The expressionS compact mass spectrometer (CMS) designed by Advion is a user-friendly single quadrupole mass spectrometer that delivers excellent performance, provides improved scan speed, and enables in-source fragmentation and on-line polarity switching. When compared to other mass spectrometer systems available on the market, the CMS is cost-effective and has a small footprint. This means, it can be easily accommodated in laboratories having limited space, In addition, more and more laboratories can realize the analytical advantages of mass spectrometry than ever before.
The cannabis sativa plant contains active compounds, which need to be analyzed for law enforcement where these compounds are prohibited. Such compounds are also used as a tool to control and optimize products in the growing number of lawful markets for cannabis plant-based products. Mass spectrometry serves as a suitable method when easy, clear, and legally defensible analysis techniques are needed to detect and determine plant metabolites. This article describes two easy workflows for studying cannabinoids, namely the psychoactive ingredient of cannabis sativa, tetrahydrocannabinol (THC), the naturally occurring cannabinol (CBN), and the degradation product cannabidiol (CBD). Figure 1 shows the chemical structures of the three compounds studied.
Figure 1. Chemical structures of the three compounds studied.
CMS/HPLC for quantitative determination of THC and FIA/TLC/CMS for qualitative detection of cannabinoids show the additional advantage of mass spectrometry for studying natural products.
Thin layer chromatography (TLC) was used in this analysis. First, cannabinoids were isolated on Merck’s TLC silica gel 60 F254 using a run solvent of 80/20 Petrolether (60-80 bp) /Dioxane supplied by Sigma Aldrich. For TLC/FIA/MS analysis, Advion’s Plate Express™ sample extraction device with a solvent flow rate of 200µL/min methanol 0.1 vol% formic acid was used (Figure 2). For HPLC analysis, specimens were examined by means of a 1220 HPLC system including Agilent’s UV detector on Sigma-Aldrich’s Supelco Titan 2.1mm column at 5 min gradient from 50 to 90% Acetonitrile 0.1 vol% formic acid and a flow rate of 350µL/min. For MS analysis, polarity switching as well as in-source CID was used to scan a mass range of m/z 100 to m/z 1000. Subsequently, SIM scanning kept in negative ion mode MS was utilized at m/z 309.2 (CBN) and m/z 313.2 (CBD and THC) for the quantitative analysis approach. The expressionS CMS mass spectrometer was utilized by both MS techniques.
Figure 2. A compact mass spectrometer (expression, Advion, NY, 1a) connected to the Plate Express (1b and 1c), a device for the extraction of samples from a TLC plate. CMS provides valuable additional information in natural product analysis workflows.
The Plate Express sample extraction device is capable of analyzing a lane on a TLC plate in an individual or sequential manner, extracting the compounds present, and transporting the same to the CMS for additional analysis, as shown in Figure 3. A standard TLC separation of an analytical mixture of CBD, CBN and THC at 1µg material on the lane is shown in Figure 3a. A qualitative TLC/FIA/MS analysis of THC is also illustrated in Figure 3a, with the ensuing negative ion mode in-source CID MS (Figure 3b) to clearly establish that THC is present in this sample (concurrently acquired data for in-source CID MS and positive ion mode MS data are not shown). TLC/FIA/MS analysis of the Rf region of THC (Rf=0.47) reveals an intense MS TIC signal with a pronounced negative ion signal at m/z 313.2 and the typical in-source CID fragments of THC (Figure 3c). Another method for studying the entire TLC lane demonstrates that CBD, CBN and THC are not baseline isolated during TLC analysis (Figure 3d), and hence, HPLC/MS must be used for quantitative analysis.
Figure 3. TLC/FIA/MS analysis of cannabinoids.
In spite of having a different molecular structure, CBD and THC exhibit the same isotopic mass and fragment similarly in positive ion mode ESI/MS. Conversely, in negative ion mode, in-source CID leads to the same m/z fragments, but at different relative intensities, revealing a clear difference between CBD and THC in negative ion mode MS. In order to illustrate a quantitative workflow, an HPLC/CMS technique was set-up to identify THC, CBD and CBN depending on SIM scanning within a short separation time and detection in negative ion mode, as shown in Figure 4a. When cannabinoid standards were subjected to triplicate analysis, calibration functions with good linearity were observed in the range of 2.5 to 250ng on column. This is adequate to measure from cannabis plant material having just 0.1 % w/w content such as leaves, stems and roots of the plant.
The expressionS CMS not only improves the data content acquired from natural products, but also aids in the quality control of products based on cannabis sativa, in addition to law enforcement.
Figure 4. 4a shows a typical HPLC/CMS chromatogram for all three analytes in negative ion mode SRM with the upper trace being the XIC of m/z 313.2 and the lower trace XIC of m/z 309.2. Good linearity calibration functions can be obtained for all three compounds (4b: THC, 4c: CBD and 4d: CBN) covering a range from 250 to 2.5ng analyte on the 2.1mm ID column used – sufficient to analyze plant material with as little as 0.1 % w/w THC content.
The expressionS CMS provides faster scan speed, while in-source CID and on-line polarity switching produce useful data for studying natural products. HPLC/CMS offers a sound and reliable quantification technique for CBD, CBN and THC, and the Plate Express sample extraction device produces targeted mass spectra from analyte spots of interest and thin layer chromatography plates.
This information has been sourced, reviewed and adapted from materials provided by Advion.
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