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Don’t Overestimate Cannabidiol During Medical Cannabis Potency Testing by Gas Chromatography

Accurate potency testing of medical cannabis with gas chromatography (GC) depends principally on choosing a column with the right selectivity; otherwise, coelutions between cannabinoids of interest may cause error in potency measurements. Cannabidiol is one of the chief cannabinoids with pharmacological value and provides relief against nausea, anxiety, and inflammation. Potency testing for medical marijuana is often done using “5-type” GC columns since they are commonly available in most labs. However, on 5-type columns cannabidiol can coelute with cannabichromene, a compound that likely also has medical value and is increasingly becoming part of potency testing. To identify and report both of these compounds accurately, a GC column with a different stationary phase is needed.

Proper Column Choice Results in More Accurate Potency Data
As shown in Figure 1, cannabinoids are aromatic compounds, meaning they will likely be better separated on a column that contains aromatics in the stationary phase because these stationary phases are more selective for aromatic-containing analytes. A fully non-aromatic stationary phase, like a “1-type” (100% dimethyl polysiloxane) column is not appropriate for this analysis since cannabichromene (CBC) and cannabidiol (CBD) will coelute completely. While 5-type columns (5% phenyl) contain some aromatic component, they generally also produce coelutions for cannabichromene and cannabidiol, depending on the conditions used. At best, CBC and CBD can be only partially resolved on 15 m 5% phenyl columns. Much better separations are obtained on higher phenyl-content phases, such as Rxi®-35Sil MS (35% phenyl type) and Rxi®-17Sil MS (50% phenyl type) columns, as they offer excellent selectivity for aromatic cannabinoids. Not only do both columns resolve cannabichromene and cannabidiol, the chromatograms in Figures 2 and 3 demonstrate that they also separate delta-8-tetrahydrocannabinol (d8-THC), delta-9- tetrahydrocannabinol (d9-THC), cannabigerol (CBG), and cannabinol (CBN). Although both columns perform well, the Rxi®-35Sil MS column is recommended because of the slightly faster analysis time and greater space overall between the peaks of interest.

While stationary phase selectivity is the most important factor in choosing a GC column for cannabinoid analysis, there are some additional aspects of this work that will benefit labs doing medical marijuana potency testing. First, cost savings were achieved by using a 15 m column. When a column with the proper selectivity is used, a 15 m column easily provides the separating power needed for this analysis at about half the cost of a 30 m column. Also, the 0.25 mm x 0.25 µm format has good sample loading capacity and is robust, especially when a proper split injection is used with a Restek Premium Precision split liner with wool. Finally, hydrogen carrier gas was used here instead of helium. Using hydrogen provides a faster analysis, increasing sample throughput. Hydrogen carrier gas is a convenient way to speed up run times, increase productivity, and reduce the cost and availability concerns associated with using helium carrier gas.

Figure 1:  Since cannabinoids are aromatic compounds, a GC column that contains aromatics in the stationary phase will provide much better separations than a column with a non-aromatic phase.


Figure 2:  The Rxi®-35Sil MS column provides both the best separation and the fastest analysis time, making it the ideal GC column choice for medical cannabis potency testing.

Peaks
1.Cannabichromene
2.Cannabidiol
3.delta-8-Tetrahydrocannabinol
Peaks
4.delta-9-Tetrahydrocannabinol
5.Cannabigerol
6.Cannabinol
Cannabis Potency Compounds on Rxi®-35Sil MS
GC_FF1248
ColumnRxi®-35Sil MS, 15 m, 0.25 mm ID, 0.25 µm (cat.# 13820)
SampleCannabinoids standard (cat.# 34014)
Cannabichromene (cat.# 34092)
delta-8-Tetrahydrocannabinol (THC) (cat.# 34090)
Cannabigerol (cat.# 34091)
Injection
Inj. Vol.:1 µL split (split ratio 50:1)
Liner:Premium 4 mm Precision® liner w/wool (cat.# 23305.5)
Inj. Temp.:250 °C
Oven
Oven Temp.:225 °C (hold 0.1 min) to 330 °C at 35 °C/min (hold 0.9 min)
Carrier GasH2, constant flow
Flow Rate:2.5 mL/min
DetectorFID @ 350 °C
Constant Column + Constant Make-up:50 mL/min
Make-up Gas Type:N2
Hydrogen flow:40 mL/min
Air flow:450 mL/min
Data Rate:20 Hz
InstrumentAgilent/HP6890 GC

Figure 3:  Cannabinoids can be effectively separated on an Rxi® 17Sil MS column, but with slightly less resolution than that obtained with the optimal selectivity of the Rxi®-35Sil MS column.

Peaks
1.Cannabichromene
2.Cannabidiol
3.delta-8-Tetrahydrocannabinol
Peaks
4.delta-9-Tetrahydrocannabinol
5.Cannabigerol
6.Cannabinol
Cannabis Potency Compounds on Rxi®-17Sil MS
GC_FF1247
ColumnRxi®-17Sil MS, 15 m, 0.25 mm ID, 0.25 µm (cat.# 14120)
SampleCannabinoids standard (cat.# 34014)
Cannabichromene (cat.# 34092)
delta-8-Tetrahydrocannabinol (THC) (cat.# 34090)
Cannabigerol (cat.# 34091)
Injection
Inj. Vol.:1 µL split (split ratio 50:1)
Liner:Premium 4 mm Precision® liner w/wool (cat.# 23305.5)
Inj. Temp.:250 °C
Oven
Oven Temp.:225 °C (hold 0.1 min) to 330 °C at 35 °C/min (hold 0.9 min)
Carrier GasH2, constant flow
Flow Rate:2.5 mL/min
DetectorFID @ 350 °C
Constant Column + Constant Make-up:50 mL/min
Make-up Gas Type:N2
Hydrogen flow:40 mL/min
Air flow:450 mL/min
Data Rate:20 Hz
InstrumentAgilent/HP6890 GC

Adjusting Conditions for 5-Type Columns
While using an Rxi®-35Sil MS column provides the best selectivity and speed for cannabinoid analysis, cannabidiol potency can be determined in medical cannabis using a 5-type column under certain conditions. If you already have a 5-type column for this work, you can vary the GC conditions, especially carrier flow and oven temperature program, and still separate cannabichromene and cannabidiol, just not as quickly or easily as with the Rxi®-35Sil MS column. Figures 4 and 5 show this analysis on Rxi®-5ms and Rxi®-5Sil MS columns, respectively. Again, the 0.25 mm x 0.25 µm format was used here because it offers better efficiency than wider bore columns (e.g., 0.32 mm and 0.53 mm IDs), which may not separate cannabichromene and cannabidiol under any operational conditions.

Figure 4:  The selectivity of a 5-type column is not sufficient to fully separate cannabichromene and cannabidiol, resulting in less accurate medical marijuana potency testing.

Peaks
1.Cannabichromene
2.Cannabidiol
3.delta-8-Tetrahydrocannabinol
Peaks
4.delta-9-Tetrahydrocannabinol
5.Cannabigerol
6.Cannabinol
Cannabis Potency Compounds on Rxi®-5ms
GC_FF1254
ColumnRxi®-5ms, 15 m, 0.25 mm ID, 0.25 µm (cat.# 13420)
SampleCannabinoids standard (cat.# 34014)
Cannabichromene (cat.# 34092)
delta-8-Tetrahydrocannabinol (THC) (cat.# 34090)
Cannabigerol (cat.# 34091)
Injection
Inj. Vol.:1 µL split (split ratio 50:1)
Liner:Premium 4 mm Precision® liner w/wool (cat.# 23305.5)
Inj. Temp.:250 °C
Oven
Oven Temp.:250 °C (hold 0.1 min) to 330 °C at 35 °C/min (hold 0.6 min)
Carrier GasH2, constant flow
Flow Rate:1.6 mL/min
DetectorFID @ 350 °C
Constant Column + Constant Make-up:50 mL/min
Make-up Gas Type:N2
Hydrogen flow:40 mL/min
Air flow:450 mL/min
Data Rate:20 Hz
InstrumentAgilent/HP6890 GC

Figure 5:  Rxi®-5Sil MS columns offer better resolution of key cannabinoids than standard 5-type columns, but the incomplete separation and longer analysis time mean further optimization is needed for accurate reporting.

Peaks
1.Cannabidiol
2.Cannabichromene
3.delta-8-Tetrahydrocannabinol
Peaks
4.delta-9-Tetrahydrocannabinol
5.Cannabigerol
6.Cannabinol
Cannabis Potency Compounds on Rxi®-5Sil MS
GC_FF1253
ColumnRxi®-5Sil MS, 15 m, 0.25 mm ID, 0.25 µm (cat.# 13620)
SampleCannabinoids standard (cat.# 34014)
Cannabichromene (cat.# 34092)
delta-8-Tetrahydrocannabinol (THC) (cat.# 34090)
Cannabigerol (cat.# 34091)
Injection
Inj. Vol.:1 µL split (split ratio 50:1)
Liner:Premium ® 4 mm Precision® liner w/wool (cat.# 23305.5)
Inj. Temp.:250 °C
Oven
Oven Temp.:150 °C (hold 0.1 min) to 330 °C at 25 °C/min (hold 0.7 min)
Carrier GasH2, constant flow
Flow Rate:1.6 mL/min
DetectorFID @ 350 °C
Constant Column + Constant Make-up:50 mL/min
Make-up Gas Type:N2
Hydrogen flow:40 mL/min
Air flow:450 mL/min
Data Rate:20 Hz
InstrumentAgilent/HP6890 GC

Note that even though these are both 5-type columns, the elution order of cannabichromene and cannabidiol changed. This is due to two things. The first is that Rxi®-5ms and Rxi®-5Sil MS columns differ slightly in selectivity for certain compounds; even though they are both considered 5-type columns, they contain different stationary phases that retain some compounds differently. The second reason is that the GC oven programs are different for the columns, which means that the compounds are eluting at different temperatures. You may be able to further optimize the separation of cannabichromene and cannabidiol on a 5-type column, but the selectivity and faster analysis that can be obtained using a high-phenyl content Rxi®-35Sil MS column make it ideal for potency determinations in medical cannabis.

To sum things up, proper column choice is essential for accurate and robust cannabis potency testing. Using the right column not only gives you more confidence in your potency values, but it also saves you time and money. Switching to hydrogen carrier gas can reduce your costs even further, while increasing sample throughput.

Visit www.restek.com/medical-cannabis for Restek® GC and LC columns, accessories, reference standards, and other products and resources for medical marijuana analysis.