WO2019090137A1 - Methods, kits and systems for sample homogenization and analysis - Google Patents

Abstract

The disclosure relates to methods, systems and kits for homogenizing samples and extracting analytes of interest from the homogenate, including, performing analytical measurements of the composition and/or the content of the analytes in the sample and fingerprinting the samples based on such measurements.

Description

METHODS, KITS AND SYSTEMS FOR SAMPLE HOMOGENIZATION

AND ANALYSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Patent Application No. 62/581,334, filed on November 3, 2017, the entire contents of which are incorporated herein by reference.

BACKGROUND

[0002] Sample homogenization attempts to bring a sample to a state where all fractions of the sample have equal composition. Homogenization is an important step in analytical testing at least because by removing sample matrices, the shielded analytes are made accessible to the probes and/or instruments that are employed in their detection. Additionally, by spreading the analytes uniformly throughout the sample, homogenization aids in the efficient extraction of analytes in analytical procedures that involve analyte extraction prior to detection. See, Rhode et al , BIOMED Research International, Article ID 145437, 2015.

[0003] Several methods of homogenization exist, e.g. , mechanical disruption using rotors, liquid homogenization (e.g., Potter-Elvehjem homogenizer, French presses, and the dounce homogenizer), sonication, manual grinding, etc. However, the cryogenic method of homogenization has been found to be both efficient and precise (evaluated using repeatability precision, relative standard deviation (RSD) and homogeneity factor (H-factor)). See, Krejcova et al , Food Chemistry, 109, 848-854, 2008. Cryogenic grinding is any method which involves use of freezing the sample to aid in homogenization. The cold temperature is also useful in preventing degradation of biological specimen due to heat that is built up during the grinding process. Cryogenic mills, which are used in this process, are well known in the art and are used in a variety of scientific applications, e.g. , DNA analysis, testing food- borne pathogens, etc. Such cryogenic mills typically operate rapid freezing in a cryogen (e.g. , liquid N₂, liquid He, liquid Ar, liquid O2, or the like), followed by mechanical grinding to generate a pulverized sample, which is then utilized in scientific analyses.

[0004] The recent push for the legalization of recreational and/or medical marijuana has led to the development of high throughput assay techniques for analyzing the different constituents of Cannabis. The information obtained from these assay procedures are used in chemical testing, developing new cultivars, and also fingerprinting for new, hybrid Cannabis varieties. See, Giese et al, Journal of AOAC International, 98, 6, 2015. Use of high throughput assays in the analysis of metabolites produced by animal tissues is discussed in Romisch-Margl et al. (Metabolomics 8, 133-142, 2012). However, in contrast to livestock metabolomics, analytical screening of Cannabis is complex as over 545 constituents have been identified in Cannabis, including a host of cannabinoids and terpenoids. See, Brenneisen, R. in Marijuana and the Cannabinoids, M.A. ElSohly (Ed.), Humana Press, Totowa, NJ, pp 137-156, 2007. Additionally, high throughput homogenizers that are currently used for Cannabis assays do not provide the requisite level of precision for drug testing as repeatability precision (RSD) values of these conventional homogenizers are lower than the acceptable predicted reproducibility standard deviation (PRSD). Moreover, interday precision of the conventional homogenizers is usually not satisfactory for the testing of volatile monoterpenes present in Cannabis. See, Giese et al , supra.

[0005] There is therefore a need for improved methods and systems for processing of biological samples, such as Cannabis, prior to conducting analytical assays for identifying the composition and accurately measuring the levels of various volatile and non-volatile components contained in such samples.

SUMMARY

[0006] The present disclosure provides methods for homogenizing samples by employing cryogenic grinding and high throughput homogenization. Although the two steps can be practiced in any order, preferably, the method involves use of cryogenic grinding followed by high throughput homogenization. The instant methods are advantageous over existing methods, e.g. , stand-alone high throughput homogenization, at least because they allow for enhanced recovery of analytes whose concentrations in the biological samples are in trace amounts (e.g. , part-per-million or even part-per-billion). The instant methods also provide for improved selectivity, better accuracy, and higher repeatability precision (RSD) compared to stand-alone homogenization methods.

[0007] The instant disclosure also relates to devices and systems for homogenizing samples. Ideally, the devices and systems are adapted for high throughput assays. The devices and systems allow sample disruption and sample homogenization steps to be carried out in a single vessel. This minimizes the risk of sample cross-contamination and/or loss of sample during the individual steps of mechanical grinding and homogenization. Moreover, the devices and the systems of the disclosure are also compatible with other auxiliary methods for the extraction, purification and/or analysis of the analyte from the homogenized sample. For example, the homogenized sample may be subjected to lysis solutions, extraction solvents and/or purification reagents. As illustrated in detail in the non-limiting Examples, the components of the disclosed homogenization system are compatible with reagents for a first extraction comprising, e.g., liquid-liquid extraction with acetonitrile and water and/or a second extraction comprising, e.g. , dispersive solid-phase extraction (dSPE) or cartridge solid phase extraction (cSPE).

[0008] In an embodiment, the disclosure relates to methods for homogenizing a sample, comprising, (a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids; (b) adding a cryogenic liquid to the container whereby the sample is frozen; (c) allowing the cryogenic liquid to completely vaporize; and (d) agitating the container to produce a homogenate. Preferably, steps (b) through (d) are repeated one or more times to achieve a homogenate with the desired degree of uniformity and/or to maintain the freezing temperature. Especially, the method is performed in a single container.

[0009] In some embodiments, the disclosure relates to methods for homogenizing a plant sample or a product thereof, comprising, (a) providing a container that includes a plant sample or a product thereof to be homogenized and two or more physical homogenization aids; (b) adding a cryogenic liquid to the container whereby the sample is frozen; (c) allowing the cryogenic liquid to completely vaporize; and (d) agitating the container to produce a homogenate. In one particular embodiment, the plant sample comprises a Cannabis sample. In another embodiment, the product comprises marijuana-infused product (MIP). Preferably, steps (b) through (d) are repeated one or more times to achieve a homogenate with the desired degree of uniformity and/or to maintain the freezing temperature. Especially, the method is performed in a single container.

[0010] In some embodiments, the disclosure relates to methods for homogenizing a sample, e.g., a plant sample (e.g. , Cannabis) or a product thereof (e.g. , MIP), comprising, (a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids; (b) adding a cryogenic liquid to the container whereby the sample is frozen to a temperature of less than about -80^°C; (c) allowing the cryogenic liquid to completely vaporize; and (d) agitating the container to produce a homogenate. In one particular embodiment, the cryogenic liquid is liquid nitrogen, liquid helium, liquid argon, liquid oxygen or a mixture thereof. Preferably, steps (b) through (d) are repeated one or more times to achieve a homogenate with the desired degree of uniformity and/or to maintain the temperature below about -80^° C. Especially, the method is performed in a single container.

[0011] In some embodiments, the disclosure relates to methods for homogenizing a sample, e.g., a plant sample (e.g. , Cannabis) or a product thereof (e.g. , MIP), comprising, (a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids; (b) adding a cryogenic liquid to the container whereby the sample is frozen, e.g. , to a temperature of less than about -80^°C; (c) allowing the cryogenic liquid to completely vaporize; and (d) agitating the container by vortexing to produce a homogenate. Preferably, steps (b) through (d) are repeated one or more times to achieve a homogenate with the desired degree of uniformity and/or to maintain the temperature below about -80^°C. Especially, the method is performed in a single container.

[0012] In some embodiments, the disclosure relates to methods for homogenizing a sample, e.g., a plant sample (e.g. , Cannabis) or a product thereof (e.g. , MIP), comprising, (a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids comprising beads or bearings; (b) adding a cryogenic liquid to the container whereby the sample is frozen, e.g. , to a temperature of less than about -80^° C; (c) allowing the cryogenic liquid to completely vaporize; and (d) agitating the container to produce a homogenate. Preferably, steps (b) through (d) are repeated one or more times to achieve a homogenate with the desired degree of uniformity and/or to maintain the temperature below about -80^° C. Especially, the method is performed in a single container.

[0013] In some embodiments, the disclosure relates to methods for homogenizing a sample, e.g., a plant sample (e.g. , Cannabis) or a product thereof (e.g. , MIP), comprising, (a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids comprising beads or bearings; (b) adding a cryogenic liquid to the container whereby the sample is frozen, e.g. , to a temperature of less than about -80^° C; (c) allowing the cryogenic liquid to completely vaporize; (d) agitating the container by vortexing to produce a homogenate; and (e) centrifuging the resulting homogenate in the tube that contains the physical homogenization aids. Preferably, steps (b) through (d) are repeated one or more times to achieve a homogenate with the desired degree of uniformity and/or to maintain the temperature below about -80^° C. Especially, the method is performed in a single container.

[0014] In some embodiments, the disclosure relates to methods for homogenizing a sample, e.g., a plant sample (e.g. , Cannabis) or a product thereof (e.g. , MIP), comprising, (a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids comprising beads or bearings; (b) adding a cryogenic liquid to the container whereby the sample is frozen, e.g. , to a temperature of less than about -80^°C; (c)(1) allowing the cryogenic liquid to completely vaporize; (c)(2) optionally digesting the frozen sample without removal of homogenization aids; (d)(1) agitating the container to produce a homogenate; and (d)(2) optionally digesting the homogenate. Preferably, at least one digestion step (c)(2) or (d)(2) are performed. In some embodiments, the digestion step is not performed. In some embodiments, the method comprises performing at least one digestion step and further comprises (e) centrifuging the resulting digested homogenate in the tube that contains the physical homogenization aids.

[0015] In some embodiments, the disclosure relates to methods for homogenizing a sample, e.g., a plant sample (e.g. , Cannabis) or a product thereof (e.g. , MIP), comprising, (a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids comprising beads or bearings; (b) adding a cryogenic liquid to the container whereby the sample is frozen, e.g. , to a temperature of less than about -80^° C; (c) allowing the cryogenic liquid to completely vaporize; (d) agitating the container to produce a homogenate; (e) centrifuging the resulting homogenate in the tube that contains the physical homogenization aids; and (f) extracting the homogenate. In some embodiments, the method comprises performing at least one extraction step and further includes extraction step is performed using an extractant, e.g., an organic liquid, an aqueous liquid or a mixture thereof. In an embodiment, the extractant comprises acetonitrile. Preferably, the extraction step is performed in the presence of salts, e.g., magnesium sulfate, and/or buffering agents, e.g. , citrate. In some embodiments, the method further includes (g) performing solid phase extraction (SPE) on a crude extract to produce a clean extract. Especially, the SPE comprises mixing the extractant with an SPE sorbent which is CI 8, a primary -secondary amine, graphitized carbon or a variant thereof. Preferably, the SPE comprises dispersive SPE. [0016] In some embodiments, the disclosure relates to methods for extracting an analyte in a sample, e.g. , a plant sample (e.g. , Cannabis) or a product thereof (e.g., MIP), comprising, (a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids comprising beads or bearings; (b) adding a cryogenic liquid to the container whereby the sample is frozen, e.g. , to a temperature of less than about -80^° C; (c) allowing the cryogenic liquid to completely vaporize; (d) agitating the container to produce a homogenate; (e) centrifuging the resulting homogenate in the tube that contains the physical homogenization aids; and (f) extracting the analyte from the homogenate, e.g. , with an extractant comprising acetonitrile. Preferably, the extraction step is performed in the presence of salts e.g., magnesium sulfate, and/or buffering agents, e.g., citrate, to induce liquid phase separation and stabilize acid and base labile groups in the analyte. In some embodiments, the method further includes (g) performing SPE on a crude extract to produce a clean extract. Especially, the SPE comprises mixing the extractant with an SPE sorbent which is CI 8, a primary-secondary amine, graphitized carbon or a variant thereof. Preferably, the SPE comprises dispersive SPE.

[0017] In some embodiments, the disclosure relates to methods for extracting an analyte in a sample, e.g. , a plant sample (e.g. , Cannabis) or a product thereof (e.g., MIP), comprising, (a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids comprising beads or bearings; (b) adding a cryogenic liquid to the container whereby the sample is frozen, e.g. , to a temperature of less than about -80^° C; (c) allowing the cryogenic liquid to completely vaporize; (d) agitating the container to produce a homogenate; (e) centrifuging the resulting homogenate in the tube that contains the physical homogenization aids; (f) extracting the analyte from the homogenate, e.g. , with an extractant comprising acetonitrile and salts e.g. , magnesium sulfate, and/or buffering agents, e.g. , citrate; (g) optionally performing SPE, e.g. , dispersive or cartridge SPE, on a crude extract to produce a clean extract; and (h) further optionally filtering the clean extract, e.g., through a PVDF membrane.

[0018] In some embodiments, the disclosure relates to methods for analyzing an analyte in a sample, e.g. , a plant sample (e.g. , Cannabis) or a product thereof (e.g., MIP), comprising, (a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids comprising beads or bearings; (b) adding a cryogenic liquid to the container whereby the sample is frozen, e.g. , to a temperature of less than about -80^° C; (c) allowing the cryogenic liquid to completely vaporize; (d) agitating the container to produce a homogenate; (e) centrifuging the resulting homogenate in the tube that contains the physical homogenization aids; (f) extracting the analyte from the homogenate, e.g. , with an extractant comprising acetonitrile and salts e.g. , magnesium sulfate, and/or buffering agents, e.g. , citrate; (g) optionally performing SPE, e.g. , dispersive SPE, on a crude extract to produce a clean extract containing the analyte; (h) further optionally filtering the clean extract containing the analyte, e.g., through a PVDF membrane; and (i) analyzing the analyte, e.g. , via HPLC, GC, LC-MS/MS or GC -MS/MS. Particularly, the analysis comprises detection and optionally quantification of the analyte. Especially, the method provides improved recovery in less time of the analyte from the sample or improved repeatability precision (RSD) compared to a stand-alone high throughput method.

[0019] In some embodiments, the disclosure relates to methods for analyzing an analyte comprising a cannabinoid or a terpene/terpenoid in a sample comprising Cannabis or an extract thereof, comprising, (a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids comprising beads or bearings; (b) adding a cryogenic liquid to the container whereby the sample is frozen, e.g. , to a temperature of less than about -80^°C; (c) allowing the cryogenic liquid to completely vaporize; (d) agitating the container to produce a homogenate; (e) centrifuging the resulting homogenate in the tube that contains the physical homogenization aids; (f) extracting the analyte from the homogenate, e.g. , with an extractant comprising acetonitrile and salts e.g. , magnesium sulfate, and/or buffering agents, e.g. , citrate; (g) optionally performing SPE, e.g. , dispersive SPE, on a crude extract to produce a clean extract containing the analyte; (h) further optionally filtering the clean extract containing the analyte, e.g. , through a PVDF membrane; and (i) analyzing the analyte, e.g., via HPLC, GC, LC-MS/MS or GC-MS/MS. Particularly, the method provides improved recovery of the analyte from the sample or improved RSD compared to a stand-alone high throughput method.

[0020] In some embodiments, the disclosure relates to use of a system for homogenizing and extracting an analyte from a sample, e.g. , a plant sample (e.g. , Cannabis) or a product thereof (e.g. , MIP), the system comprising (a) a container and two or more mechanical homogenization aids for homogenizing a sample; (b) a cryogenic liquid for freezing the sample; (c) an agitator for agitating the frozen sample to generate a homogenate; (d) a centrifuge for separating a supernatant from the homogenate and (e) an extractant for extracting the analyte from the homogenate comprising a solvent, e.g., acetonitrile and salts e.g., magnesium sulfate, and/or buffering agents, e.g., citrate; optionally together with reagents for cleaning-up the extract comprising (f) SPE systems, e.g., dispersive SPE; and/or (g) filtration systems. Preferably, the system further includes a system for analyzing, e.g. , detecting or quantitating the analyte in the sample, comprising HPLC, GC, LC-MS/MS or GC-MS/MS.

[0021] In some embodiments, the disclosure provides tangible media comprising a fingerprint of a sample comprising a plurality of analytes, wherein the composition of the each analyte and the amount thereof is determined with the analytical method of the disclosure. Preferably, the fingerprint comprises one or more cannabinoids and one or more terpenes or terpenoids present in a Cannabis sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings/tables and the description below. Other features, objects, and advantages of the disclosure will be apparent from the drawings/tables and detailed description, and from the claims.

[0023] FIG. 1 shows a flowchart of a representative extraction/analytical method of the disclosure.

DETAILED DESCRIPTION

[0024] Various aspects now will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0025] Where a range of values is provided in this disclosure, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is also included. For example, if a range of 1 μΜ to 8 μΜ is stated, it is intended that 2 μΜ, 3 μΜ, 4 μΜ, 5 μΜ, 6 μΜ, and 7 μΜ are also expressly disclosed. [0026] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "amino acid" includes a single amino acid as well as two or more of the same or different amino acids; reference to an "excipient" includes a single excipient as well as two or more of the same or different excipients, and the like.

[0027] The word "about" means a range of plus or minus 10% of that value, e.g., "about 50" means 45 to 55, "about 25,000" means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example in a list of numerical values such as "about 49, about 50, about 55, "about 50" means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g. , more than 49.5 to less than 52.5. Furthermore, the phrases "less than about" a value or "greater than about" a value should be understood in view of the definition of the term "about" provided herein.

[0028] The various embodiments of the disclosure are described in detail below: [0029] I. METHODS

[0030] This disclosure relates to improved systems and methods to homogenize a sample. The methods disclosed herein can provide homogenates in which 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or substantially all, of the analytes of interest in a sample are recovered from the sample matrix. The resulting homogenate can then, for example, be analyzed to identify and/or quantify its constituents with greater accuracy and precision than is achieved using homogenates prepared in other ways. The methods described herein are readily employed to produce a homogenate of a single sample or several or multiple samples concurrently, such as in a high throughput system.

[0031] The homogenization method comprises cryogenic grinding of a sample to produce a homogenate in the container in which the homogenate will be further processed, such as by digestions and/or extraction, for analysis. This provides several advantages over conventional methods, including eliminating the possibility of sample cross-contamination, reduced down time due to clean up of equipment to prevent cross-contamination, and reduced loss of and separation of components to the surfaces of the homogenization apparatus in the homogenization process. [0032] The method for homogenizing a sample, comprises, a) providing a container that includes a sample to be homogenized and two or more physical or mechanical homogenization aids (e.g., crushers); b) adding a cryogen to the container; c) allowing the cryogen to completely vaporize; and d) agitating the container to produce a homogenate. In the practice of the method, steps b) - d) can be performed once, or two or more time, to maintain the temperature of the sample at a desired level and/or to obtain a homogenate with the desired degree of uniformity.

[0033] The term "homogenize" is a term of art that is understood to refer to a process for making a sample uniform or homogenous in composition and/or properties.

[0034] The systems and methods disclosed herein can be used to homogenize any sample. Typically, samples homogenized using the method include, but are not limited to biological sample (e.g., a sample containing cells, tissues, biological fluid), food sample (e.g., raw or processed food for human, plant or animal use), environmental sample (e.g. , a sample obtained from sea, soil, air, matrix, or flora), synthetic sample (e.g., sample containing fermentation products of cells or a non-biological industrial process for the chemical synthesis or degradation of a compound), drugs (e.g. , tablets, capsules and other drug forms), an extract (e.g. , obtained by separation techniques), or a combination thereof.

[0035] The samples may be in any form, e.g. , solutions, emulsions, suspensions, gels, sols, colloids, and solids. The term "solution" refers to a liquid mixture in which the minor component (e.g., the analyte) is uniformly distributed within the major component (e.g., buffer). "Emulsions" refer to a fine dispersion of minute droplets of one liquid in another in which it is not soluble or miscible (e.g., oil and water). "Suspensions" refer to heterogeneous mixtures in which the solute particles do not dissolve but get suspended throughout the bulk of the medium. "Gels" refer to solid jelly-like material that can have properties ranging from soft and weak to hard and tough and are defined as a substantially dilute cross-linked system, which exhibits no flow. "Sols" refer to colloidal suspensions of very small solid particles in a continuous liquid medium.

[0036] The homogenization method disrupts the structure of the sample to release analytes from the sample matrix. For instance, wherein the sample is a biological sample, the cell membranes, cell walls, subcellular organelles, extracellular matrix, etc. , of the sample is disrupted by freezing and mechanical grinding to produce the homogenate. Typically, the macroscopic structure to the sample is destroyed allowing efficient DNA extraction.

[0037] The method disclosed herein preferably employs a plurality of physical homogenization aids, e.g., balls, bearings and/or beads, which act to aid in grinding the sample when the sample is agitated. Balls, bearings or beads for homogenizing the sample may be made of any suitable material, such as garnet, glass, ceramic, steel (stainless steel), polytetrafluoroethylene, silica, zirconium silicate, zirconium oxide and the like. Preferably, bearings and beads are made of stainless steel. The diameter of the bearings can be in the range of 2 mm to about 50 mm, e.g. , 5 mm to 20 mm, preferably 8 mm to 15 mm. Similarly, beads the diameter of beads can be in the range of 0.05 mm to 20 mm, e.g. , 0.5 mm to 10 mm, including 2 mm to 8 mm. However, any size bead which bests suits the container and sample size may be used. Beads adapted for homogenizing plant tissue can be purchased commercially, e.g. , BEADBEATER series manufactured by Biospec, Inc. (Bartlesville, OK, USA). Variations thereof, e.g. , dental mixers, or amalgamators, which have been adapted to mechanically beat samples, may also be used. Principally, agitation of the sample, for example by rapid shaking or oscillation of the container that contains the sample, is used to impart impact energy to the physical homogenization aids (e.g. , beads and/or bearings) and these strike the samples repeatedly to disrupt the macrostructures so that the analytes therein are released. Agitation can be carried out multiple times, e.g. , 2, 3, 4, 5, or more times, and for a sufficient amount of time to achieve the desired uniformity or consistency in the homogenate.

[0038] The methods and systems of the disclosure are useful for homogenizing heterogeneous samples which are characteristically non-uniform and may comprise a plurality of macroscopic substructures, e.g. , an organ comprising several tissues, each of which comprise a variety of cell types; a matrix with a variety of components dispersed throughout the matrix. For instance, a heterogeneous biological sample may include a mixture of materials having a wide variability in size (e.g. , a flower of Cannabis sp. is about 0.5 inches, while leaflets of Cannabis may be up to 12 inches long and 1 inch wide). The samples may also exhibit heterogeneity in chemical composition (e.g., inflorescences of Cannabis may have different compositions of cannabinoids and terpenoids in different parts thereof, such as, stems, stalks, bracts, buds, and flowers). [0039] In the practice of the method, the sample is frozen, and preferable is maintained at a cryogenic temperature (e.g. , below about -80°C) to facilitate homogenization, prevent degradation of analytes and/or prevent loss of volatile analytes. Cryogenic temperature is used herein to mean a temperature less than about -80°C, e.g. , less than about -100°C, less than -120°C, less than -140°C, less than -160°C, less than -180°C, less than -190°C or a lower temperature.

[0040] Preferably, the cryogenic homogenization method steps of the disclosure are carried out at a temperature below 100 K (-173°C) and more preferably at or below 77 K (-196°C). Preferably the cryogen used in the method is a liquid cryogen, such as liquid nitrogen, liquid helium, liquid argon, liquid oxygen or mixtures thereof.

[0041] In the practice of the method, a cryogen is added to the sample and allowed vaporize, e.g., via evaporation, boiling, or a combination thereof. Typically, the wait time for vaporization is short, e.g., about 15 mins, about 10 mins, about 5 mins, about 3 mins, about 1 min, or less.

[0042] The homogenization method of the disclosure comprises agitating the sample to produce a homogenate. Any suitable method of agitation can be used, but typically the samples are vortexed. Alternatively, sample can be agitated using, for example, a suitable shaker or other suitable device. The samples are agitated (e.g., vortexed) for a period of time sufficient to achieve a homogenate with the desired degree of uniformity. For example, a sample may be agitated (e.g., vortexed) for a duration of about 4 mins to about 40 mins, preferably for a duration of 10 mins to 20 minutes. Any suitable vortexer (e.g., VWR multi- tube vortexer Catalog Nos. 58816-115, 58816-116 or 58816-190) may be used to vortex the sample.

[0043] In some embodiments, the method of the disclosure includes use of a plurality of bearings or beads or a combination thereof. The sample may be subjected to pulses of mechanical treatment, such as one or more, e.g., two or more, 3 or more, four or more pulses, and 8 or less, 6 or less, including 4 or less pulses. The pulse of a mechanical treatment may have a duration in the range of 10 to 60 seconds, e.g. , 15 to 50 seconds, including 20 to 45 seconds. In some embodiments, the mechanical treatment comprises vortexing or pulsing. [0044] In some embodiments, the homogenization methods further comprises centrifugation. The centrifugation step can be used to precipitate insoluble material in the homogenate, e.g. , cell wall, membranes, chromatin and/or other debris. Wherein the homogenization is carried out in an organic solvent, e.g. , acetonitrile, methanol, or chloroform, the debris may include any molecule that is not typically soluble therein, e.g. , polypeptides, proteins, carbohydrates, glycoproteins or proteoglycans, etc. The centrifugation step may be carried out at 1000 rpm to 7000 rpm, preferably at 2500 to 5000 rpm, e.g. , 3000 rpm, 3250 rpm, 3500 rpm, 4000 rpm, or greater rpm. A variety of centrifugation systems may be employed, e.g., THERMO- FISHER Scientific Sorvall ST 40 centrifuge (Catalog # 75004525).

[0045] In some embodiments, after the homogenate is obtained from the sample, the various components in the homogenate may be further extracted. Accordingly, in some embodiments, the methods of the disclosure comprise producing a raw extract comprising an analyte. For example, the method may comprise extracting an analyte from the sample with a solution that comprises an extractant. Non-limiting examples of extractants include chemicals used in phase separation, for example, liquid-liquid interfaces, e.g., water-acetonitrile, water- methanol, water-chloroform, etc. The two components of the system may be used in any ratio between 1 :99 to 99: 1, e.g. , a 30:70 mixture, a 40:60 mixture, a 50:50 mixture, a 60:40 mixture, a 70:30 mixture, etc. Preferably, a 1 : 1 mixture of an aqueous phase (e.g. , water) an organic phase (e.g. , acetonitrile) is used. The ratio of acetonitrile (or other non-polar solvents) in the mixture can be lowered if the sample is not rich in oils. Conversely, where the sample is expected to be rich in polar compounds, e.g., terpenes/terpenoids or cannabinoids, the ratio of acetonitrile (or other polar solvent) can be increased.

[0046] In a specific embodiment, the extractant comprises a salt which is sodium chloride, sodium acetate, magnesium sulfate, sodium sulfate or a combination thereof. The preferred extractant comprises a polar aprotic solvent, e.g., acetone, ethyl acetate, or acetonitrile, dimethylformamide (DMF), tetrahydrofuran (THF), ethyl acetate, dimethyl sulfoxide (DMSO), nitromethane or propylene carbonate although a polar protic solvent such as, e.g. , methanol, ethanol, or n-butanol, may also be used. See, Rejczak et al, Open Chem., 13: 980-1010, 2015. Most preferably, the extractant comprises acetonitrile as it provides extraction of the broadest range of organic compounds without co-extraction of large amounts of lipophilic material and is highly compatible with GC/MS and LC/MS applications showing the least interference. [0047] The extractant may further comprise a buffer, e.g., an acetate buffer or a citrate buffer. Representative examples include, e.g. , trisodium citrate dehydrate, disodium hydrogen citrate sesquihydrate and sodium acetate (NaAc). See, Lehotay et al. , Journal of Chromatography A, 1217, 2548-2560, 2010. Preferably, the extractant comprises a citrate buffer.

[0048] In some embodiments, after the extractant is added to the homogenate, the resulting mixture is centrifuged. Any type of centrifuge machine, e.g., microcentrifuge or high-speed centrifuge, may be employed. For instance, the mixture comprising the homogenate and the extractant may be centrifuged in a THERMO-FISHER Scientific Sorvall ST 40 centrifuge for about 2 mins to about 10 mins, preferably for about 5 mins. The mixture may be centrifuged at a speed between 2000 rpm to about 6000 rpm, preferably between 3000 rpm to about 4000 rpm, e.g. , about 3700 rpm. Following centrifugation, the supernatant comprising the analyte is transferred to a fresh container and the pellet may be discarded. Optionally, the pellet may be processed to one or more rounds of homogenization, extraction and centrifugal separation steps outlined above.

[0049] Optionally, depending on the sample, the sample may be lysed with a lysis solution, although this step is not necessary with the high throughput method of the present disclosure. The lysis solution preferably comprises a buffer and optionally a reducing agent. Representative examples of plant lysis buffer include, e.g. , 50 mM TRIS-HCl, 20 mM EDTA, 1% SDS, pH 8.0. See, WO 1998/024929. The reducing agent preferably comprises β- mercaptoethanol. In another embodiment the lysis solution includes a chaotropic salt, non- ionic detergent (e.g. , non-ionic surfactant) and reducing agent. With preference said chaotropic salt is guanidine HC1. Preferably said non-ionic agent is selected from triethyleneglycol monolauryl ether, (octylplhenoxy)polyethoxyethanol, sorbitan monolaurate, T-octylphenoxyployethoxyethanol, or a combination thereof. Preferably the non-ionic detergent or combination thereof is in the range of 0.1-10%. With preference the reducing agent is 2-aminoethanethiol, tris-carboxyethylphosphine (TCEP), or β-mercaptoethanol.

[0050] In some embodiments, the lysis solution may contain one or more lytic enzymes. Representative examples of such enzymes include, e.g. , cellulase (Sigma- Aldrich catalog # C2605), pectinase (Sigma catalog # P2611) and/or pectolyase (Sigma catalog # P5431).

[0051] In some embodiments, the raw extract is cleaned up using sorbent trap. A variety of solid-phase extraction (SPE) reagents can be used as pre-concentrators and have been employed previously for Cannabis assays. For sorbent traps, the use of several pre- concentrating materials can be used to determine the effectiveness for the absorption of THC in the raw extract. These materials include, but are not limited to, organic polymers (e.g. , C- 18 polymer, a primary-secondary amine (PSA) polymer), carbon-based molecular sieves and graphitized carbon black (GCB). Preferably, the sorbent trap comprises a dispersive solid phase extraction (SPE). Typically, dispersive SPE comprises suspending a sorbent (e.g. , PSA or GCB) in the sample solution to trap interference from the solution, and the refined solution is analyzed for the analyte. Reagents used in dispersive SPE can be purchased commercially, e.g., Waters Catalog # 186008073 (comprising 1200 mg MgS0₄, 400 mg PSA and 400 mg C18 in 15 mL tube) or 186004834 (comprising 900 mg MgS0₄ & 150 mg PSA, C18, in 15mL tube) (Waters Corp., Milford, MA, USA).

[0052] After the sorbent is allowed to mix with the raw extract, the mixture may be centrifuged to pellet the sorbent and the supernatant may be filtered to remove any particulate matter. For example, the centrifugation step may be carried out at about 500 rpm to about 2000 rpm, preferably at about 1000 rpm for a period between 0.5 min and 3 mins, preferably for about 1 min. Any centrifugation device may be used, e.g., Sorvall ST 49 centrifuges (Thermo-Fisher). For filtration, any membrane filters may be employed, e.g. , filters having a porosity of about 0.1 μΜ to about 1 μΜ, e.g. , 0.1 μιη (Sigma Catalog # VVLP01300), 0.22 μΜ (Sigma # GVWP01300), 0.45 μιη (Sigma # HVLP01300), 0.65 μιη (Sigma # DVPP01300) or the like.

[0053] Purely as a representative example, in some embodiments, the extraction step comprises implementation of Quick Easy Cheap Effective Rugged Safe (QUECHERS) procedure. The original QUECHERS method has been described in Anastassiades et al. (J. O AC Int. 86(2): 412-31, 2003), the disclosure in which is incorporated herein by reference. QUECHERS generally involves two steps: the first step is the production of a raw extract, while the second step is a clean-up step to remove various contaminants. The first step involves mixing with agitation a sample with a solution comprising an extractant (e.g. , acetonitrile) and various salts (e.g. , magnesium sulfate) and optionally buffers (e.g. , sodium citrate) to produce the raw extract. The second step involves removing various contaminants, e.g., non-polar contaminants, from the raw extract by, e.g. , solid phase extraction (SPE) to generate a solution containing the analyte of interest. The analyte of interest can then be subsequently detected and quantified by, e.g. , gas chromatography and mass spectrometry (GC/MS) or liquid chromatography and mass spectrometry (LC/MS).

[0054] Alternately, the raw extract may be processed using cartridge solid phase extraction (cSPE) cleanup. The process is very similar to the dispersive SPE described above. An anhydrous salt, e.g. , MgSC>4, is added into an SPE cartridge (Sigma- Aldrich catalog # 57056) and rinsed with one cartridge-full of acetonitrile. The solvent is eluted until the cartridge is dry. A sample containing raw extract (e.g. , Cannabis extract) is added and the sample is allowed to gravity elute. Once the sample has eluted, "wash" eluent, e.g. , acetonitrile, is passed through the cartridge. Once the eluent has almost filled the tube, the eluent is added to the concentration vessel. Ideally, the extract is clear in color or a hint of a yellow/green tint compared to a blank. The eluent is dried in a standard dryer (e.g. , TURBOVAP II, BIOTAGE, and Charlotte, NC, USA) at about 35 °C to about 80 °C (preferably at about 50°C). The dried sample is then reconstituted with vehicle acetonitrile and the reconstituted sample may be analyzed via GC, LC, or HPLC, optionally together with tandem mass spectrometry (MS/MS).

[0055] The above-mentioned methods of homogenization, extraction and purification are preferably performed under 1 hour, particularly under 50 mins, and especially under 40 mins.

[0056] In some embodiments, the methods and/or assays of the disclosure may be multiplexed. The disclosure relates to multiplex methods for rapid fingerprinting of Cannabis containing samples based on markers that provide information on molecular relationships of one or more Cannabis species or varieties. As used herein, the term "multiplexing" refers to performance of simultaneous, multiple determinations in a single assay protocol and "multiplexed assay" refers to a process that is designed to implement such a capability. Preferably, the multiplexed assays of the disclosure are performed using gas chromatography or liquid chromatography coupled to tandem mass spectrometry (GC -MS/MS or LC- MS/MS). See, Citti et al , Phytochem Anal, Sep 15, 2017 (PMID: 28915313); Salomone et al , J Mass Spectrom., 47(5):604-10, 2012; Chu et al. J Anal Toxicol, 26(8):575-81, 2002 and Andrenyak et al. , JAnal Toxicol, 41(4):277-288, 2017.

[0057] In some embodiments, the fingerprint comprises information on the composition and abundance of 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or more analyte markers which can be analyzed using the methods described herein. Preferably, the fingerprint comprises at least one cannabinoid analyte marker and/or at least one terpene/terpenoid marker. The fingerprint can be used to label raw Cannabis as well as marijuana-infused products (MIP) or marijuana-infused foods (MIF).

[0058] The above-mentioned methods are used in detecting and/or quantitating analytes present in a variety of samples. As used herein, the term "sample" refers to any composition of matter containing an analyte of interest. In some embodiments, the sample is a biological sample, e.g. , sample containing cells or products derived therefrom (e.g. , carbohydrates, proteins, lipids, nucleic acids, metabolites, etc). In some embodiments, the sample is a plant cell or an animal cell or a tissue containing a plurality of said cells. Samples include, for example, primary cells, cultured cells, frozen cells and transformed cells (e.g. , E. coli or yeast transformed with a nucleic acid encoding hexanoyl Co A synthetase and/or olivetolic acid cyclase (OAC), which produces delta-9-tetrahydrocannbinol (THC)).

[0059] In some embodiments, the sample includes, for example, cells or a mixture thereof. In some embodiments, the sample comprises at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of a cell population of interest, e.g. , cells which produce an analyte of interest, such as, for example, THC.

[0060] In a particular embodiment, the biological sample is a plant tissue. As used herein, the term "plant tissue" refers to any part of a plant. Examples of plant organs include, e.g. , the leaf, stem, root, tuber, seed, branch, pubescence, nodule, leaf axil, flower, pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, bract, fruit, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen, and leaf sheath. The disclosure includes ovules and pollens of plants. As used herein when discussing plants, the term "ovule" refers to the female gametophyte, whereas the term "pollen" means the male gametophyte. Representative types of plant tissue include, e.g. , dermal tissue (for example, epidermis), vascular tissue (for example, xylem, phloem) or ground tissue (for example, collenchyma, parenchyma, sclerenchyma, fibers, sclerids, etc.).

[0061] In some embodiments, wherein the sample comprises a plant, the sample includes a plant part. As used herein, the term "plant part" refers to any part of a plant including but not limited to the embryo, shoot, root, stem, seed, stipule, leaf, petal, flower bud, flower, ovule, bract, trichome, branch, petiole, internode, bark, pubescence, tiller, rhizome, frond, blade, ovule, pollen, stamen, and the like. The two main parts of plants grown in some sort of media, such as soil or vermiculite, are often referred to as the "above-ground" part, also often referred to as the "shoots", and the "below-ground" part, also often referred to as the "roots." Both parts are included by this definition.

[0062] In some embodiments, the plant is a flowering plant such as Cannabis. Cannabis, more commonly known as marijuana, is a genus of flowering plants that includes at least three species, Cannabis sativa, Cannabis indica, and Cannabis ruderalis as determined by plant phenotypes and secondary metabolite profiles. In practice however, cannabis nomenclature is often used incorrectly or interchangeably. Cannabis literature can be found referring to all cannabis varieties as "sativas" or all cannabinoid producing plants as "indicas." Indeed the promiscuous crosses of indoor cannabis breeding programs have made it difficult to distinguish varieties, with most cannabis being sold in the United States having features of both sativa and indica species.

[0063] Wherein the sample comprises a Cannabis plant, the sample may comprise any part of the Cannabis plant, including, but not limited to, flower, buds, cola, trichomes, calyx, pistil, fan leaves, sugar leaves, seed, stem, node, or root). Plant part may also include certain extracts such as kief or hash which includes cannabis trichomes or glands. In some embodiments, the plant part may include inflorescences comprising, e.g., complete flower head of a plant including stems, stalks, bracts, and flowers. See, U.S. Pat. No. 9,095,554 for a detailed disclosure on the anatomical parts of Cannabis sativa.

[0064] The disclosure also relates non-biological sample such as, e.g. , marijuana-infused products. The term includes, e.g. , marijuana-infused food (MIF). As used herein, "marijuana-infused product" refers to edibles, beverages, botanicals, tinctures, Cannabis kitchens, that contain marijuana, which product is not intended to be used or consumed by smoking.

[0065] The methods and systems of the disclosure are useful in the extraction of analytes from one or more of the aforementioned samples. Non-limiting examples of analytes include the following genera of analytes: (a) cellular products (e.g. , Δ-9-THC, CBD, THCA, CBDA from Cannabis); (b) micro-toxins (e.g., cymoxanil and/or famoxadone); (c) pesticides (e.g. , bifenazate, bifenthrin, cyfluthrin, etoxazole, imazalil, imidacloprid, myclobutanil, spiromesifen, trifloxystrobin), (d) microorganisms (e.g. , bacteria (such as, gram-negative bacteria, coliform), yeast) including cellular macromolecules (e.g. , nucleic acids and/or proteins), (e) my co-toxins (e.g. , type A- trichothecenes or type B-trichothecenes), alternariol, zearalenone (e.g., a-zearalanol or 13-zearalanol), aflatoxins (e.g., Bl, B2, Gl, or G2) and ochratoxins (e.g. , ochratoxin A); and/or (f) metal contaminants (e.g. , arsenic, cadmium, lead, or mercury).

[0066] Particularly, the analyte is a compound or a molecule that is derived from a plant. For instance, wherein the biological sample is a Cannabis plant, the analyte may comprise one or more cannabinoids or one or more terpenes/terpenoids.

[0067] Preferably, the analyte is a terpeno-phenolic compound produced by Cannabis plants. Cannabinoids, terpenoids, and other compounds are secreted by glandular trichomes that occur most abundantly on the floral calyxes and bracts of female plants. As a drug it usually comes in the form of dried flower buds (marijuana), resin (hashish), or various extracts collectively known as hashish oil. There are at least 483 identifiable chemical constituents known to exist in the Cannabis plant (Brenneisen et al , Chemistry and Analysis of Phytocannabinoids and other Cannabis Constituents, In Marijuana and the Cannabinoids, ElSohly, Ed. Humana Press, Totowa, New Jersey, 2007) and at least 85 different cannabinoids have been isolated from the plant (El-Alfy et al , Pharmacology Biochemistry and Behavior 95 (4): 434-42, 2010). The two cannabinoids usually produced in greatest abundance are cannabidiol (CBD) and/or Δ⁹ -tetrahydrocannabinol (THC). THC is psychoactive while CBD is not. See, ElSohly, Ed. (supra).

[0068] In some embodiments, the analyte comprises one or more cannabinoids. Cannabinoids are the most studied group of secondary metabolites in cannabis and generally exist in two forms- in acid form and in neutral (decarboxylated) form. The acid form is designated by an "A" at the end of its acronym (e.g. , THCA). The phytocannabinoids are synthesized in the plant as acid forms, and while some decarboxylation does occur in the plant, it increases significantly post-harvest and the kinetics increase at high temperatures. See Sanchez et al , Plant Cell Physiol., 49(12): 1767-82, 2008. The neutral forms are usually consumed by humans. Decarboxylation is usually achieved by thorough drying of the plant material followed by heating it, often by combustion, vaporization, or heating or baking in an oven. Unless otherwise noted, references to cannabinoids in a plant include both the acidic and decarboxylated versions (e.g. , CBD and CBD A). [0069] In some embodiments, the analytes include cannabinoids of cannabis plants. Representative types of cannabinoid analytes include, but are not limited to, Δ⁹- Tetrahydrocannabinol (A⁹-THC), A⁸-Tetrahydrocannabinol (A⁸-THC), Cannabichromene (CBC), Cannabicyclol (CBL), Cannabidiol (CBD), Cannabielsoin (CBE), Cannabigerol (CBG), Cannabinidiol (CBND), Cannabinol (CBN), Cannabitriol (CBT), and their propyl homologs, including, but are not limited to cannabidivarin (CBDV), Δ⁹- Tetrahydrocannabivarin (THCV), cannabichromevarin (CBCV), and cannabigerovarin (CBGV). See Holley et al. (J. Pharm. Set , 64:892-894, 1975) and De Zeeuw et al. {Science, 175:778-779). Non-THC cannabinoids can be collectively referred to as "CBs", wherein CBs can be one of CBDV, CBGV, CBCV, CBD, CBC, CBE, CBG, CBN, CBND, and CBT cannabinoids, or a combination thereof.

[0070] In some embodiments, the analytes include cannabinoid acids (e.g. , cannabidiolic acid (CBDA)). When the herbal product is dried, stored, or heated, the acids decarboxylize gradually or completely into neutral forms (e.g. , CBDA→CBD).

[0071] In particular embodiments, the analyte comprises THC. Known as delta-9- tetrahydrocannabinol (A9-THC), THC is the principal psychoactive constituent (or cannabinoid) of the cannabis plant. In the cannabis plant, THC occurs mainly as tetrahydrocannabinolic acid (THCA, 2-COOH-THC). In other specific embodiments, the analyte comprises CBD. CBD is a cannabinoid found in cannabis which is produced through the same metabolic pathway as THC, until the last step, where CBDA synthase catalyzes the formation of CBD from its precursor. See Marks et al , Journal of Experimental Botany, 60 (13): 3715-3726, 2009. Many natural and/or synthetic variants of CBD and THC are found in marijuana-infused products (MIP), e.g. , food. In yet another embodiment, the analyte comprises CBG. CBG is a non-psychoactive cannabinoid found in the Cannabis genus of plants. In another embodiment, the analyte comprises cannabigerol. Cannabigerol is found in higher concentrations in hemp rather than in varieties of Cannabis cultivated for high THC content. In another embodiment, the analyte comprises CBN. CBN is a psychoactive substance cannabinoid found in Cannabis sativa and Cannabis indica/afghanica. It is also a metabolite of tetrahydrocannabinol (THC). In another embodiment, the analyte comprises CBC. CBC bears structural similarity to the other natural cannabinoids, including tetrahydrocannabinol, tetrahydrocannabivarin, cannabidiol, and cannabinol, among others. Evidence has suggested that it may play a role in the anti-inflammatory and anti-viral effects of cannabis, and may contribute to the overall analgesic effects of cannabis. In another embodiment, the analyte comprises cannabivarin or cannabivarol or CBV. Cannabivarin (CBV) is a non-psychoactive cannabinoid found in minor amounts in the hemp plant Cannabis sativa. It is an analog of cannabinol (CBN). CBV is an oxidation product of tetrahydrocannabivarin (THCV, THV). In some embodiments, the analyte comprises CBDV, which is a non-psychoactive cannabinoid found in Cannabis. CBDV is a homolog of cannabidiol (CBD). Plants with relatively high levels of CBDV have been reported in feral populations of C. indica from northwest India, and in hashish from Nepal. In some embodiments, the analyte comprises THCV (or THV), which is a homologue of tetrahydrocannabinol (THC). THCV is a terpeno-phenolic compound is found naturally in Cannabis, sometimes in significant amounts. In yet other embodiments, the analyte is cannabicyclol (CBL), which is a non-psychotomimetic cannabinoid found in Cannabis. CBL is a degradative product like cannabinol.

[0072] In some embodiments, the analyte is a terpene or a terpenoid. Terpenes are a large and diverse class of organic compounds, produced by a variety of plants. They are often strong smelling and thus may have had a protective function. Terpenes are derived biosynthetically from units of isoprene, which has the molecular formula C₅H₈. The basic molecular formulae of terpenes are multiples of that, (C₅H₈)_n where n is the number of linked isoprene units. The isoprene units may be linked together "head to tail" to form linear chains or they may be arranged to form rings. Non-limiting examples of terpenes include hemi terpenes, monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, sesquarterpenes, tetraterpenes, polyterpenes, and norisoprenoids. Terpenoids, also known as isoprenoids, are a large and diverse class of naturally occurring organic chemicals similar to terpenes, derived from five-carbon isoprene units assembled and modified in thousands of ways. Most are multicyclic structures that differ from one another not only in functional groups but also in their basic carbon skeletons. Plant terpenoids are used extensively for their aromatic qualities. They play a role in traditional herbal remedies and are under investigation for antibacterial, antineoplastic, and other pharmaceutical functions. Well-known terpenoids include citral, menthol, camphor, salvinorin A in the plant Salvia divinorum, and the cannabinoids found in Cannabis. Non-limiting examples of terpenoids include, Hemiterpenoids, 1 isoprene unit (5 carbons); Monoterpenoids, 2 isoprene units (IOC); Sesquiterpenoids, 3 isoprene units (15C); Diterpenoids, 4 isoprene units (20C) (e.g. ginkgolides); Sesterterpenoids, 5 isoprene units (25C); Triterpenoids, 6 isoprene units (30C) (e.g. sterols); Tetraterpenoids, 8 isoprene units (40C) (e.g. , carotenoids); and Polyterpenoid with a larger number of isoprene units. Terpenoids are mainly synthesized in two metabolic pathways: mevalonic acid pathway and MEP/DOXP pathway.

[0073] In some embodiments, the analytes include terpenes. Over 120 different terpenes are produced naturally by Cannabis (Russo et al , British Journal of Pharmacology, 163: 1344- 1364, 201 1). Within the context and verbiage of this document the terms "terpenoid" and "terpene" are used interchangeably. Cannabinoids are odorless, so terpenoids are responsible for the unique odor of cannabis, and each variety has a slightly different profile that can potentially be used as a tool for identification of different varieties or geographical origins of samples. Generally speaking, terpenes are considered to be pharmacologically relevant when present in concentrations of at least 0.05% in plant material (Hazekamp et al , Phytochemistry, 2058-73, 2010; Russo et al , British Journal of Pharmacology, 163: 1344- 1364, 201 1). Thus, although there are an estimated 120 different terpenes, only a few are produced at high enough levels to be detectable, and fewer still which are able to reach pharmacologically relevant levels.

[0074] In some embodiments, the analyte further comprises D-limonene. D-limonene is a monoterpenoid that is widely distributed in nature and often associated with citrus. In some embodiements, the analyte further comprises β-Myrcene is a monoterpenoid also found in cannabis, and has a variety of pharmacological effects. It is often associated with a sweet fruit like taste. D-Linalool is a monoterpenoid with very well-known anxiolytic effects. It is often associated with lavender, and frequented used in aromatherapy for its sedative impact. a-Pinene is a monoterpene common in nature, also with a plethora of effects on mammals and humans. It acts as an acetylcholinesterase inhibitor which aids memory and counteracts the short-term memory loss associated with A₉-THC intoxication. β-Caryophyllene is often the most predominant sesquiterpenoid in cannabis. It is less volatile than the monoterpenoids, thus it is found in higher concentrations in material that has been processed by heat to aid in decarboxylation. Caryophyllene oxide is another sesquiterpenoid found in cannabis, which has antifungal and anti-platelet aggregation properties. Nerolidol is a sesquiterpene that is often found in citrus peels that exhibits a range of interesting properties. It acts as a sedative, inhibits fungal growth, and has potent anti-malarial and anti-leishmanial activity. [0075] In some embodiments, the disclosure relates to a fingerprint comprising a plurality of terpenes and/or a plurality of cannabinoids. In some embodiments, the fingerprint includes anywhere between 2 to 30, preferably about 10-20, and especially about 17 of the most expressed terpenes: terpinolene, alpha phelladrene, beta ocimene, carene, limonene, gamma terpinene, alpha pinene, alpha terpinene, beta pinene, fenchol, camphene, alpha terpineol, alpha humulene, beta caryophyllene, linalool, cary oxide, and myrcene. In some embodiments, the fingerprint includes anywhere between 2 to 30, preferably about 10-20, and especially about 17 of the most expressed cannabinoids, e.g., A⁹-THC, A⁸-THC, CBDV, CBGV, CBCV, CBD, CBC, CBE, CBG, CBN, CBND, and CBT.

[0076] The methods and systems of the disclosure allow detection of one or more analytes in the sample at a detection limit that is equal or lower than a threshold detection limit. The term "threshold detection limit", as used herein, refers to a target detection limit for a given analyte. In some embodiments, the threshold detection limit is the regulatory limit for a given non-polar analyte in a given country or a state of the United States. In some embodiments, the threshold limit may be 1000 ppb or less, 950 ppb or less, 900 ppb or less, 850 ppb or less, 800 ppb or less, 750 ppb or less, 700 ppb or less, 650 ppb or less, 600 ppb or less, 550 ppb or less, 500 ppb or less, 450 ppb or less, 400 ppb or less, 350 ppb or less, 300 ppb or less, 250 ppb or less, 200 ppb or less, 150 ppb or less, 100 ppb or less, 90 ppb or less, 80 ppb or less, 70 ppb or less, 60 ppb or less, 50 ppb or less, 40 ppb or less, 30 ppb or less, 20 ppb or less, 10 ppb or less, 9 ppb or less, 8 ppb or less, 7 ppb or less, 6 ppb or less, 5 ppb or less, 4 ppb or less, 3 ppb or less, 2 ppb or less or 1 ppb or less.

[0077] In a specific embodiment, the threshold detection limit for an analyte comprising cannabinoid or terpene/terpenoid or a derivative thereof is the regulatory limit for the analyte in a given country or a state of the United States. In some embodiments, the threshold detection limit for the analyte is 50 ppb or less, 45 ppb or less, 40 ppb or less, 35 ppb or less, 30 ppb or less, 25 ppb or less, 20 ppb or less, 15 ppb or less, 10 ppb or less, 9 ppb or less, 8 ppb or less, 7 ppb or less, 6 ppb or less, 5 ppb or less, 4 ppb or less, 3 ppb or less, 2 ppb or less or 1 ppb or less.

[0078] The disclosure further relates to homogenization of a sample, coupled with the detection of analytes therein in a multi-step process which is practiced in a high throughput format. The term "high throughput" includes, e.g. , conducting a plurality of cryomilling, homogenization, extraction, and purification (preferably all of the procedures) in a single vessel, thereby eliminating the need for separation, transfer, and/or concentration of the homogenate.

[0079] The disclosure further relates to methods for detecting one or more of the aforementioned analytes. Chemical analyses of the parental and progeny specialty Cannabis varieties of the present disclosure may be carried out using standard chemical separation techniques well known in the art. Initial field analyses of cannabinoids may also be carried out using thin layer chromatography (TLC) as described in "Cannabis Inflorescence & Leaf QC" from The American Herbal Pharmacopeia, 2013. Chromatographic methods, as described in U.S. Pat. No. 9,370,164; U.S. Pat. No 9,035,130; and U.S. Pub. No. 2013/0337477, may also be used.

[0080] In particular embodiments, qualitative identification of cannabinoids and terpenes may be carried out by gas chromatography or liquid chromatography. Each chromatographic technique may be coupled with tandem mass spectrometry (GC-MS or LC-MS). Quantitative analysis may be performed with gas chromatograph-flame ionizing detection (GC-FID) and/or HPLC-PDA (photo diode array). The assays for cannabinoids include orthogonal methods of GC-FID and HPLC for the highest level of accuracy.

[0081] The homogenization, extraction, purification and/or analysis steps may be multiplexed.

[0082] In some embodiments, the methods disclosed herein may be practiced according to the steps outlined in FIG. 1. A sample is submitted to a cryogenic lysis step. Preferably, this mechanical lysis is performed at least at -80°C (preferably at least at -170°C) with a vortexer. Samples, e.g. , whole marijuana fluorescence and/or marijuana-infused products, are partially lysed by cryogenic grinding. Preferably, the cryogenic lysis step is performed such that at least about 50%, 60%, 70%, 80%, 90%, or greater % of the cells are lysed indiscriminately. The crude extract is then subjected to an extraction step comprising mixing with 1 : 1 H₂O/CH₃CN; adding a salt (e.g., QUECHERS HYPERSEP™); vortexing the mixture; and centrifuging the mixture to pellet the salt. Next, the supernatant of the centrifuged extract is subject to one or more purification steps comprising solid phase extraction cleanup; centrifuging to remove debris; and optionally filtering through a gauze (e.g. , PVDF membrane). Lastly, the filtrate may be subjected to an analytical procedure e.g. , HPLC, LC- MS/MS and/or GC-MS/MS analysis, to determine the composition of the various analytes in the sample.

[0083] II. KITS/SYSTEMS

[0084] The disclosure further relates to kits comprising, in one or more packages, components for cryomilling and components for high throughput homogenization, optionally together with components for using the components for homogenizing samples and/or recovering analytes therefrom. In some embodiments, the kits comprise, in one or more packages, components for cryomilling comprising liquid N₂ together with beads and vials together with components for HTH comprising, beads, vessels, extraction reagents (e.g., 1^st extraction reagent kit comprising acetonitrile and/or acetate together with salts, buffers, and optionally lysis reagents; and 2^nd extraction reagent comprising cleanup reagents for dSPE or cSPE) and optionally purification components (e.g., PVDF membranes). The instructions may include, for example, information about the duration and the particulars of one or more steps, e.g., centrifugation step, liquid phase extraction step, cleanup step and/or analytical step.

[0085] The disclosure further relates to tangible media comprising a fingerprint of a biological sample comprising a plurality of analytes, wherein the composition of the each analyte and the amount thereof is determined using the aforementioned analytical method(s). For example, the tangible media may comprise a fingerprint of a Cannabis plant, wherein the fingerprint includes a parameter selected from amounts (in actual weight or specific weight) and/or concentrations of at least one cannabinoid or at least one terpene/terpenoid present in a sample. Preferably, the parameter is detected and optionally further quantified using the analytical methods of the present disclosure, e.g., homogenization comprising cryomilling and bead homogenization, liquid phase extraction, QUECHERS clean-up/purification, and GC/LC-tandem mass spectrometry analytical procedure described above.

[0086] The disclosure now being generally described, it will be more readily understood by reference to the following examples, which are included merely for the purpose of illustration of certain aspects and embodiments of the disclosure, and are not intended to limit the disclosure. EXAMPLES

[0087] The structures, materials, compositions, and methods described herein are intended to be representative examples of the disclosure, and it will be understood that the scope of the disclosure is not limited by the scope of the examples. Those skilled in the art will recognize that the disclosure may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the ambit of the disclosure.

[0088] Example 1: General preparative methods for extraction of samples

[0089] A sample containing an analyte of interest is accurately weighed, at room temperature, in an appropriately labelled centrifuge tube. Two clean stainless steel bearings, each roughly 11 mm in diameter, and three clean stainless steel beads, each about 4.8mm in diameter, are placed into the tube. The size and number of beads may be optimized for the sample type and amount. Also, depending on preference, the homogenization beads may be made from made from materials other than steel, e.g. , ceramic, glass, etc. The tube containing sample is then placed in a sample rack and carefully filled with liquid nitrogen to freeze the sample, during which time, the sample tube caps are kept separate and uncontaminated. After allowing the liquid nitrogen to boil off completely, the tube is capped and loaded onto a multi-sample vortexer to mechanically homogenize the sample. The speed and time required to homogenize the sample depends on the size and physical properties of the sample. The aforementioned freeze-boil-vortex steps may be repeated iteratively until the desired sample consistency is achieved. Finally, the sample may be optionally digested, e.g., enzymatically digested, leaving the homogenization beads in the sample tube with the sample.

[0090] Example 2: Extraction of Cannabis product (flower, edibles, and concentrates) for analysis. This procedure establishes the process of extraction of marijuana flower, edibles, and concentrates for analysis in the analytical laboratory.

[0091] Three clean stainless steel bearings (e.g. , diameter of 4.0 mm ± 1 mm) are placed into a clearly labelled 50 ml Falcon tube (cap is labelled as well). The resin or concentrate of marijuana plant (e.g. , 0.1 g) is weighed in the Falcon tube. Alternately the sample may contain edible sample, for example, marijuana-infused food product (e.g. , 1 g). Two clean stainless steel bearings (e.g. , 11 mm) are placed into the tube on top of the sample. The Falcon tube is inserted into a rack and the tubes are filled with liquid gas (e.g., liquid N₂), after which the liquid nitrogen is allowed to boil off (in about 5 mins). The Falcon tubes are capped and loaded onto a vortexer (e.g. , multi-tube vortexer) to mechanically homogenize the sample. After 10 to 20 minutes of vortexing, the sample is homogenized. A mixture of water and acetonitrile (e.g. , preferably 1 : 1 ratio) is added to the homogenized sample and the resulting mixture is again vortexed in the vortexer (about 2 mins). To the thoroughly mixed sample, a salt packet is added and the mixture is again vortexed (approx. 5 mins). Particularly for this step, quick-easy-cheap-effective-rugged-safe extraction salts, e.g. , QUECHERS HYPERSEP™ Dispersive SPE multipack (see, Lehotay et al , J AO AC Int., 88, 630-638, 2005), may be employed. The sample is then centrifuged for 5 mins (e.g., 3700 rpm in a THERMO-FISHER Scientific Sorvall ST 40 centrifuge). A portion of the supernatant is then transferred to a vessel (e.g. , a 15 ml disposable polyproylene conical tube) with pre-made QUECHERS HYPERSEP™ Dispersive SPE clean-up and briefly vortexed with the vortex mixer. The sample is centrifuged on the Sorvall ST 49 centrifuge (e.g. , 1 min @ 1000 rpm) and the extract is filtered through a < 0.5 μιτι PVDF filter and transferred into a labelled HPLC vial. The sample is optionally diluted as needed, prior to HPLC analysis. For instance, concentrates may be diluted 1 : 10 in methanol; while marijuana-infused products (MIP) are preferably not diluted.

[0092] Example 3: Analysis of Cannabis product (flower, edibles, and concentrates) via high performance liquid chromatography (HPLC).

[0093] This procedure establishes a process for the analysis of marijuana product for the detection and/or quantification of various components therein using HPLC. This method may be used for determining the potency of the sample. First, samples containing Cannabis product are homogenized and extracted using the procedure described in detail in Examples 1 and/or 2. The processed samples are then analyzed by HPLC.

[0094] For HPLC, mobile phase A and phase B are created. For mobile phase A, 1 L of pure water from the lab water system is placed in a graduated cylinder and transferred into a HPLC mobile phase bottle and 1.0 ml formic acid is added to this mobile phase. The combination is mixed and degassed for 5 mins. For mobile phase B, 1 L of acetonitrile is placed in a graduated cylinder and transferred into an HPLC mobile phase bottle and 1.0 ml formic acid is added to this mobile phase. The combination is mixed and degassed for 5 mins. For instrumentation, the amount of mobile phases A and B is checked to insure there is ample supply of each phase to complete the run. In certain runs, each instrument run will use approximately 1.8 ml of mobile phase A and 8.2 ml of mobile phase B. The volume of mobile phases A and B that are required for a run may be computed using routine techniques (Note: the wash method uses different amounts than a normal run). Excess mobile phases A and B are placed in the bottles so as to ensure that the chromatograph is not run dry. Samples are eluted through a standard C-18 column Restek Raptor ARC-18 (a C18, octadecylsilane column manufactured by Restek Corporation, Bellefonte, PA, USA) according to the manufacturer's instructions.

[0095] Example 4: Protocol for the quantification of pesticides in a sample via GC-MSMS and LC-MSMS.

[0096] This procedure establishes a process for the analysis of marijuana inflorescence for pesticide quantification using GC-MSMS and LC-MSMS. First, samples containing Cannabis product are homogenized using the procedure described in detail in Examples 1 and/or 2. To the homogenate, ultrapure water (e.g. , 10 mL) is added and the mixture is vortexed for about 10 mins. Replicates are created from this sample. A trace amount of pesticide (e.g. , 10 ng pesticide/1 g Cannabis, which is equivalent to 10 ppb) is spiked into one of the sample replicates and all the replicates are vortexed for about 1 minute. To the vortexed sample, acetonitrile (e.g. , 5 mL) and ethyl acetate (e.g., 5 mL) is added and the mixture is vortexed again (e.g. , about 15 minutes). The vortexed mixture is chilled at - 20° C for about 10 mins and salt packets (e.g., QUECHERS salt comprising, e.g. , 4 mg MgSC^, lg NaCl, lg trisodium citrate 2H₂0; 0.5 g Na bis-citrate) are added to partition water and acetonitrile. The mixture is vortexed (about 1 min) and centrifuged at 3000 RPM for about 5 minutes, after which the entire acetonitrile layer is transferred into a clean falcon tube. To compute recovery rates, the spiked replicate containing 1 ppb (10 ng pesticide/10 mL acetonitrile) is used as a standard.

[0097] For cartridge solid phase extraction (cSPE) cleanup, MgSC^ (about 0.5 cm) is added into a cartridge SPE cartridge containing 500 mg of primary secondary amine (PSA) and 500 mg of graphitized carbon black (GCB) (about 6 mL) and rinsed with one cartridge-full of acetonitrile/ethyl acetate 50:50 (about 4 mL). The solvent is eluted at 2 drops/second until the cartridge is almost dry. A sample containing Cannabis extract (about 2 mL) is added and the sample is allowed to elute at a rate of about 1 drop per 3-5 seconds using a vacuum elution manifold. Once the sample has eluted, about 5 cartridge volumes of acetonitrile/ethyl acetate 50:50 (about 20 mL) is passed through the cartridge at 1 drop/second. The eluent is added to the concentration vessel. Ideally, the extract is clear in color (the sample extract may have a hint of a yellow tint compared to a blank). The eluent is dried in a standard dryer (e.g., TURBOVAP II, BIOTAGE, and Charlotte, NC, USA) at 50°C with a nitrogen gas flow of 3 mL/minute. The dried sample is then reconstituted with 0.5 mL of acetonitrile/ethyl acetate 50:50 by rinsing the concentration vessel about 20 times via pipette aspiration. The reconstituted sample is then transferred to auto-sampler vials equipment with limited volume inserts. In one example, the extract is split between two sample vial inserts - one for liquid chromatography-tandem mass spectrometry (LC-MS/MS) and one for gas chromatography- tandem mass spectrometry (GC-MS/MS) and the samples were analyzed. Assuming 100% recovery, the final pesticide concentration will be about 4 parts per billion (ppb). Matrix matched calibration curves are created using un-spiked cannabis taken through the entire above process to quantify recovery of spiked sample.

[0098] Example 5: Protocol for analysis of micro-toxins in a sample

[0099] This procedure establishes a process for the analysis of marijuana product for the detection and/or quantification of various microtoxins using LC/MS analysis. First, samples containing Cannabis product are homogenized and extracted using the procedure described in detail in Examples 1 and/or 2. Then, micro-toxins in the sample, e.g. , cymoxanil and/or famoxadone, are analyzed using analytical techniques. For instance, cymoxanil content may be analyzed using SPE, wherein, the acetonitrile aliquot is back extracted with hexane, concentrated, diluted with water and passed through a conditioned anion exchange solid phase extraction (SPE) cartridge stacked on a conditioned carbon black SPE cartridge. Cymoxanil passes through the SAX cartridge and is retained on the carbon black cartridge, and selectively eluted off the cartridge and dissolved in a hexane/ethyl acetate mixture. The resulting solutions are passed through silica SPE cartridges. Samples are filtered and analyzed by HPLC with UV detection. For the analysis of famoxadone, the acetonitrile aliquot is back-extracted with hexane, and the acetonitrile fraction is concentrated and carefully dried. The residue is dissolved in 10% ethyl ether/90% hexane (v/v) and passed through a GC column packed with a layer of sodium sulfate, an absorobent (e.g. , FLORISIL), and sodium sulfate. The columns are washed with additional eluent. The eluate is concentrated, carefully dried, reconstituted in acetonitrile and water and analyzed by LC/UV. Further details on the method of analysis of these micro-toxins is provided in the SPEX Sample Prep product brochure on GENOGRINDER 2010 by Tucker et al ,

[0100] Example 6: Protocol for analysis of my co-toxins in a sample

[0101] Mycotoxins are toxic natural secondary metabolites produced by several species of fungi on agricultural products. More than 300 mycotoxins, of varying toxicities, have been identified. Mycotoxins are chemically stable and are usually unaltered during food processing and/or by heat treatment. As such, myco-toxin content in food is an important barometer of adherence to food-safety and health regulation and also trade compliance. The analysis of mycotoxins is challenging due to the large number of compounds to be detected and the wide physicochemical properties they possess. Additionally, agricultural products are complex substrates, which may be contaminated with several mycotoxins at low very concentrations- thereby making detection difficult.

[0102] This procedure establishes a process for the analysis of marijuana product for the detection and/or quantification of various mycotoxins using LC-MS/MS analysis. First, samples containing Cannabis product are homogenized and extracted using the procedure described in detail in Examples 1 and/or 2. Then, my co-toxins in the sample, e.g. , type A- and B-trichothecenes, ochratoxin A, alternariol, zearalenone (e.g. , a-zearalanol and 13- zearalanol) and aflatoxins (e.g. , Bl, B2, Gl, or G2), may be analyzed using LC-MS/MS analysis detailed above. Other downstream analytical techniques, such as the protocols described in UCT Product Brochure entitled "Determination of Mycotoxin Residues by LC- MS/MS Featuring Two Alternate Sample Extraction Procedures" (Catalog # 4109-01-01, published: 2014) may also be used.

[0103] Example 7: Protocol for analysis of yeast biomolecules and/or metabolites.

[0104] Yeast (e.g., Saccharomyces cerevisiae or Schizosaccharomyces pombe) is a popular host for gene expression studies and the production of recombinant proteins. However, partly due to their cell walls, yeast products such as mRNA and intracellular proteins are often difficult to extract using traditional enzymatic methods. This procedure establishes a process for the analysis of yeast products, e.g., nucleic acids and/or proteins, for the detection and/or quantification of various analytes, such as, mRNA transcripts or polypeptide products, including sugars and/or lipids that are turned over by the modulation of various metabolic pathways. First, samples containing yeast, e.g. , S. cerevisiae or S. pombe are homogenized and extracted using the procedure described in detail in Examples 1 and/or 2. Then, the various analytes of interest are analyzed using routine biochemical techniques, e.g. , ELISA (for proteins), PCR and/or electrophoresis (for nucleic acids), spectroscopy or chromatography (for carbohydrates and other metabolites). The extraction techniques of the disclosure are therefore advantageous over art-known "yeast mills" such as those manufactured by SPEX Sample Prep (2013).

[0105] Example 8: Protocol for analysis of nucleic acids from cells.

[0106] Nucleic acid molecules, e.g. , DNA (both genomic and mitochondrial) and RNA (e.g. , mRNA and miRNA), are central to various biological processes and also serve as markers of many diseases such as cancer. Extraction and analysis of nucleic acids is often a bottleneck in many biochemical assays, both from standpoint of analyte recovery as well as efficiency. The present disclosure therefore provides a method for improving, with efficiency, recovery of nucleic acid analytes from biological and/or environmental samples.

[0107] In accordance with the generic principle described in Examples 1 and 2, cryogenic freezing of biological samples lyses the cellular barriers (e.g. , cell wall, cell membrane and/or other membranes such as chloroplast or mitochondrial membrane), thereby suspending the nucleic acids in the homogenate, which are then extracted using the high throughput methods of the instant disclosure. Depending on the downstream processes, the extracted nucleic acids can be analytically employed in various applications, e.g. , genotyping the sample and testing for various microbial contaminants, such as, bacteria, fungi and other fauna.

[0108] The instantly disclosed method greatly simplifies the process of nucleic acid extraction and analysis compared to conventional techniques that rely on the use of lysis buffer and elution reagents.

[0109] Example 9: Protocol for analysis of pesticides

[0110] This procedure establishes a process for the analysis of marijuana product for the detection and/or quantification of various pesticides using LC/MS analysis. First, samples containing Cannabis product are homogenized and extracted using the procedure described in detail in Examples 1 and/or 2. Then, pesticides in the sample, e.g. , flutianil and/or etoxazole, are analyzed using analytical techniques.

[0111] Extracts of samples suspected of containing one or more pesticides is optionally diluted and dissolved in acetonitrile containing 1% acetic acid. The dissolved extracts are analyzed with gas chromatography (e.g. , HP 5890 GC) coupled to a mass selective detector.

[0112] The analytes are separated on a capillary column The GC unit may be coupled to a mass spectrometer. The MS unit may be operated in electron impact ionization mode.

[0113] Example 10: Protocol for the testing of analytes contained in samples as per government regulation or requirement

[0114] The systems and methods of the disclosure can be applied upstream to any method used to extract and test analytes that are requested by Federal agencies such as the EPA and FDA. Additionally, state governments each require laboratories to measure an assigned list of analytes to certain levels. Medical cannabis products are tested for many analytes which, at very low levels, may cause subjects (who likely have compromised immune systems) to become sick. Therefore, measurement of metals, microbial, pesticide, mycotoxin, and solvent levels in cannabis products is detrimental to the patient and public health. The technique at hand allows for more efficient extraction of these analytes from cannabis and cannabis infused products.

[0115] This procedure establishes a process for the analysis of marijuana product for the detection and/or quantification of various analytes contained therein, which will allow determinations to be made about the safety and/or legal conformity of the product. First, samples containing Cannabis product are homogenized and extracted using the procedure described in detail in Examples 1 and/or 2. Then, analytes in the sample, e.g., the molecules listed under Table 1, are analyzed using one or more of the aforementioned analytical techniques.

[0116] Table 1 : Various categories of molecules (including a representative listing of specific molecules) that are tested under Massachusetts law. Similar regulations exist in other states where medical and adult use cannabis products are legal.

1 ,2-Dichloroethene 1870

Dichloromethane 600

1,2- 100

Dimethoxy ethane

N,N - 1090

Dimethylacetamide

N,N - 880

Dimethylformamide

Dimethyl sulfoxide 5000

1,4-Dioxane 380

Ethanol 5000

2-Ethoxyethanol 160

Ethyl acetate 5000

Ethylene glycol 620

Ethyl ether 5000

Ethyl formate 5000

Formamide 220

Formic acid 5000

Heptane 5000

Hexane 290

Isobutyl acetate 5000

Isopropyl acetate 5000

Methanol 3000 2-Methoxyethanol 50

Methyl acetate 5000

3 -Methyl- 1-butanol 5000

Methylbutyl ketone 50

Methylcyclohexane 1180

Methylethyl ketone 5000

Methylisobutyl 5000 ketone

2-Methyl-l- 5000 propanol

N- 530

Methylpyrrolidone

Nitromethane 50

Pentane 5000

1-Pentanol 5000

1-Propanol 5000

2-Propanol 5000

Propyl acetate 5000

Pyridine 200

Sulfolane 160

Tetrahydrofuran 720

Tetralin 100

Toluene 890 1,1,2- 80

Trichloroethene

1,1,1- NA

Trichloroethane

Xylene 2170

Propane 1 n-Butane 1

Iso-Butane 1

[0117] Other embodiments: The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions described elsewhere in the specification for those used in the preceding examples.

[0118] From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of the methods and, without departing from the spirit and scope thereof, can make various changes and modifications to adapt it to various usages and conditions.

[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described in the foregoing paragraphs. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. In case of conflict, the present specification, including definitions, will control.

[0120] All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All published references, documents, manuscripts, scientific literature cited herein are hereby incorporated by reference. All identifier and accession numbers pertaining to scientific databases referenced herein (e.g. , PUB MED, NCBI) are hereby incorporated by reference.

Claims

We claim:

1. A method for homogenizing a sample, comprising, a) providing a container that includes a sample to be homogenized and two or more physical homogenization aids; b) adding a cryogenic liquid to the container whereby the sample is frozen; c) allowing the cryogenic liquid to completely vaporize; and d) agitating the container to produce a homogenate.

2. The method of claim 1, wherein the sample comprises a plant sample or a product thereof.

3. The method of claim 2, wherein the plant sample comprises a Cannabis sample and wherein the product comprises a marijuana-infused product (MIP) or marijuana-infused food (MIF).

4. The method of any one of claims 1-3, wherein the sample is frozen to a temperature of less than about -80^° C.

5. The method of any one of claims 1-4, wherein the cryogenic liquid is liquid nitrogen, liquid helium, liquid argon, liquid oxygen or mixtures thereof.

6. The method of any one of claims 1-5, wherein the agitating comprises vortexing the sample.

7. The method of any one of claims 1-6, wherein the physical homogenization aids are beads or bearings.

8. The method of claim 7, wherein the agitating comprises vortexing to produce a homogenate and further comprising e) centrifuging the resulting homogenate in the tube that contains the physical homogenization aids.

9. The method of any one of claims 1-8, wherein steps b) through d) are repeated one or more times to achieve a homogenate with the desired degree of uniformity and/or to maintain the temperature below about -80^° C.

10. The method of any one of claims 1-9, wherein the method is performed in a single container.

11. The method of any one of claims 1-10, further comprising digesting or extracting the homogenate.

12. The method of claim 11, wherein the homogenate is extracted using an extractant.

13. The method of claim 12, wherein the extractant is an organic liquid, an aqueous liquid or a mixture thereof.

14. The method of claim 13, wherein the extractant comprises acetonitrile.

15. The method of any one of claims 12-14, comprising extracting one or more analytes contained in the homogenate into an extractant comprising acetonitrile in the presence of high amounts of salts and/or buffering agents to induce liquid phase separation and stabilize acid and base labile groups in the analyte.

16. The method of claim 15, wherein the salt is sodium chloride or magnesium sulfate and the buffer is citrate.

17. The method of any one of claims 12-16, further comprising removing the extractant and optionally performing solid phase extraction (SPE) on the extractant to produce a clean extract.

18. The method of claim 17, wherein the SPE comprises mixing the extractant with an SPE sorbent which is C I 8, a primary-secondary amine, graphitized carbon or a variant thereof.

19. The method of claim 17 wherein the variant of SPE sorbent comprises dispersive SPE.

20. The method of any one of claims 17-19, further comprising filtering the clean extract.

21. The method of claim 20 wherein the clean extract is filtered through a PVDF membrane.

22. The method of any one of claims 1-21, further comprising analyzing the homogenate, extractant or clean extract for the presence and/or amount of one or more analytes.

23. The method of claim 22, wherein the analyzed comprises high performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) or gas chromatography coupled to tandem mass spectrometry (GC-MS/MS).

24. The method of claim 20, wherein the analysis comprises detection and optionally quantification of an analyte.

25. The method of any one of claims 1-24, wherein the sample comprises a Cannabis sample or a sample containing a Cannabis extract, a cannabinoid or a terpene/terpenoid or a combination thereof, and the analyte comprises a cannabinoid or a terpene/terpenoid or a combination thereof.

26. The method according to any one of the foregoing claims which provides improved recovery of an analyte from the sample or improved repeatability precision (RSD) compared to a stand-alone high throughput method.

27. The method of claim 23, wherein the sample comprises Cannabis and the method comprises improved recovery of at least one component of Cannabis compared to the recovery thereof with stand-alone high-throughput homogenization.

28. The method of claim 24, wherein the component comprises at least one cannabinoid or at least one terpenoid.

29. Use of a system for homogenizing a sample, the system comprising (a) a container and two or more mechanical homogenization aids for homogenizing a sample; (b) a cryogenic liquid for freezing the sample; (c) an agitator for agitating the frozen sample to generate a homogenate; (d) a centrifuge for separating a supernatant from the homogenate and (e) an extractant for extracting the analyte from the homogenate comprising a solvent and salts and/or buffering agents; optionally together with reagents for cleaning-up the extract comprising (f) SPE systems; and/or (g) filtration systems.

30. A tangible media comprising a fingerprint of a sample comprising a plurality of analytes, wherein the composition of the each analyte and the amount thereof is determined with the analytical method of any one of claims 19 to 21.

31. The tangible media of claim 30, wherein the sample comprises Cannabis and the fingerprint comprises one or more cannabinoids and one or more terpenes or terpenoids.