WO2023049969A1 - An analysis method and uses thereof - Google Patents

Abstract

The present disclosure relates generally to a method for evaluating one or both of the presence and concentration of at least one cannabinoid in a sample. In certain embodiments, the method is useful for monitoring cannabis plants for a change in its chemotypic profile and selecting growing conditions that favor the development of a cannabis plant with a desirable chemotypic profile.

Description

AN ANALYSIS METHOD AND USES THEREOF

RELATED APPLICATIONS

[0001] This application claims priority from Australian Provisional Patent Application No. 2021903143 filed on 1 October 2021, the entire content of which is incorporated by reference.

FIELD

[0002] The present disclosure relates generally to a method for evaluating one or both of the presence and concentration of at least one cannabinoid in a sample. In certain embodiments, the method is useful for monitoring Cannabis plants for a change in its chemotypic profile and selecting growing conditions that favor the development of a Cannabis plant with a desirable chemotypic profile.

BACKGROUND

[0003] Cannabis sativa L. is one of the earliest domesticated and cultivated plants with records of its use in central Asia dating back more than 6000 years. Cannabis belongs to the Cannabaceae family and has been used for millennia for its source of fibre, seed oil, food and medicinal purposes.

[0004] It is estimated that Cannabis plants produce more than 550 different molecules, including cannabinoids, terpenes and other phenolics. Cannabinoids, such as A⁹- tetrahydrocannabinol (THC) and cannabidiol (CBD), are the most commonly known and researched molecules produced by cannabis plants. CBD and THC are naturally present in their acidic forms, tetrahydrocannabinolic acid (THCA-A) and cannabidiolic acid (CBDA), which are alternative products of a shared precursor cannabigerolic acid (CBGA). Other cannabinoids of interest include cannabigerol (CBG), cannabichromene (CBC), cannabinol (CBN) and tetrahydrocannabivarin (THCV), which are found at low or trace amounts in Cannabis.

[0005] Accurate quantitation of cannabinoids different samples (e.g., plant derived samples) is essential in understanding the pharmacological benefits of using products containing one or multiple cannabinoids as a medicine. However, the rapid, high-throughput analysis of cannabinoids has been limited due to the structural similarity of some cannabinoids, which makes it difficult to accurately distinguish different cannabinoids using currently available analysis methods. For example, A⁸-THC, is a structural isomer and derivative of A⁹-THC, which has an identical molecular weight, similar fragmentation ions and retention time, with the only difference being a shift in the position of the double bond. Due to the higher abundance of A⁹-THC in some cultivars, rapid analysis methods can often result in co-elution, which makes it difficult to accurately determine the concentration of A⁸- THC. Additionally, A⁸-THC tends to be in low abundance in cannabis plant material, such that quantitation software may not be sensitive enough to distinguish between the A⁹-THC peaks and A⁸-THC peaks. As a consequence, many analytical methods for the quantitation of cannabinoids only validate using major cannabinoid compounds, which are well characterized with robust isolated cannabinoids available for use in calibration.

[0006] The limitations associated with the quantitation of cannabinoids are observed across a number of different methods that are currently used for the analysis of cannabinoids. Gas chromatography (GC) or liquid chromatography (LC) in tandem with a mass spectrometer (MS) and/or a diode-array detector (DAD) are currently the most common methods for the analysis of cannabinoids. For example, high-performance liquid chromatography with a diode-array detector (HPLC-DAD) has been used to quantify cannabinoid compounds in both Cannabis oils and Cannabis plant material, but lacks the selectivity to distinguish between structurally similar, co-eluting compounds, resulting in unacceptably high limit of quantitation (LOQ; Bumier et al., 2019, Taianta, 192: 135-41). GC and GC-MS are more specific methods of cannabinoid analysis, however, GC methods are limited by the inability to directly quantitate acidic cannabinoids without derivatization, as the operating temperature of the injection port can decarboxylate the acidic cannabinoids into their neutral derivatives (Cardenia et al., 2018, Journal of Food and Drug Analysis., 26(4): 1283-92).

[0007] There remains, therefore, a need for improved methods for the rapid and high- throughput analysis of the chemical components of Cannabis, including the detection of cannabinoids in samples, such as extracts from Cannabis plant material.

SUMMARY

[0008] In an aspect of the present disclosure there is provided a method for evaluating one or both of the presence and concentration of at least one cannabinoid in a sample, the method comprising: a. obtaining absorbance wavelength data from the sample, wherein the absorbance wavelength data is obtained at a wavelength of from about 10 nm to about 400 nm; b. obtaining spectrometric data from the sample, wherein the spectrometric data is obtained by triple quadrupole mass spectrometry (QQQ-MS); c. comparing the spectrometric data and the absorbance wavelength data obtained in step (a) and (b) with a reference value; and d. based on the comparison in (c), evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample.

[0009] In another aspect, the present disclosure provides a method for monitoring a Cannabis plant for a change to its chemotypic profde, the method comprising: a. evaluating, in accordance with the method described herein, one or both of the presence and concentration of at least one cannabinoid in a first sample from a Cannabis plant; b. evaluating, in accordance with the method described herein, one or both of the presence and concentration of at least one cannabinoid in a second sample from the Cannabis plant, wherein the second sample is taken from the Cannabis plant at a time point subsequent to the first sample; and c. comparing one or both of the presence and concentration of the at least one cannabinoid evaluated in step (a) and step (b) to determine whether there has been a change in the chemotypic profile of the Cannabis plant.

[0010] In another aspect, the present disclosure provides a method for selecting growing conditions that favor the development of a Cannabis plant with a desirable chemotypic profile, the method comprising: a. exposing a first Cannabis plant to a first set of selected growing conditions for a period of time; b. exposing a second Cannabis plant to a second set of selected growing conditions for a period of time, wherein the second set of selected growing conditions is different from the first set of selected growing conditions; c. optionally, repeating step (b) for a subsequent set of growing conditions that is different from the first and second sets of selected growing conditions; d. evaluating, in accordance with the method described herein, one or both of the presence and concentration of at least one cannabinoid in a sample from each of the Cannabis plants exposed to the set of selected growing conditions of steps (a)-(c); and e. selecting from the set of growing conditions of steps (a)-(c) one or more sets of selected growing conditions that favor the development of a Cannabis plant with a desirable chemotypic profile based on one or both of the presence and concentration of the at least one cannabinoid evaluated in step (d).

BRIEF DESCRIPTION OF THE DRAWING

[0011] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the accompanying drawing.

[0012] Figure 1 is a schematic representation of the elution profile of the cannabinoids cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidolic acid (CBDA), cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiol (CBD), tetrahydrocannabidivarin (THCV), tetrahydrocannabidivarinic acid (THCVA), cannabinol (CBN), cannabinolic acid (CBNA), tetrahydrocannabinol (THC), delta-8- tetrahydrocannabinol (A8-THC), cannabicyclol (CBL), cannabichromene (CBC), tetrahydrocannabinolic acid (THCA-A), and cannabichromenic acid (CBCA) standards using the liquid chromatography triple-quadrupole mass spectrometer (LC-QQQ-MS).

DETAILED DESCRIPTION

[0013] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information. [0014] Unless otherwise indicated the molecular biology, cell culture, laboratory, plant breeding and selection techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), Glover and Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present); Janick (2001) Plant Breeding Reviews, John Wiley & Sons, 252 p.; Jensen ed. (1988) Plant Breeding Methodology, John Wiley & Sons, 676 p., Richard, A.J. ed. (1990) Plant Breeding Systems, Unwin Hyman, 529 p.; Walter ed. (1987) Plant Breeding, Vol. I, Theory and Techniques, MacMillan Pub. Co.; Slavko ed. (1990) Principles and Methods of Plant Breeding, Elsevier, 386 p.; and Allard, R.W. ed. (1999) Principles of Plant Breeding, John-Wiley & Sons, 240 p. The ICAC Recorder, Vol. XV no. 2: 3-14; all of which are incorporated by reference. The procedures described are believed to be well known in the art and are provided for the convenience of the reader. All other publications mentioned in this specification are also incorporated by reference in their entirety.

[0015] The articles "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an inflorescence" includes a single inflorescence, as well as two or more inflorescence; reference to "a sample" includes a single sample, as well as two or more samples; and so forth.

[0016] In the context of this specification, the term “about” in relation to a numerical value or range is intended to cover numbers falling within ± 10% of the specified numerical value or range.

[0017] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment, unless expressly stated otherwise.

[0018] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to mean the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. [0019] The term “optionally” is used herein to mean that the subsequent described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiment in which the event or circumstance occurs as well as embodiments in which it does not.

[0020] The present invention is based in part on the surprising observations made in the experiments described herein that cannabinoids can be accurately and specifically detected and quantitated using liquid chromatography, an ultra-violet diode array detector (UV-DAD) and triple quadrupole mass spectrometry (LC-QQQ-MS). The methods described herein can, in some instances, advantageously reduce the total analysis time to less than 15 minutes across a panel of different cannabinoids. This is twice as fast as previously described methods for the detection of cannabinoids, and is reproducible across samples derived from Cannabis plants with different chemotypic profiles.

[0021] Accordingly, in an aspect, the present disclosure provides a method for evaluating one or both of the presence and concentration of at least one cannabinoid in a sample, the method comprising: a. obtaining absorbance wavelength data from the sample, wherein the absorbance wavelength data is obtained at a wavelength of from about 10 nm to about 400 nm; b. obtaining spectrometric data from the sample, wherein the spectrometric data is obtained by triple quadrupole mass spectrometry (QQQ-MS); c. comparing the spectrometric data and the absorbance wavelength data obtained in step (a) and (b) with a reference value; and d. based on the comparison in (c), evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample.

Cannabis

[0022] Cannabis is an erect annual herb with a dioecious breeding system, although monoecious plants exist. Wild and cultivated forms of Cannabis are morphologically variable, which has resulted in difficulty defining the taxonomic organization of the genus. [0023] As used herein, the term "Cannabis" means a plant, plant part, seed or product derived therefrom of the species Cannabis sativa, Cannabis indica and Cannabis ruderalis.

[0024] The terms "plant", "cultivar", "variety", "strain" or "race" are used interchangeably herein to refer to a plant or a group of similar plants according to their structural features and performance (i.e., morphological and physiological characteristics).

[0025] The reference genome for C. sativa is the assembled draft genome and transcriptome of "Purple Kush" or "PK" (van Bakal et al. 2011, Genome Biology, 12:R102). C. sativa, has a diploid genome (2n = 20) with a karyotype comprising nine autosomes and a pair of sex chromosomes (X and Y). Female plants are homogametic (XX) and males heterogametic (XY) with sex determination controlled by an X-to-autosome balance system.

[0026] Cannabis is often divided into categories based on the abundance of THC and CBD, in particular, Type I Cannabis is THC -predominant, Type II Cannabis contains both THC and CBD, and Type III is CBD-predominant. It follows, therefore, that the detection of cannabinoids in a sample derived from Cannabis plant material may be used to classify the plant material into Type I (THC/THCA-enriched), Type II (THC/THCA- and CBD/CBDA-enriched) and/or Type III (CBD/CBDA-enriched) Cannabis plant material.

[0027] As used herein the term "enriched" means that the referenced cannabinoid(s) is/are the main cannabinoid(s) in the Cannabis plant material.

[0028] The term "plant" as used herein refers to a whole plant, parts thereof obtained from or derived from, such as, e.g., leaves, stems, roots, flowers, single cells (e.g., pollen), seeds, plant cells and the like. The term "plant part" also includes any material listed in the Plant Part Code Table as approved by the Australian Therapeutic Goods Administration (TGA) Business Services (TBS).

Cannabinoids

[0029] The term "cannabinoid", as used herein, refers to a family of terpeno-phenolic compounds, of which more than 100 compounds are known to exist in nature. Cannabinoids will be known to persons skilled in the art, illustrative examples of which are provided in Table 1, including acid and neutral (i.e., decarboxylated) forms thereof.

[0030] Cannabinoids are synthesized in cannabis plants as carboxylic acids. While some decarboxylation may occur in the plant, decarboxylation typically occurs post-harvest and is increased by exposing plant material to heat (Sanchez and Verpoote, 2008, Plant Cell Physiology, 49(12): 1767-82). Decarboxylation is usually achieved by drying, heating and/or curing (z. e. , heating for a specific time and temperature to ensure maximum decarboxylation) the plant material. Persons skilled in the art would be familiar with methods by which decarboxylation of cannabinoids can be promoted, illustrative examples of which include combustion, vaporization, curing, drying, heating and baking.

[0031] The precursors of cannabinoids originate from two distinct biosynthetic pathways: the polyketide pathway, giving rise to olivetolic acid (OLA) and the plastidal 2- C-methyl-D-erythritol 4-phosphate (MEP) pathway, leading to the synthesis of geranyl diphosphate (GPP). OLA is formed from hexanoyl-CoA, derived from the short-chain fatty acid hexanoate, by aldol condensation with three molecules of malonyl-CoA. This reaction is catalyzed by a polyketide synthase (PKS) enzyme and an olivetolic acid cyclase (OAC). The geranylpyrophosphate :olivetolate geranyltransferase catalyzes the alkylation of OLA with GPP leading to the formation of CBGA, the central precursor of various cannabinoids. Three oxidocyclases are responsible for the diversity of cannabinoids: tetrahydrocannabinolic acid synthase (THCAS) converts CBGA to THCA, while cannabidiolic acid synthase (CBDAS) forms CBDA, and cannabichromenic acid synthase (CBCAS) produces CBCA. Propyl cannabinoids (cannabinoids with a C3 side-chain, instead of a C5 side-chain), such as tetrahydrocannabivarinic acid (THCVA), are synthesized from a divarinolic acid precursor.

[0032] " A-9-tetrahydrocannabinolic acid" or "THCA-A" is synthesized from the CBGA precursor by THCA synthase. The neutral form "A-9-tetrahydrocannabinol" or "THC" is associated with psychoactive effects of Cannabis, which are primarily mediated by its activation of CBlG-protein coupled receptors, which result in a decrease in the concentration of cyclic AMP (cAMP) through the inhibition of adenylate cyclase. THC also exhibits partial agonist activity at the cannabinoid receptors CB1 and CB2. CB1 is mainly associated with the central nervous system, while CB2 is expressed predominantly in the cells of the immune system. As a result, THC is also associated with pain relief, relaxation, fatigue, appetite stimulation, and alteration of the visual, auditory and olfactory senses, furthermore, more recent studies have indicated that THC mediates an anti-cholinesterase action, which may suggest its use for the treatment of Alzheimer's disease and myasthenia (Eubanks et al., 2006, Molecular Pharmaceuticals, 3(6): 773-7). [0033] Cannabidiolic acid" or "CBDA" is also a derivative of cannabigerolic acid (CBGA), which is converted to CBDA by CBDA synthase. Its neutral form, "cannabidiol" or "CBD" has antagonist activity on agonists of the CB1 and CB2 receptors. CBD has also been shown to act as an antagonist of the putative cannabinoid receptor, GPR55. CBD is commonly associated with therapeutic or medicinal effects of Cannabis and has been suggested for use as a sedative, anti-inflammatory, anti-anxiety, anti-nausea, atypical antipsychotic, and as a cancer treatment. CBD can also increase alertness, and attenuate the memory impairing effect of THC.

Chemotypic profile

[0034] The terms "chemotypic profile" or "chemotype" are used interchangeably herein to refer to a representation of the type, amount, level, ratio and/or proportion of cannabinoids that are present in the Cannabis plant or part thereof, as typically detected from plant material derived from the plant or plant part, including an extract therefrom.

[0035] The chemotypic profile in a Cannabis plant will typically predominantly comprise the acidic form of the cannabinoids, but may also comprise some decarboxylated (i. e. , neutral) forms thereof, at various concentrations or levels at any given time (i. e. , at propagation, growth, harvest, drying, curing, etc.).

[0036] In an embodiment, the chemotypic profile evaluates at least one cannabinoid selected from the group consisting of cannabidolic acid (CBDA), tetrahydrocannabinolic acid (THCA-A), cannabidivarinic acid (CBDVA), cannabigerolic acid (CBGA), tetrahydrocannabidivarinic acid (THCVA), cannabinolic acid (CBNA), cannabichromenic acid (CBCA), cannabidivarin (CBDV), cannabidiol (CBD), delta-8-tetrahydrocannabinol (A8-THC), tetrahydrocannabinol (A9-THC), cannabigerol (CBG), cannabinol (CBN), cannabicyclol (CBL), cannabichromene (CBC) and tetrahydrocannabidivarin (THCV).

[0037] In another embodiment, the chemotypic profile evaluates CBDA, THCA-A, CBDVA, CBGA, THCVA, CBNA, CBCA, CBDV, CBD, A8-THC, A9-THC, CBG, CBN, CBL, CBC and THCV.

[0038] In an embodiment, the chemotypic profile evaluates at least one cannabinoid in acid form and at least one cannabinoid in neutral form. [0039] In an embodiment, the chemotypic profile evaluates at least one cannabinoid in neutral form, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six or more preferably at least seven cannabinoids in neutral form.

[0040] In an embodiment, the chemotypic profile evaluates at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six or more preferably at least seven cannabinoids in neutral form selected from the group consisting of CBD, A8-THC, A9-THC, CBC, CBG, CBDV, THCV and CBN.

[0041] In an embodiment, the chemotypic profile evaluates at least one cannabinoid in acid form, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six or more preferably at least seven cannabinoids in acid form.

[0042] In an embodiment, the chemotypic profile evaluates at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six or more preferably at least seven cannabinoids in acid form selected from the group consisting of CBDA, THCA-A, CBDVA, CBGA, THCVA, CBNA and CBCA.

[0043] In an embodiment, the chemotypic profile evaluates at least two closely-related cannabinoids. By “closely-related cannabinoids” as used herein is meant two cannabinoids that (i) elute together under the LC conditions used, eluting either simultaneously or having overlapping elution profiles and/or (ii) are structurally similar. Structurally similar cannabinoids include those that share a specific ring configuration, for example, a ring configuration having three fused rings such as A9-THC, A8-THC, THCA, THCV, THCVA, CBN and CBNA; or a ring configuration having two rings attached by a single bond such as CBD, CBA, CBDV and CBDVA; or a ring configuration having two fused rings such as CBC and CBCA; or a ring configuration having a single ring such as CBG and CBGA See Table 1). In an embodiment, the chemotypic profile evaluates at least two, at least three, at least four, at least five, at least six, at least seven, at least 8, at least 9, at least 10, at least 11 or at least 12 closely-related cannabinoids.

Methods for evaluating the presence and/or concentration of cannabinoids

[0044] As enabled herein, the method for evaluating one or both of the presence and concentration of at least one a cannabinoid in a sample is achieved by the combination of wavelength absorbance detection and QQQ-MS. In some embodiments, the wavelength absorbance data, such as UV absorbance data, is obtained before the mass spectral data.

[0045] The term "sample" as used herein may comprise a single cannabinoid or multiple cannabinoids. The source the sample comprising the cannabinoid may be natural or synthetic. Suitable sources of the sample would be known to persons skilled in the art, illustrative examples of which include Cannabis plant material (e.g., cells, tissues, cultures (or subcultures thereof) and extracts or fractions thereof. As described elsewhere herein, it is also contemplated that the sample is derived from a separation processes, such as chromatographic separation.

[0046] In an embodiment, the sample is derived from Cannabis plant material. In an embodiment, the sample is an extract of Cannabis plant material.

[0047] The term "Cannabis plant material" as used herein refers to any part of the Cannabis plant, including the leaves, stems, roots, and buds, or parts thereof, as described elsewhere herein.

[0048] In an embodiment, the Cannabis plant material is an inflorescence.

[0049] The term “inflorescence” as used herein means the complete flower head of the Cannabis plant, comprising stems, stalks, bracts, flowers and trichomes (i.e., glandular, sessile and stalked trichomes).

[0050] In an embodiment, the Cannabis plant material is from a female cannabis plant.

[0051] In an embodiment, the Cannabis plant material is at least partially dried.

[0052] The term "drying" as used herein refers to any method for drying the plant material. Illustrative examples include air-drying, curing, and heat drying. In an embodiment, the Cannabis plant material is dried in a temperature, light and humidity controlled environment, such as a temperature of about 21 °C and a humidity of from about 38% and 45% RH. In another embodiment, heat is applied to the Cannabis plant material during the drying process to cure the dried plant material. Temperatures suitable for curing dried plant material would be known to persons skilled in the art, illustrative examples of which include a temperature from about 60°C to about 225 °C, preferably from about 100°C to about 150°C, preferably from about 110°C to about 130°C, or more preferably about 120°C. In an embodiment, the dried Cannabis plant material is cured by heating the dried cannabis plant material at about 120°C for 2 hours.

[0053] It is to be understood that the terms “dry”, "dried", “drying”, and the like are not intended to mean the absence of moisture in the Cannabis plant material, and therefore includes any state in which at least some moisture has been removed from the Cannabis plant material. Persons skilled in the art will be familiar with the extent to which Cannabis plant material can be dried to allow for concentration of compound(s), e.g., decarboxylated (i.e., neutral) cannabinoids. In an embodiment, the Cannabis plant material is dried under conditions and for a period of time that gives rise to a loss of at least 5%, preferably at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, or more preferably at least 99% of the moisture content of the plant material at the time of harvest.

[0054] In an embodiment, the Cannabis plant material is not heat treated or dried, with a view to preserving the acid forms of cannabinoids in the sample.

[0055] In another embodiment, the sample is an extract from Cannabis plant material.

[0056] The term "extract", as used herein, is to be understood as including a whole Cannabis plant extract, such as resin, hash and keif, a solution comprising solubilized cannabinoids obtained from a Cannabis plant or the Cannabis plant material, as well as substantially purified compounds isolated from the harvested Cannabis plant material, such as cannabinoids, terpenes and/or flavonoids.

[0057] As used herein, "substantially purified" refers to a compound or molecule that has been isolated from other components with which it is typically associated in its native state (i.e., within the plant material). Preferably, the substantially purified molecule is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. [0058] In an embodiment, the extract is prepared using a solvent selected from the group consisting of water, methanol, ethanol, acetone, acetonitrile and combinations of the foregoing. In another embodiment, the solvent is methanol.

[0059] Methods for solvent-based extraction for plant material would be known to persons skilled in the art, illustrative examples of which include maceration, percolation, decoction, and the methods described by Abubakar and Haque (2020, Journal of Pharmacy and Bioallied Sciences, 12(1): 1-10.

[0060] It is generally understood that the sample undergoes chromatographic separation prior to UV absorbance measurements and QQQ-MS. Accordingly, in an embodiment, the sample is obtained by chromatographic separation.

[0061] Methods for chromatographic separation would be known to persons skilled in the art, illustrative examples of which include paper chromatography (PC), thin-layer chromatography (TLC), column chromatography (CC), liquid chromatography (LC) and gas chromatography (GC).

[0062] In an embodiment, the chromatographic separation is performed by liquid chromatography (LC). In some embodiments, the liquid chromatography is carried out using a C18 column with a particle size of about 5 pm, about 3 pm or about 1.6 pm, preferably about 1.6 pm. In an embodiment, the column internal diameter is from about 1 mm to about 22 mm (e.g, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, about 20 mm, about 21 mm or about 22 mm), preferably from about 1 mm to about 4.6 mm, more preferably from about 1 mm to about 3 mm, e.g, about 2.1 mm. In some embodiments, the mobile phase comprises water with 1% formic acid and acetonitrile with 1% formic acid and the gradient is from about 60% to about 80% (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79% or about 80%) acetonitrile with formic acid for the first 5 minutes, preferably from about 65% to about 75%, and more preferably about 75%. To minimize decarboxylation, the liquid chromatography step may be maintained at a temperature below about 45 °C, e.g., at about 40°C. [0063] As described elsewhere herein, the methods described herein unexpectedly reduced the total analysis time (i. e. , total run time) for evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample to less than 15 minutes. This rapid analysis time is particularly used for high throughput methods for the detection of cannabinoids in commercial and research environments.

[0064] The term "total analysis time" as used herein refers to the time for both chromatographic separation (i. e. , elution) and evaluating one or both of the presence and concentration of the at least one cannabinoid per sample.

[0065] In an embodiment, the time for (i) chromatographic separation, and (ii) evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample is less than 15 minutes (e.g., 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute).

[0066] Thus, in an embodiment, the time for (i) chromatographic separation, and (ii) evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample is less than about 15 minutes, preferably about 15, preferably about 14, preferably about 13, preferably about 12, preferably about 11, preferably about 10, preferably about 9, preferably about 8, preferably about 7, preferably about 6, preferably about 5, preferably about 4, preferably about 3, preferably about 2 or more preferably about 1 minute. In another embodiment, the time for (i) chromatographic separation, and (ii) evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample is from about 1 minute to about 30 minutes. Thus, in an embodiment, the time for ((i) chromatographic separation, and (ii) evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample is from about 1 minute to about 30 minutes, preferably from about 2 minutes to about 25 minutes, preferably from about 5 minutes to about 20 minutes, preferably from about 5 minutes to about 15 minutes, or more preferably from about 10 minutes to about 15 minutes.

[0067] In an embodiment, the time for (i) chromatographic separation, and (ii) evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample is from about 5 minutes to about 10 minutes. In another embodiment, the time for (i) chromatographic separation, and (ii) evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample is about 8 minutes. [0068] The present disclosure provides methods which comprise obtaining spectrometric data by "triple quadrupole mass spectrometry", "QQQ-MS", "TQ/MS" or "QqQ-MS".

[0069] QQQ-MS consists of three quadrupoles arranged in series with the first and third quadrupole acting as MSI and MS2 respectively and the CID taking place in the second quadrupole.

[0070] The term "spectrometric data" as used herein refers to a spectrum or spectra measured in either reflection or transmission. QQQ-MS spectra can be used to identify single chemical characteristics of a certain chemical group (e.g., cannabinoids) and more complex characteristics, such as the chemical, structural, sensory or functional qualities of different samples. As described elsewhere herein, QQQ-MS spectra can be used to obtain the mass- to-charge ratio (m/z) of different product ions and their associated collision energy (CE). Beneficially, QQQ-MS allows for multiplexed detection of multiple cannabinoids in a sample, also referred to as "multiple reaction monitoring" or "MRM".

[0071] In an embodiment, the method detects at least one cannabinoid selected from the group consisting of CBDA, THCA-A, CBDVA, CBGA, THCVA, CBNA, CBCA, CBDV, CBD, A8-THC, A9-THC, CBG, CBN, CBL, CBC and THCV.

[0072] By "at least one" means 1, 2, 3, 4, 5, 6, 7, and so on. In an embodiment, the detects at least 2, preferably 2, preferably 3, preferably 4, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10, preferably 11, preferably 12, preferably 13, preferably 14, preferably 15, or more preferably 16 cannabinoids selected from the group consisting of CBDA, THCA-A, CBDVA, CBGA, THCVA, CBNA, CBCA, CBDV, CBD, A8-THC, A9-THC, CBG, CBN, CBL, CBC and THCV.

[0073] In an embodiment, the method detects all of CBDA, THCA-A, CBDVA, CBGA, THCVA, CBNA, CBCA, CBDV, CBD, A8-THC, A9-THC, CBG, CBN, CBL, CBC and THCV.

[0074] The methods of the present disclosure further comprise obtaining absorbance wavelength data at a wavelength of from about 10 nm to about 400 nm (z. e. , within the ultra violet (UV) range; e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,

118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,

136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,

154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,

172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,

190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,

208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,

226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,

244, 245, 246, 247, 248, 249, 250, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,

261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 272, 272, 273, 274, 275, 276, 277, 278,

279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,

297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,

315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,

333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,

351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,

369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,

387, 388, 389, 390, 393, 392, 393, 394, 395, 396, 397, 398, 399, or 400 nm).

[0075] In an embodiment, the absorbance wavelength data is acquired at a wavelength of from about 200 nm to about 300 nm.

[0076] Absorbance wavelength data acquired in the UV range may be obtained using methods and apparatus which would be known to persons skilled in the art, illustrative examples of which include an UV-UV-VIS detector and an UV- diode array detector (DAD).

[0077] In an embodiment, the absorbance wavelength data is acquired by UV-diode array detector (DAD).

[0078] As described elsewhere herein, absorbance wavelength data can be used to determine the concentration of a detected cannabinoid in the sample.

[0079] The terms "level", "content", "concentration" and the like, are used interchangeably herein to describe an amount of the cannabinoid, and may be represented in absolute terms (e.g., mg/g, mg/mL, pg/mL, etc.) or in relative terms, such as a cannabinoid content relative to a reference value, ratio to any or all of the other compounds in the cannabis plant material or as a percentage of the amount (e.g., by weight) of any or all of the other compounds in the cannabis plant material.

[0080] In an embodiment, the concentration of the at least one cannabinoid in the sample is from about 0.001 pg/mL to about 250 pg/mL (e.g., 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,

67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90,

91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,

112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,

130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,

148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,

166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,

184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,

202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,

220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,

238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250 pg/mL).

[0081] Thus, in an embodiment, the concentration of the at least one cannabinoid in the sample is from about 0.001 pg/mL to about 250 pg/mL, preferably about 0.001, preferably about 0.002, preferably about 0.003, preferably about 0.004, preferably about 0.005, preferably about 0.006, preferably about 0.007, preferably about 0.008, preferably about 0.009, preferably about 0.01, preferably about 0.02, preferably about 0.03, preferably about 0.04, preferably about 0.05, preferably about 0.06, preferably about 0.07, preferably about 0.08, preferably about 0.09, preferably about 0.1, preferably about 0.2, preferably about 0.3, preferably about 0.4, preferably about 0.5, preferably about 0.6, preferably about 0.7, preferably about 0.8, preferably about 0.9, preferably about 1, preferably about 2, preferably about 3, preferably about 4, preferably about 5, preferably about 6, preferably about 7, preferably about 8, preferably about 9, preferably about 10, preferably about 11, preferably about 12, preferably about 13, preferably about 14, preferably about 15, preferably about 16, preferably about 17, preferably about 18, preferably about 19, preferably about 20, preferably about 21, preferably about 22, preferably about 23, preferably about 24, preferably about 25, preferably about 26, preferably about 27, preferably about 28, preferably about 29, preferably about 30, preferably about 31, preferably about 32, preferably about 33, preferably about 34, preferably about 35, preferably about 36, preferably about 37, preferably about 38, preferably about 39, preferably about 40, preferably about 41, preferably about 42, preferably about 43, preferably about 44, preferably about 45, preferably about 46, preferably about 47, preferably about 48, preferably about 49, preferably about 50, preferably about 51, preferably about 52, preferably about 53, preferably about 54, preferably about 55, preferably about 56, preferably about 57, preferably about 58, preferably about 59, preferably about 60, preferably about 61, preferably about 62, preferably about 63, preferably about 64, preferably about 65, preferably about 66, preferably about 67, preferably about 68, preferably about 69, preferably about 70, preferably about 71, preferably about 72, preferably about 73, preferably about 74, preferably about 75, preferably about 76, preferably about 77, preferably about 78, preferably about 79, preferably about 80, preferably about 81, preferably about 82, preferably about 83, preferably about 84, preferably about 85, preferably about 86, preferably about 87, preferably about 88, preferably about 89 , preferably about 90, preferably about 91, preferably about 92, preferably about 93, preferably about 94, preferably about 95, preferably about 96, preferably about 97, preferably about 98, preferably about 99, preferably about 100, preferably about 101, preferably about 102, preferably about 103, preferably about 104, preferably about 105, preferably about 106, preferably about 107, preferably about 108, preferably about 109, preferably about 110, preferably about 111, preferably about 112, preferably about 113, preferably about 114, preferably about 115, preferably about 116, preferably about 117, preferably about 118, preferably about 119, preferably about 120, preferably about 121, preferably about 122, preferably about 123, preferably about 124, preferably about 125, preferably about 126, preferably about 127, preferably about 128, preferably about 129, preferably about 130, preferably about 131, preferably about 132, preferably about 133, preferably about 134, preferably about 135, preferably about 136, preferably about 137, preferably about 138, preferably about 139, preferably about 140, preferably about 141, preferably about 142, preferably about 143, preferably about 144, preferably about 145, preferably about 146, preferably about 147, preferably about 148, preferably about 149, preferably about 150, preferably about 151, preferably about 152, preferably about 153, preferably about 154, preferably about 155, preferably about 156, preferably about 157, preferably about 158, preferably about 159, preferably about 160, preferably about 161, preferably about 162, preferably about 163, preferably about 164, preferably about 165, preferably about 166, preferably about 167, preferably about 168, preferably about 169, preferably about 170, preferably about 171, preferably about 172, preferably about 173, preferably about 174, preferably about 175, preferably about 176, preferably about 177, preferably about 178, preferably about 179, preferably about 180, preferably about 181, preferably about 182, preferably about 183, preferably about 184, preferably about 185, preferably about 186, preferably about 187, preferably about 188, preferably about 189, preferably about 190, preferably about 191, preferably about 192, preferably about 193, preferably about 194, preferably about 195, preferably about 196, preferably about 197, preferably about 198, preferably about 199, preferably about 200, preferably about 201, preferably about 202, preferably about 203, preferably about 204, preferably about 205, preferably about 206, preferably about 207, preferably about 208, preferably about 209, preferably about 210, preferably about 211, preferably about 212, preferably about 213, preferably about 214, preferably about 215, preferably about 216, preferably about 217, preferably about 218, preferably about 219, preferably about 220, preferably about 221, preferably about 222, preferably about 223, preferably about 224, preferably about 225, preferably about 226, preferably about 227, preferably about 228, preferably about 229, preferably about 230, preferably about 231, preferably about 232, preferably about 233, preferably about 234, preferably about 235, preferably about 236, preferably about 237, preferably about 238, preferably about 239, preferably about 240, preferably about 241, preferably about 242, preferably about 243, preferably about 244, preferably about 245, preferably about 246, preferably about 247, preferably about 248, preferably about 249 or more preferably about 250 pg/mL.

[0082] In an embodiment, the concentration of the cannabinoid in the sample is from about 0.01 pg/mL to about 100 pg/mL.

[0083] In an embodiment, the concentration of the cannabinoid in the sample is from about 0.01 pg/mL to about 10 pg/mL.

[0084] Limit of detection" or "LOD" refers to the lowest amount of analyte in the sample that can be detected, but not quantified.

[0085] In an embodiment, the LOD of the method is from about 0.01 pg/mL to about 0.3 pg/mL.

[0086] In another embodiment, the LOD is about 0.1 pg/mL. [0087] Accordingly, in an embodiment, the concentration of the cannabinoid in the sample is from about 0.1 pg/mL to about 100 pg/mL. In another embodiment, the concentration of the cannabinoid in the sample is from about 0.1 pg/mL to about 10 pg/mL.

[0088] Limit of quantification" or "LOQ" refers to lowest concentration of the analyte which can be determined quantitatively.

[0089] In an embodiment, the LOQ of the method is from about 0.01 pg/mL to about 1 pg/mL. In another embodiment, the LOQ is from about 0.08 pg/mL to about 0.71 pg/mL.

[0090] Accordingly, in an embodiment, the concentration of the cannabinoid in the sample is from about 0.08 pg/mL to about 100 pg/mL. In another embodiment, the concentration of the cannabinoid in the sample is from about 0.0.08 pg/mL to about 10 pg/mL.

[0091] It should be appreciated that the endogenous levels of one or more of the cannabinoids in the sample may be taken into consideration when calculating the relative cannabinoid concentration in samples. For example, major cannabinoids, such as THCA-A, A9-THC, CBDA and CBD are known to be present at higher concentrations in some cannabis plants as compared to minor cannabinoids, such as CBDVA, CBGA, THCVA, CBNA, CBCA, CBDV, A8-THC, CBG, CBN, CBL, CBC and THCV. The wavelength used for quantitation may be adjusted for better accuracy based on the abundance of the cannabinoids detected.

[0092] In an embodiment, the at least one cannabinoid is one or more or all of CBDA, THCA-A, CBD and A9-THC (i.e., a major cannabinoid). In accordance with this embodiment, the absorbance wavelength data is acquired at a wavelength of from about 250 nm to about 300 nm. In another embodiment, the absorbance wavelength data is acquired at a wavelength of about 280 nm.

[0093] In another embodiment, the at least one cannabinoid is one or more or all of CBDVA, CBGA, THCVA, CBNA, CBCA, CBDV, A8-THC, CBG, CBN, CBL, CBC and THCV (i.e., a minor cannabinoid). In accordance with this embodiment, the absorbance wavelength data is acquired at a wavelength of from about 200 nm to about 250 nm. In another embodiment, the absorbance wavelength data is acquired at a wavelength of about [0094] The term "reference value" as used herein refers to a characteristic (e.g., mass- to-charge ratio (m/z) of at least one product ion, the collision energy of at least one product ion, concentration, molecular weight and retention time) that may be used to compare the QQQ-MS spectra and/or absorbance wavelength data of the sample as described herein.

[0095] In an embodiment, the reference value will reflect characteristics of a Cannabis plant with a known chemotypic profde or type. The skilled person will appreciate that where a comparison is made between the Cannabis plants or Cannabis plant material derived from Cannabis plants (e.g., Cannabis plants with an unknown chemotypic profde) and those which have a known chemotypic profde or type, the comparison is performed with plants grown under essentially identical growing conditions, growth time, temperature, water and nutrient supply, etc., and for Cannabis plant material, samples, extracts or fractions obtained from such plants.

[0096] In an embodiment, the reference value is a known or predetermined characteristic that may be used to detect and quantify the concentration of a cannabinoid in a sample, e.g., the reference value may be a pre-determined mass-to-charge ratio (m/z) of at least one product ion, the collision energy of at least one product ion, concentration, molecular weight and retention time of a primary standard for one, or multiple cannabinoids. Such primary standards may be isolated cannabinoids derived from plant material or synthetic cannabinoids.

[0097] In an embodiment, the one or both of the presence and concentration of the at least one cannabinoid in the sample is determined by comparison to a reference value of the at least one cannabinoid, wherein the reference value is a predetermined value selected from the group consisting of (i) mass-to-charge ratio (m/z) of at least one product ion, (ii) collision energy of at least one product ion, (iii) concentration, (iv) molecular weight and (v) retention time.

[0098] In an embodiment, the at least one product ion is selected from the group consisting of a precursor ion, a quantifier ion and a qualifier ion.

[0099] The m/z of one or more or all of the precursor ion, a quantifier ion and a qualifier ion may be used to define transitions that are characteristic of a cannabinoid, which may be used to simultaneously detect multiple cannabinoids in a sample. Such transitions may be referred to as "MRM transitions". [0100] In an embodiment, the concentration of at least one cannabinoid in the sample is evaluated with a relative standard deviation (RSD) of from about 1% to about 10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%).

[0101] Thus, in an embodiment, the concentration of at least one cannabinoid in the sample is evaluated with a relative standard deviation of from about 1% to about 10%, preferably about 1%, preferably about 2%, preferably about 3%, preferably about 4%, preferably about 5%, preferably about 6%, preferably about 7%, preferably about 8%, preferably about 9%, or more preferably about 10%).

[0102] In another aspect, there is provided a method for evaluating one or both of the presence and concentration of at least one cannabinoid in a sample, the method comprising: a. obtaining absorbance wavelength data from the sample, wherein the absorbance wavelength data is obtained at a wavelength of from about 10 nm to about 400 nm; followed by b. obtaining spectrometric data from the sample, wherein the spectrometric data is obtained by triple quadrupole mass spectrometry (QQQ-MS); c. comparing the spectrometric data and the absorbance wavelength data obtained in step (a) and (b) with a reference value; and d. based on the comparison in (c), evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample.

Methods for monitoring a cannabis plant

[0103] The methods disclosed herein may suitably be used to monitor changes to the chemotypic profde of Cannabis plants, for example, during their growth cycle. This advantageously allows breeders, cultivators and the like to monitor their crop to ensure their plants retain / maintain the desired chemotype(s) or chemotypic profile(s) and, where necessary, remove and/or discard plants with an undesirable chemotype or chemotypic profile.

[0104] Thus, in another aspect of the present disclosure, there is provided a method for monitoring a Cannabis plant for a change to its chemotypic profile, the method comprising: a. evaluating, in accordance with the method described herein, one or both of the presence and concentration of at least one cannabinoid in a first sample from a Cannabis plant; b. evaluating, in accordance with the method described herein, one or both of the presence and concentration of at least one cannabinoid in a second sample from the Cannabis plant, wherein the second sample is taken from the Cannabis plant at a time point subsequent to the first sample; and c. comparing one or both of the presence and concentration of the at least one cannabinoid evaluated in step (a) and step (b) to determine whether there has been a change in the chemotypic profile of the Cannabis plant.

Methods for selecting growing conditions

[0105] The methods disclosed herein may also suitably be used to select growing conditions (e.g., frequency of watering, water quantity and/or quality; amount and/or type of fertilizer used; etc.) that give rise to or promote the development of Cannabis plants with a desired chemotypic profile. This advantageously allows breeders, cultivators and the like to optimize growing conditions to produce Cannabis plants with desired chemotype(s) or chemotypic profile(s).

[0106] Thus, in another aspect disclosed herein, there is provided a method for selecting growing conditions that favor the development of a cannabis plant with a desirable chemotypic profile, the method comprising: a. exposing a first Cannabis plant to a first set of selected growing conditions for a period of time; b. exposing a second Cannabis plant to a second set of selected growing conditions for a period of time, wherein the second set of selected growing conditions is different from the first set of selected growing conditions; c. optionally, repeating step (b) for a subsequent set of growing conditions that is different from the first and second sets of selected growing conditions; d. evaluating, in accordance with the method described herein, one or both of the presence and concentration of at least one cannabinoid in a sample from each of the Cannabis plants exposed to the set of selected growing conditions of steps (a)-(c); and e. selecting from the set of growing conditions of steps (a)-(c) one or more sets of selected growing conditions that favor the development of a Cannabis plant with a desirable chemotypic profile based on one or both of the presence and concentration of the at least one cannabinoid evaluated in step (d). [0107] The term "selecting" as used herein means the selection of a particular growing condition from one or more different growing conditions based on the chemotypic profile of the Cannabis plants that develop following exposure to each growing condition evaluated in accordance with the methods disclosed herein.

[0108] All publications mentioned in this specification are herein incorporated by reference. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0109] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the present disclosure without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0110] The present disclosure will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the disclosure.

EXAMPLES

Example 1 - General materials and methods

Reagents and standards

[oni] All reagents, water with 0.1% formic acid (mobile phase A), acetonitrile with 0.1% formic acid (mobile phase B) and methanol were HPLC grade and obtained from Fisher Scientific (Fair Lawn, NJ, USA). Primary standards for CBDA and THCA-A in acetonitrile, and CBDVA, CBDV, CBGA, CBG, CBD, THCV, THCVA, CBN, CBNA, A9- THC, A8-THC, CBL, CBC, and CBCA in methanol, at 1000 pg/mL, were commercially purchased from Novachem Pty Ltd. (Heidelberg West, Australia) as distributor for Cerilliant Corporation (Round Rock, Texas, USA).

[0112] Two separate mixed standards were prepared: one working standard at 100 pg/mL CBDA, CBD, CBN, A9-THC, CBC and THCA-A in methanol; and the other 100 pg/mL CBDVA, CBDV, CBGA, CBG, THCV, THCVA, CBNA, A8-THC, CBL and CBCA in methanol. The working standard concentrations were 0.001, 0.01, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 50 and 100 pg/mb prepared as serial dilutions from their respective 100 pg/mL working standard. All standards were stored at -80°C until required for analysis.

Sample preparation and extraction

[0113] Dried and ground Cannabis inflorescences were obtained from the Victorian Government Medicinal Cannabis Cultivation Facility. Samples used are referred to as Strain- 2, Strain-3, Strain-4, Strain-5 and Strain-6 (Table 2). Samples were placed in liquid nitrogen for 1 min and ground to a fine powder using a SPEX Sample Prep 2010 Geno/Grinder for 1 min at 1500 rpm. After grinding, 10 mg of each sample was weighed into 2 m Axygen microtube and extracted with 1 m of methanol, vortexed for 30 sec, sonicated for 5 min and centrifuged at 13,000 rpm for 5 min. The supernatant was transferred to a 2 mb amber HPEC vial and diluted 1 in 10 to ensure responses were within the quantitative range of the instrument.

Pre-extraction spike preparation

[0114] Recovery samples were prepared by spiking 10 mg of sample with 100 ph of 100 pg/mb standard and then prepared to 1 mb in methanol. A 1: 10 dilution of the extract was performed to achieve a concentration of 1 pg/mb. The high spike (HS) was prepared by adding 500 ph of 100 pg/mb standard and followed the steps mentioned previously to achieve concentrations of 5 pg/mb. Samples were sonicated, vortexed and transferred into 2 mb amber HPEC vials as previously described. A further 1 in 2 dilution was required to ensure all cannabinoids fit within the calibration curve, making the total dilution 1 in 20. Final spikes concentrations were 0.5 pg/mb (low spike, ES); and 2.5 pg/mb (high spike, HS).

Post-extraction spike preparation

[0115] To determine matrix effect, samples were extracted with methanol as detailed above, 100 ph of the extract was transferred to a 2 mb amber HPEC vial, spiked with 10 pb of 100 pg/mb standard and made to a final volume of 1 mb with methanol to achieve a concentration of 1 pg/mb. The HS used 50 ph of 100 pg/mb standard and prepared as described to achieve a concentration 5 pg/mb. A further 1 in 2 dilution was required to ensure all cannabinoids fit within the calibration curve, making the total dilution 1 in 20. Final spike concentrations were 0.5 pg/mb (ES); and 2.5 pg/mb (HS). Instrumentation parameters

[0116] Analysis was performed on an Agilent Triple Quadrupole Mass Spectrometer 6460 coupled with an Agilent High-performance liquid chromatography 1290 Infinity II LC System equipped with a degasser, binary pump, temperature controlled autosampler, column compartment and UV-DAD. Agilent Mass Hunter Data Acquisition Version 10 was used for instrument control. The column used was a Phenomenex Luna Omega Cis 150 x 2.1 mm x 1.6 pm column with an injection volume of 5 pL. The mobile phases consisted of (A) water with 0.1 % formic acid and (B) acetonitrile with 0.1% formic acid. Separation was achieved using the following gradient parameters: 0-5 min, 70% B; 5-7 min; 100% B, 7-7. 1 min 70% B followed by equilibration to initial conditions at a flow rate of 0.4 mL/min. The total runtime is less than 8 minutes with all compounds eluted in less than 5 minutes. The autosampler was maintained at 15 °C with the column temperature maintained at 40°C. The UV-DAD was set up to acquire spectra at wavelengths of 280 nm and 214 nm. The QQQ- MS parameters were as follows: the gas temperature was set at 300°C; the gas flow at 5 L/min; the nebulizer pressure at 45 psi; the sheath gas temperature at 250°C and the sheath gas flow at 11 L/min. The ion spray voltage was at 3500 V at the capillary and 500 V at the nozzle.

Data processing

[0117] Limit of detection (LOD) and limit of quantitation (LOQ) were determined using the LINEST function of excel and data from the Agilent Mass Hunter Quantitative Analysis where a signal ratio of 3.3: 1 from baseline was used for LOD and LOQ was determined using signal ratio of 10: 1 from baseline. R² values and equations were calculated using Agilent Mass Hunter Quantitative Analysis software, where the calibration curves’ fit origin was forced through zero.

Example 2 - Compound separation

[0118] The analysis was performed using LC-QQQ-MS, precursor, quantifier, qualifier ions and collision energies as detailed in Table 3, with all compounds eluted within 8 minutes. Baseline separation for all cannabinoids was achieved except for compounds which exhibited the same retention time and molecular weight, which were: A8-THC, A9-THC and CBC; CBL could be characterized and quantified using different product ions (Figure 1). Example 3 - Linearity, LOD and LOQ

[0119] The calibration curves consisted of 11 working standards prepared in methanol. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be approximately 0.1 pg/mL and 0.08 to 0.71 pg/mL. respectively. The R² values for each cannabinoid was 0.990 or better (Table 4). Due to the high abundance of CBDA and THCA- A in the samples, UV-DAD was used for quantitation, which has been reported to be accurate up to 250 pg/mL by Elkins et al. (2019, Journal of Chromatography B, 1109: 76-83).

Example 4 - Accuracy and precision

[0120] Accuracy and precision of the method was assessed by calculating the mean result of seven injections and determining the percent relative standard deviation (%RSD) of the repeat injections. This was performed on both standards and seven independent extracts of different Cannabis strains. Repeated injections of the 0.25, 1 and 5 pg/mL standards resulted in a %RSD < 2.0 for all analytes that are within the linear range of the method (Table 5). Mean cannabinoid content and %RSD for each Cannabis strain was determined with values ranging between 2 and 6% (Table 6). THCA was found to be highly abundant across all four strains, with CBDA also prominent in strains with an even ratio of THC and CBD (i.e., Strain-2 and Strain-3). CBGA, CBG, THCVA, CBNA, THC and CBCA were found in relatively low abundance in all four strains, with CBGA, THC and CBCA the most prominent. CBDVA, CBD, and CBC were only observed in Strain-2 and Strain-3 indicating a pathway link to CBDA. There were no cannabinoids exclusive to the high THC strains (Strain-4 and Strain-5). CBDV, THCV, CBN and CBL were not detected in any samples.

Example 5 - Recovery and matrix effect

[0121] Working standards were used to spike samples for pre- and post-spikes to evaluate recovery and matrix effect at 0.5 and 2.5 pg/mL concentrations. Matrix effect was defined as ion suppression. Recovery (RE) and matrix effect (ME) was calculated using the formula:

%ME or %RE = spiked sample - no spike sample * 100 spike level

[0122] Pre -extraction recovery values ranged from 73.0% to 126.2% across all cannabinoids at both spike concentrations in all cannabis strains (Table 7). The LS for CBD, THC and CBCA yielded higher than expected results ranging from 131.4% to 158.1%, this was not observed for the HS and is likely due to high endogenous levels in the samples. CBDA and THCA-A were quantified by UV with recovery values ranging from 71.2% to 101.0%. Recovery values for CBDA, determined by MS for Strain-4 and Strain-5, ranged from 105% to 108.8%. This confirms that at low levels the method is robust enough to accurately quantify samples with low concentrations of CBDA. A8-THC and THCA-A was spiked on a separate low THC strain to determine method efficiency. Recovery values ranged between 93.9% and 115.5% (Table 8).

[0123] Post-extraction values ranged from 72.5% to 120.6% across all cannabinoids at both spike concentrations (Table 9). Again, CBDA matrix effect was determined by MS for Strain-4 and Strain-5, with values ranging from 98.1 to 112.1%. CBDA and THCA-A were quantified using UV where values ranged from 85.9% to 102.4%. A8-THC and THCA-A was spiked in a separate low THC strain with post extraction values ranging from 96.7% to 115.2% (Table 10).

[0124] The extraction efficiency of the method is very good with most of the matrix effect and recovery samples within 10% RSD and returning values between 90 and 110% indicative of full extraction. CBDV, CBDA and THCV are a few examples of a full extraction across all spikes in all four Cannabis strains.

Discussion

[0125] These data enable a rapid, high-throughput method for the quantitation of major and minor cannabinoids in Cannabis plant material or extracts thereof. The high-throughput method described herein unexpectedly reduced the analysis time to eight minutes per sample, which represents a significant improvement on previous methods. This improved analysis method been fully validated for 16 different cannabinoids (i.e., CBDVA, CBDV, CBDA, CBGA, CBG, CBD, THCV, THCVA, CBN, CBNA, A9-THC, A8-THC, CBL, CBC, THCA-A and CBCA), and maintains consistent and acceptable RSD values across the linearity, limit of detection, limit of quantitation, accuracy, precision, spikes for matrix effect and recoveries.

[0126] As described elsewhere herein, one of the difficulties associated with the quantitation of cannabinoids is the structural similarities in the molecule structures of cannabinoids, which can make it difficult to distinguish between related, but different molecules. In particular, A8-THC and A9-THC co-elute due to their structural similarity and high endogenous levels of A9-THC as compared to A8-THC. Such limitations have been overcome using the method described herein, without the need for extended analysis time or by incorporating multistep gradients, which reduce throughput. Accordingly, the analysis method disclosed herein is a rapid and cost effective method for the high-throughput analysis of major and minor cannabinoids, which is particularly useful in commercial or research environments.

Table 1. Cannabinoids

Table 2. Description of cannabis strains

Table 3. Precursor, qualifier, quantifier ions and collision energy of each respective cannabinoid compound

Precursor (m/z)

Compound [M+H]⁺ Quantifier (m/z) CE(eV) Qualifier (m/z) CE(eV)

CBDVA 331.2 313.1 10 191 26

CBDV 287.2 123 18 165 35

CBDA 359.2 341 10 219 30

CBGA 361.2 219 15 343 22

CBG 317.3 193 10 123 35

CBD 315.2 193.1 18 123 30

THCV 287.2 123 30 165 18

THCVA 331.2 313.1 10 191 30

CBN 311.2 208 34 195.1 22

CBNA 355.2 337.1 10 235.1 30

THC 315.2 217.2 22 165.1 22

A8-THC 315.2 119 35 159.7 30

CBL 315.2 235.1 20 165 26

CBC 315.2 259 20 217 18

THCA-A 359.2 219 6 233 26

CBCA 359.2 219 15 233 22

Table 4. Linear concentration range, correlation coefficient, limit of detection and limit of quantification for cannabinoid standards test

Compound Concentration Range Equation R² LOD LOQ

(iig/mL) (pg/mL) (pg/mL)

CBDVA 0.001-5 y=14085*x 0.998 0.10 0.24

CBDV 0.001-5 y=786*x 0.999 0.05 0.10

CBDA 0.001-10 y=23687*x 0.999 0.05 0.16

CBDA* 1-100 y=12*x 0.997 0.10 0.24

CBGA 0.001-5 y=5237*x 0.995 0.10 0.08

CBG 0.001-5 y=2516*x 0.999 0.10 0.20

CBD 0.001-10 y=1841*x 0.995 0.12 0.35

THCV 0.001-2.5 y=1103*x 0.998 0.13 0.40

THCVA 0.001-2.5 y=12252*x 0.999 0.10 0.25

CBN 0.001-10 y=588*x 0.998 0.10 0.27

CBNA 0.001-5 y=8428*x 0.991 0.10 0.25

THC 0.001 - 10 y=182*x 0.990 0.23 0.71

A8-THC 0.001-10 y=88*x 0.995 0.10 0.25

CBL 0.001-5 y=3369*x 0.999 0.10 0.11

CBC 0.001-10 y=350*x 0.999 0.12 0.35

THCA-A 0.001 - 10 y=2079*x 0.999 0.02 0.07

THCA-A* 1-100 y=18*x 0.998 0.12 0.37

CBCA 0.001-10 y=1680*x 0.990 0.10 0.16

*Results calculated using UV-DAD set to 280 nm due to high endogenous levels in samples.

Table 5. Relative standard deviation (%RSD) values for 16 cannabinoid compounds in standard solution at 0.25, 1 and 5 pg/mL

Compound RT(min) 0.25 pg/mL 1 pg/mL 5 pg/mL

CBDVA 2.35 1.70 1.97 0.99

CBDV 2.49 1.96 2.06 2.00

CBDA 2.95 1.37 2.07 1.90

CBDA* 3.08 0.58 0.63 1.37

CBGA 3.08 2.07 1.99 1.36

CBG 3.15 1.83 1.68 0.71

CBD 3.24 2.65** 1.71 1.33

THCV 3.35 5.07** 2.09 1.51

THCVA 3.79 1.29 1.95 1.10

CBN 3.91 3.67** 0.87 1.07

CBNA 4.23 1.81** 1.78 0.78

THC 4.30 6.22** 1.86 2.02

A8-THC 4.30 26.5** 1.87 1.98

CBL 4.56 1.31 1.12 1.33

CBC 4.63 1.83** 1.03 1.38

THCA-A 4.73 1.89 2.08 1.54

THCA-A* 4.73 0.82** 0.86 1.47

CBCA 4.90 1.76 1.97 0.92

*Results calculated using UV-DAD set to 280 nm due to high endogenous levels in samples.

**Values marked are equal or below LOQ on the MS. RT, retention time (minutes).

Table 6. Concentration and RSD values for 16 cannabinoid compounds in different cannabis strains

Strain-2 Strain-3 Strain-4 Strain-5

Compound Cone. %RSD Cone. %RSD Cone. %RSD Cone. %RSD

CBDVA 0.35 3.27 0.20 4.30 <LOQ N/A <LOQ N/A

CBDV <LOQ N/A <LOQ N/A <LOQ N/A <LOQ N/A

CBDA* 77.2 4.30 101 2.53 <LOQ N/A <LOQ N/A

CBGA 2.21 2.03 2.41 3.71 3.08 2.48 3.26 4.88

CBG 0.56 4.94 0.66 3.40 1.88 4.07 1.60 5.39

CBD 2.16 3.78 2.95 3.05 <LOQ N/A <LOQ N/A

THCV <LOQ N/A <LOQ N/A <LOQ N/A <LOQ N/A

THCVA 0.34 3.70 0.19 3.86 0.91 3.51 0.90 5.92

CBN <LOQ N/A <LOQ N/A <LOQ N/A <LOQ N/A

CBNA 0.10 3.19 0.08 5.80 0.09 4.49 0.15 4.70

THC 2.36 4.22 3.06 3.44 5.62 4.23 5.42 4.94

CBL <LOQ N/A <LOQ N/A <LOQ N/A <LOQ N/A

CBC 0.20 4.43 0.30 2.34 <LOQ N/A <LOQ N/A

THCA-A* 44.2 4.22 49.6 2.65 107 4.31 119 5.39

CBCA 5.23 3.94 5.75 2.46 5.18 2.99 5.34 3.58

*Results calculated using UV-DAD set to 280 nm due to high endogenous levels in samples.

Concentration units (Cone.) are in pg/mL.

Table 7. Pre-extraction spikes for recovery values in different cannabis strains

Strain-2 Strain-3 Strain-4 Strain-5

Compound LS HS LS HS LS HS LS HS

CBDVA 88.0 99.7 94.0 97.6 99.9 102.8 101.1 101.2

CBDV 92.6 95.2 94.2 93.0 96.9 92.8 96.2 89.7

CBDA* ND 71.2 ND 91.5 84.8 91.8 88.5 90.4

CBDA N/A N/A N/A N/A 105.0 108.8 107.0 106.0

CBGA 81.7 89.1 88.7 78.8 106.4 81.5 103.7 67.9

CBG 88.4 85.1 97.5 82.0 103.5 78.3 98.8 76.8

CBD 153.7 95.0 158.1 92.0 106.4 104.4 110.8 99.4

THCV 96.5 91.4 91.6 89.5 94.3 91.7 95.1 90.1

THCVA 95.2 95.7 89.5 95.1 97.6 91.3 96.5 89.1

CBN 89.4 92.6 92.5 89.4 95.7 91.1 94.8 92.0

CBNA 90.1 93.0 77.2 89.2 76.1 ND 76.3 ND

THC 102.9 88.7 131.4 100.4 142.0 79.1 157.9 65.6

CBL 104.6 99.2 95.7 103.0 104.3 102.0 109.0 99.9

CBC 88.5 92.5 79.6 89.3 88.0 97.5 95.6 93.0

THCA-A* ND 80.9 ND 101.0 ND 91.7 ND 68.6

CBCA 80.8 83.8 95.1 81.9 103.8 97.8 121.6 100.2

*Results calculated using UV-DAD set to 280 nm due to high endogenous levels in samples.

LS, low spike. HS, high spike.

Table 8. Pre-extraction spikes for recovery for A8-THC and THCA-A in a low THC cannabis strain

Strain-6

Compound LS HS

A8-THC 97.0 93.9

THCA-A* 115.5 99.8

*Results calculated using UV-DAD set to 280 nm due to high endogenous levels in samples.

LS, low spike. HS, high spike. Table 9. Post-extraction spikes for matrix effect in different cannabis strains

Strain-2 Strain-3 Strain-4 Strain-5

Compound LS HS LS HS LS HS LS HS

CBDVA 84.1 96.4 92.3 93.5 97.5 92.9 89.0 92.4

CBDV 87.8 102.7 84.2 98.3 92.2 92.6 86.6 95.0

CBDA* ND 99.5 ND 107 100.8 96.0 85.9 93.3

CBDA N/A N/A N/A N/A 111.7 109.4 98.1 112.1

CBGA 73.4 88.7 82.8 77.7 125.9 89.7 99.8 79.1

CBG 78.6 95.6 88.9 92.5 123.1 93.7 100.3 92.4

CBD 100.9 91.6 102.1 82.3 85.6 98.1 83.1 102.9

THCV 91.4 103.3 94.6 99.1 99.8 99.8 92.4 96.1

THCVA 78.2 103.4 87.1 102.2 94 97.9 87.4 96.7

CBN 84.6 96.4 76.1 98.7 92.1 95.6 82.5 95.2

CBNA 112 91.7 98 85.7 85.4 70.6 90.8 79.6

THC 98.9 86.7 81.6 98.3 78.1 72.5 97.6 89.9

CBL 87.8 94.1 89.3 90.6 99.9 91.7 87.2 87.3

CBC 82.1 96.1 101.2 107.8 109.2 108.4 101.3 105.6

THCA-A* ND 99.8 ND 100.2 ND 271 ND 102.4

CBCA 85.1 88.4 89.3 79.1 120.6 104.0 97.3 90.0

*Results calculated using UV-DAD set to 280 nm due to high endogenous levels in samples.

LS, low spike. HS, high spike.

Table 10. Post-extraction spikes for matrix effect for A8-THC and THCA-A in a low

THC cannabis strain

Strain-6

Compound LS HS

A8-THC 115.2 104.6

THCA-A* 108.1 96.7

*Results calculated using UV-DAD set to 280 nm due to high endogenous levels in samples.

LS, low spike. HS, high spike.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for evaluating one or both of the presence and concentration of at least one cannabinoid in a sample, the method comprising: a. obtaining absorbance wavelength data from the sample, wherein the absorbance wavelength data is obtained at a wavelength of from about 10 nm to about 400 nm; b. obtaining spectrometric data from the sample, wherein the spectrometric data is obtained by triple quadrupole mass spectrometry (QQQ-MS); c. comparing the spectrometric data and the absorbance wavelength data obtained in step (a) and (b) with a reference value; and d. based on the comparison in (c), evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample.

2. The method of claim 1, wherein the sample is derived from Cannabis plant material.

3. The method of claim 2, wherein the cannabis plant material is from a female Cannabis plant.

4. The method of claim 2 or claim 3, wherein the Cannabis plant material is an inflorescence.

5. The method of any one of claims 2 to 4, wherein the sample is an extract from the Cannabis plant material.

6. The method of claim 5, wherein the sample is extracted from the Cannabis plant material using a solvent selected from the group consisting of methanol, ethanol, acetone and acetonitrile.

7. The method of claim 6, wherein the solvent is methanol.

8. The method of any one of claims 2 to 7, further comprising classifying the Cannabis plant material into Type I, Type II or Type III Cannabis plant material based on one or both of the presence and concentration of the at least one cannabinoid in the sample.

9. The method of any one of claims 1 to 8, wherein the sample is obtained by chromatographic separation. The method of claim 9, wherein the time for (i) chromatographic separation, and (ii) evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample is less than about 15 minutes. The method of claim 10, wherein the time for (i) chromatographic separation, and (ii) evaluating one or both of the presence and concentration of the at least one cannabinoid in the sample is from about 5 minutes to about 10 minutes. The method of any one of claims 1 to 11, wherein the at least one cannabinoid is selected from the group consisting of CBDA, THCA-A, CBDVA, CBGA, THCVA, CBNA, CBCA, CBDV, CBD, A8-THC, A9-THC, CBG, CBN, CBL, CBC and THCV. The method of claim 12, wherein the at least one cannabinoid comprises CBDA, THCA-A, CBDVA, CBGA, THCVA, CBNA, CBCA, CBDV, CBD, A8-THC, A9- THC, CBG, CBN, CBL, CBC and THCV. The method of any one of claims 1 to 13, wherein the concentration of the at least one cannabinoid in the sample is from about 0.001 pg/mL to about 250 pg/mL. The method of any one of claims 1 to 14, wherein the absorbance wavelength data is acquired at a wavelength of from about 200 nm to about 300 nm. The method of claim 1, wherein the at least one cannabinoid is selected from the group consisting of CBDA, THCA-A, CBD and A9-THC. The method of claim 16, wherein the concentration of the at least one cannabinoid in the sample is from about 0.01 pg/mL to about 100 pg/mL. The method of claim 16 or claim 17, wherein the absorbance wavelength data is obtained at a wavelength of from about 250 nm to about 300 nm. The method of claim 18, wherein the absorbance wavelength data is obtained at a wavelength of about 280 nm. The method of claim 1, wherein the at least one cannabinoid is selected from the group consisting of CBDVA, CBGA, THCVA, CBNA, CBCA, CBDV, A8-THC, CBG, CBN, CBL, CBC and THCV. The method of claim 20, wherein the concentration of the at least one cannabinoid in the sample is from about 0.01 pg/mL to about 10 pg/mL. The method of claim 20 or claim 21, wherein the absorbance wavelength data is obtained at a wavelength of from about 200 nm to about 250 nm. The method of claim 22, wherein the absorbance wavelength data is obtained at a wavelength of about 214 nm. The method of any one of claims 1 to 23, wherein one or both of the presence and concentration of two or more closely-related cannabinoids is evaluated. The method of any one of claims 1 to 24, wherein the one or both of the presence and concentration of the at least one cannabinoid in the sample is determined by comparison to a reference value of the at least one cannabinoid, wherein the reference value is a predetermined value selected from the group consisting of (i) mass-to- charge ratio (m/z) of at least one product ion, (ii) collision energy of at least one product ion, (iii) concentration, (iv) molecular weight and (v) retention time. The method of claim 25, wherein the at least one product ion is selected from the group consisting of a precursor ion, a quantifier ion and a qualifier ion. The method of claim 25 or claim 26, wherein the concentration of the at least one cannabinoid in the sample is evaluated with a relative standard deviation of from about 1% to about 10%. A method for monitoring a Cannabis plant for a change to its chemotypic profile, the method comprising: a. evaluating, in accordance with the method of any one of claims 1 to 27, one or both of the presence and concentration of at least one cannabinoid in a first sample from a Cannabis plant; b. evaluating, in accordance with the method of any one of claims 1 to 27, one or both of the presence and concentration of at least one cannabinoid in a second sample from the Cannabis plant, wherein the second sample is taken from the Cannabis plant at a time point subsequent to the first sample; and c. comparing one or both of the presence and concentration of the at least one cannabinoid evaluated in step (a) and step (b) to determine whether there has been a change in the chemotypic profde of the Cannabis plant. A method for selecting growing conditions that favor the development of a Cannabis plant with a desirable chemotypic profde, the method comprising: a. exposing a first Cannabis plant to a first set of selected growing conditions for a period of time; b. exposing a second Cannabis plant to a second set of selected growing conditions for a period of time, wherein the second set of selected growing conditions is different from the first set of selected growing conditions; c. optionally, repeating step (b) for a subsequent set of growing conditions that is different from the first and second sets of selected growing conditions; d. evaluating, in accordance with the method of any one of claims 1 to 27, one or both of the presence and concentration of at least one cannabinoid in a sample from each of the Cannabis plants exposed to the set of selected growing conditions of steps (a)-(c); and e. selecting from the set of growing conditions of steps (a)-(c) one or more sets of selected growing conditions that favor the development of a Cannabis plant with a desirable chemotypic profile based on one or both of the presence and concentration of the at least one cannabinoid evaluated in step (d).