AOAC 2018 Methods

( 6 ) Filter with 0.22 µm PTFE filter into amber 2 mL microcentrifuge tube. ( 7 ) Store at 4°C prior to analysis and analyze within 2 days of preparation. G. LC Analysis ( a ) Setup .—( 1 ) Set detector to 220 nm, column temperature to 25°C, injection volume to 5 µL, autosampler temperature to 4°C, and flow rate to 0.6 mL/min. ( 2 ) Program the gradient as described in Table 2018.10B . ( 3 ) Equilibrate column with initial gradient conditions: 52% mobile phase B. ( b ) Procedure .—( 1 ) Make single injection of one set of calibration standards (Std 1–7). ( 2 ) Make single injections of each LC sample. ( 3 ) After approximately every 10 samples injections, and the end of the run, reinject one of the calibration standards for QC purposes. H. Calculations ( a ) Calibration standards .—( 1 ) Measure the peak areas for all 10 cannabinoids: THCA, THC, CBDA, CBD, CBGA, CBG, THCV, CBC, CBN, and CBDVA in the set of calibration standards and the reinjected QC calibration standards. ( 2 ) Ensure that the reinjected calibration standard peak areas are within 5% of the initial calibration standard peak areas. ( 3 ) Construct a plot of the analyte concentration ( x -axis) versus individual peak area ( y -axis) for each cannabinoid. Use least- squares analysis to determine slope, intercept, and correlation coefficient (r 2 ) for the best-fit line for each analyte. ( b ) Unknown samples .—( 1 ) From the standard curves and peak areas of each analyte in the samples, calculate the concentration for each cannabinoid in each sample solution. If the peak area of any analyte is above the standard curve for that analyte, dilute the extract 1:20 in the extraction solvent, then filter and repeat the analysis for that extract.

( 2 ) Calculate the amount of each cannabinoid in the original sample as follows:

where concentration = concentration of analyte (μg/L) from linear regression analysis, volume = initial volume (mL), mass = mass of sample (mg), D = dilution factor, and 10 = conversion from mg/g to %w/w. ( 3 ) Report the cannabinoid concentrations in the original samples as mean concentrations with 95% confidence intervals as follows: where x = mean of the triplicate values, t αν = t critical value and S x = size of the standard error [ S x = s √ n , where s = SD and n = number of replicates]. For triplicate measurements and α = 0.05, the t critical value is 4.303. I. Typical Chromatograms Typical chromatograms are shown in Figure 2018.10 for a mixed calibration standard solution and a cannabis dried flower sample. References: Mudge, E.M., Murch, S.J., & Brown, P.N. (2017) Anal. Bioanal. Chem . 409 , 3153 DOI: 10.1007/s00216-017-0256-3Analytical and

Bioanalytical Chemistry J. AOAC Int . (future issue)

AOAC SMPR 2017.002 Quantitation of Cannabinoids in Dried Plant Materials J. AOAC Int . 100 , 1204(2017) DOI: 10.5740/jaoacint.SMPR2017.002

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