Liquid Chromatography– Time-of-Flight Mass Spectrometry for Cannabinoid Profiling and Quantitation in Hemp Oil Extracts

February 28, 2018
Figure 1: Commonly analyzed cannabinoids native to C. sativa.
Click to enlarge, Figure 1: Commonly analyzed cannabinoids native to C. sativa.
Table I: Cannabinoids, physicochemical information, and retention times
Click to enlarge, Table I: Cannabinoids, physicochemical information, and retention times
Table II: List of commercially available products purchased for the analysis
Click to enlarge, Table II: List of commercially available products purchased for the analysis (product names masked)
Abstract / Synopsis: 

The method presented here allows for the accurate, precise, and robust speciation, profiling, and quantification of cannabinoids in hemp oil
extracts and commercial cannabinoid products for research and development laboratories.

Analysis of hemp extracts has increasingly become an area of scientific interest. Hemp is a fibrous variety of C. sativa with a low psychoactive Δ9-tetrahydrocannabinol (THC) component and contains as many as 60 nonpsychoactive cannabinoid compounds (1). Hemp oil and hemp extracts therefore offer a source of cannabinoids that can be used for research purposes in many laboratories.

Common analyses for cannabis and cannabinoid products include residual solvent analysis, terpene profiling and quantitation, pesticide residue analysis, toxic metal analysis, and cannabinoid profiling and quantitation. Traditional analytical platforms for profiling and quantification of cannabis and cannabinoid products include thin layer chromatography (TLC), gas chromatography–flame ionization detection (GC–FID), gas chromatography–mass spectrometry (GC–MS), and high performance liquid chromatography with ultraviolet (HPLC–UV) detection. The method presented herein allows for the profiling and quantification of cannabinoids in hemp oil products via liquid chromatography with time-of-flight mass spectrometry detection (LC–TOF-MS). Using LC–TOF-MS improves cannabinoid analysis through high resolution, accurate mass information for speciation and increased linear dynamic range compared to traditional fixed-wavelength UV detection.

Experimental

An Agilent 1290 Infinity II ultrahigh-pressure liquid chromatography (UHPLC) system and 6230B LC–TOF-MS system (Agilent Technologies) were used for these analyses. The LC–TOF-MS data were compared to data collected on the UHPLC system with diode-array detection (DAD) for range and linearity comparisons.

UHPLC-DAD Conditions
The UHPLC system used for both the UHPLC–DAD investigations and the LC–TOF-MS work included a quaternary pump, a multisampler automated sampling system with flush port, a multicolumn thermostated column oven, and a diode-array detector. The analytical column was a 100 mm x 2.1 mm, 1.8-µm dp Zorbax Bonus RP column (Agilent Technologies). The column oven and autosampler temperatures were 50 °C and 23 °C, respectively. The needle wash was set to 3.5 s at the flush port with a solution of 25:25:50 water–isopropanol–methanol and the injection volume was 0.05 µL. The diode-array detector was fixed to a wavelength of 280 nm. Mobile-phase A was water, B was methanol, and C was 0.1% (v/v) formic acid plus 2.2 mL of 5.0 M ammonium formate per liter in water. The flow rate was set to 0.50 mL/min and the mobile-phase gradient started at 23% A, 72% B, and 5% C increasing B to 95% linearly from time = 0.00 min to time = 12.5 min while holding C constant at 5%. The method stop time was 15 min and the reequilibration time was 5.0 min, resulting in an overall cycle time of 20 min. It should be noted that the analytical run time can be reduced to 7.25 min (including a 1.0-min post time) by using a 50 mm x 2.1 mm, 1.8-µm dp Zorbax Bonus RP column (Agilent Technologies) and decreasing the gradient time from 12.5 min to 6.25 min, keeping all other parameters as above. Figure 1 shows the chemical structures for the compounds used in this work. (See upper right for figure, click to enlarge, caption: Figure 1: Commonly analyzed cannabinoids native to C. sativa.)

TOF-MS Conditions
The experimental conditions for the LC–TOF-MS system were as follows: dual electrospray ionization (ESI) source in positive ionization mode, drying gas (nitrogen) at 10 L/min, drying gas temperature at 350 °C, nebulizer gas (nitrogen) at 40 psi, capillary voltage at 4000 V, and fragmentor voltage at 70 V. The spectral acquisition rate was 1.0 Hz over 100–1700 m/z. The internal reference mass was enabled using 121.0508730 m/z and 922.0097980 m/z as the target ions to correct for TOF drift over time.

Calibrator Preparation
Commercially available cannabinoid standards prepared at 1.0mg/mL in organic solvent were purchased from Cerilliant Corporation and used for method development and unknown analysis. Table I illustrates the empirical formula, accurate mass, and retention time for these compounds. (See upper right for Table I, click to enlarge.) Seven samples of commercially available hemp oil products were purchased from four manufacturers for unknown analysis, profiling, and quantification. Table II lists these with product descriptions and masked identifiers. (See upper right for Table II, click to enlarge.)

For LC–DAD analyses, calibrators containing a mixture of the commercial standard solutions were prepared over a range of 50–1000 µg/mL. For LC–TOF-MS analyses, calibrators containing a mixture of the commercial standard solutions were prepared over a range of 100–5000 µg/mL for method development and 10–1000 µg/mL for the analysis of the unknown samples. A second lower concentration calibration curve was created to establish THC limits of detection via LC–TOF-MS for samples ostensibly containing very low levels of this compound. The THC curve ranged from 12.5 ng/mL to 1000 ng/mL in solvent.

Disclosures: 

This article was originally published in "Advancing The Analysis of Medical Cannabis," a supplement to LCGC North America and Spectroscopy magazines in May 2017.

References: 
  1. Bacca, What’s the Difference Between Hemp and Marijuana? www.Alternet.org. Retrieved April 17, 2017 from: http://www.alternet.org/drugs/whats-difference-between-hemp-and-marijuana.