitive study was performed to determine the timeline
of detectablity of ingested THC. It is generally purported that the ingestion of marijuana is
detectable in urine for weeks or even months after ingestion. It has been reported that
occasional users purge any detectable levels of THC from their system within 48-72 hours. It
is widely accepted that other ingested illicit substances including Cocaine,
Methamphetamine, and MDMA are not detectable after 24-48 hours of ingestion, thereby
making THC the only illicit drug widely believe to be reliably tested for long term using a
urinalysis. This study casts doubt on the validity of drug testing as a reliable means for
determining ingestion of illicit substances at all due to the relatively short detection times of
any drug.
The study tested one subject using a common and readily available IC urinalysis(ICA)
test. On day one(D1) an initial test was performed to ensure that the subject was free of
detectable levels of THC. On day two(D2) the subjected ingested THC using the common
procedure of smoking dried cannabis using a pipe. Approximately two 'bowls' were smoked

over the course of a day, and the subject ingested his last 'hit' at approximately 8pm on day
two(D2). After the initial ingestion the subject abstained from any further contact with THC
or marijuana for the duration of the study. No special cleansing products were used, no
unusual amounts of liquids were consumed, and no adulterants were introduced into the
specimen.
The subject was then tested every day until the ICA(*1) tested negative (-) for
detectable levels of THC(D4). The subject was then tested for one additional day(D5) to
ensure the reliability of the previous test(D4).
The subject is a 21 year old male 5'7' aprox. 145lbs - occasional marijuana smoker
(~1/wk-1/mo) who prior to initial testing abstained from ingesting marijuana(THC) for
approximately two weeks before the initial ICA(*1) urinalysis on which he tested negative(-).

IC Assay Overview:
The ICA(*1) is a competitive binding immunoassay in which drug and drug
metabolites in a urine sample compete with immobilized drug conjugate for limited labeled
antibody binding sites. By utilizing antibodies that are specific to different drug classes, the
test permits independent, simultaneous detection of two drugs from a single sample. The
approximate run time is 5 minutes.
In the assay procedure, urine mixes with labeled antibody-dye conjugate and migrates
along a porous membrane. When the concentration of a given drug is below the detection
limit of the test, unbound antibody-dye conjugate binds to antigen conjugate immobilized on
the membrane, producing a rose-pink color band in the appropriate Test Zone for that drug.
Conversely, when the drug level is at or above the detection limit, free drug competes with
the immobilized antigen conjugate on the membrane by binding to antibody-dye conjugate,
forming an antigen- antibody complex, preventing the development of a rose-pink color band.
Regardless of the drug levels in the sample, a rose-pink color band is produced in each
Control Zone (marked “C”) by a parallel immunochemical reaction. These bands serve as
built-in quality control measures by demonstrating antibody recognition, verifying that the
reagents are chemically active. Each ICA(*1) contains dye-conjugated antibody and
immobilized antigen in protein matrix with sodium azide. The samples were tested by
immersing the ICA(*1) test strips in the urine for ~10 seconds congruent with package
labeling.



The test is a two panel test purported to detect at the NIDA cutoff levels:
 THC(tetrahydrocannabinol) at 50ng/ml
 METH(methamphetamine) at 500ng/ml.

The ICA(*1) consisted of two panels, one that tested for the presence of METH, and
one for the presence of THC. Each panel had two lines, the first line being the metabolite
detection line, the second line being the urine detection line. The second line alerts you to the
fact that the urine has in fact passed over the metabolite detection line, allowing you to
determine that the sample has traveled over the metabolite detection line and has in fact been
tested.
 A positive detection of THC was determined by the lack of a detection line.
 A negative detection of THC was determined by the presence of a detection
line.
According to the package, the width and opacity of the detection lines indicate
nothing about the intherent presence of THC or METH in the urine, though we found on day
three a slight presence of a detection line, it was still determined to be a failing test, as the
line was so faint as to be inconsequential. It was noted that the on THC panel the urine
detection and THC detection lines were consistently lighter than the METH panel lines.

Study Analysis:
Days on which tests are passed are denoted by color, green is a passing test, red is a failed
test.
Day One (Thursday) (D1): Initial test to determine passing levels of THC in the urine.
Day Two (Friday) (D2): Day of ingestion
Day Three (Saturday) (D3): T+24
Day Four (Sunday) (D4): First Passing Day, approximately two days (48 hours) after
initial ingestion
Day Five (Monday) (D5): Consistent with the previous day, the subject passed the
ICA(*1) test.
Study Summary:
The study showed that in an occasional marijuana smoker that detection times for THC are
approximately 24 hours, within 48 hours of ingestion the subject was testing clean (-)
Detection times were determined to be so short that it would be possible to ingest THC on a

friday and be clean by monday, thereby negating any test given after 48 hours of initial
ingestion.

Figure: THC detection urine tests



















Reference
California NORML Guide Interpreting Drug Test Results, 2012. Drug Test Detection.
Available online at: http://www.canorml.org/healthfacts/drugtestguide/drugtest
detection.html. Accessed on November 4, 2013.

Forensic Fluids Laboratories, 2011. Oral Fluid vs. Urine Drug Testing. Available online
at: http://www.forensicfluids.com/oral_v_urine.htm. Accessed on November 4, 2013.

Microgen Bioproducts. Toxicology. Available online at: http://www.microgenbioproduct
s.com/pdf/Product%20Brochures_Individual/Toxilab.pdf. Accessed on November 4,
2013.

M.J. Bogusz, 2008. Handbook of Analytical Separations Volume 6 Forensic Science 2
nd

Edition. UK: Elsevier. Page 213.

Marijuana. Drug Test Detection Times. Available online at: http://www.marijuana.com/
news/drug-test/detection-time/. Accessed on November 4, 2013.

ProCog.org, 2013. Pharmaceutical Drugs Based on Cannabis. Available at: http://medical
marijuana.procon.org/view.resource.php?resourceID=000883.Accessed on November
4, 2013.

Quality Manager of Idaho State Police, 2008. Urine Toxicology. Available online at:
http://www.isp.idaho.gov/forensics/documents/archivedAMs/Toxicology/2.2.4%20T
OXI-LAB%20c-THC%20rev%205.pdf. Accessed on November 4, 2013.