Development of Pure Certified Reference Material of Cannabidiol

Abstract

Cannabidiol (CBD) is the major functional component in hemp and has a broad range of pharmacological applications, such as analgesic, anti-epileptic, anti-anxiety, etc. Currently, CBD is widely used in pharmaceuticals, cosmetics, and food. To ensure the quality and safety of the products containing CBD, more and more related sample testing is being conducted, and the demand for CBD-certified reference material (CRM) has also sharply increased. However, there is currently a lack of relevant reference materials. In this paper, a simple method for preparing CBD CRM was established based on preparative liquid chromatography using crude hemp extract as a raw material. A qualitative analysis of CBD was performed using techniques such as ultraviolet absorption spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR), and differential scanning calorimetry (DSC). High-performance liquid chromatography (HPLC) was used for the homogeneity and stability tests, and the data were analyzed using an F-test and a T-test, respectively. Then, eight qualified laboratories were chosen for the determination of a certified value using HPLC. The results show that the CBD CRM had excellent homogeneity and good stability for 18 months. The certified value was 99.57%, with an expanded uncertainty of 0.24% (p = 0.95, k = 2). The developed CBD CRM can be used for the detection and quality control of cannabidiol products.

Keywords: cannabidiol; certified reference material; preparative liquid chromatography.

Figure 1

Structural equation of cannabis phenolic compounds.

Figure 2

The total development process for the CBD CRM.

Figure 3

(a) Chromatogram of five cannabinoid standards. (b) Chromatogram of the CBD crude extract.

Figure 4

Preparative liquid chromatography of the CBD crude extract. Separation conditions: column: preparation of separation column (C18, HT-ODS-P-10 µm, 20 mm × 250 mm, PAT-201207-1). Mobile phase: 85% methanol +15% water; flow rate: 1.5 mL/min; column temperature: 30 °C; detection wavelength: 220 nm; injected volume: 0.5 mL; CBD concentration: 500 mg/mL.

Figure 5

(a) Chromatogram of the purified cannabidiol product based on the column from Elite (Su persil-ODS2, 250 mm × 4.6 mm, 5 μm). (b) Chromatogram of the purified cannabidiol product based on the column from Agilent (ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm). Separation conditions: column temperature: 35 °C; injection volume: 10 μL; mobile phase: 0.1% formic acid in water: 0.1% formic acid in acetonitrile = 20:80 (v/v); flow rate: 1 mL/min; detection wavelength: 220 nm.

Figure 6

Three-dimensional spectral scan of the purified CBD product. Separation conditions: column: ZOBRAX SB-C18, (250 mm × 4.6 mm, 5 μm); column temperature: 35 °C; injection volume: 10 μL; mobile phase: 0.1% formic acid in water: 0.1% formic acid in acetonitrile = 20:80 (v/v); flow rate: 1 mL/min; detection wavelength: 220 nm.

Figure 7

Chromatogram of the purified cannabidiol product based on the column from Agilent (ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm). Separation conditions: column temperature: 35 °C; injection volume: 10 μL; mobile phase: 0.1% formic acid in water: 0.1% formic acid in acetonitrile = 20:80 (v/v); flow rate: 1 mL/min; detection wavelength: 220 nm. Gradient program: 0–30 min, 10%B–90%B; 30–35 min, 90%B–90%B; 35.1 min, 10%B; 35.1–45 min, 10%B–10%B.

Figure 8

TG curve of cannabidiol determination.

Figure 9

(a) Gas chromatograms of the methanol standard, ethanol standard, and n-propanol standard. (b) Gas chromatograms of the purified cannabidiol product.

Figure 10

The structural equation of CBD CRM.

Figure 11

The trend in the purity of the CBD CRM.