No. SSI-LCMS-108

Liquid Chromatography Mass Spectrometry Quantitative Analysis of 16 with Complete Baseline Separation of Δ8 and Δ9-THC Utilizing the Triple Quad LCMS-8050

Asra Gilani1, Shelby Grinnan2 No. LCMS-108 1Shimadzu Scientific Instruments, Durham, NC; 2Cannabusiness (Aquatic resources management), Lexington, KY

■ Background Growing interest in analyzing the inherently complex The method demonstrates ideal chromatographic - and -based products necessitate the conditions utilizing the UHPLC in conjunction with analytical benefits of LC-MSMS such as higher the ideal diffusion properties of the column delivering sensitivity, selectivity, and resolution for the complete baseline separation of all cannabinoids determination of cannabinoids. Several methods have including Delta-8 (Δ8-THC) and been developed for optimal results of resolution, Delta-9 Tetrahydrocannabinol (Δ9- THC). sensitivity, and throughput but this method introduces the list of 16 cannabinoids being analyzed ■ Method and Materials by triple quadruple LCMS. To assist in optimizing for A mixture of 16 cannabinoids was prepared using an high throughput while maintaining sensitivity and 11-component mixture (Shimadzu 220-91239-21) resolution, this application demonstrates a sub-five- with five additional individual cannabinoids obtained minute gradient method utilizing the Nexera UHPLC from Sigma Aldrich (Cerilliant). The stock solution system and triple quad LCMS-8050. included the following Cannabinoids as well as their acidic forms: Cannabidivarinic acid (CBDVA), ■ Introduction (CBDV), Cannabidiolic acid (CBDA), The following method application is driven by the (CBGA), (CBG), potential terpenes interferences with THC (CBD), (THCV), quantitation when analyzing fortified Cannabis Tetrahydrocannabivarinic acid (THCVA), products on an HPLC system with a UV or PDA (CBN), Cannabinolic acid (CBNA), Delta 9- detector. Generally, terpene interference is not a Tetrahydrocannabinol (Δ9-THC), Delta 8- problem as naturally occurring terpenoid content Tetrahydrocannabinol (Δ8-THC), Cannabicyclol (CBL), does not exceed 0.2% of the total and terpenes are (CBC), Tetrahydrocannabinolic lost in the baseline under the currently run gradient acid A (THCA-A), and Cannabichromenic acid conditions with HPLC-UV or UHPLC-PDA1. However, a (CBCA). sample that has been fortified with excess terpene content may face co-elution issues of the terpenes The column was obtained from Restek.2 LCMSgrade mobile with individual cannabinoids because the terpenes are phase solvent were obtain wed from Honeywell. A stock also quite hydrophobic and exhibit similar retention concentration of 1000 ppb for each standard was on the column as the cannabinoids. used for MRM optimization. The standards were prepared in 25:75 LCMS grade Water: Methanol This application employs the highly selective solvent in varying concentrations ranging from 1 capabilities of Shimadzu Scientific Instruments’ ng/ml – 2000 ng/ml. For quantification, samples were LCMS-8050 triple quad as well as the efficacy of this diluted in the ratio of 1:9, 1:99, 1:999 v/v Cannabis instrumentation in achieving chromatographic speed, extract to ethanol. The samples were loaded onto the analyte selectivity and quantitative sensitivity of all Shimadzu Nexera UHPLC with LCMS-8050 for cannabinoids. analysis using the analytical conditions listed in Table 1. No. SSI-LCMS-108

Table 1: Analytical Conditions

HPLC- Shimadzu Nexera UHPLC ■ Results and Discussion The chromatographic method that was developed Column Temperature 40 °C yielded baseline separation of all 16 cannabinoids Injection Volume 5 uL (Figure 1) including the Delta-8 THC and Delta-9 THC (Figure 2 and 3). A twelve-point calibration curve was prepared ranging from 1 to 2000 ng/ml. LCMS-8050 Triple Quad Mass Spectrometer Due to the wide linear range a best-fit weighting 2 Ionization Heated ESI method was used (1/C ). Calibration curves for the all Cannabinoids exhibited R2 values ≥ 0.99. All the Nebulizing Gas 3 L/min chromatograms show the lowest calibrator of the Drying Gas 10 L/min linear range (Figure 4). All standards were run in series of triplicates as well as calibration checks that Heating Gas 10 L/min were also run in replicates of six injections. The DL Temperature 250 °C statistical results can be seen in Table 2. Heat Block Temperature 400 °C

Interface Temperature 370 °C

1x10^4 CBCA 150 Δ9-THC D3

125 Δ9-THCA-A 100 CBDA Δ9 - THC CBDV CBG CBN CBL 75 THCV CBDVA CBD CBNA 50 Δ8 -THC CBGA THCVA CBC 25

0

0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 min Figure 1: MRM and TIC of simultaneous acquisition of 16 cannabinoids at 100ng/ml.

1.55e5 Δ9-THC Q 315.20>193.10 (+) 1.5e5 R1 4.34% (99.43) R2 57.61% (98.68) 1.4e5 R3 41.26% (103.55)

1.3e5

1.2e5

1.1e5

1.0e5 Δ8-THC

9.0e4

8.0e4

7.0e4

6.0e4

5.0e4

4.0e4

3.0e4

2.0e4

1.0e4

0.0e0

2.25 2.30 2.35 2.40 2.45 2.50 2.55 2.60 2.65 2.70 2.75 2.80 RT (min)

Figure 2: Chemical structure Δ8-THC and Δ9-THC Figure 3: Chemical structure Δ8-THC and Δ9-THC No. SSI-LCMS-108

Table 2: MRM Transitions, calibration range, accuracy and precision results for potency. The table shows the quantitative abilities of this method for each analyte. Values of linearity refer to the linear range specified in the table. MRM Transition MRM Transitions Compound Linear Range ng/mL Linearity r² %RSD % Accuracy (quantifier ion) (qualifying ion) CBDV 287.20>165.00 287.20>123.05 1- 2000 0.997 5.4 101.6 CBG 317.20>192.90 317.20>123.00 1- 2000 0.997 3.2 95.9 CBD 315.20>193.00 315.20>135.05 1- 2000 0.997 0.4 99.0 THCV 287.20>165.20 287.20>231.10 1- 2000 0.997 4.6 96.2 CBN 311.15>223.00 311.15>43.30 1- 2000 0.998 2.0 105.0 CBC 315.20>193.10 315.20>259.05 1- 2000 0.999 9.1 102.0 CBL 315.05>235.10 315.05>81.20 1- 2000 0.999 3.0 103.5 315.20>245.00 Δ9 - THC 315.20>193.10 315.20>123.20 1- 2000 0.998 6.7 94.5 315.20>259.10 315.20>135.10 Δ8 -THC 315.20>192.95 1- 2000 0.997 4.2 107.0 315.20>123.25 CBDA 357.00>244.95 357.00>339.25 1- 2000 0.9929 4.2 106.7 CBGA 359.10>341.20 359.10>315.20 1- 2000 0.996 2.1 101.3 329.15>216.95 CBDVA 329.15>311.25 1- 2000 0.9932 2.7 90.9 329.15>107.15 357.00>245.20 Δ9-THCA-A 357.00>313.15 1- 2000 0.9924 0.9 95.6 357.00>191.20 329.00>285.10 THCVA 329.10>217.10 1- 2000 0.996 8.9 105.5 329.00>163.15 353.10>279.20 CBNA 353.00>309.15 1- 2000 0.997 2.8 100.2 353.00>171.25 357.15>313.20 CBCA 357.15>191.20 1- 2000 0.9927 9.9 92.0 357.15>203.10

Q 287.20>165.00 (+) 2.08e4Q 317.20>192.90 (+) 2.32e4Q 315.20>193.00 (+) 2.19e4Q 287.20>165.20 (+) 2.27e4 CBDV CBG CBD THCV 2.0e4 2.0e4 2.0e4 2.0e4

1.5e4 1.5e4 1.5e4 1.5e4 1.0e4 1.0e4 1.0e4 1.0e4

5.0e3 5.0e3 5.0e3 5.0e3

0.0e0 0.0e0 0.0e0 0.0e0 0.9 1.0 1.1 1.4 1.5 1.6 1.3 1.4 1.5 1.5 1.6 Q 311.15>223.00 (+) 2.89e4Q 315.20>193.10 (+) 1.14e4Q 315.05>235.10 (+) 2.88e4Q 315.20>193.10 (+) 1.75e4 CBN CBC CBL Δ9-THC

2.5e4 1.0e4 2.5e4 1.5e4 2.0e4 8.0e3 2.0e4 1.0e4 1.5e4 6.0e3 1.5e4

1.0e4 4.0e3 1.0e4 5.0e3 5.0e3 2.0e3 5.0e3

0.0e0 0.0e0 0.0e0 0.0e0 2.0 2.1 2.2 3.1 3.2 3.3 2.8 2.9 2.4 2.5 2.6 Q 315.20>192.95 (+) 1.70e4Q 357.00>244.95 (-) 1.28e4Q 359.10>341.20 (-)CBGA 1.64e4Q 329.15>311.25 (-) 1.60e4 CBDA CBDVA 1.3e4 1.5e4 1.5e4 1.5e4 Δ8-THC 1.0e4

1.0e4 7.5e3 1.0e4 1.0e4

5.0e3 5.0e3 5.0e3 5.0e3 2.5e3

0.0e0 0.0e0 0.0e0 0.0e0 2.6 2.7 2.8 1.4 1.5 1.6 1.7 1.8 1.9 1.0 1.1 1.2 Q 357.00>313.15 (-) 5.12e4Q 329.00>285.10 (-) 5.68e3Q 353.00>309.15 (-) 1.45e4Q 357.15>313.20 (-) 4.62e4 THCVA CBNA CBCA 5.0e4 Δ9-THCA-A 5.0e3 1.3e4 4.0e4 4.0e4 4.0e3 1.0e4 3.0e4 3.0e4 3.0e3 7.5e3 2.0e4 2.0e4 2.0e3 5.0e3

1.0e4 1.0e3 2.5e3 1.0e4

0.0e0 0.0e0 0.0e0 0.0e0 3.8 3.9 4.0 2.3 2.4 2.5 3.1 3.2 4.0 4.1 4.2 Figure 4: The above chromatograms show the lowest calibrator of the linear range. No. SSI-LCMS-108

■ Conclusion ■ Acknowledgements This application demonstrates a rapid, selective and Shimadzu Scientific Instruments would like to thank robust method for the detection of various its collaborators: Cannabusiness (Aquatic Resources cannabinoids by using the Shimadzu LCMS-8050. All Management), who worked to develop the analytical sixteen cannabinoids were detected at levels as low as conditions that made this application note possible, 1 ng/mL with a minimum signal-to-noise ratio of 10 and Restek Corporations for providing us with the with the only limitation in hitting an even lower LOQ materials used for developing this method. being the wide linear range. It is important to note that these results were achieved using standards without matrix. Matrix-matched calibration standards cannot be used because of native amounts of cannabinoids; therefore, utilizing Internal Standards is recommended. This method demonstrates the ability of the Nexera X2 UHPLC and LCMS-8050 mass spectrometer to enable simultaneous, accurate measurement of many of the cannabinoids of interest. This method is suitable for quantitating cannabinoids in raw or commercial products.

■ References 1. Shimadzu Scientific instruments (2016) The Determination of CBD and General Content in Hemp Oils Using HPLC with UV Detection. 2. Restek. (2017). 16 Cannabinoids on Raptor ARC-18 1.8 μm by LC-UV. LCMS-8040 LCMS-8045 LCMS-8050 LCMS-8060 LCMS-2020 Q-TOF LCMS-9030

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