Indian J Med Res 132, August 2010, pp 201-208

Qualitative high performance thin layer chromatography (HPTLC)

analysis of cannabinoids in urine samples of Cannabis abusers

Priyamvada Sharma*, M.M. Srinivas Bharath** & Pratima Murthy*,+

Deaddiction Unit & Departments of **Neurochemistry & +Psychiatry, National Institute of Mental Health &
*

Neurosciences, Bangalore, India

Received May 12, 2009

Background & objectives: Cannabis is one of the most commonly abused drugs worldwide. There is a
distinct clinical correlation between cannabis abuse and mental disorders. However, it is essential to
establish cannabis intake in the abusers in order to establish causality between cannabis and psychiatric
illness. The limitations of current detection methods using commercial cassettes prompted us to
standardize the method of extraction and detection of cannabinoids in the urine samples of cannabis
abusers attending a de-addiction centre in south India.
Methods: In this study, diagnostic tests on 102 male patients suspected with cannabis abuse were done.
Liquid-liquid extraction of cannabinoids from urine was done and screened by Duquenois-Levine, fast
blue B salt and p-dimethylaminobenzaldehyde (p-DMAB) tests. All the results were confirmed by high
performance thin layer chromatography (HPTLC). Samples were considered positive for cannabis based
on the positive indication in colour test and by detection of 11-nor-Δ9 tetrahydrocannabinol-9-carboxylic
acid (THC-COOH) on HPTLC.
Results: Based on the colour tests and HPTLC, cannabis abuse was detected in 64 of 102 patients tested.
HPTLC method was found to be sensitive for detection and possible quantitation of THC-COOH.
Interpretation & conclusion: We report the standardization and utility of cannabinoid extraction,
screening and detection by HPTLC in the urine samples of cannabis abusers. The HPTLC method was
found to be high throughput, sensitive, reproducible and cost-effective compared to commercial kits.

Key words Cannabinoids - cannabis - deaddiction - drug abuse - thin layer chromatography

Many studies have demonstrated that psychosis,
violence, aggression and crime are closely associated
with drug abuse thereby making substance abuse
a complicated psychosocial condition1-4. Analysis
of psychotic behaviour among substance abusers
provides valuable clinical and biochemical information
regarding the mechanism of action of the compounds
and their role in psychosis5. However, it is necessary to
demonstrate the presence of the implicated substance
in order to make a more definitive diagnosis for a
substance induced disorder. To establish such a positive
correlation, it is necessary to develop and improve
methods of drug detection in patient samples.
The abuse of cannabis, one of the most common
illicit drugs and its association with psychosis has been
observed in India5-8. Cannabis (Marijuana or charas/

201
202 INDIAN J MED RES, AUGUST 2010
ganja/bhang as it is known commonly in India) is
predominantly obtained from Cannabis sativa L. It
includes all parts of the plant except the seeds and
woody material. Cannabis and cannabis resins (Hashish)
are smoked with tobacco or ingested9,10. Once ingested,
cannabis is metabolized to generate several metabolites
in the human body. ∆9-tetrahydrocannabinol (THC) is
considered as the primary psychoactive compound
in cannabis. Once ingested, THC is metabolized to
generate its hydroxylated and carboxylated metabolites
of which, 11-nor- ∆9tetrahydrocannabinol-9-carboxylic
acid (THC-COOH) is the major metabolite excreted in
urine11. Although only a few cannabinoids are detectable
in the urine, detection of THC-COOH is considered as
a confirmatory test for cannabis9.
The presence of cannabinoids is usually detected
using colour tests12, high performance liquid
chromatography (HPLC)9,13-15, gas chromatography16
and commercially available immunoassay based
cassettes17-20. Following screening tests for cannabinoid
detection, it is necessary to perform confirmatory
tests using advanced techniques such as fluorescence
polarization immunoassy21, enzyme immunoassay22
high performance thin layer chromatography
(HPTLC)23 and HPLC, which provide additional scope
for quantitative monitoring of drugs during follow up
of patients24. However, utilization of such confirmatory
tests in the de-addiction centers of India is limited.
The de-addiction centre housed in the National
Institute of Mental Health and Neurosciences
(NIMHANS), Bangalore (www.nimhans.kar.nic.in /
deaddiction), India, was started in 1994 as a nodal
centre for south India. The current study describes a
standardized method of extraction and detection of
cannabinoids in the urine of 102 suspected cannabis
abusers attending NIMHANS de-addiction centre of
which, 40 samples were also tested for cannabis in
parallel by commercial cassettes.
Material & Methods
All chemicals used were of analytical grade. Bulk
solvents and routine chemicals were obtained from
Sisco research laboratories (Mumbai, India) and Merck
& Co. Inc (Whitehouse Station, NJ, USA). Ready-to-
use silica gel coated plates were obtained from Merck.
Commercial test kits for cannabis were obtained from
Nano-Ditech corp. NJ, USA.
Receipt of urine samples: This investigation is
preliminary to a large study that involves analysis of
substance abuse in first episode psychosis patients for
which approval has been obtained from the institutional
ethics committee.
Urine samples from 102 male patients (n=102;
age 28.6 + 8.3 yr) exhibiting psychotic behaviour
with suspected cannabis abuse admitted to outpatient
department/ de-addiction centre or psychiatric wards
of NIMHANS, Bangalore between September 2008
and March 2009 were collected for detection of
cannabinoids. Each sample was accompanied with a
requisition form containing the details of the patient
including name, ward number, clinical profile,
suspected drug used and last date of abuse. A detailed
case history of individual patients was recorded during
the OPD sessions. For 40 randomly selected patients,
urine samples were analyzed by commercial test kits
according to the specifications of the manufacturer.
Approximately 200 µl of the urine sample was placed
on the test window of the cannabis cassette through a
dropper and was allowed to move along the cassette
by capillary action. Cannabis positive sample were
identified by the absence of a band against appearance
of a distinct band in the positive control. The samples
(10-20 ml per patient per evaluation) were collected
in 20 ml labelled leak-proof sterile plastic containers.
Temperature and pH of the sample was checked to
monitor any adulteration. In most cases, the samples
obtained were processed for drug test on the same day.
If not, the samples were stored at 0-4oC until extraction
and analysis.
Preparation of cannabis standard solution: Cannabis
resins were extracted from the dried parts of Cannabis
sativa plants based on the method described earlier24,26.
Briefly, the dried leaves and the flowering tops of
cannabis plant were soaked in chloroform or petroleum
ether (1 mg/ml w/v). The solvent enriched in cannabis
resins was separated, evaporated to dryness and the
residue was reconstituted in one ml methanol and used
as a positive control (cannabis standard) throughout
the study.
Extraction of cannabinoids from urine samples:
Cannabinoids were extracted from urine by specific
liquid-liquid extraction method that was slightly
modified from the protocol described previously24,27,28.
To 10 ml of urine, 1 ml of 6M NAOH was added, for
alkaline hydrolysis, vortexed and heated at 60oC for
30 min. The sample was cooled and pH adjusted to
4.0 with acetic acid to enrich for THC-COOH. For
enrichment of cannabidiol (CBD) and cannabinol
(CBN), pH was maintained in alkaline condition. In
both cases, the cannabinoids were extracted with ethyl
 SHARMA et al: DETECTION OF CANNABINOIDS IN URINE 203
acetate: iso-propanol (8.5:1.5 v/v). Total volume was
about 22-24 ml per extraction. The aqueous phase was
again extracted twice with ethyl acetate: isopropanol
(8.5:1.5v/v).The aqueous phase was discarded and the
organic phase of all the three extractions was mixed
and evaporated to dryness at 70-80oC in a water bath.
The residue was reconstituted in 500 µl methanol and
subjected to colour test and HPTLC analysis.
Colour tests for cannabis: The extracted samples were
first screened by the following standard colour indicator
based methods for the presence of cannbinoids and
were confirmed by HPTLC followed by spray test.
(i) Duquenois-Levine test - This test was carried out
based on the method previously published12. Briefly, to
100 µl extract, 200 µl of Duquenois reagent (2.5% v/v
of acetaldehyde and 2% vanillin w/v in 95% ethanol)
was added and mixed thoroughly for 1 min. To this
mixture, 200 µl concentrated hydrochloric acid (HCl)
was added and mixed gently followed by addition of
500 µl chloroform. Appearance of purple colour after a
few minutes that moved into the chloroform layer was
considered as a positive indicator of cannabinoids.
(ii) Fast blue B salt test - This was carried out based on
the method described previously29. Briefly, to 10-20 µl of
the extract, a pinch of fast blue B salt (fast blue B mixed
with anhydrous sodium sulphate in the ratio 2.5 : 100)
was added. To this mixture, 500 µl of chloroform was
added and mixed thoroughly for 1 min. Later, 20 µl of
0.1 N aqueous NaOH was added and vortexed for 2 min.
The presence of wine red colour in the chloroform layer
was considered as a positive indicator of cannabis.
(iii) p-dimethylaminobenzaldehyde (p-DMAB) test -
This test was carried out based on the method previously
published12. Briefly, 100 µl fresh p-DMAB reagent (4%
w/v of p-DMAB in 1:1 mixture of 95% ethanol and
concentrated HCl) was added to 100 µl of the extracted
sample and presence of red colour changing to violet
on dilution was considered positive for cannabis.
HPTLC based detection of cannabinoids: Following
extraction and colour tests, cannabis standard and
extracted samples (up to 20 samples/batch) were
processed on the automated HPTLC system (CAMAG,
Muttenz, Switzerland) according to the instructions of
the manufacturer. Ready-to-use silica coated plates
(Cat. No. 1.05547.0001 by Merck) were activated by
blowing hot air for 5-10 min and placed in the automatic
sample applicator. The HPTLC was programmed to
automatically spray 5-10 µl of each sample in band
form using specialized Hamilton syringe on one-side
of the TLC plate in individual tracks. The TLC plate
was developed in ethyl acetate: methanol: ammonia
(8.5:1:0.5) or petroleum ether: diethyl ether (ratio 9:1
v/v) solvent system as described earlier16,17. The plate
was developed in the automated developing chamber
(CAMAG) until the solvent front reached the maximum
distance (80 mm distance in a typical 20 x 10 cm plate).
The developed plate was dried with a plate drier and
subjected to UV analysis (wavelength: 200-600 nm) in
the dedicated UV detector. All tracks in the plate were
scanned at user-defined wavelength (278 and 350 nm
for cannabis) and individual Rf values of peaks were
obtained. These data were matched with the cannabis
standard and compared against the in-built CAMAG
drug/chemical library to identify the cannabinoids in
the urine sample.
Spray test for cannabnoids: Following HPTLC,
visualization of cannabinoids was also carried out
by spraying fast blue B solution onto the developed
plate. Appearance of red, orange and purple spots was
considered as positive evidence for the presence of
THC-COOH, CBD and CBN respectively29.
Results
This study was done to generate a standardized
protocol for detection of cannabinoids in the urine
samples of patients with cannabis abuse and associated
psychosis. A standard cannabinoid mixture (positive
control) utilizing the street sample of cannabis plant
was prepared and the presence of cannabinoids in
this mixture was confirmed by all the color indicator
based tests. The presence of cannabinoids only in
organic phase and not in aqueous fraction confirmed
the enrichment of cannabinoids by the extraction
procedure (data not shown). It was observed that the
colour tests were specific only to cannabis patients and
were not reliable in multiple drug-abusers.
To further characterize the components of cannabis,
the cannabis standard was subjected to HPTLC analysis
in different solvent systems. Based on preliminary
experiments, it was found that the cannabinoids
exhibited different Rf values in different solvent
systems (Table I). We selected THC-COOH, CBN and
CBD as major cannabis metabolites for detection in the
standard and urine samples24,30.
HPTLC based separation of cannabinoids was
done using ethylacetate: methanol: ammonia (8.5:
1 : 0.5) solvent system. Fig. 1A and 1B show the
chromatogram and spectral scanning curve (200-700
nm) for THC-COOH.
204 INDIAN J MED RES, AUGUST 2010

Table I. Rf values of cannabinoids in solvent systems: (a) ethyl acetate: methanol: ammonia (8.5:1:0.5) (b) petroleum ether: diethyl ether
(4:1) (c) cyclohexane: di-isopropyl ether: diethylamine (5.2:4:0.8) (d) heptane: diethyl ether: glacial acetic acid (80:10:4) (e) plate sprayed
with di-ethylamine; solvent system: xylene: hexane: diethylamine (2.5:1:0.1)
No. Metabolite Rf in (a) Rf in (b) Rf in (c) Rf in (d) Rf in (e)
1. THC-COOH 0.32+0.06 (n=57) 0.32 0.39 0.32 0.29
2. CBN 0.95 0.27 0.28 - 0.20
3. CBD 0.95 0.36 0.44 - 0.36
In all the solvent systems, the Rf values are subject to minor variation depending on the laboratory conditions such as temperature, humidity
and other parameters (e.g., age and quality of the patient material)
CBD, cannabidiol; CBN, cannabinol; THC-COOH, 11-nor-Δ9 tetrahydrocannabinol-9-carboxylic acid

Table II. Summary of the diagnostic tests on ten samples of cannabis abusers
Patient no. Rf for THC-COOH Duquenois-Levine Fast blue B p-DMAB Final result
+/- (colour) +/- (colour) +/- (colour)
1 0.36 + (purple) + (red) + (red) +
2 0.36 + (purple) + (red) + (red) +
3 0.36 + (purple) + (red) + (red) +
4 - - - - -
5 - - - - -
6 - - - - -
7 - - - - -
8 - - - - -
9 0.35 + (purple) + (red) + (red) +
10 - - - - -
Cannabis standard 0.35 + (purple) + (red) + (red) +

Following extraction of cannabinoids from urine

samples of all 102 patients, all the samples were
screened by Duquenois-Levine, fast blue BB salt
and p-DMAB tests. Table II shows findings of ten
representative samples from among the 102 samples
analyzed by the three screening tests. The samples
were further confirmed by HPTLC analysis. (A)
For HPTLC, 5-10 µl of each sample was spotted
on pre-activated silica gel (11 samples per plate
including one cannabis standard as positive control)
and developed in ethyl acetate-methanol-ammonia
solvent system in the automatic developing chamber
(Fig. 2). The dried plates were scanned in the TLC
scanner at wavelength 278 nm. The peaks obtained
in all the tracks were analyzed and the Rf value was
compared to the standard. The presence of a specific
peak for THC-COOH at Rf around 0.32 + 0.06 was
recorded and considered as a positive result for
cannabinoids (Fig. 3 and Table II). We observed that (B)
Wavelength (nm)
two of the samples not confirmed by the colour tests
were found to contain the THC-COOH peak indicating Fig. 1. HPTLC profile of cannabis standard run on silica plates and
developed in solvent system A (ethyl acetate: ammonia 8.5:1 : 0.5).
the level of sensitivity of the HPTLC and the necessity
(A) shows absorbance at 278 nm (A.U= arbitrary units) of THC-
for confirmation following colour based screening tests. COOH in the cannabis standard plotted against Rf value. (B) shows
Further, the absorbance (OD) value of THC-COOH the spectral scanning of THC-COOH in the cannabis standard with
varied differently in different samples indicating the absorbance (AU) plotted against wavelength.
 SHARMA et al: DETECTION OF CANNABINOIDS IN URINE 205

Fig. 2. HPTLC profile of extracted cannabinoids from patient samples developed in solvent system A
(ethyl acetate: ammonia 8.5:1 : 0.5). + corresponds to positive control (Cannabis standard). Rest of the
lanes correspond to the profile of cannabinoids extracted from patient samples (patient nos. 1 to 10 as
shown in Table II). Reference scale for Rf value is also shown.

Fig. 3. 3-dimensional representation of the scanning data of the HPTLC plate shown in Fig. 2. The HPTLC
profile of cannabinoids extracted from urine samples patients and plotted as absorbance (OD) vs. Rf value. “+”
indicates the profile of cannabis standard. The numbers 1 to 10 correspond to the HPTLC profile of samples
1-10 (shown in Table II). Arrows indicate the position of the THC-COOH peak (Rf ~0.32) in cannabis positive
samples.
206 INDIAN J MED RES, AUGUST 2010
Table III. Summary of the drug analysis carried out on urine samples of all cannabis abusers included in this study
Result Colour test
Duquenois- Fast blue
Levine test B Test
Positive 62 62
Negative 38 38
Not determined 2 2
Total
*
Immunoassay based commercial cassette screening was initially carried out on 40 randomly selected from among the 102 samples. Further
confirmation by HPTLC showed that the cassette-test results matched with the outcome of the HPTLC analysis (i.e., 26 positive and 14
negative among 40 samples) indicating the consistency of analysis
level of abuse among the patients. Specific quantitation
of THC-COOH levels would be more useful and could
be correlated with psychiatric symptoms. In this study,
of the 102 suspected cases of cannabis abuse, based on
positive result in the colour test and HPTLC analysis,
64 samples were found to be positive (Table III).
Discussion
Toxicology of substance abuse poses a great
challenge to biochemists in terms of chemical analysis
and characterization of the effects of these drugs on
the human system. There seems to be convincing
evidence that cannabis abuse is linked with subsequent
occurrence of schizophrenia and psychosis2,5. For
reliable psychiatric correlation, there needs to be well-
standardized methods for cannabinoids. Although
cannabis test based on commercially available ready-to-
use cassettes is available, it has limited utility. Because
it is relatively expensive and has limited sensitivity
threshold, and gives only qualitative and not absolute
quantification. Most of the commercial kits clearly
state that the test provides only a preliminary result
and more specific alternative testing method should be
used to confirm the immunoassay result19. This could
be by either HPTLC or GC/MS or HPLC11,18. In routine
TLC testing, the detection is only by spray method and
the Rf value is not accurately recorded30. However,
UV based scanning after developing HPTLC plate
not only provides opportunity for scanning at specific
wavelengths but could also be useful for quantitation.
As mentioned by Meatherall and Garriott23, the limit of
detection for THC-COOH by HPTLC is 5 ng/ml when
2 ml of urine is used indicating the sensitivity of the
technique. The limit of detection of THC-COOH was
in the similar range in the current study. There have
been some reports on the utilization of TLC based
qualitative and semi-quantitative analysis of cannabis
Spray test HPTLC Immunoassay*
analysis
p-DMAB Fast blue –B
Spray
62 64 64 26
38 38 38 14
2 0 0 0
102 102 102 40
and cannabinoids such as THC-COOH30-32. However,
utilization of HPTLC based detection is very limited
in Indian centers. It was observed that the HPTLC
based detection of cannabinoids is ideal for a hospital
setting involving several patients. It is a cost-effective
(operational costs approximately INR 20 per sample),
highly sensitive, accurate and less time consuming
(following extraction, for 20 samples, starting from
sample application up to the final data analysis and
reporting only one hour is required) method. Further,
with specific standards, quantification is possible which
could help correlate the progress in rehabilitation/
detoxification with the levels of cannabis in the urine.
Urine analysis for drug testing is advantageous
because it is non invasive, easy to collect with no
risk to patient. However, urine analysis has its own
limitations. For most compounds, drug abuse screening
yields qualitative data indicating either presence or
absence of a drug. Nevertheless, it is difficult to predict
the route of administration, quantity, frequency and the
date of last abuse. In addition, a positive drug test does
not reflect drug dependence but only indicates recent
consumption. However, if a patient is found repeatedly
positive for drug abuse and exhibits clinical history of
behavioural abnormality, then the tests might imply
drug dependence. This kind of monitoring during the
follow up of such patients could give better qualitative
correlation. Therefore, based on a single analysis, not
all the information related to drug abuse of a patient
can be obtained24,33,34. Therefore, in this study, clinical
history and last date of abuse were recorded to get
better association and distinction between acute and
chronic users.
It has been established that cannabis metabolites
including THC-COOH are lipid soluble and tend
to accumulate in the fat tissues of the human body.
 SHARMA et al: DETECTION OF CANNABINOIDS IN URINE 207
Consequently, its availability in the serum and urine
is limited, making its levels decline rapidly in these
body fluids after consumption. If very high levels of
cannabis metabolites are detected in the patient urine
for a long time that indicates chronic cannabis abuse10.
However, a quantitative correlation between THC-
COOH and severity of psychosis is not clear35. In the
future, quantitative comparison with known quantity of
purified THC-COOH as a standard could give a better
picture regarding its reliability in clinical correlation
with the severity of psychosis.
In the current study, cannabis consumption was
suspected based on colour test and HPTLC data and
visualization by spray test. In the samples studied, Rf
value of THC-COOH in some of the samples varied
between 0.32 and 0.39. This minor discrepancy might
be either due to chronic abuse or due to the difference
in the quality of the abused substance. The change in
Rf might also depend on the source and species of the
cannabis obtained by the user.
In conclusion, colour based tests for cannabinoids
were standardized and screened for urine samples from
patients of drug abuse. HPTLC was found to be a powerful
technique for detection and potential quantitation of
drugs and compounds in clinical samples.
Acknowledgment
The authors thank the director and technical staff of forensic
research laboratory, Madiwala, Bangalore, India, for technical help
and standardization of experimental techniques. The technical
assistance by Ms. Parul Shivhare and Ms. Veena Nambiar is
gratefully acknowledged.
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