U.S. patent application number 14/771592 was filed with the patent office on 2016-01-21 for methods for cannabinoid quantification.
This patent application is currently assigned to Compassionate Analytic Inc.. The applicant listed for this patent is COMPASSIONATE ANALYTICS INC.. Invention is credited to Caleb Eades, Philippe Lucas.
Application Number | 20160018424 14/771592 |
Document ID | / |
Family ID | 51427430 |
Filed Date | 2016-01-21 |
United States Patent
Application |
20160018424 |
Kind Code |
A1 |
Lucas; Philippe ; et
al. |
January 21, 2016 |
METHODS FOR CANNABINOID QUANTIFICATION
Abstract
A method for quantification of the concentration of one or more
cannabinoid compounds in a liquid sample is provided. The method
involves contacting the liquid sample with at least one
cannabinoid-sensitive visualization reagent, allowing the at least
one cannabinoid-sensitive visualization reagent to develop for a
defined amount of time; and comparing the resulting color change of
the at least one cannabinoid-sensitive visualization reagent to a
calibrated quantification reference chart.
Inventors: |
Lucas; Philippe; (Victoria,
CA) ; Eades; Caleb; (Gabriola Island, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
COMPASSIONATE ANALYTICS INC. |
Victoria |
|
CA |
|
|
Assignee: |
Compassionate Analytic Inc.
Victoria
CA
|
Family ID: |
51427430 |
Appl. No.: |
14/771592 |
Filed: |
February 28, 2014 |
PCT Filed: |
February 28, 2014 |
PCT NO: |
PCT/CA2014/000157 |
371 Date: |
August 31, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61771263 |
Mar 1, 2013 |
|
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61827128 |
May 24, 2013 |
|
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61884409 |
Sep 30, 2013 |
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Current U.S.
Class: |
436/93 ;
436/131 |
Current CPC
Class: |
G01N 33/52 20130101;
G01N 33/523 20130101; G01N 21/78 20130101; G01N 33/948 20130101;
G01N 33/5302 20130101 |
International
Class: |
G01N 33/94 20060101
G01N033/94 |
Claims
1. A method for quantification of the concentration of one or more
cannabinoid compounds in a liquid sample, the method comprising: a)
contacting the liquid sample with at least one
cannabinoid-sensitive visualization reagent; b) allowing the at
least one cannabinoid-sensitive visualization reagent to develop
for a defined amount of time; and c) comparing the resulting color
change of the at least one cannabinoid-sensitive visualization
reagent to a calibrated quantification reference chart.
2. The method of claim 1, wherein the at least one
cannabinoid-sensitive visualization reagent is present in a liquid
form.
3. The method of claim 1, wherein the at least one
cannabinoid-sensitive visualization reagent is present in a solid
form.
4. The method of claim 1 or 2, wherein the liquid sample is
contacted separately with the at least one cannabinoid-sensitive
visualization reagent.
5. The method of claim 3, wherein the at least one
cannabinoid-sensitive visualization reagent is impregnated in a
test strip.
6. The method of claim 5, wherein the test strip comprises a porous
matrix uniformly impregnated with the at least one
cannabinoid-sensitive visualization reagent.
7. The method of any one of claims 1-6, wherein the at least one
cannabinoid-sensitive visualization reagent reacts with THC, CBD,
or CBN.
8. The method of claim 7, wherein the cannabinoid is THC.
9. The method of claim 7, wherein the cannabinoid is CBD.
10. The method of claim 8, wherein the at least one
cannabinoid-sensitive visualization reagent is one or more
diazonium salts, a Duquenois reagent, a Ghamrawy reagent, or a
modified Ghamrawy reagent.
11. The method of claim 10, wherein the one or more diazonium salts
are: Fast Blue B, Fast Blue BB, Fast Red B, Fast Red GG, Fast
Orange GR, Fast Corinth V, Fast Garnet GC, Fast Red AV, or Fast
Bordeaux GP.
12. The method of claim 9, wherein the at least one
cannabinoid-sensitive visualization reagent is a strong base.
13. The method of claim 13, wherein the strong base is potassium
hydroxide or sodium hydroxide.
14. The method of claim 7-13, wherein the at least one
cannabinoid-sensitive visualization reagent is impregnated in a
test strip.
15. The method of claim 14, wherein the test strip comprises a
porous matrix uniformly impregnated with the at least one
cannabinoid-sensitive visualization reagent.
16. A method for quantification of one or more cannabinoid
compounds in a solid test sample, the method comprising: 1)
contacting the solid test sample with an extraction solvent,
wherein one or more cannabinoids are extracted from the solid
sample into an extraction solvent resulting in a
cannabinoid-containing liquid extraction solution; 2) contacting
the cannabinoid-containing liquid extraction solution with one or
more cannabinoid-sensitive visualization reagents; and 3) comparing
the intensity of the resulting color change of the one or more
cannabinoid-sensitive visualization reagents to a calibrated
quantification reference chart.
17. An apparatus comprising a test strip impregnated with at least
one cannabinoid-sensitive visualization reagent.
18. The apparatus of claim 17 wherein the at least one
cannabinoid-sensitive visualization reagent is one or more
diazonium salts, a Duquenois reagent, a Ghamrawy reagent, a
modified Ghamrawy reagent, and potassium hydroxide.
19. A kit for quantification of the concentration of one or more
cannabinoid compounds in a sample, the kit comprising: 1) at least
one cannabinoid sensitive visualization reagent; and 2) a
calibrated quantification reference chart.
20. The kit of claims 19 wherein the at least one
cannabinoid-sensitive visualization reagent is present in a liquid
form or a solid form.
21. The kit of claim 19 or 20, wherein the at least one
cannabinoid-sensitive visualization reagent is pre-measured as a
dry reagent in one or more reaction containers.
22. The kit of any one of claims 19-21, wherein the at least one
cannabinoid-sensitive visualization reagent is impregnated into a
test strip.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application Ser. No. 61/771,263 filed on Mar. 1, 2013,
entitled "Cannabidiol Quantification"; U.S. Provisional Patent
Application Ser. No. 61/827,128 filed on May 24, 2013 entitled
"Cannabinoid Quantification, In Solution"; and U.S. Provisional
Patent Application Ser. No. 61/884,409 filed on Sep. 30, 2013
entitled "Cannabinoid Quantification", all of which are expressly
incorporated by reference herein to the extent permitted by
law.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
for accurate quantification of cannabinoid compounds in a
sample.
BACKGROUND OF THE INVENTION
[0003] The legal production, sale and use of cannabis for medical
purposes is becoming more prevalent in many countries, including
the United States and Canada. It is important for medical cannabis
growers, dispensaries and end-users to know the concentration of
certain cannabinoids, particularly delta-9-tetrahydrocannabinol
(THC) in specific plant samples--such information can be important
for strain development, for optimizing production/growing cycles,
for complying with jurisdiction-specific legal requirements, and
for other quality control purposes. Quantification of cannabinoids
in a sample generally requires a laboratory test, for instance
using gas chromatography, which can be expensive and generally
requires time to send the sample to an appropriate lab and wait for
the results. Home-based tests, such as Alpha-Cat, have been
developed using thin-layer chromatography, however this test is
still quite complex for the average untrained user, and the
quantification resolution of the test is limited.
[0004] Reagents are known in the art that react chemically with
CBD, under specific reaction conditions, to create a color change.
Mechoulam (Tetrahedron 24(16): 5615-5624, 1968) teaches that CBD,
when contacted with 5% ethanolic potassium hydroxide (KOH) is
converted to quinone, which has a purple color.
[0005] U.S. Pat. No. 4,771,005, issued to Spiro, teaches methods
for positive/negative cannabinoid detection in human sample using a
diazonium salt, for instance Fast Blue BB.
[0006] WO/1989/009395 (published in the name of Fraser and Johnson)
teaches a test paper for positive/negative cannabinoid detection
using diazonium salt, for instance Fast Blue BB.
[0007] U.S. Pat. No. 8,124,420, issued in the name of Amisar,
teaches a test paper and kit for detection and/or identification of
a range of drugs of abuse, which may include cannabinoids, and the
test paper may include a diazonium salt, for instance Fast Corinth
V.
[0008] Fischedick et al. (2009 Phytochem Anal 20:421-6.) teaches a
method for quantifying cannabinoids in a sample using high
performance thin layer chromatography (HPTLC).
SUMMARY OF THE INVENTION
[0009] In one aspect of the invention, a method for quantification
of the concentration of one or more cannabinoid compounds in a
liquid sample is provided. The method involves contacting the
liquid sample with at least one cannabinoid-sensitive visualization
reagent; allowing the at least one cannabinoid-sensitive
visualization reagent to develop for a defined amount of time; and
comparing the resulting color change of the at least one
cannabinoid-sensitive visualization reagent to a calibrated
quantification reference chart. The at least one
cannabinoid-sensitive visualization reagent may be present in a
liquid form. The at least one cannabinoid-sensitive visualization
reagent may be present in a solid form. The liquid sample may be
contacted separately with the at least one cannabinoid-sensitive
visualization reagent. The at least one cannabinoid-sensitive
visualization reagent may be impregnated in a test strip. The test
strip may include a porous matrix uniformly impregnated with the at
least one cannabinoid-sensitive visualization reagent. The at least
one cannabinoid-sensitive visualization reagent reacts with
cannabinoids, which can include THC, CBD, or CBN. Optionally, the
cannabinoid may be solely THC, CBD or another cannabinoid. The at
least one cannabinoid-sensitive visualization reagent may include
one or more diazonium salts, a Duquenois reagent, a Ghamrawy
reagent, or a modified Ghamrawy reagent. The one or more diazonium
salts may include Fast Blue B, Fast Blue BB, Fast Red B, Fast Red
GG, Fast Orange GR, Fast Corinth V, Fast Garnet GC, Fast Red AV, or
Fast Bordeaux GP. Optionally, the at least one
cannabinoid-sensitive visualization reagent may be a strong base,
which may be potassium hydroxide or sodium hydroxide. Further, the
at least one cannabinoid-sensitive visualization reagent may be
impregnated in a test strip. Further still, the test strip may
include a porous matrix uniformly impregnated with the at least one
cannabinoid-sensitive visualization reagent.
[0010] In another aspect of the invention, a method for
quantification of one or more cannabinoid compounds in a solid test
sample is provided. The method involves contacting the solid test
sample with an extraction solvent, wherein one or more cannabinoids
are extracted from the solid sample into an extraction solvent
resulting in a cannabinoid-containing liquid extraction solution;
contacting the cannabinoid-containing liquid extraction solution
with one or more cannabinoid-sensitive visualization reagents; and
comparing the intensity of the resulting color change of the one or
more cannabinoid-sensitive visualization reagents to a calibrated
quantification reference chart.
[0011] In another aspect of the invention, an apparatus which
includes a test strip impregnated with at least one
cannabinoid-sensitive visualization reagent is disclosed.
Optionally the at least one cannabinoid-sensitive visualization
reagent may be one or more diazonium salts, a Duquenois reagent, a
Ghamrawy reagent, a modified Ghamrawy reagent, and potassium
hydroxide.
[0012] In another aspect of the invention, a kit for quantification
of the concentration of one or more cannabinoid compounds in a
sample is disclosed. The kit includes at least one cannabinoid
sensitive visualization reagent, and a calibrated quantification
reference chart. Optionally, the at least one cannabinoid-sensitive
visualization reagent is present in a liquid form or a solid form.
Further and optionally, the at least one cannabinoid-sensitive
visualization reagent is pre-measured as a dry reagent in one or
more reaction containers. Further still, the at least one
cannabinoid-sensitive visualization reagent is impregnated into a
test strip.
[0013] Aspects of the present invention are based, in part, on the
finding that certain cannabinoid-sensitive visualization reagents
may be used to quantify the cannabinoid concentration in a sample.
Herein, it has been demonstrated that the use of one or more
cannabinoid-sensitive visualization reagents which cause an
absorbance shift (color change) in a solution upon contact with
cannabinoids including THC and/or CBD, may be utilized to quantify
the concentration of such cannabinoids in a given sample.
Quantification is achieved by contacting the one or more
cannabinoid-sensitive visualization reagents with a liquid
cannabinoid-containing sample, or alternatively with a liquid
extraction from a solid cannabinoid-containing sample, and
comparing the resulting absorbance shift to that caused by samples
with known cannabinoid concentrations. Herein, examples have been
provided of such assays to determine the concentration of THC, CBD,
and/or other cannabinoids in a solid plant test sample, or a liquid
test sample. Examples have been provided of a calibrated
quantification reference chart, useful in said assay, calibrated to
determine the cannabinoid concentration in a solid plant test
sample, and suitable for solid plant test samples having
cannabinoid concentration ideally between 0-25%, but potentially
higher. In some embodiments, the calibrated quantification
reference chart has been optimized for this cannabinoid
concentration range by extracting cannabinoids from series of solid
samples having known cannabinoid concentrations ranging from 0% to
25%, using a consistent and uniform extraction solvent composition,
volume, and extraction time for all of the samples, and contacting
the resulting cannabinoid-containing extraction liquid with a
defined amount of one or more cannabinoid-sensitive visualization
reagents. The resulting absorbance shift, or color change, caused
by each of the samples of known cannabinoid concentration provides
a calibrated quantification reference chart for use in the
experimental assay for the test sample--by extracting the
cannabinoids from the unknown test sample using the exact same
extraction solvent composition, volume, and extraction time;
contacting the resulting cannabinoid-containing extraction liquid
with the same amount/concentration of the one or more
cannabinoid-sensitive visualization reagents; and comparing the
resulting absorbance shift(s), or color change(s), with the
calibrated quantification reference chart, one may thus determine
the cannabinoid concentration in the unknown solid test sample. In
the examples provided herein, the extraction solvent composition,
volume, and extraction time have been optimized to ensure that the
absorbance shift caused by contacting the resulting
cannabinoid-containing extraction solution with the one or more
cannabinoid-sensitive visualization reagents is in the optimized
range of the visualization reagent, such that the absorbance, or
color change intensity, is proportional to the cannabinoid
concentration in the test sample. In certain aspects of the
invention, the cannabinoid that may be quantified is one or more of
THC, CBD and/or CBN, and the cannabinoid-sensitive visualization
reagent one or more of: i) diazonium salt, for instance Fast Blue
B, Fast Blue BB, Fast Blue RR, Fast Corinth V, Fast Garnet, Fast
Bordeaux, and the like; ii) a Duquenois reagent; and iii) a
Ghamrawy reagent or modified Ghamrawy reagent. In certain aspects
of the invention, the cannabinoid that may be quantified is CBD,
and the cannabinoid-sensitive visualization reagent is a strong
base for instance potassium hydroxide or sodium hydroxide, wherein
the medium in which the strong base contacts the CBD is methanol,
ethanol, propanol, or another lower alcohol solvent.
[0014] Examples have been provided of a variety of visualization
reaction compositions and methods suitable for the present
invention. In certain aspects of the invention, the visualization
reaction may occur in a liquid solution, wherein the one or
cannabinoids of the liquid test sample, or extracted from the solid
test sample, are contacted with the one or more
cannabinoid-sensitive visualization reagents in solution, for
instance using a solvent composition that is permissive for the
visualization reaction. In certain aspects, the cannabinoids of the
test sample are separately contacted with each of the one or more
cannabinoid-sensitive visualization reagents in separate reaction
vessel. In other aspects of the invention, the visualization
reaction may be accomplished using a test paper, wherein the test
paper is impregnated with a cannabinoid-sensitive visualization
reagent. In certain aspects, the test paper may be a test strip
comprising one or more test pads, wherein each test pad is
impregnated with a different cannabinoid-sensitive visualization
reagent. In this regard, one of unique features of the present
invention is based in part on the finding that certain
cannabinoid-sensitive visualization reagents have different, and
potentially overlapping, quantification ranges and/or
sensitivities. Thus, in certain aspects of the present invention,
the use of more than one cannabinoid-sensitive visualization
reagent may allow for an extended range and accuracy of
quantification. Another feature is that certain cannabinoid
sensitive visualization reagents may be impregnated on a test strip
and included in a kit in such a way that the activity of the
visualization reagent is retained when the strip is eventually used
by the end-user.
BRIEF DESCRIPTION OF THE FIGURES
[0015] Two sets of identical figures are being submitted herewith:
a first set rendered in black and white, and a second set is
rendered in colour.
[0016] FIG. 1 depicts a calibrated quantification reference chart
for THC samples having an ideal range of 5-20%. The quantification
reference chart was generated using Fast Blue BB. The chart shows a
light yellow colour at the left side, changing to a darker
yellow/orange on the right side.
[0017] FIG. 2 depicts an extended range calibrated quantification
reference chart. The chart includes overlapping ranges for 2
different cannabinoid-sensitive visualization reagents, to allow
for quantification from 0% to over 25% THC in a solid sample.
[0018] FIG. 3 depicts a calibrated quantification reference chart
for CBD samples having ideal range 0-15%. Moving from left to
right, the figure shows the color changing from very light purple
at the lower end of the scale to a much darker purple at the higher
end of the scale.
[0019] FIG. 4 depicts a calibrated reference chart for Fast BB
impregnated test strips. The chart shows that, moving from left to
right, the color change of the strip goes from a very light
orange/red at the low end of the scale to a much darker red at the
higher end of the scale.
[0020] FIG. 5 depicts representative test papers prepared using 30%
KOH (left), 10% KOH (middle), and 1% KOH (right).
[0021] FIG. 6 depicts a CBD Calibrated Reference Chart. Moving from
left to right on the chart, the color change shown on the chart
goes from a very light violet/purple for the lower CBD % to a much
darker purple for higher CBD %.
[0022] FIG. 7 depicts a THC Calibrated Reference Chart using
modified Ghamrawy reagent impregnated test papers. Moving from left
to right on the chart, the color change shown on the chart goes
from a very light violet/purple for the lower THC % to a much
darker purple for higher THC %.
DETAILED DESCRIPTION
[0023] Any terms not directly defined herein shall be understood to
have the meanings commonly associated with them as understood
within the art of the invention. As employed throughout the
specification, the following terms, unless otherwise indicated,
shall be understood to have the following meanings.
[0024] The term `sample` means a complex substance that may be
tested or analyzed for the presence of certain compounds. A
`sample` may be a liquid sample or a solid sample. A liquid sample
may comprise a bodily fluid such as urine or blood. A liquid sample
may comprise a solvent extract of a solid sample, wherein various
compounds from the solid sample are extracted into the liquid
solvent using methods known in the art. A solid sample may include
plant material, for instance cannabis plant material.
[0025] The term `cannabis` means a genus of flowering plants that
includes three putative species, Cannabis sativa, Cannabis indica,
and Cannabis ruderalis. The term `cannabis` may also refer to plant
material derived or extracted from the cannabis plant, for instance
the leaves, stem, seeds, flowering bodies, or other portions of the
plant.
[0026] The term `cannabinoid` or `cannabinoids` means a class of
chemical compounds which include the phytocannabinoids
(oxygen-containing C21 aromatic hydrocarbon compounds found in the
cannabis plant), and chemical compounds which mimic the actions of
phytocannabinoids or have a similar structure (e.g.,
endocannabinoids, found in the nervous and immune systems of
animals and that activate cannabinoid receptors). Phytocannabinoids
are known to occur in significant quantities in the cannabis plant,
and may include, but are not limited to tetrahydrocannabinol (THC),
cannabidiol (CBD), cannabinol (CBN), and cannabigerol (CBG).
[0027] The term `THC` means tetrahydrocannabinol and may include
different isoforms and variants, such as
delta-9-Tetrahydrocannabinol (.DELTA.9-THC) and
delta-8-tetrahydrocannabinol (.DELTA.8-THC). The inventors herein
disclose methods and an apparatus for quantification of THC and/or
other cannabinoids from a sample, for instance from a solid
cannabis sample.
[0028] The term `CBD` means cannabidiol, a cannabinoid often found
in cannabis, and having a CAS registry number 13956-29-1.
Cannabidiol is known to have many beneficial medicinal
qualities.
[0029] The term `extraction` means to transfer compounds from a
sample into another medium, for instance into a liquid solvent. The
solvent may be chosen such that certain desired compounds are
soluble in the solvent, and thus when the sample is contacted with
the solvent, the desired compounds are transferred to the solvent.
Extraction of solid samples may be aided or enhanced by grinding,
macerating or otherwise pulverizing the sample material. Extraction
is aided by shaking, vortexing or otherwise mixing the solvent with
the sample. Uniform extraction yields may be achieved by using
defined extraction methods, including duration of extraction,
solvent composition, and the like. In certain embodiments, a solid
sample may be heated prior to extraction--this may serve to
chemically convert certain compounds that are not soluble in the
extraction solvent into compounds that are soluble in the
extraction solvent. Furthermore, the converted compounds may be
more reactive with visualization reagents. In certain embodiments
of the invention, solid cannabis samples are heated prior to
extraction in order to decarboxylate the acid form of THC, CBD,
etc. into non-acid forms which are more readily extracted and more
readily react with the visualization reagents of the present
invention. Ideally, the heat conversion is carried out a
temperature that is below the volatilization temperature of the
cannabinoids, but above the decarboxylation temperature. In this
way, any THC-A and CBD-A in the solid sample, may be converted to
THC and CBD prior to extraction, and the subsequent visualization
reactions of the invention will give a more accurate quantification
of total THC and/or total CBD in the sample.
[0030] A `visualization reagent` means a reagent or compound that
changes color upon contact with a particular analyte or class of
analytes, or alternatively causes the analyte to change color upon
contact with the visualization reagent, or alternatively causes a
color change in a reaction medium containing a particular analyte
or class of analytes. The change in color will result in a change
in the spectral absorbance of the reaction medium, detection of
which may be visible to the naked eye, or may be more accurately
quantified using a device such as a colorimeter, spectrometer,
spectrophotometer, or the like. Visualization reagents may
alternatively be referred to as colorimetric reagents. There are
numerous types of visualization reagents described in the art.
Certain visualization reagents may be suitable for quantification
of analytes, while others may not. Suitable visualization reagents
may cause a color change that is proportional to the concentration
of the analyte, over a specific analyte concentration
range--sometimes referred to as the `linear range` or `optimal
range` of the visualization reagent. The optimal range of the
visualization reagent for the specific analyte must be wide enough
to provide quantification information across a sufficient range to
be a useful visualization reagent. A visualization reaction may
require contacting the visualization reagent with the analyte in
reaction conditions suitable to cause the appropriate chemical
reaction. For instance, a suitable solvent may be used to
facilitate the visualization reaction. The suitable solvent of the
present invention may be an alcohol such as methanol, ethanol,
propanol and the like.
[0031] A `diazonium salt`, or `diazonium compound`, is a compound
belonging to a group of organic compounds sharing a common
functional group R--N2+X-- where R can be any organic residue such
alkyl or aryl and X is an inorganic or organic anion such as a
halogen. Diazonium salts, especially those where R is an aryl
group, are important intermediates in the organic synthesis of azo
dyes. Diazonium salts are often used as visualization reagents by
conversion of the diazonium salt into an azo dye, such conversion
causing a change in absorbance. Examples of diazonium salts may
include, but are not limited to, Fast Blue B
(3,3'-dimethoxybenzidine (o-dianisidine)), Fast Blue BB
(4-Benzoylamino-2,5-diethoxyaniline), Fast Red B
(2-Methoxy-4-nitroaniline), Fast Red GG (4-Nitroaniline), Fast
Orange GR (2-Nitroaniline), Fast Corinth V
(2-methoxy-5-methyl-4-(4-methyl-2-nitrophenyl)-azobenzene-diazonium),
Fast Garnet GC (4-(m-Tolylazo)-3-methylaniline), Fast Red AV, and
Fast Bordeaux GP.
[0032] A `Duquenois reagent` is a reagent used in a
Duquenois-Levine test for detecting cannabis (see: P. Duquenois and
H. N. Moustapha, J. Egypt. Med. Ass., 1938, 21, 224.) A Duquenois
reagent may comprise a mixture of vanillin and acetaldehyde, along
with hydrochloric acid. In certain embodiments, the hydrochloric
acid may be substituted for another acid. In certain embodiments,
the strong acid may be present as a dry form, for instance
p-toluenesulfonic acid, and may be impregnated in a test
paper/strip.
[0033] A `Ghamrawy reagent` refers to a combination of compounds
that may be used for detection of THC and CBD. A Ghamrawy reagent
is further described in: Kovar, Karl-Artur and Martina Laudszun.
(Chemistry and Reaction Mechanisms of Rapid Tests for Drugs of
Abuse and Precursors Chemicals, United Nations Scientific and
Technical Notes v.89-51669, Germany. February 1989. The Ghamrawy
reagent consists of p-dimethylaminobenzaldehyde (p-DMAB) along with
concentrated sulfuric or hydrochloric acid. In certain embodiments,
the Ghamrawy reagent may be modified--the inventors have identified
other strong acids that may be useful for catalyzing the color
reaction with p-DMAB. For instance, p-toluenesulfonic acid that may
be used instead of hydrochloric acid or sulfuric acid. This acid is
advantageous for use in test strips, by virtue of the fact that
p-toluenesulfonic acid may be present as a solid, whereas
hydrochloric and sulfuric acid generally are not. Furthermore
p-toluenesulfonic acid is non-corrosive and thus will not degrade
the test strip. This surprising finding has enabled the use of test
papers/strips impregnated with the modified Ghamrawy reagent for
quantification of THC and/or CBD. Thus, a `modified Ghamrawy
reagent` comprises p-DMAB and a strong acid, for instance
p-toluenesulfonic acid. Other strong acids that may be present as a
solid may also be used in a modified Ghamrawy reagent.
[0034] The term `KOH` means `potassium hydroxide`. Potassium
hydroxide may have the CAS registry number 1310-58-3. The term
`NaOH` means `sodium hydroxide`. Sodium hydroxide may have the CAS
registry number 1310-73-2.
[0035] The term `cannabinoid-sensitive visualization reagent`
refers to a reagent such as a visualization reagent that undergoes
a change in properties, such as spectral absorbance, upon contact
with a cannabinoid. In the context of the present invention, the
change in chemical properties may occur due to a change in either
reactant in the visualization reaction, or even a change in the
reaction medium itself. Alternatively, the change in chemical
properties may occur due to the formation of a new compound, such
as a reaction by-product. A cannabinoid-sensitive visualization
reagent may be a diazonium salt. Certain diazonium salts are known
to cause a change of color in the presence of cannabinoids such as
THC, CBD and/or CBN--such diazonium salts include, but are not
limited to, Fast Blue B, Fast Blue BB, Fast Corinth V, and Fast
Garnet GC. A cannabinoid-sensitive visualization reagent may be a
Duquenois reagent. A cannabinoid-sensitive visualization reagent
may be a Ghamrawy reagent or a modified Ghamrawy reagent. A
cannabinoid-sensitive visualization reagent may be potassium
hydroxide or sodium hydroxide.
[0036] The term `permissive` or `permissive solvent` refers to
solvents that have a composition that allow the desired chemical
reaction to occur. For instance, certain chemical reactions may
proceed much more readily in an alcohol than in water. In such
case, the alcohol would be a permissive solvent and would be the
desired solvent for the reaction. In various embodiments, the use
of permissive solvents is important for the utility of the
invention.
[0037] The term `calibrated quantification reference chart` means a
chart, graph, or other visual representation showing the specific
absorbance shift or color change of one or more specific
visualization reagents caused by a specific analyte across a range
of specific concentration, under specific conditions, such that
comparison of an analyte at an unknown concentration, using the
same one or more specific visualization reagents under the same
specific conditions, to the calibrated quantification reference
chart will allow one to determine the concentration of that analyte
in the unknown test sample. The specific absorbance shift or color
change may be determined for instance using a test paper, test
strip, a colorimeter, a spectrometer, or by visual inspection. The
present invention utilizes a calibrated quantification reference
chart to determine the concentration of cannabinoids in a sample.
In certain embodiments of the invention, a calibrated
quantification reference chart may be used or created for
determining the cannabinoid concentration in an unknown solid
sample by extracting cannabinoids from series of solid samples
having known cannabinoid concentrations, using a uniform extraction
solvent composition, volume, and extraction time for all of the
samples, and contacting the resulting cannabinoid-containing
extraction liquid with a defined amount of one or more
cannabinoid-sensitive visualization reagents. The resulting
absorbance shift(s), or color change(s), caused by each of the
samples of known cannabinoid concentration provides a calibrated
quantification reference chart for use in the experimental assay
for the unknown test sample. By then extracting the cannabinoids
from the unknown test sample using the exact same extraction
solvent composition, volume, and extraction time; contacting the
resulting cannabinoid-containing extraction liquid with the same
amount/concentration of one or more cannabinoid-sensitive
visualization reagents; and comparing the resulting absorbance
shift(s), or color change(s), with the calibrated quantification
reference chart, one may thus determine the cannabinoid
concentration in the unknown solid sample.
[0038] The term `porous matrix` refers to a solid material, ie. a
matrix, that is permeated with pores or small holes to allow
absorbance of a fluid into the matrix. A porous matrix may be a
type of paper or filter, such as blotting paper. Examples of a
porous matrix include Whatman paper, CF1, CF2, CF3, cellulose
paper, and the like.
[0039] The term `test paper` refers to a porous matrix which is
impregnated with a diagnostic reagent, for instance a
cannabinoid-sensitive visualization reagent such as a diazonium
salt, Duquenois reagent, Ghamrawy reagent, or modified Ghamrawy
reagent. Impregnation of the porous matrix with the reagent may be
accomplished in several different ways. The reagent to be
impregnated into the porous matrix to form the test paper may first
be dissolved in a suitable solvent, and the porous matrix then
contacted with or submersed in the resulting solution containing
the dissolved reagent. For instance, the solvent may be an alcohol
such as methanol, ethanol or propanol. Preferably, the solvent may
be a ketone such as acetone or methyl ethyl ketone--these solvent
may prevent degradation of the dye and reduce background coloration
on the test strips. Evaporation of the solvent will result in a dry
test strip impregnated with the reagent. Suitable solvents should
thus be chosen such that the reagent is miscible in the solvent,
and such that the solvent is volatile enough to effectively
evaporate away from the porous matrix. Examples of suitable
solvents include, but are not limited to, water, methanol, ethanol,
isopropanol, petroleum ether, methyl ethyl ketone, acetone,
dimethylchloride, hexane. In certain embodiments, the visualization
reagent may be heated after impregnation of the test paper, or
during the drying of the test paper. Such heating may activate the
visualization reagent. In certain embodiments the test paper may be
subsequently sealed into an air tight package, for instance by
vacuum sealing. This may preserve the activation or activity of the
visualization reagent. A test paper may be contacted with a test
compound, for instance a cannabinoid, in a suitable solvent, and
the reaction of the test compound with the diagnostic reagent may
cause the test paper to change color. In various embodiments of the
invention, the color change of the test paper may be proportional
to the concentration of one or more cannabinoids in the solution,
and may thus be used to quantify the cannabinoid concentration.
[0040] The term `test strip` refers to a strip of material to which
is attached one or more test papers. A test strip may be elongated
to allow for ease of contacting with the test solution, for
instance by dipping into a test tube or the like. The test strip
may include i) a backing material, which may be absorbent or
non-absorbent, and is preferentially but not necessarily inert; ii)
one or more test papers comprising one or more
cannabinoid-sensitive visualization reagents; and iii) an adhesive
to attach the one or more test papers to the backing material.
Quantification of Cannabinoid Concentration in Liquid or Solid
Samples Using One or More Cannabinoid-Sensitive Visualization
Reagents
[0041] Herein, the inventors describe methods for determination of
the cannabinoid concentration in liquid or solid samples using one
or more cannabinoid-sensitive visualization reagents. The inventors
have surprisingly determined that certain cannabinoid-sensitive
visualization reagents can be utilized in conjunction with a
calibrated reference chart to determine the actual concentration of
cannabinoids in a sample, rather than merely detecting the presence
or absence of cannabinoids, and are thus suitable for cannabinoid
quantification. In certain embodiments of the invention, such one
or more cannabinoid-sensitive visualization reagents are first
contacted with a cannabinoid-containing liquid sample, or with a
liquid extraction of a cannabinoid-containing solid sample, under
conditions which cause a color change or absorbance shift of the
one or more visualization reagents, wherein such color change or
absorbance shift is proportional to the cannabinoid concentration.
The color change or absorbance shift of the visualization reagent
is then compared to a calibrated quantification reference chart,
wherein the calibrated quantification reference chart is created by
testing a series of liquid or solid sample of known cannabinoid
concentration under the exact same test conditions as the sample of
unknown concentration--ideally the calibrated quantification
reference chart would show the color change or absorbance shift at
a number of known cannabinoid concentrations. In such a way, one
may compare the color change or absorbance shift of the sample of
unknown cannabinoid concentration to the calibrated quantification
reference chart to determine the cannabinoid concentration in the
unknown sample. Determination of the color change or absorbance
shift may be accomplished by numerous means, for instance using a
colorimeter or spectrophotometer, or by visual inspection. In
certain embodiments, the one or more cannabinoid-sensitive
visualization reagents is a diazonium salt, such as Fast Blue B,
Fast Blue BB, Fast Corinth V, Fast Garnet GC, and the like, which
are useful for quantification of THC, CBD and/or CBN. In other
embodiments, the cannabinoid-sensitive visualization reagent is a
strong base, for instance potassium hydroxide or sodium hydroxide,
useful for quantification of CBD. In other embodiments, the one or
more cannabinoid-sensitive visualization reagents is a Duquenois
reagent, a Ghamrawy reagent, or a modified Ghamrawy reagent.
[0042] In one embodiment of the invention, there is provided a
method for quantification of the concentration of one or more
cannabinoid compounds from a liquid test sample, the method
involves: 1) contacting the cannabinoid-containing liquid sample
with a defined amount of one or more cannabinoid-sensitive
visualization reagents; 2) allowing the resulting visualization
reaction(s) to develop for a defined amount of time; and 3)
comparing the intensity of the resulting color change, or
absorbance shift, of the one or more cannabinoid-sensitive
visualization reagents to a calibrated quantification reference
chart, wherein such comparison allows determination of the
cannabinoid concentration in the test sample. The calibrated
quantification reference chart may be produced, for instance, by
contacting a series of calibrated liquid samples having
pre-determined cannabinoid concentrations with the same
amount/composition of one or more cannabinoid-sensitive
visualization reagents under the exact same conditions as to be
used for the unknown test sample, including using the same one or
more cannabinoid-visualization reagents and the same color
development time as to be used for the test sample. In certain
embodiments wherein two or more cannabinoid-sensitive visualization
reagents are used, each visualization reagent may be separately
contacted with the cannabinoid(s) of the test sample. In certain
embodiments, the method may be useful for the quantification of
cannabinoids THC, CBD and/or CBN in a liquid sample. The one or
more cannabinoid-sensitive visualization reagents may be a
diazonium salt. The one or more cannabinoid-sensitive visualization
reagents may be chosen from the following: Fast Blue B, Fast Blue
BB, Fast Blue RR, Fast Corinth V, Fast Garnet, Fast Bordeaux. The
diazonium salt(s) and the cannabinoids may be contacted in
permissive solvent. The one or more cannabinoid-sensitive
visualization reagents may be a Duquenois reagent, a Ghamrawy
reagent, and/or a modified Ghamrawy reagent. In other embodiments,
the method may be useful for the quantification of CBD in a liquid
sample. The cannabinoid-sensitive visualization reagent may be a
strong base, for instance potassium hydroxide, sodium hydroxide.
The strong base, for instance potassium hydroxide, and/or
cannabinoid(s) may be contacted in a permissive solvent such as a
lower alcohol, for instance methanol, ethanol, propanol, and the
like.
[0043] In another embodiment of the invention, there is provided a
method for quantification of one or more cannabinoid compounds in a
solid test sample, the method involves: 1) contacting a defined
amount of solid test sample with a defined volume of an extraction
solvent for a defined amount of time, wherein one or more
cannabinoids are extracted from the solid sample into an extraction
solvent resulting in a cannabinoid-containing liquid extraction
solution; 2) contacting the resulting cannabinoid-containing liquid
extraction solution with defined amount of one or more
cannabinoid-sensitive visualization reagent; 3) allowing the
resulting visualization reaction(s) to develop for a defined amount
of time; and 4) comparing the intensity of the resulting color
change, or absorbance shift, of the one or more
cannabinoid-sensitive visualization reagents to a calibrated
quantification reference chart, wherein such comparison allows
determination of the cannabinoid concentration in the solid test
sample. The calibrated quantification reference chart may be
produced by performing the same assay method on a series of solid
samples having known cannabinoid concentrations and recording the
resulting absorbance changes. For instance, the calibrated
quantification reference chart may be produced by contacting a
series of solid samples having pre-determined cannabinoid
concentrations with the same defined amount of the same defined
extraction solvent composition, and then contacting a defined
amount of resulting cannabinoid-containing extraction solution with
one or more cannabinoid-sensitive visualization reagents under the
exact same conditions as to be used for each test sample, including
using the same one or more cannabinoid-visualization reagent
compositions and concentrations, and the same color development
time as to be used for the test sample. In certain embodiments
wherein two or more cannabinoid-sensitive visualization reagents
are used, each visualization reagent is separately contacted with
the cannabinoid(s) of the test sample. In certain embodiments, the
method may be useful for quantification of the concentration
cannabinoids THC, CBD and/or CBN in a solid test sample. The one or
more cannabinoid-sensitive visualization reagent may be a diazonium
salt. The cannabinoid-sensitive visualization reagent may be chosen
from the following: Fast Blue B, Fast Blue BB, Fast Blue RR, Fast
Corinth V, Fast Garnet, Fast Bordeaux. The diazonium salt(s) and
the cannabinoids may be contacted in permissive solvent. The
cannabinoid-sensitive visualization reagent may be a Duquenois
reagent, a Ghamrawy reagent, or a modified Ghamrawy reagent. In
certain embodiments, the method may be useful for the
quantification of CBD in a solid test sample. The
cannabinoid-sensitive visualization reagent may be a strong base,
for instance potassium hydroxide, sodium hydroxide. The strong
base, for instance potassium hydroxide, and/or cannabinoid(s) may
be contacted in a permissive solvent such as a lower alcohol, for
instance methanol, ethanol, propanol, isopropanol, etc.
[0044] Extended Range Of Quantification
[0045] Herein, cannabinoid quantification assays are described
which have an extended range of quantification. The cannabinoid
quantification assays described in various embodiments of the
invention all have a lower and upper limit of quantification, below
and above which quantification is relatively ineffective. Below the
lower limit of quantification, the cannabinoid concentration is too
low to cause a significant absorbance shift, or color change, from
the cannabinoid-sensitive visualization reagent. Above the upper
limit of quantification, the absorbance shift, or color change, may
become saturated such that no further color change may be detected,
even with an increased concentration of the test analyte. Optimal
quantification results are thus achieved between the lower and
upper limits of quantification, and the greater the distance
between these two values, the more useful the assay may
be--suitable visualization reagents must be tested and selected in
order to obtain a useful quantification assay. Since cannabinoids
may exist at wide range different concentrations in a particular
sample, it would be desirable to develop assays with extended
ranges. In order to accomplish this, the present inventors take
advantage of the fact that different visualization reagents, such
as cannabinoid-sensitive visualization reagents, often have
different sensitivity to, and/or optimal ranges for, the test
compound in question, for instance cannabinoids. These different
visualization reagents may have different lower and upper limits of
quantification, and thus different effective quantification ranges.
In fact, certain visualization reagents may have overlapping
quantification ranges, which may be useful for developing an
extended range assay method using multiple visualization
reagents.
[0046] In certain embodiments of the invention, there is provided
an extended range cannabinoid quantification assay method involving
the use of two or more cannabinoid-sensitive visualization
reagents, useful for quantification of cannabinoids in a liquid or
solid test sample. The extended range cannabinoid quantification
assay method may have certain advantages over assay methods using a
single visualization reagent. The assay method may be carried out
using the same steps as described in other aspects of the invention
that use a single visualization reagent (described herein), except
that two or more visualization reactions are carried out in
parallel, for instance in separate tubes or on separate test
papers, and the absorbance shift, or color change, of each of the
visualization reactions is compared to a calibrated reference
chart, wherein the calibrated reference chart shows the calibrated
quantification values for each of the two or more
cannabinoid-sensitive visualization reagents used in the assay. In
certain embodiments of the invention, the assay method may be
useful for quantification of cannabinoid concentrations across an
extended range. In certain embodiments of the invention, the two or
more cannabinoid-sensitive visualization reagents have differing
cannabinoid sensitivity and or optimal quantification ranges. In
certain embodiments of the invention, the optimal quantification
ranges of each of the one or more visualization reagents are
overlapping. The overlapping ranges allows for the quantification
of cannabinoids in a sample across a wider range, which may improve
the utility of the assay. For instance, and by way of non-limiting
example, user may have a number of solid samples with expected THC
%'s between 0 and 25%; the use of first single visualization
reagent may allow optimal quantification of THC between 0-10% in
the solid sample. The use of a second visualization reagent with an
optimal quantification range of 10-25% would extend the overall
optimal range of the assay to 0-25%. The use of additional
cannabinoid-sensitive visualization reagents having even different
quantification ranges may be used to further extend the
quantification range of the assay method. It should be noted that
the use of more than one visualization reagent can also improve the
accuracy of quantification of cannabinoids in the overlapping
region of the optimal quantification ranges of the visualization
reagents, due to multiple readouts from the two or more
visualization reagents. In certain embodiments of the invention,
the two or more cannabinoid-sensitive visualization reagents may be
diazonium salts and/or Duquenois-Levine reagent and/or Ghamrawy
reagent and/or modified Ghamrawy reagent. In certain embodiments of
the invention, the diazonium salts may be selected from: Fast Blue
B, Fast Blue BB, Fast Blue RR, Fast Corinth V, Fast Garnet, Fast
Bordeaux, or other cannabinoid-sensitive diazonium salts.
[0047] Visualization
[0048] In certain embodiments of the invention, methods are
described for contacting cannabinoids from a test sample with one
or more cannabinoid-sensitive visualization reagents, which results
in an absorbance shift, or color change, suitable for
quantification of the cannabinoids in the test sample. As
exemplified in various working examples provided herein, the
invention provides different compositions and methods useful for
carrying out the visualization reaction. In certain embodiments,
the visualization reaction may be carried out in a liquid solution,
for instance in a reaction vessel such as a test tube, microtube,
sample container, etc. A liquid solution of the
cannabinoid-sensitive visualization reagent may be added to a
liquid cannabinoid-containing solution, according to the methods of
the present invention, and the color reaction may thus occur in
solution. In certain embodiments of the invention, the
cannabinoid-sensitive visualization reagent solution may be
prepared fresh at the time of use.
[0049] An aspect of the present invention is to provide simple
assays for cannabinoid quantification, and thus there are herein
provided additional methods for reducing the number of required
steps to complete the disclosed assays. These simplified assays are
thus easier to use, have reduced handling which can increase
accuracy, and are more cost effective. In certain cases, the
simplification of the assays makes them more suitable for
developing a test kit, as the modified steps may increase the shelf
life of the reagents in the test kit. For instance, it is known
that diazonium salt dyes such as Fast Blue B, Fast Blue BB, Fast
Corinth V, etc. are inherently unstable in solution, particularly
in aqueous solutions, and that this property has hindered the
development of usable cannabinoid tests with this reagent, even for
screening purposes. Thus, in certain embodiments of the invention,
the diazonium salt visualization reagent may be provided in its dry
powder form, and in certain aspects may be pre-measured in a
defined amount in the reaction tube, or in a separate pouch to be
added to the reaction tube. Other visualization reagents may
similarly be provided in dry form. The user may then carry out the
first steps of the quantification assay method in a first tube,
thus extracting the cannabinoids from a solid sample in a defined
amount of extraction solvent composition and volume, and then
transfer a defined amount of the resulting cannabinoid-containing
extraction liquid into a second tube containing a dry, pre-measured
visualization reagent, and then mixing to dissolve the
visualization reagent in the cannabinoid-containing extraction
liquid, and comparing the resulting absorbance shift to a
calibrated quantification reference chart. In the case of more than
one visualization reagent, each may be provided in separate tubes.
In certain embodiments of the invention, the diazonium salt may be
stabilized as a salt of BF3, picric acid, sodium perchlorate, or
other salts known in the art to stabilize such diazonium compounds,
for instance as described in U.S. Pat. No. 4,771,005 and U.S. Pat.
No. 8,124,420. In certain embodiments of the invention, the dry
reagent may also include a solid diluent, preferable an inert solid
diluent. The solid diluent may be useful for accurate measurement
purposes, and may also serve as a dessicant and/or light protectant
to aid in the stability of the dry reagent. In certain embodiments,
the solid diluent may be soluble in the extraction solution to be
used in the visualization reaction.
[0050] In another aspect of the invention, there is provided a
simplified assay for quantification of CBD in a sample. Potassium
hydroxide and sodium hydroxide are cannabinoid-sensitive
visualization reagents that are specific for CBD. In general, to
obtain an absorbance shift, the potassium hydroxide must be
contacted with CBD in an ethanolic medium. The inventors have
disclosed herein that an absorbance shift may also occur with
potassium hydroxide in a methanolic medium, or other lower
alcoholic media. Thus, in order to reduce the number of steps for
the CBD quantification assays of the present invention, it would be
desirable to extract the CBD from the sample using an extraction
solvent such as methanol, ethanol, propanol, or other lower
alcohols. In this way, the subsequent visualization reaction may be
easily accomplished, by adding, for instance, solid potassium
hydroxide in a pre-measured amount, or by adding a small amount of
a concentrated potassium hydroxide solution, without a requirement
to change the reaction medium, since the extracted CBD will be in a
suitable solvent that is permissive for the visualization reaction
with potassium hydroxide. This simplified assay may be particularly
suitable for development of a test kit, since the visualization
reagent, for instance potassium hydroxide or sodium hydroxide, may
be provided in a non-alcoholic stock solution, such as an aqueous
solution, that is more suitable for shipping and storage.
Similarly, the visualization reagent, potassium hydroxide or sodium
hydroxide may be provided in a concentrated form, in either
alcoholic or non-alcoholic solution, thus allowing the addition of
a very small amount, such as a drop or a few drops, without
requiring the CBD containing extraction solution to be exchanged to
a lower alcohol to allow the visualization reaction.
[0051] Test Papers And Strips
[0052] Certain embodiments of the invention are based, in part, on
the surprising finding that test papers impregnated with certain
cannabinoid-sensitive visualization reagents may be used to
quantify the cannabinoid concentration in a sample. The inventor
has provided examples of such test papers suitable for quantifying
cannabinoids such as THC, CBD and/or CBN in a sample. Certain test
papers are particularly useful for quantifying CBD in a test
sample. Certain test papers are particularly useful for quantifying
THC in a test sample. In these embodiments, the previously
described methods for quantifying cannabinoids using
cannabinoid-sensitive visualization reagents is accomplished by
carrying out the visualization reaction on the test paper or test
strip, rather than in solution in a reaction vessel.
[0053] CBD Test Papers and Strips
[0054] In certain embodiments, the cannabinoid-sensitive
visualization reagent, for instance KOH or NaOH, is specific for
CBD, and is present on the test paper in a sufficient amount to
cause a colorimetric reaction on the test paper when the test strip
is contacted with CBD in a permissive solvent. The permissive
solvent may be an alcohol such as ethanol, methanol, or
isopropanol. The inventors disclose that test strips impregnated
with a CBD-specific cannabinoid-sensitive visualization reagent
such as KOH, which undergoes an absorbance shift (changes color)
upon contact with CBD in an appropriate solvent, may be utilized to
quantify the concentration of such CBD in a given sample.
Quantification is achieved by contacting the said test strip with a
liquid CBD-containing sample (in a permissive solvent), or
alternatively with a liquid extraction from a solid CBD-containing
sample (in a permissive solvent), and comparing the resulting
absorbance shift to that caused by samples with known CBD
concentrations. The inventors have provided an example of such an
assay to determine the concentration of CBD in a solid plant
sample. The inventors have provided an example of a quantification
reference chart, useful in said assay, calibrated to determine the
CBD concentration in a solid plant sample, and suitable for solid
plant samples having CBD concentration between 0-15%, and possibly
higher. The calibrated reference chart has been optimized for this
CBD concentration range by extracting cannabinoids from a series of
solid samples having known CBD concentrations ranging from 0% to
15%, using a uniform extraction solvent composition, volume, and
extraction time for all of the samples, and contacting the
resulting CBD-containing extraction liquid with test strips
impregnated with KOH. The resulting absorbance shift, or color
change, caused by each of the samples of known CBD concentration
provides a reference chart for use in the experimental assay for
the unknown sample--by extracting the CBD from the unknown sample
using the exact same extraction solvent composition, volume, and
extraction time; contacting the resulting CBD-containing extraction
liquid with the test papers impregnated with KOH; and comparing the
resulting absorbance shift, or color change, with the calibrated
quantification reference chart, one may thus determine the CBD
concentration in the unknown solid sample. In an Example provided
herein, the extraction solvent composition, volume, and extraction
time have been optimized to ensure that the absorbance shift caused
by contacting the resulting CBD-containing extraction solution with
test papers impregnated with KOH is in the linear range of the
visualization reagent, such that the absorbance, or color change
intensity, is proportional to the CBD concentration. An important
feature of the quantification method using the KOH-impregnated test
papers is that the solvent used to extract or otherwise dissolve
the CBD is of an appropriate composition to allow the visualization
reaction to occur on the test paper. In an Example detailed herein,
for instance, the KOH is dried onto the paper, and the
CBD-containing test samples are dissolved in methanol or propanol,
thus when the liquid test sample is contacted with the test strip,
the methanol or propanol solvent creates an appropriate environment
for the visualization reaction to occur between the KOH and CBD. In
these various embodiments, the test papers may be affixed to a
solid support, for instance to form a test strip, colorimetric
strip, dipstick, and the like. As used herein, the terms `test
paper` and `test strip` may be used interchangeably.
[0055] In one aspect of the invention, there is provided an
apparatus including a test strip wherein the test strip comprises a
porous matrix uniformly impregnated with a CBD-specific cannabinoid
sensitive visualization reagent. The CBD-specific
cannabinoid-sensitive visualization reagent may be a strong base.
The CBD-specific cannabinoid-sensitive visualization reagent may be
KOH or NaOH. In certain embodiments, the CBD-specific
cannabinoid-sensitive visualization reagent is present on the test
strip in sufficient amount to cause a colorimetric chemical
reaction when contacted with CBD and a permissive solvent. The
permissive solvent may be an alcohol. The permissive solvent may be
ethanol, methanol, or propanol. In some embodiments, the
CBD-specific cannabinoid-sensitive visualization reagent is present
in the test paper in sufficient amount to allow quantification of
CBD in a CBD containing sample.
[0056] In another aspect of the invention, there is provided a
method for quantification of the concentration of CBD in a liquid
sample, the method involves: 1) contacting the liquid sample with a
test strip comprising a porous matrix uniformly impregnated with a
CBD-specific cannabinoid-sensitive visualization reagent, wherein
the liquid sample comprises a solvent of an appropriate composition
to allow a colorimetric reaction between the CBD and the
CBD-specific cannabinoid-sensitive visualization reagent; 2)
removing the test strip from the liquid sample and allowing it to
develop for a defined amount of time; and 3) comparing the
intensity of the resulting color change, or absorbance shift, of
the test strip to a calibrated quantification reference chart, such
that comparison of the color intensity change or absorbance shift
of the test sample to calibrated quantification reference chart
allows determination of the CBD concentration in the test sample.
The calibrated quantification reference chart may be produced, for
instance, by contacting a series of calibrated samples having
pre-determined CBD concentrations with a series of test strips
under the exact same conditions as to be used for the test sample,
including using the same CBD-specific cannabinoid-sensitive
visualization reagent, the same liquid solvent, and the same color
development time as to be used for the test sample. The
CBD-specific cannabinoid-sensitive visualization reagent may be a
strong base. The CBD-specific cannabinoid-sensitive visualization
reagent may be a KOH. The permissive liquid solvent may be an
alcohol. The appropriate liquid solvent may be methanol, ethanol,
or isopropanol.
[0057] In another aspect of the invention, there is provided a
method for quantification of CBD in a solid sample, the method
involves: 1) contacting a defined amount of solid sample with a
defined volume of an extraction solvent for a defined amount of
time, wherein CBD is extracted from the solid sample into the
extraction solvent resulting in a CBD-containing liquid extraction
solution, and wherein the extraction solvent has a composition
suitable to allow the reaction of the extracted CBD with a
CBD-specific cannabinoid-sensitive visualization reagent; 2)
contacting the resulting liquid extraction solution with a test
strip comprising a porous matrix uniformly impregnated with a
CBD-specific cannabinoid-sensitive visualization reagent; 3)
removing the test strip from the liquid extraction solution and
allowing it to develop for a defined amount of time; and 4)
comparing the intensity of the resulting color change, or
absorbance shift, of the test strip to a calibrated quantification
reference chart, such that comparison of the color intensity change
or absorbance shift of the test sample to a calibrated
quantification reference chart allows determination of the CBD
concentration in the test sample. The calibrated quantification
reference chart may be created by performing the identical method
as used on the unknown solid sample on a series of solid samples of
known CBD concentration, and recording the resultant color change,
or absorbance shift, caused by the known CBD concentrations. The
CBD-sensitive visualization reagent may be a strong base. The
CBD-specific cannabinoid-sensitive visualization reagent may be a
KOH. The extraction solvent may be an alcohol. The extraction
solvent may be methanol, ethanol, or isopropanol.
[0058] The inventors further disclose that test papers or strips
impregnated with a strong base such as KOH rapidly lose
effectiveness towards CBD when exposed for only a short time to
ambient air. This means that such strips may only be effective when
freshly prepared, which is a significant limitation for the
applicability in field tests or in kits. The inventors have
successfully extended the shelf life of such test strips by vacuum
sealing to remove any air and/or moisture from contacting the
strips, allowing their use days, weeks, or months after
preparation. In certain embodiments, there is provided a
CBD-sensitive test paper and/or strip comprising a porous matrix
uniformly impregnated with a CBD-sensitive visualization reagent,
wherein the test paper and/or strip is provided in a sealed
container or package. The sealed container or package may include a
dessicant.
[0059] THC Test Papers and Strips
[0060] The inventors also provide examples of compositions and
methods for quantifying cannabinoids THC, CBD and/or CBN in a test
sample, using test strips comprising a porous matrix impregnated
with cannabinoid-sensitive visualization reagents. In certain
embodiments of the invention, the test strip comprises multiple
test papers wherein each test paper is impregnated with a different
cannabinoid-sensitive visualization reagent.
[0061] In other aspects of the invention, the one or more
cannabinoid-sensitive visualization reagents is a diazonium salt.
In other embodiments, one of the cannabinoid-sensitive
visualization reagents is a Ghamrawy reagent or a modified Ghamrawy
reagent. The inventors have developed a method for manufacturing
test papers with a modified Ghamrawy reagent. Such reagents have
been described as requiring hydrochloric acid or sulfuric acid to
be effective. As these acids are generally only present in liquid
form, these reagents have previously only been suitable for liquid
based reactions. The inventors have identified strong acids that
may also exist as a solid, for instance p-toluenesulfonic acid.
Thus, in certain embodiments, the modified Ghamrawy reagent
comprises p-DMAB and p-toluenesulfonic acid. In certain
embodiments, the test paper is manufactured by dissolving the
modified Ghamrawy reagents in a solvent such as methanol--either
together or separately--and then contacting the test paper with the
reagent and allowing to dry. The test strip may be heated during
the drying process, which the inventors have shown to improve the
effectiveness of the test papers. In other embodiments, the test
strips may comprise a Duquenois reagent.
[0062] In other aspects of the invention, there is thus provided an
apparatus which includes a test strip, wherein the test strip
comprises a porous matrix uniformly impregnated with one or more
cannabinoid-sensitive visualization reagents. In certain
embodiments, the one or more cannabinoid-sensitive visualization
reagents are present on the test strip in sufficient amount to
cause a colorimetric chemical reaction when contacted with
cannabinoids and an appropriate solvent. In some embodiments, the
cannabinoid-sensitive visualization reagents are present in
sufficient amount to allow quantification of THC and/or CBD and/or
CBN in a cannabinoid containing sample. In certain embodiments, the
one or more visualization reagents are present in different
discrete regions of the test strip, for instance on separate test
papers that are affixed to the test strip. In certain embodiments,
the cannabinoid-sensitive visualization reagents are diazonium
salts such as Fast Blue B, Fast Blue BB, Fast Garnet, and/or Fast
Corinth V. In certain embodiments, the cannabinoid-sensitive
visualization reagent may also be a Duquenois reagent. In certain
embodiments, the cannabinoid-sensitive visualization reagent may be
a Ghamrawy reagent or a modified Ghamrawy reagent. In certain
embodiments, the test strip further comprises an inert support.
[0063] In another aspect of the invention, there is provided a
method for quantification of the concentration of one or more
cannabinoid compounds in a liquid sample, the method involves: 1)
contacting the liquid sample with a test strip comprising a porous
matrix uniformly impregnated with one or more cannabinoid-sensitive
visualization reagents; 2) removing the test strip from the liquid
sample and allowing it to develop for a defined amount of time; and
3) comparing the intensity of the resulting color change, or
absorbance shift, of the one or more cannabinoid-sensitive
visualization reagents to a calibrated quantification reference
chart, such that comparison of the color intensity change or
absorbance shift of the test sample to the calibrated
quantification reference chart allows determination of the
cannabinoid concentration in the test sample. The calibrated
quantification reference chart may be produced, for instance, by
contacting a series of calibrated samples having pre-determined
cannabinoid concentrations with a series of test strips under the
exact same conditions as to be used for the test sample, including
using the same one or more cannabinoid-visualization reagents and
the same color development time as to be used for the test sample.
In certain embodiments, the test strip comprises one or more test
papers, each individually impregnated with different
cannabinoid-sensitive visualization reagents. The
cannabinoid-sensitive visualization reagent may be a diazonium
salt. The cannabinoid-sensitive visualization reagent may be chosen
from the following: Fast Blue B, Fast Blue BB, Fast Garnet and Fast
Corinth V. The cannabinoid-sensitive visualization reagent may be a
Duquenois reagent. The cannabinoid compound to be quantified may be
THC. In certain embodiments, the cannabinoid-sensitive
visualization reagent may be a Ghamrawy reagent or a modified
Ghamrawy reagent.
[0064] In another aspect of the invention, there is provided a
method for quantification of one or more cannabinoid compounds in a
solid sample, the method involves: 1) contacting a defined amount
of solid sample with a defined volume of an extraction solvent for
a defined amount of time, wherein cannabinoids are extracted from
the solid sample into the extraction solvent resulting in a
cannabinoid-containing liquid extraction solution; 2) contacting
the resulting liquid extraction solution with a test strip
comprising a porous matrix uniformly impregnated with one or more
cannabinoid-sensitive visualization reagents; 3) removing the test
strip from the liquid extraction solution and allowing it to
develop for a defined amount of time; and 4) comparing the
intensity of the resulting color change, or absorbance shift, of
the cannabinoid-sensitive visualization reagent to a calibrated
quantification reference chart, such that comparison of the color
intensity change or absorbance shift of the test sample to a
calibrated quantification reference chart allows determination of
the cannabinoid concentration in the test sample. The calibrated
quantification reference chart may be created by performing the
identical method as used on the unknown solid sample on a series of
solid samples of known cannabinoid concentration, and recording the
resultant color change, or absorbance shift, caused by the known
cannabinoid concentrations. In certain embodiments, the test strip
comprises one or more test papers, each individually impregnated
with different cannabinoid-sensitive visualization reagents. The
cannabinoid-sensitive visualization reagent may be a diazonium
salt. The cannabinoid-sensitive visualization reagent may be chosen
from the following: Fast Blue B, Fast Blue BB, Fast Garnet and Fast
Corinth V. The cannabinoid compound to be quantified may be THC.
The cannabinoid-sensitive visualization reagent may be a Duquenois
reagent. The cannabinoid compound to be quantified may be THC. In
certain embodiments, the cannabinoid-sensitive visualization
reagent may be a Ghamrawy reagent or a modified Ghamrawy
reagent
[0065] Kits
[0066] In another aspect of the invention, there is provided a kit
for quantification of the concentration of one or more cannabinoid
compounds in a sample, the kit comprising: 1) optionally, a solvent
for extraction of cannabinoid compounds from a solid sample; 2) one
or more cannabinoid-sensitive visualization reagent; and 3) a
calibrated quantification reference chart. In a further embodiment,
the calibrated quantification reference chart may be replaced by an
instruction or set of instructions guiding or directing to a
calibrated quantification reference chart, for instance via web
link, URL, email address, or other means. In certain embodiments,
the extraction solvent has a composition that is permissive for a
visualization reaction with the cannabinoid-sensitive visualization
reagents. In certain embodiments, the kit is useful for the
quantification of THC in a test sample, and the one or more
cannabinoid-sensitive visualization reagents is chosen from: a
diazonium salt, for instance Fast Blue B, Fast Blue BB, Fast
Corinth V, and Fast Garnet GC; a Duquenois reagent; a Ghamrawy
reagent; and/or a modified Ghamrawy reagent. In certain embodiments
of the invention the kit is useful for the quantification of CBD in
a test sample, and the cannabinoid-sensitive visualization reagent
is a strong base, for instance potassium hydroxide or sodium
hydroxide. In certain embodiments, the cannabinoid-sensitive
visualization reagent is provided in a pre-measured amount suitable
for single test reactions. In certain embodiments, the pre-measured
cannabinoid-sensitive visualization reagent is provided in dry
form. The pre-measured cannabinoid-sensitive visualization reagent
may be provided in a ready to use reaction vessel, for instance a
test tube, plastic tube, eppendorf tube, and the like, or in a
separate pouch or container. In certain embodiments, the dry
reagent may further include a solid diluent. In certain
embodiments, the extraction solvent may have a composition that is:
1) suitable for dissolving the dry pre-measured
cannabinoid-sensitive visualization reagent; and 2) permissive for
a visualization reaction between the cannabinoid and the
cannabinoid-sensitive visualization reagent.
[0067] In another aspect of the invention, there is provided a kit
for extended range quantification of the concentration of one or
more cannabinoid compounds in a sample, the kit includes: 1)
optionally, a solvent for extraction of cannabinoid compounds from
the sample; 2) two or more cannabinoid-sensitive visualization
reagents; and 3) a calibrated quantification reference chart,
wherein the calibrated quantification reference chart comprises
calibrated quantification reference for each of the two or more
cannabinoid-sensitive visualization reagents. In a further
embodiment, the calibrated quantification reference chart may be
replaced by an instruction or set of instructions guiding or
directing to a calibrated quantification reference chart, for
instance via web link, URL, email address, or other means. In
certain embodiments, the extraction solvent has a composition that
is permissive for a visualization reaction with the two or more
cannabinoid-sensitive visualization reagents. In certain
embodiments, the kit is useful for the quantification of THC in a
test sample, and the cannabinoid-sensitive visualization reagents
are chosen from: a diazonium salt, for instance Fast Blue B, Fast
Blue BB, Fast Corinth V, and Fast Garnet GC; a Duquenois reagent; a
Ghamrawy reagent; and/or a modified Ghamrawy reagent. In certain
embodiments, the cannabinoid-sensitive visualization reagent is
provided in a pre-measured amount suitable for a single test
reaction. In certain embodiments, the pre-measured
cannabinoid-sensitive visualization reagent is provided in dry
form. The dry reagent may further include a solid diluent The
pre-measured cannabinoid-sensitive visualization reagent may be
provided in a ready to use reaction vessel, for instance a test
tube, plastic tube, eppendorf tube, and the like, or may be
provided in a separate pouch or container. In certain embodiments,
the extraction solvent may have a composition that is 1) suitable
for dissolving the dry pre-measured cannabinoid-sensitive
visualization reagent; and 2) permissive for a visualization
reaction between the cannabinoid and the cannabinoid-sensitive
visualization reagent.
[0068] The following examples are provided for illustrative
purposes, and are not intended to be limiting.
EXAMPLES
Example 1--Quantification of THC Content of an Unknown Solid
Cannabis Sample Using the Compositions and Methods of the
Invention--Visualization in Solution
[0069] A. Preparation of Cannabinoid-Sensitive Visualization
Reagents
[0070] Fast Blue BB diazonium salt was dissolved in methanol to a
final concentration of 0.1%.
[0071] B. Production of Calibrated Quantification Reference
Chart
[0072] Solid cannabis samples with known THC concentration were
used to create the calibrated quantification reference chart. These
cannabis samples were known to have negligible concentrations of
other cannabinoids that might also react with the
cannabinoid-sensitive visualization reagent, such as CBD or CBN.
For each sample, the following protocol was followed to extract the
THC into a liquid THC containing solution. The solid sample was
macerated into small pieces, and a portion was wrapped in aluminum
foil. This was heated at 325.degree. F. for exactly 5 minutes. The
resulting solid sample was removed from the foil, crumbled, and a
0.1 g portion was placed into a plastic tube. Exactly 5.0 mL of
methanol or isopropanol was placed into the plastic tube. The tube
with the 0.1 g solid sample and 5.0 mL of methanol or isopropanol
was shaken vigorously for 30 seconds. A 1.0 mL aliquot of the
resulting THC containing extraction solution was transferred to an
eppendorf tube. One drop (.about.20 uL) of cannabinoid-sensitive
visualization reagent from step A was added, and the reaction was
allowed to develop for exactly 10 minutes. This procedure was
completed in parallel with a number of solid cannabis samples
having different known THC concentrations. After the colour
development step, all the resulting color reactions were used to
create the calibrated quantification reference chart shown in FIG.
1. The colors from each tube are then easily transferred to a
suitable medium, for instance onto a reference card and the like,
for ease of comparison at any future point. More specifically, as
shown in FIG. 1, the calibrated quantification reference chart for
THC samples is disclosed having an ideal range of 5-20%. The
quantification reference chart was generated using Fast Blue BB.
The chart shows a light yellow colour at the left side, changing to
a darker yellow/orange on the right side.
[0073] C. Determination of THC Concentration in Unknown Solid
Cannabis Sample
[0074] A solid cannabis sample with unknown THC concentration (and
known to have low concentration of other cannabinoids that might
react with the cannabinoid-sensitive visualization reagent, such as
CBD or cannabinol) was prepared using the exact same methodology as
the samples in Step B. The resulting THC-containing extraction
liquid was contacted with cannabinoid-sensitive visualization
reagent from Step A and color development for 10 minutes, exactly
as in Step B. The resulting color change was then compared to the
calibrated quantification reference chart from Step B to determine
the THC concentration in the unknown sample.
Example 2--Quantification of THC Content of an Unknown Solid
Cannabis Sample Using an Extended Range Calibrated Quantification
Reference Chart-Visualization in Solution
[0075] A. Preparation of Two Cannabinoid-Sensitive Visualization
Reagents
[0076] Fast Blue BB salt was dissolved in methanol to a final
concentration of 0.1%. Fast Corinth V was dissolved in methanol to
a final concentration of 0.1%
[0077] B. Production of Calibrated Quantification Reference
Chart
[0078] Solid cannabis samples with known THC concentration were
used to create the calibrated quantification reference chart. These
cannabis samples were known to have negligible concentrations of
other cannabinoids that might also react with the
cannabinoid-sensitive visualization reagent, such as CBD or
cannabinol. For each sample, the following protocol was followed to
extract the THC into a liquid THC containing solution. The solid
sample was macerated into small pieces, and a portion was wrapped
in aluminum foil. This was heated at 325 degrees Celsius for
exactly 5 minutes. The resulting solid sample was removed from the
foil, crumbed, and a 0.1 g portion was placed into a plastic
container. Exactly 5.0 mL of methanol or isopropanol was placed
into the plastic container. The container with the 0.1 g solid
sample and 5.0 mL of methanol or isopropanol was shaken vigorously
for 10 seconds. A 1.0 mL aliquot of the resulting THC containing
extraction solution was transferred to a first eppendorf tube, and
a 1.0 mL aliquot of the resulting THC containing extraction
solution was transferred to a second eppendorf tube. One drop
(.about.20 uL) of Fast Blue BB cannabinoid-sensitive visualization
reagent from step A was added to the first eppendorf tube, and one
drop (.about.20 uL) of Fast Corinth V cannabinoid-sensitive
visualization reagent from step A was added to the second eppendorf
tube, and the reaction in each tube was allowed to develop for
exactly 10 minutes. This procedure was completed in parallel with a
number of solid cannabis samples having different known THC
concentrations. After the colour development step, all the
resulting color changes for Fast Blue BB, and the resulting color
changes for Fast Corinth V were used to create the extended range
calibrated quantification reference chart (FIG. 2). More
specifically, as shown in FIG. 2, an extended range calibrated
quantification reference chart is detailed therein. The chart
includes overlapping ranges for 2 different cannabinoid-sensitive
visualization reagents, to allow for quantification from 0% to over
25% THC in a solid sample. The top row of colors is generated using
Fast Corinth V. The bottom row of colors is generated using Fast
Blue BB. The top row, moving from left to right, shows the color
change starting as light orange at the bottom of the range and
getting progressively more red at the top of the range. The ideal
quantification range for the top row is 0-14%. The bottom row,
moving from left to right, shows the color change starting at light
yellow at the bottom of the range, and moving to darker orange at
the top of the range. The ideal quantification range is 11-25%. The
overlapping ideal ranges provides good quantification between 0-25%
THC.
[0079] C. Determination of THC Concentration in Unknown Solid
Cannabis Sample
[0080] A solid cannabis sample with unknown THC concentration (and
known to have low concentration of other cannabinoids that might
react with the cannabinoid-sensitive visualization reagent, such as
CBD or cannabinol) was prepared using the exact same methodology as
the samples in Step B. The resulting THC-containing extraction
liquid was contacted with cannabinoid-sensitive visualization
reagent from Step A and colour development for 10 minutes, exactly
as in Step B. The resulting colour change was then compared to the
extended range calibrated quantification reference chart from Step
B to determine the THC concentration in the unknown sample.
Example 3--Quantification of CBD Content of an Unknown Solid
Cannabis Sample Using the Compositions and Methods of the
Invention-Visualization in Solution
[0081] A. Preparation of Cannabinoid-Sensitive Visualization
Reagent
[0082] Potassium hydroxide was dissolved in water to a final
concentration of 20%.
[0083] B. Production of Calibrated Quantification Reference
Chart
[0084] Solid cannabis samples with known CBD concentration were
used to create the calibrated quantification reference chart. For
each sample, the following protocol was followed to extract the CBD
into a liquid CBD containing solution. The solid sample was
macerated into small pieces, and a portion was wrapped in aluminum
foil. This was heated at 325 degrees Celsius for exactly 5 minutes.
The resulting solid sample was removed from the foil, crumbed, and
a 0.1 g portion was placed into a plastic container. Exactly 3.0 mL
of methanol or isopropanol was placed into the plastic container.
The container with the 0.1 g solid sample and 3.0 mL of methanol or
isopropanol was shaken vigorously for 10 seconds. A 1.0 mL aliquot
of the resulting THC containing extraction solution was transferred
to an eppendorf tube. Two drops (.about.40 uL) of
cannabinoid-sensitive visualization reagent potassium hydroxide
from step A was added, and the reaction was allowed to develop for
exactly 10 minutes. This procedure was completed in parallel with a
number of solid cannabis samples having different known CBD
concentrations. After the colour development step, all the
resulting reactions were lined up in order of increasing THC
concentration and the colors used to create the calibrated
quantification reference chart (FIG. 3). The colors from each tube
are then easily transferred to a suitable medium, for instance onto
a reference card and the like, for ease of comparison at any future
point. More specifically, as shown in FIG. 3, a calibrated
quantification reference chart for CBD samples is depicted
demonstrating an ideal range of 0-15%. Moving from left to right,
the figure shows the color changing from very light purple at the
lower end of the scale to a much darker purple at the higher end of
the scale.
[0085] C. Determination of THC concentration in unknown solid
cannabis sample
[0086] A solid cannabis sample with unknown CBD concentration was
prepared using the exact same methodology as the samples in Step B.
The resulting CBD-containing extraction liquid was contacted with
cannabinoid-sensitive visualization reagent from Step A and colour
development for 10 minutes, exactly as in Step B. The resulting
colour change was then compared to the extended range calibrated
quantification reference chart from Step B to determine the CBD
concentration in the unknown sample.
Example 4--Quantification of THC Content of an Unknown Solid
Cannabis Sample Using the Compositions and Methods of the
Invention
[0087] A. Preparation of Test Strips Impregnated with Fast Blue
BB
[0088] Fast Blue BB salt was dissolved in methanol to a final
concentration of 1%. Strips of Whatman paper (CF2) were submersed
in the 1% Fast Blue BB methanol solution, removed, and allowed to
dry.
[0089] B. Production of Calibrated Quantification Reference
Chart
[0090] Solid cannabis samples with known THC concentration were
used to create the calibrated quantification reference chart. For
each sample, the following protocol was followed to extract the THC
into a liquid THC containing solution. The solid sample was
macerated into small pieces, and a portion was wrapped in aluminum
foil. This was heated at 325 degrees Celsius for exactly 5 minutes.
The resulting solid sample was removed from the foil, crumbed, and
a 0.1 g portion was placed into a plastic container. Exactly 15 mL
of methanol or isopropanol was placed into the plastic container.
The container with the 0.1 g solid sample and 15 mL of methanol or
isopropanol was shaken vigorously for 10 seconds. A 1.5 mL aliquot
of the resulting THC containing extraction solution was transferred
to an eppendorf tube. An unused Fast Blue impregnated test strip
from Part A was dipped briefly into the THC containing extraction
solution, and excess liquid was shaken off the test strip. The test
strip was allowed to air dry and the colour was allowed to develop
for exactly 10 minutes from the point where the strip first touched
the THC containing extraction solution. This procedure was
completed in parallel with a number of solid cannabis samples
having different known THC concentrations. After the colour
development step, all the resulting test strips were lined up in
order of increasing THC concentration, and a picture taken to
create the calibrated quantification reference chart--see FIG. 4.
More specifically, as shown in FIG. 4, a calibrated reference chart
for Fast BB impregnated test strips is depicted. The chart shows
that, moving from left to right, the color change of the strip goes
from a very light orange/red at the low end of the scale to a much
darker red at the higher end of the scale.
[0091] C. Determination of THC Concentration in Unknown Solid
Cannabis Sample
[0092] A solid cannabis sample with unknown THC concentration was
prepared using the exact same methodology as the samples in Step B.
The resulting THC-containing extraction liquid was tested by
dipping a Fast Blue BB impregnated test strip from Step A briefly
into the THC containing extraction liquid, removing excess liquid,
and allowing air drying and colour development for 10 minutes,
exactly as in Step B. The resulting colour change was then compared
to the calibrated quantification reference chart from Step B to
determine the THC concentration in the unknown sample.
Example 5--Quantification of CBD Content of an Unknown Solid
Cannabis Sample Using the Compositions and Methods of the
Invention
[0093] A. Preparation of Test Strips Impregnated with KOH
[0094] KOH was dissolved in methanol to a final concentration
between 1-30% methanol (1%, 10%, 30%). Strips of Whatman paper
(CF1) were submersed in the KOH solution, removed, and allowed to
dry. Dried test strips were dipped in a methanol extract of a CBD
containing Cannabis sample. The Cannabis sample contained 15% CBD
and the extract was prepared by i) macerating the sample and
heating at 325 degrees Fahrenheit for 5 minutes to convert CBD-A to
CBD; ii) adding 0.1 g of the heated sample to 1.0 mL of methanol;
and iii) shaking vigorously for 20 seconds. After the test strips
were dipped in the CBD liquid extract, they were allowed to develop
for 5 minutes. FIG. 5 shows that impregnation of the test strips
using 1% or 10% KOH in methanol did not result in a significant
color change in the subsequent reaction with the CBD liquid
extract, however using 30% did allow for a significant colorimetric
reaction to occur. More specifically, as shown in FIG. 5,
representative test papers are depicted having been prepared using
30% KOH (left), 10% KOH (middle), and 1% KOH (right).
[0095] B. Production of Calibrated Quantification Reference
Chart
[0096] Solid cannabis samples with known CBD concentration were
used to create the calibrated quantification reference chart. For
each sample, the following protocol was followed to extract the CBD
into a liquid CBD containing solution. The solid sample was
macerated into small pieces, and a portion was wrapped in aluminum
foil. This was heated at 325 degrees Fahrenheit for exactly 5
minutes. The resulting solid sample was removed from the foil,
crumbled, and a 0.1 g portion was placed into a plastic container.
Exactly 1.0 mL of methanol or isopropanol was placed into the
plastic container. The container with the 0.1 g solid sample and
1.0 mL of methanol or isopropanol was shaken vigorously for 20
seconds. An aliquot of the resulting CBD containing extraction
solution was transferred to an eppendorf tube. A KOH impregnated
test strip was prepared as in Part A, except that the impregnation
of the test strips was performed with KOH dissolved in methanol at
a concentration of 20% w/v. An unused KOH impregnated test strip
was dipped briefly into the CBD containing extraction solution, and
excess liquid was shaken off the test strip. The test strip was
allowed to air dry and the color was allowed to develop for exactly
5 minutes from the point where the strip first touched the CBD
containing extraction solution. This procedure was completed in
parallel with a number of solid cannabis samples having different
known CBD concentrations. After the color development step, all the
resulting test strips were lined up in order of increasing CBD
concentration, and a picture taken to create the calibrated
quantification reference chart--see FIG. 6. It can be seen that the
color intensity increases in direct correlation with the increasing
CBD concentration in the sample. More specifically, as shown in
FIG. 6, a CBD Calibrated Reference Chart is depicted. Moving from
left to right on the chart therein, the color change shown on the
chart goes from a very light violet/purple for the lower CBD % to a
much darker purple for higher CBD %.
[0097] C. Determination of CBD Concentration in Unknown Solid
Cannabis Sample
[0098] A solid cannabis sample with unknown CBD concentration was
prepared using the exact same methodology as the samples in Step B.
The resulting CBD-containing extraction liquid was tested by
dipping a KOH impregnated test strip from Step A briefly into the
CBD containing extraction liquid, removing excess liquid, and
allowing air drying and color development for 5 minutes, exactly as
in Step B. The resulting color change was then compared to the
calibrated quantification reference chart from Step B to determine
the CBD concentration in the unknown sample.
Example 6--Quantification of THC Content of an Unknown Solid
Cannabis Sample Using the Compositions and Methods of the
Invention
[0099] A. Preparation of Test Papers Impregnated with Modified
Ghamrawy Reagent
[0100] A 10% solution of p-dimethylaminobenzaldehyde (p-DMAB) in
methanol was prepared in a tube. In a separate tube, a 5 M solution
of p-toluenesulfonic acid in methanol was prepared. Squares of CF4
paper (GE Healthcare) were immersed in the p-DMAB solution and
allowed to dry, and then quickly dipped in the p-toluenesulfonic
acid solution. The strips were then placed in an oven at 200
degrees Fahrenheit for 2 minutes until the strips were completely
dry.
[0101] B. Preparation of Calibrated Reference Chart
[0102] Solid cannabis samples with known THC concentration were
used to create the calibrated quantification reference chart. For
each sample, the following protocol was followed to extract the THC
into a liquid THC containing solution. The solid sample was
macerated into small pieces, and a portion was wrapped in aluminum
foil. This was heated at 325 degrees Celsius for exactly 5 minutes.
The resulting solid sample was removed from the foil, crumbed, and
a 0.1 g portion was placed into a plastic container. Exactly 15 mL
of methanol or isopropanol was placed into the plastic container.
The container with the 0.1 g solid sample and 15 mL of methanol or
isopropanol was shaken vigorously for 10 seconds. A 1.5 mL aliquot
of the resulting THC containing extraction solution was transferred
to an eppendorf tube. An unused modified Ghamrawy reagent
impregnated test strip from Part A was dipped briefly into the THC
containing extraction solution, and excess liquid was shaken off
the test strip. The test strip was allowed to air dry and the
colour was allowed to develop for exactly 10 minutes from the point
where the strip first touched the THC containing extraction
solution. This procedure was completed in parallel with a number of
solid cannabis samples having different known THC concentrations.
After the colour development step, all the resulting test strips
were lined up in order of increasing THC concentration, and a
picture taken to create the calibrated quantification reference
chart--see FIG. 7. More specifically, as shown in FIG. 7, a THC
Calibrated Reference Chart using modified Ghamrawy reagent
impregnated test papers is depicted. Moving from left to right on
the chart therein, the color change shown on the chart goes from a
very light violet/purple for the lower THC % to a much darker
purple for higher THC %.
[0103] C. Quantification of THC Concentration in a Test Sample of
Unknown Concentration
[0104] A solid cannabis sample with unknown THC concentration (and
known to have low concentration of other cannabinoids that might
react with the cannabinoid-sensitive visualization reagent, such as
CBD) was prepared using the exact same methodology as the samples
in Step B. The resulting THC-containing extraction liquid was
contacted with cannabinoid-sensitive visualization reagent from
Step A and colour development for 10 minutes, exactly as in Step B.
The resulting colour change was then compared to the calibrated
quantification reference chart from Step B to determine the THC
concentration in the unknown sample.
[0105] While specific embodiments of the invention have been
described and illustrated, such embodiments should be considered
illustrative of the invention only and not as limiting the
invention as construed in accordance with the accompanying claims.
Other features and advantages of the invention will be apparent
from the following description of the drawings and the invention,
and from the claims.
* * * * *