The “Green Rush” of cannabis and hemp continues to increase because of the medicinal and health benefits of these two plants. The two major beneficial compound classes are the cannabinoids and terpenes. This article explores the various techniques for conducting analysis of these compounds, including: integrated versus modular high performance liquid chromatography (HPLC), HPLC versus ultrahigh-pressure liquid chromatography (UHPLC), UHPLC-ultraviolet (UV) versus UHPLC-photodiode array (PDA) detectors, HPLC-UV versus quadrupole liquid chromatography mass spectrometry (LC–MS) versus triple quadrupole mass spectrometry (LC–MS/MS), quadrupole time-of-flight mass spectrometers (QTOF-MS) versus matrix-assisted laser desorption or ionization time-of-flight mass spectrometers (MALDI-TOF-MS), LC versus gas chromatography (GC) systems, and sample preparation for LC- and GC-based methods.
UHPLC-UV Versus UHPLC-PDA Detectors
The acronym UHPLC implies the system can be utilized either as an HPLC or UHPLC system. There are multiple types of detectors that may be used for cannabinoid analysis. This section focuses on systems based on absorbance detection in the ultraviolet-visible (UV-vis) region of the electromagnetic spectrum (190–900 nm wavelength range).
For cannabinoid analysis the most important part of the spectrum is in the UV region of 190–350 nm. The HPLC-UV is typically used to measure only a couple of specific wavelengths, such as 220 nm or 228 nm. With UV detection, confirmation of the specific cannabinoid is based on the retention time, as shown in Figures 1 and 2. The photodiode array (PDA) detector, also known as a diode array detector (DAD), can measure the entire wavelength range simultaneously, which may provide other advantages. Figure 3 shows an example of the cannabinoid spectral absorbance profiles, which can provide a second form of identity confirmation. Notice the neutral cannabinoids (delta-9-tetrahydrocannabinol [∆9-THC], ∆8-THC, tetrahydrocannabivarin [THCV], cannabidiol [CBD], cannabidivarin [CBDV], and cannabigerol [CBG]) in the blue traces have similar spectral profiles that differ from the acidic forms (tetrahydrocannabinolic acid [THCA], cannabidiolic acid [CBDA], and cannabigerolic acid [CGBA]) shown in the red traces.
The carboxyl group (-COOH) of the acidic cannabinoids provides additional conjugation of the electron structure of the molecule. This results in longer wavelengths of the peak maximums. The PDA could be used to distinguish the neutral cannabinoids from the acidic forms, but may not be sufficiently reliable to confirm cannabinoids within the same class. Also, other cannabinoids (cannabinol [CBN] and cannabichromene [CBC] shown in the green traces) have substantially different spectral profiles based on their structures.
PDA detection has other advantages in that the spectral profile may assist in determining an unknown peak in the chromatograms, such as another cannabinoid or terpene. Full confirmation analysis should be performed by a mass detector-based system, as discussed later in this article. In the pharmaceutical industry, a PDA detector is often used to determine peak purity of the target compound. The absorbance spectra are compared at multiple points across the peak, as shown in Figure 4. Differences in the spectra can be indicative of coelution in the chromatographic peak. A peak purity index of 1.000000 indicates a pure compound is eluting. As the peak purity index becomes lower, it can be concluded that coelution of multiple compounds exists.
Peak deconvolution is another possibility with PDA detection systems. A PDA detector collects time information (the chromatogram) and spectral information (UV spectrum), shown in Figure 5. By using both sets of information, it is possible to deconvolute the data and determine the quantity of each analyte in a coeluting peak.
Deconvolution relies on sound scientific principles, not estimation based on Gaussian modeling, which has been used in the past. Figure 6 shows an example of five cannabinoid peaks being deconvoluted using Shimadzu’s Intelligent Peak Deconvolution Analysis (i-PDeA) software. Two forms of peak identification, including retention time and absorbance spectral i.d., are now easily obtained. A third confirmation of mass spectral i.d. can be obtained by adding a mass spectrometer as described in the next section.
Comparing HPLC–UV, LC–MS, and LC–MS/MS
Instead of an absorbance spectral detection of an HPLC-UV or HPLC-PDA, the analyst may prefer a mass spectrometry-based system, such as a single quadrupole liquid chromatography-mass spectrometry (LC–MS) system or a triple quadrupole mass spectrometer (LC–MS/MS), also known as tandem mass spectrometry. These types of mass spectrometers are referred to as high-speed mass spectrometers. They provide a fingerprint of the eluting chromatographic peak, especially with LC–MS/MS. A simplistic difference between LC–MS and LC–MS/MS is that the latter can assist in removing compound interferences and provide more confidence in compound identification.
Mass spectrometers can provide the analyst valuable insight with information about molecular weight and structures. For scientists in the research and development (R&D) arena, a mass spectrometer is often considered vital in the pursuit of critical molecular knowledge. LC–MS and LC–MS/MS can also be combined with a UV-vis or PDA detector to provide three forms of compound identification: retention time, absorbance spectra, and mass spectra.
Each of these techniques have a certain cost. For example, an HPLC-UV costs in the range of $40–$50K while an HPLC-PDA system is in the $50–$60K range. UHPLC-based systems usually cost about 20% more. Most quality assurance (QA) and quality control (QC) laboratories believe these instruments are sufficient. High-speed LC–MS systems are in the $100–$120K range, while LC–MS/MS systems are in the $300–$400K range and often are reserved for researchers. Many states permit using an HPLC-UV for cannabinoid analysis. An exception is Tennessee, where the Department of Agriculture requires LC–MS/MS. In addition to a clear instrument cost advantage, conventional HPLC holds numerous other advantages, including a better linear dynamic range, better detector stability, reduced frequency of calibration, and a lower operator salary level (that is, technician versus chemist).
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About the Authors
Bob Clifford, PhD, is the General Manager of Marketing at Shimadzu Scientific Instruments in Columbia, Maryland. Craig Young is the HPLC Product Manager at Shimadzu Scientific Instruments. Alan Owens is a Senior GCMS Product Specialist at Shimadzu Scientific Instruments. Jim Mott, PhD, is a Field Tech Support Supervisor at Shimadzu Scientific Instruments. Rachel Lieberman, PhD, is the Forensics Marketing Manager at Shimadzu Scientific Instruments. Direct correspondence to: [email protected].
How to Cite This Article
B. Clifford, C. Young, A. Owens, J. Mott, and R. Lieberman, Cannabis Science and Technology 3(1), 34-42 (2020).