[ APPLICATION NOTE ]

Separation of 16 in Flower and Extracts Using a Reversed-Phase Isocratic HPLC Method Andrew J Aubin, Catharine Layton, and Shawn Helmueller Waters Corporation, Milford, MA, USA

APPLICATION BENEFITS INTRODUCTION The Alliance™ HPLC System is a highly As the legalization of cannabis for both medicinal and recreational use reliable and robust instrument that can continues to advance, the need for simple and reliable analytical methods be used for routine analysis of cannabis for the analysis of these products is desired by many industry stakeholders and cannabis extracts. (producers, regulators, and consumers). The cannabis plant () is a complex natural product that is known to produce at least 100 different cannabinoids.1 Traditionally, laboratories have focused their analysis on five primary compounds: delta-9- (d9-THC), delta-9-tetrahydrocannabinolic acid (THC-A), cannabidiolic acid (CBD-A), (CBD), and (CBN). Recently, more attention has been paid to some of the other minor cannabinoids, particularly in the medicinal market, which requires methods that are capable of higher resolution separations. Many of the minor cannabinoids have shown some medicinal effects so it is important to have the ability to separate and identify them. It is also desirable to have methods that minimize peak co-elutions of minor and major cannabinoids and provide accurate quantitative results. Several industry standardization bodies are currently developing harmonized methods for profiling to minimize the inherent variability in sampling, extraction, and instrumental analysis with an aim to improve potency result accuracy. High Performance Liquid Chromatography (HPLC) is the preferred methodology for potency determination (Total THC, Total CBD, or THC/CBD ratio) as it can identify and measure the structurally similar cannabinoids and their different forms (e.g. free form and corresponding acid forms of THC and CBD) in a single analysis. This application note presents a simple isocratic separation of 16 cannabinoids WATERS SOLUTIONS that can be used for the analysis of both plant material and concentrates to Alliance HPLC System ensure the quality and safety of cannabis products. Empower™ 3 Chromatography Data Software (CDS) 0.34 0.32 2998 Photodiode Array (PDA) Detector 0.30 CBD- A - 6.917 0.28 CBN - 9.376 0.26 CORTECS™ Columns 0.24 0.22 0.20 0.18 CBD - 6.331 AU 0.16

0.14 .088 0.12

KEYWORDS THC- A - 17.445 0.10 D9-THC - 11 - 3.898 Cannabis, cannabinoid, marijuana, 0.08 -A .846 - 8.591

0.06 CBDV -A

0.04 CBDV - 3.447 THCV CBG- A - 10.277 cannabinoid potency, cannabidiol, CBD, THCV - 5.516 CBC - 15.798 CBG - 7.393 D8-THC - 11

0.02 CB L - 13.823 CBL- A - 21.628 CBC- A - 23.374 Figure 1. Isocratic tetrahydrocannabinol, THC, 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 separation of Minutes 16 cannabinoids.

1 [ APPLICATION NOTE ]

EXPERIMENTAL

LC conditions Standard preparation LC system: Alliance HPLC Linearity of major cannabinoids d9-THC, THC-A, CBD, CBD-A, Column: CORTECS Shield RP18 and CBN was determined for 10 concentrations between 2.7 µm, 4.6 mm × 150 mm 0.004 mg/mL and 1.000 mg/mL, prepared via serial dilution (p/n: 186008685) in methanol using DEA exempt standards.

Analytical flow rate: 2.0 mL/min Sample preparation Mobile phase A: Water with 0.1% TFA Four representative, pre-prepared samples were obtained Mobile phase B: Acetonitrile from a local cannabis testing laboratory and one from a hemp processing laboratory. Samples were prepared as follows: For Isocratic: 41:59 mobile phase A/mobile phase B flower, a portion of homogenized plant material (Table 2) was Oven temp.: 35 °C added to acetonitrile or ethanol and sonicated for 20 minutes. Detector: 2998 PDA The subsequent extract was filtered through a 0.22 µm syringe tip filter, diluted with methanol, and placed into a 2 mL sample Detection wavelength: 228 nm at 4.8 nm resolution vial ready for analysis. Concentrates were prepared similarly with isopropanol being used as the extraction solvent. Injection volume: 5 µL

Software: Empower 3 CDS Table 2. Samples used in the study.

Extraction Extraction Standard description Sample Dilution factor weight (mg) volume (mL) US DEA exempt reference standard solutions were High CBDA flower 304.6 4 (ACN) 5 obtained from Cerilliant Corporation, Round Rock, TX. High THCA flower 200.1 4 (ACN) 5 These pre-dissolved solutions have been previously shown CBDA concentrate 17.5 4 (IPA) 5 THCA concentrate 15.9 4 (IPA) 5 to be suitable for the generation of calibration curves2 Hemp flower 91.0 20 (EtOH) 1 when handled in an appropriate manner.3 Table 1 lists the cannabinoid standards used in this application note.

Table 1. Cannabinoids used in the separation.

Cannabinoid No. Cannabinoid CAS number abbreviation 1 Delta-9-tetrahydrocannabinol d9-THC 1972-08-03 2 Delta-9-tetrahydrocannabinolic acid THC-A 23978-85-0 3 Cannabidiol CBD 13956-29-1 4 Cannabidiolic acid CBD-A 1244-58-2 5 Cannabinol CBN 521-35-7 6 Delta-8-tetrahydrocannabinol d8-THC 5957-75-5 7 CBG 25654-31-3 8 CBG-A 25555-57-1 9 CBC 20675-51-8 10 Cannabichromenic acid CBC-A 185505-15-1 11 THCV 31262-37-0 12 Tetrahydrocannabivarinic acid THCV-A 39986-26-0 13 CBDV 24274-48-4 14 Cannabidivarinic acid CBDV-A 31932-13-5 15 Cannabicyclol CBL 21366-63-2 16 Cannabicyclolic acid CBL-A 40524-99-0

Separation of 16 Cannabinoids in Cannabis Flower and Extracts Using a Reversed-Phase Isocratic HPLC Method 2 [ APPLICATION NOTE ]

RESULTS AND DISCUSSION 16 cannabinoids were fully separated in 26 minutes by HPLC using a reversed-phase isocratic method as shown in Figure 1. Resolution of all 16 compounds was calculated 2.5 or greater (Table 3).

Table 3. Compound resolution and retention times from Figure 1. Table 4. Linear regression of five major cannabinoids (CBD, d9-THC, CBD-A, THC-A, and CBN). RT Peak # Name Resolution (min) Cannabinoid R2 1 CBDV 3.44 – CBD 0.9997 2 CBDV-A 3.90 4.78 d9-THC 0.9998 3 THCV 5.52 13.97 CBD-A 0.9995 4 CBD 6.33 5.8 THC-A 0.9993 5 CBD-A 6.92 3.66 CBN 0.9995 6 CBG 7.39 2.75 7 THCV-A 8.59 6.35 8 CBN 9.38 3.75 9 CBG-A 10.28 3.88 10 d9-THC 11.09 3.22 11 d8-THC 11.85 2.86 12 CBL 13.82 6.71 13 CBC 15.80 5.80 14 THC-A 17.44 4.28 15 CBL-A 21.63 9.24 16 CBC-A 23.37 3.34

2.60

2.40 CBD- A - 6.828 2.20

2.00

1.80

1.60

1.40 AU 1.20

1.00

0.80

0.60 CBD - 6.319

.100

0.40

- 3.900 0.20 -A CBC - 15.802 D9-THC - 11 CBG- A - 10.282 CBC- A - 23.360 THC- A - 17.461 CBDV CBG - 7.397 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.0 0 14.00 16.0 0 18.00 20.0 0 22.00 24.00 26.00 Figure 2. High CBD-A Minutes flower sample.

1.40

1.30 .023

1.20 D9-THC - 11 1.10

1.00 THC- A - 17.35 0 0.90 0.80

AU 0.70 0.60 0.50

0.40 0.30 CBG- A - 10.24 9

0.20 - 8.60 7 0.10 -A CBG - 7.40 6 CBN - 9.395 CBC - 15.80 3 CBD- A - 6.94 0 THCV - 5.52 6 THCV CBC- A - 23.369 CB L - 14.480 CBD - 6.274 0.00 0.00 2.00 4.00 6.00 8.00 10.0 0 12.0 0 14.0 0 16.0 0 18.0 0 20.0 0 22.0 0 24.0 0 26.0 0 Figure 3. High THC-A

Minutes flower sample.

Separation of 16 Cannabinoids in Cannabis Flower and Extracts Using a Reversed-Phase Isocratic HPLC Method 3 [ APPLICATION NOTE ]

1.00 0.95 0.90

0.85 CBD- A - 6.89 5 0.80 0.75 0.70 0.65 0.60 0.55

AU 0.50 0.45 0.40 0.35 0.30 0.25

0.20

.085

0.15

- 3.90 1 -A 0.10 CBD - 6.336

0.05 THC- A - 17.43 0 CBG- A - 10.27 3 D9-THC - 11 CBDV CBC - 15.775 CBC- A - 23.328 CBG - 7.39 3 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.0 0 14.0 0 16.0 0 18.0 0 20.0 0 22.0 0 24.0 0 26.0 0 Figure 4. High CBD-A Minutes concentrate sample.

0.44 0.42 0.40 0.38 THC- A - 17.404 0.36 0.34 0.32 0.30 0.28 0.26 0.24

AU 0.22 0.20 0.18 0.16 0.14 0.12

0.10 .099

0.08

0.06 - 8.60 2 -A 0.04 D9-THC - 11 THCV 0.02 CBG- A - 10.28 7 CBD - 6.28 0 CBG - 7.407

CBD- A - 6.94 1 CBC- A - 23.354 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.0 0 14.0 0 16.0 0 18.0 0 20.0 0 22.0 0 24.0 0 26.0 0 Figure 5. High THC-A Minutes concentrate sample.

0.80

0.75 0.70 0.65 CBD- A - 6.89 2 0.60 0.55 0.50 0.45

AU 0.40 0.35 0.30 0.25 0.20

0.15

0.10 - 3.89 6 - 10.26 0 -A 0.05 CBD - 6.327 CBC- A - 23.322 THC- A - 17.42 4 CBDV CBG- A CBC - 15.76 2 CB L - 14.427 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.0 0 14.0 0 16.0 0 18.0 0 20.0 0 22.0 0 24.0 0 26.0 0 Figure 6. Hemp Minutes flower sample.

Separation of 16 Cannabinoids in Cannabis Flower and Extracts Using a Reversed-Phase Isocratic HPLC Method 4 [ APPLICATION NOTE ]

6 x 106

5 x 106

4 x 106

3 x 106

2 x 106

1 x 106

0 Area (µV*sec)

-1 x 106 0.00 0.10 .020 .030 0.40 0.50 .060 .070 0.80 0.90 1.00 Figure 7. Standard curve for CBD (mg/mL) at 10 concentrations between 0.004 mg/mL and 1.000 mg/mL.

Many HPLC methods have been published as the cannabis industry has continued to expand.4,5,6 These methods are presented using a variety of columns, mobile phase combinations, pH adjustments, and gradient profiles, but more importantly, with only a subset of target cannabinoids. It is possible for any cannabinoid to be present in any cannabis sample. In addition, any cannabis sample can have any cannabinoid as a major or minor constituent; therefore good chromatographic resolution of all cannabinoids of interest is essential. For HPLC methods it is recommended that a resolution of >2 between the peak of interest and the closest potential interfering peak is achieved.7 To ensure the most accurate and reliable results, every effort should be made to separate as many cannabinoids as possible in a given analysis. Further, methods that are simple, with easy to prepare reagents, reduce operator errors which in turn generates higher quality results. In the method presented here, the mobile phase consists of 0.1% trifluoroacetic acid (TFA) in water and acetonitrile which are mixed using the automated solvent blending capability of the Waters™ Alliance System.8 This significantly reduces variability that can happen when manually preparing mixed mobile phases. Detection is accomplished using a Waters 2998 Photodiode Array (PDA) Detector operated in single wavelength mode (228 nm) for simplicity. Even when operated in this mode, full scan PDA data is collected and is available for library matching or peak purity assessment when desired.9 Separations were achieved using a CORTECS Shield RP18 Column (p/n: 186008685). This solid-core column provides alternative selectivity when compared to typical C18 bonded chemistries, especially for phenolic compounds. The instrument was fully controlled and all data were collected and processed using Empower 3 Chromatography Data Software (CDS).

Table 5. Quantitative analysis results of representative samples presented as %dry weight. ND=Not Detected.

CBD CBD-A CBN THC THCA High CBD-A flower 3.34 11.77 ND 0.35 0.19 High THC-A flower 0.06 0.06 0.10 24.57 15.69 CBD-A concentrate 8.48 65.41 ND 1.19 3.07 THC-A concentrate 0.13 ND ND 7.58 64.84 Hemp flower 0.28 8.83 ND ND 0.32

Table 6. Qualitative analysis results of representative samples. Green shading indicates found, grey shading indicates not found.

CBDV CBDV-A THCV CBG THCV-A CBG-A D8-THC CBL CBC CBL-A CBC-A High CBD-A flower High THC-A flower CBD-A concentrate THCA concentrate Hemp flower

Separation of 16 Cannabinoids in Cannabis Flower and Extracts Using a Reversed-Phase Isocratic HPLC Method 5 [ APPLICATION NOTE ]

CONCLUSIONS References ■■ The Alliance HPLC-UV System combined with the CORTECS Shield 1. MM Radwan, A S Wanas, S Chandra, M A ElSohly. Natural Cannabinoids of Cannabis and Methods RP18 Column provided an isocratic separation of 16 cannabinoids in of Analysis. In: Chandra S, Lata H, ElSohly M (eds) less than 26 minutes. Cannabis sativa L. Botany and Biotechnology. Springer, Cham (2017). ■■ All 16 compounds were separated with baseline resolution >2.5. 2. C E Layton, A J Aubin. Method Validation for Assay ■■ Linearity of the five major cannabinoids was shown at 10 concentration Determination of Cannabidiol Isolates. Journal of 2 Liquid Chromatography & Related Technologies. levels with R values >0.999. 41:3 (2018). ■■ This method was demonstrated on five samples representing the most 3. C E Layton, A J Aubin. Considerations When cannabinoid containing sample types. Cannabinoid amounts were Handling DEA-Exempt Cannabinoid Reference Standard Preparations Used For Potency generated for those samples. Determination. Presented at the 4th Annual Emerald Conference. February 15–16, 2018 in San Diego, CA. Acknowledgments 4. R Upton. (Eds.) Cannabis inflorescence: Cannabis The authors thank ProVerde Labs and Cattis LLC for the donation spp.; Standards of Identity, Analysis, and Quality of the prepared samples. Control. American Herbal Pharmacopoeia, Scotts Valley CA (2014). 5. B Patel, D Wene, Z T Fan. Qualitative and Quantitative Measurement of Cannabinoids in Cannabis using a Modified HPLC/DAD Method. J Pharm Biomed Anal. Nov 30;146:15–23 (2017). 6. Giese et al. Development and Validation of a Reliable and Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis. Journal of AOAC International. 98:6 (2015). 7. M E Swartz, I S Krull. Handbook of Analytical Validation. Boca Raton, FL: CRC Press (2012). 8. Automated Solvent Blending Enhances HPLC Performance. Waters Performance PerSPECtives no: 720000294EN. (2003). 9. Waters 996 Photodiode Detector: Peak Purity I: What is Peak Purity Analysis? Waters Performance PerSPECtives no: wpp16. (1996).

Disclaimer Waters does not support, encourage or promote the use of its products or services in connection with an illegal use, cultivation, or trade of cannabis or cannabis products. Waters products are intended to be used for cannabis related purposes only in compliance with all applicable laws in a manner that promotes public safety and/or in connection with federally approved research, or state approved medical research.

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