CROSS-REFERENCE TO RELATED APPLICATIONS

[00011 This application claims the benefit of U.S. Prov. No. 62/607,730, filed on December 19, 2017, the entire content of which is incorporated herein by reference.

BACKGROUND

[0002] Gas chromatography (GC)is a powerful analytical tool, and the theoretical limit of quantitation (LOQ) of cyfluthrin in cannabis flower using GC has been determined to be about 5 ppb (Tran et al, Technical publication number: RUO-MKT-02-6729-A, January 2017; SCIEX, Inc., Framingham, MA). However, this level of quantitation is not achieved in practice, and the actual lower limit of detection for cyfluthrin in cannabis flower using GC is much higher, e.g., about 50 ppb or higher. This deviation for the theoretical LOQ is related to the complex makeup of cannabis flower and difficulty separating cyfluthrin from components of cannabis flowers (e.g., oils and pigments contained in the flowers). Additionally, some regulators of medical or recreational cannabis require an ability to detect and measure cyfluthrin in cannabis below 10 ppb. There is therefore an unmet need for systems and methods that allow for the detection and or quantitation of cyfluthrin in cannabis below 10 ppb.

DETAILED DESCRIPTION

[0003] A method for the determination of cyfluthrin in cannabis inflorescence down to 10 ppb is described. Cyfluthrin is extracted from homogenized cannabis inflorescence using two cleanup steps: The first involves a liquid-liquid extraction via QuEChERS dispersive solid phase extraction, which was modified from the European Committee for Standardization (CEN) Method 1S662 (Document # EN 15662 version 2.2, APR 2008). The original CEN protocol does not disclose a method for analyzing cyfluthrin at the desired threshold level of detection (e.g., 10 ppb or less) in plant samples, such as, for example, cannabis samples.

[0004] In accordance with the methods of the disclosure, the liquid-liquid extraction step and or SPE cleanup yields an organic layer, and a portion of the organic layer is further processed using a customized cartridge cleanup which contains several substrates capable of retaining the uniquely high concentrations of pigment and fatty material present in cannabis without significant retention of the pesticide, cyfluthrin. The final extract is adjusted to reach an on-column concentration within the instrument detection limit in matrix. Quantification of cyfluthrin is carried out using gas chromatography with tandem mass spectrometry (GC-MS/MS).

[0005] The method of the disclosure provides improved recovery and also matrix purity. Recoveries in test cannabis samples fortified at 10 ppb range between 92% and 137% with an average of 104%. The relative standard deviation of replicate, spiked sample preparations on different days by different analysts is 18% when calculated from concentrations resulting from quantification via in-matrix calibration curve, and is 10% when calculated using peak area.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] 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.

[0007] FIG. 1 shows total ion count chromatogram of spiked versus neat cannabis, as determined using the exemplary methods of the disclosure. The bottom curve (black) represents neat cannabis; the top curve (blue) represents curve of spiked (10 ppb) cannabis.

[0008] FIG. 2 shows total ion count chromatogram of cyfluthrin in matrix calibration. The bottom curve (black) represents 2.S ppb matrix-matched sample; the middle curve (blue) represents S ppb matrix-matched sample; and the top curve (pink) represents 10 ppb matrix- matched sample.

EXAMPLES

[0009] Example 1:

[0010] Protocol to ensure compliance with regulations for pesticide levels in medical cannabis

[0011] Until this point, the inventors are aware of no peer-reviewed publications demonstrating the ability to detect cyfluthrin in cannabis below 50 ppb. The exemplary methods disclosed herein provides for the detection and/or analysis of cyfluthrin in cannabis with a detection limit below 10 ppb, e.g., about 9 ppb, about 8 ppb, about 7 ppb, about 6 ppb, about 5 ppb, 4 ppb, about 3 ppb, about 2 ppb, about 1 ppb, about 0.5 ppb.

[0012] Equipment

[0013] GC-MS/MS benchtop system e.g., Thermo Scientific TRACE 1310 Gas Chromatograph, TSQ 8000 Evo Triple Quadropole Mass Spectrometer, TriPlus RSH Autosampler (Thermo Fisher Scientific Inc., Waltham, MA).

[0014] Concentrator e.g., Turbo Vap II, Turbo Vap II Concentration Workstation (Zymark Corporation, Hopkinton, MA).

[0015] Solid phase extraction vacuum manifold e.g., Resprep 24-Port PSE Manifold (Restek Corporation, Bellefomte, PA).

[0016] Analytical reagents/chemicals

[0017] Cyfluthrin - mix of isomers (P-354S), ACCUSTANDARD®, 10 ug/mL in methanol;

[0018] Imazalil-dS Sulfate (1268S02) (Toronto Research Chemicals Incorporated, North York, ON, Canada) 2.5 mg;

[0019] Mass spectrometry grade acetonitrile, ethyl acetate and ultrapure water (18 ohm);

[0020] High purity hydrogen, compressed air helium and argon [0021] Consumables

[0022] GC column, e.g., Rxi-5Sil MS (Restek Corporation, Bellefonte, PA).

[0023] Cartridge cleanup, e.g., 6 mL Combo SPE Cartridge, Cat. # 26194 (Restek Corporation,

Bellefonte, PA).

[0024] Magnesium sulfate anhydrous, e.g., Fisher Chemical, Cat. # M65-S00 (Thermo Fisher).

[0025] QUECHERS Mylar Pouches (4 mg MgSCVlg NaCl/lg Na3Citrat.2H ₂ O/0.5g Na ₂ HCitrat.) 4g - Thermo Scientific, Cat. # 60105-344.

[0026] Conical 50 mL tubes, e.g., Thermo Scientific, Cat. # 339653.

[0027] Autosampler vials, e.g., Cert surestop clr I-D vial kit, Thermo Scientific National Certified Vial Kit

[0028] Autosampler vial inserts, e.g., 300 μL target polyspring insert conical silanized, Thermo Scientific

[0029] Experimental methods:

[0030] (a) Solvent Standard is prepared as follows

[0031] The spiking standard (1 ppm pesticide) comprises 980 μL acetonitrile and 20 μL of 100 μg/mL pesticide standard. The internal standard (1 ppm) comprises 990 μL acetonitrile + 10 μL of 100 ug/mL imazalil-d5. The 100 ppb intermediate standard (used only to create the check standard) comprises 900 μL acetonitrile + 100 μL 1 ppm spiking standard. The check standard (5 ppb) comprises 950 μL acetonitrile + 50 μL of 100 ppb intermediate standard.

[0032] (b) Sample preparation

[0033] First samples are homogenized and water content in the sample is determined. Cannabis inflorescence is cryogenically ground to a fine powder using a ceramic mortar and pestle and is allowed to equilibrate to room temperature.

[0034] Approximately 1 gram of homogenized cannabis is weighed into a 50 mL conical tube and the actual weight is noted to the ten-thousandths place. Approximately 150 mg of homogenized cannabis is weighed into a Karl Fischer sampling vial. Gas-phase Karl Fischer analysis is carried out on this sample portion to determine water content which is later subtracted from the sample weighed out for pesticide analysis to allow calculation of the dry-weight percent of cyfluthrin in the sample.

[0035] Next, the pesticides are extracted using liquid-liquid extraction. Ultrapure water (10 mL) is added to hydrate the cannabis sample. All samples are spiked with an internal standard (10 ng of deuterated imazalil) while all control samples are spiked with internal standard as well as the pesticide/s being analyzed (10 ng).

[0036] The hydrated sample is then vortexed for about 10 minutes. A 50:50 mix of acetonitrile/ethyl acetate (10 mL) is added to the vortexed sample followed by vortexing for an additional 10 minutes. [0037] Next, QUECHERS salts are added to separate the aqueous from organic solvents and the mixture is vortexed for about 1 minute. The sample is centrifuged at about 3S00 revolutions per minute for about 5 minutes.

[0038] The aforementioned steps can be performed iteratively.

[0039] (c) Cartridge cleanup

[0040] The vacuum manifold ports are rinsed with 50:50 mix of acetonitrile/ethyl acetate to ensure cleanliness. To each Resprep 6 mL Combo SPE Cartridge (packed with 500 mg CarboPrep 90/500 mg primary secondary amine, with polyethylene frits), approximately ¼" of magnesium sulfate is added and the column is tapped to pack and secure to the vacuum manifold. Approximately one cartridge-full (about 5 mL) of 50:50 mix of acetonitrile and ethyl acetate is eluted through each cartridge to rinse the substrate at 3 drops/second until dry. The vacuum manifold is then turned off. An additional 50:50 mix of acetonitrile and ethyl acetate is added to each cartridge to submerge the sorbents. The collection tubes are then placed under cartridges.

[0041] Exactly 2 mL of cannabis extract (prepared as above) is added to the cartridge using a micropipette and pass through at approximately 3 drops/second. Once the extract has reached sorbent-level, approximately 5 mL of 50:50 mix of acetonitrile/ethyl acetate is added and the sample is eluted at 2 drops/second until dry. Additional 5 mL of 50:50 mix of acetonitrile/ethyl acetate is passed through the cartridge at 3 drops/second until dry. The eluent is dispensed into evaporation vessel. To clean the cartridge, two additional 5 mL portions are passed through each cartridge and the eluents are dispensed into existing evaporation vessel.

[0042] The evaporation vessels are then placed into the Turbovap II (set at 50°C with a nitrogen flow of 3.5 mL/min) and evaporated until dryness (about 30 minutes). The dried substrate is reconstituted with exactly 500 μL of acetonitrile and the reconstituted substrate is aspirated and rinsed 10 times at the bottom of the concentration vessel using a micropipette. The sides of the vessel are also rinsed similarly 10 times.

[0043] (d) Matrix-matched Calibration Standard Preparation

[0044] Approximately 1 mL of un-spiked cannabis extract that has gone through the above two sample preparation steps is set aside. The following 4 levels of in-matrix calibration standards are created inside of 300 μL limited volume inserts placed auto-sampler vials.

[0045] 100 ppb matrix matched standard (std) - 90 μL extract + 10 μL pesticide std. at 1 ppm.

[0046] 10 ppb matrix matched standard - 270 μL extract + 30 μL 100 ppb matrix matched std.

[0047] 5 ppb matrix matched standard - 150 μL extract + 150 μL 10 ppb matrix matched std.

[0048] 2.5 ppb matrix matched standard - 100 μL extract + 100 μL 5 ppb matrix matched std.

[0049] (e) Tuning, operation of the GC -MS/MS and analysis of data

[0050] The analytical instrument is tuned and operated as per the manufacturer's instructions. The peaks are analyzed using standard software, e.g., Chromeleon™ 7.2 Chromatography Data System

(CDS) Software (ThermoFisher Scientific, Waltham MA). [0051] Example 2: Determination of cyfluthrin levels in cannabis inflorescence

[0052] Apparatus and Instrumentation:

[0053] GC-MS/MS benchtop system - Thermo Scientific TRACE 1310 Gas Chromatography TSQ 8000 Evo Triple Quadropole Mass Spectrometer, TriPlus RSH Autosampler (Thermo Fisher Scientific Inc., Waltham, MA).

[0054] Turbo Vap -Turbo Vap II Concentration Workstation (Zymark Corp., Hopkinton, MA).

[0055] Solid phase extraction vacuum manifold -Resprep 24-Port PSE Manifold (Restek Corp., Bellefomte, PA).

[0056] GC column -Rxi-SSil MS (Restek Corp., Bellefomte, PA).

[0057] Sample homogenization and determination of cannabis dry weight:

[0058] Cannabis inflorescence is ground to a fine powder in a mortar and pestle with liquid nitrogen. After the sample has equilibrated to room temperature, a portion is weighed for gas- phase Karl Fischer analysis to determine water content. At the same instance, portions of sample are weighed out for pesticide analysis. This ensures that the water content of the sample is accurately represented and accounted for in the final ppb calculation (ng of pesticide/g of dry cannabis).

[0059] Sample cleanup:

[0060] Ground cannabis sample is hydrated with 10 mL of ultrapure water, spiked with a known amount of analyte, and vortexed. A mix of organic solvents is then added to the sample followed by additional vortexing. QUECHERS salts are then introduced to separate water and organic layers. Centrifugation is employed to enhance the separation of water and organic layers. A portion of the organic layer is passed through a cartridge cleanup containing versions of graphitized carbon black (for the removal of pigments and sterols), primary-secondary amine (for the removal of sugars and fatty acids), and magnesium sulfate (for the removal of any residual water in the organic layer). The cartridge is rinsed with several volumes of organic solvent to ensure high recovery of anah/tes. No matrix breakthrough is visually apparent, as resulting extract is nearly- clear in color even after concentration.

[0061 ] Calibration and Analysis:

[0062] Each batch of samples is accompanied by an in-matrix calibration curve which includes concentrations above and below the theoretical on-column concentration of a sample originally containing 10 ppb cyfluthrin. One sample spiked at 10 ppb and taken through the entire process is also included with each batch. A low concentration standard in solvent is run periodically throughout the batch to indicate when the system requires maintenance (replace injection liner, trim the GC column, and/or clean the MS ion source). Solvent blanks are injected periodically to ensure carryover is not present. Quantitation is carried out using the in-matrix calibration curve.

Alternatively, if a pass/fail criterion is acceptable, the peak area of the unknown sample can be compared to that of the spiked sample. If the peak area of the unknown is found to be less than that of the spike at the maximum residue limit, then the sample is compliant. See, Herbal Medicine Compendium's bulletin # 467 on Chemical Tests-Residual Solvents U.S. Pharmacopeial Convention # 40 (last accessed: December 18, 2017).

[0063] Exemplary results are provided in FIG. 1 and FIG. 2.

[0064] In accordance with the present methods, the levels of various pesticides (e.g., bifenazate, bifenthrin, cyfluthrin, etoxazole, imazalil, imidacloprid, myclobutanil, spiromesifen, trifloxystrobin) in biological samples, may be analyzed. Table 1 provides a list of pesticides and the threshold concentrations (in ppb) that are tested in Massachusetts.

[0065] Table 1: Various types of pesticides that are tested under Massachusetts law. Similar regulations exist in other states where medical and adult use cannabis products are legal.

[0066] 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.

[0067] 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.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in die 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. [0069] 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., PUBMED, MCBI) are hereby incorporated by reference.