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HPLC Cannabis Testing: Which Column is Right for You?

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Introduction

The potency testing of cannabis products is of vital importance to the cannabis industry and, on the surface, can seem straightforward. But different labs have different needs, and there are many unique requirements and obstacles to overcome depending upon state regulations. While some labs may be interested in only a few cannabinoids to meet their required testing standards, others may be interested in offering as many cannabinoids as possible to outperform their competitors. In addition to the analyte list, there are other pertinent factors for laboratories to consider, such as solvent consumption, organic solvent type, run time, and instrument capabilities/limitations. In this article, each of these topics will be discussed to help method developers choose the best potency method to meet their unique HPLC cannabis testing requirements.

Method Development

When starting method development for cannabis potency testing, the vast number of column dimension offerings can be overwhelming. In general practice, a C18-type column chemistry is utilized for potency testing, but other column chemistries, such as Fluorophenyl, can be useful when utilized for the rapid separation of challenging isomers. Check out our blog on looking for isomers when analyzing for THC concentrates [1].

The Raptor ARC-18 column is sterically hindered to resist harsh low-pH mobile phases and offers consistent retention, peak shape, and robustness for cannabinoid analyses. This column is available in a wide variety of column dimensions that can be tailored to your laboratory’s cannabis testing needs. It is important to understand the advantages that different column dimensions offer, and how they can best be used to meet different goals. In general, large column dimensions, such as 150 mm x 4.6 mm, will offer higher resolving capabilities while smaller column dimensions oftentimes offer the advantage of lower solvent consumption and shorter run times.

Choosing the Right Column Dimensions

Using shorter column dimensions often allows for faster run times, but is using a shorter column always advantageous? Since potency testing is routinely performed by HPLC-UV, larger internal dimension columns, such as 3.0 mm or 4.6 mm, are more robust and easily transferrable from instrument to instrument over a 2.1 mm internal dimension column. This is due to the effects of instrument extra-column volume. Extra-column volume is the volume from the injector to the detector (excluding the analytical column) and should be minimized when possible. Larger column dimensions are less effected by the extra-column volume because of the larger overall volume of the column and its associated impact on peak volume. Read more about extra-column volume and the effects on potency testing in our technical article [2].

Smaller dimensions are ideal for rapid analysis, especially when an analyte list is limited. In the example below, a 50 mm x 3 mm, 2.7 µm Raptor ARC-18 column was used to resolve 7 analytes using simple mobile phase additives and gradient conditions with an overall cycle time of 8 minutes. This rapid analysis allows for high throughput and uses methanol as the organic modifier, which is more cost-effective than acetonitrile. The conditions for this analysis are outlined in Table I, and the chromatogram for the 7 cannabinoids is displayed in Figure 1. This method was applied to hemp oil and CBD hemp flower to demonstrate its applicability in matrix samples (Figure 2A and Figure 2B).

Table I: Method conditions for the analysis of 7 cannabinoids.

Column: Raptor ARC-18 EXP guard column cartridge
50 mm x 3.0 mm ID, 2.7 µm (cat.# 9314A5E)
Guard Column: Raptor ARC-18 EXP guard column cartridge
5 mm x 3.0 mm ID, 2.7 µm (cat.# 9314A0253)
Mobile phase A: Water, 5 mM ammonium formate, 0.1% formic acid
Mobile phase B: Methanol, 0.1% formic acid
Column Temp: 30 °C
Sample Concentration: 50 ppm
Injection Volume: 3 µL
Detector: UV/Vis monitored at 228 nm
Flow Rate: 0.8 mL/min
  Time (min) %B
  0.00 75
  5.00 75
  5.50 95
  6.50 95
  6.51 75
  8.00 75
 

Figure 1: Chromatogram obtained for 7 cannabinoids in solvent by the method conditions outlined in Table I.

 

Figure 2A: Chromatogram obtained from conditions outlined in Table I for hemp oil.

Hemp oil was prepared by aliquoting 50 µL of oil and adding 950 µL of acetonitrile. After vortexing for 30 seconds, 750 µL was transferred to a vial and 250 µL of water added. The sample was vortexed, and a 1:20 dilution was performed. The analytes were spiked at 50 ppm.

Figure 2B: Chromatogram obtained from conditions outlined in Table I for CBD hemp flower.

Flower was prepared by weighing 500 mg in a centrifuge tube and extracted with 10 mL of 80:20 methanol:water. Samples were vortexed for 15 seconds and sonicated for 5 minutes for 3 cycles. Flower was centrifuged at 4000 rpm for 5 minutes. Supernatant was diluted at 1:50 and all analytes spiked at 50 ppm, except CBDA which was measured at endogenous levels.

 

The method above is ideal for labs interested in a limited number of cannabinoids that are required to be monitored to meet compliance regulations, such as hemp testing labs. For labs that are interested in using a 50 mm x 3 mm column dimension but want to monitor more cannabinoids, a method for 13 analytes was developed. This method uses the same column dimension and mobile phases but requires a higher flow rate and two additional minutes of run time (Table II). The chromatogram for 13 analytes run under these conditions is displayed in Figure 3. This method was also used to analyze hemp oil and CBD hemp flower to demonstrate applicability to real-world samples (Figure 4A and 4B).

Table II: Method conditions for the analysis of 13 cannabinoids.

Column: Raptor ARC-18 50 mm x 3.0 mm ID, 2.7 µm (cat.# 9314A5E)
Guard Column: Raptor ARC-18 EXP guard column cartridge 5 mm x 3.0 mm ID, 2.7 µm (cat.# 9314A0253)
Mobile phase A: Water, 5 mM ammonium formate, 0.1% formic acid
Mobile phase B: Methanol, 0.1% formic acid
Column Temp: 50 °C
Sample Concentration: 50 ppm
Injection Volume: 3 µL
Detector: UV/Vis monitored at 228 nm
Flow Rate: 1.0 mL/min
  Time (min) %B
  0.00 65
  5.00 70
  6.50 70
  7.50 80
  8.50 80
  8.51 65
  10.00 65
 

Figure 3: Chromatogram obtained for 13 cannabinoids in solvent by the method conditions outlined in Table II.

 

Figure 4A: Chromatogram obtained from conditions outlined in Table II for hemp oil.

Hemp oil was prepared by aliquoting 50 µL of oil and adding 950 µL of acetonitrile. After vortexing for 30 seconds, 750 µL was transferred to a vial and 250 µL of water added. The sample was vortexed, and a 1:20 dilution was performed. Analytes were spiked at 50 ppm.

Figure 4B: Chromatogram obtained from conditions outlined in Table II for CBD hemp flower.

Flower was prepared by weighing 500 mg in a centrifuge tube and extracted with 10 mL of 80/20 methanol/water. Samples were vortexed for 15 seconds and sonicated for 5 minutes for 3 cycles. Flower was centrifuged at 4000 rpm for 5 minutes. Supernatant was diluted at 1:50 and all analytes spiked at 50 ppm, except CBDA which was measured at endogenous levels.

 

This methodology is useful for labs that want to offer a rapid, extended panel to their customers using a 50 x 3 mm column dimension. It also includes both delta-10-THC compounds and achieves great resolution between the two epimers.

Advantages of Larger Column Dimensions

Resolving difficult neighboring compounds in a potency panel sometimes requires using larger column dimensions, which offer increased column efficiencies compared to shorter column dimensions. Despite popular belief, a longer column does not always equate to a longer analysis time. To demonstrate the advantage of larger column dimension, and thus higher resolving power, a 150 mm x 3 mm, 2.7 µm column dimension was utilized under the conditions outlined in Table III. This developed method allowed for the full resolution of 15 cannabinoids in just 10 minutes (Figure 5).

Table III: Method conditions for the analysis of 15 cannabinoids.

Column: Raptor ARC-18 150 mm x 3.0 mm ID, 2.7 µm (cat.# 9314A6E)
Guard Column: Raptor ARC-18 EXP guard column cartridge 5 mm x 3.0 mm ID, 2.7 µm (cat.# 9314A0253)
Mobile phase A: Water, 6 mM ammonium formate, 0.1% formic acid
Mobile phase B: Acetonitrile, 0.1% formic acid
Column Temp: 30 °C
Sample Concentration: 50 ppm
Injection Volume: 3 µL
Detector: UV/Vis monitored at 228 nm
Flow Rate: 0.8 mL/min
  Time (min) %B
  0.00 70
  8.00 74
  8.01 70
  10.00 70
 
 

Figure 5: Chromatogram obtained for 15 cannabinoids in solvent by the method conditions outlined in Table III.

 

In this example, there are several key factors that allowed for the separation of all 15 analytes and, in particular, the exo-THC and CBNA cannabinoids. In addition to using a larger column dimension, the organic modifier was changed to acetonitrile (rather than methanol, which was used in the previous examples), and the buffer concentration was increased. Exo-THC can be difficult to add into potency methods because it is an isomer of THC and is often challenging to resolve from THC, especially on smaller dimension columns. Switching the organic modifier from methanol to acetonitrile helped drive the selectivity change needed to resolve exo-THC and delta-9-THC. Buffer concentration is another parameter at play. CBNA, and other acidic cannabinoids to some degree, are very sensitive to buffer concentration. Analytes will elute later in the chromatogram when less buffer is utilized and earlier when more buffer is used. Here, the buffer concentration was optimized to allow for full separation in under 10 minutes. This same method was then applied to hemp oil and CBD hemp flower to further demonstrate its applicability to these samples (Figure 6A and Figure 6B).

 

Figure 6A: Chromatogram obtained from conditions outlined in Table III for hemp oil.

Hemp oil was prepared by aliquoting 50 µL of oil and adding 950 µL of acetonitrile. After vortexing for 30 seconds, 750 µL was transferred to a vial and 250 µL of water added. The sample was vortexed and a 1:20 dilution performed. Analytes were spiked at 50 ppm.

Figure 6B: Chromatogram obtained from conditions outlined in Table III for CBD hemp flower.

Flower was prepared by weighing 500 mg in a centrifuge tube and extracted with 10 mL of 80:20 methanol:water. Samples were vortexed for 15 seconds and sonicated for 5 minutes for 3 cycles. Flower was centrifuged at 4000 rpm for 5 minutes. Supernatant was diluted at 1:50 and all analytes spiked at 50 ppm, except CBDA which was measured at endogenous levels.

 

Although not ideal, sometimes it is necessary to extend run times, especially when dealing with hydrophobic compounds that tend to stick to the stationary phase. Tetrahydrocannabinol acetate, or THCO, is one such compound that has gained a lot of attention in the industry lately. This compound can be tricky to add into methods due to its affinity for the column, typically requiring a high organic mobile phase to elute. Here, a method was developed with the conditions outlined in Table IV using the previous analyte list with the additional THCO acetate with overall cycle time of 12 minutes.

Table IV: Method conditions for the analysis of 16 cannabinoids.

Column: Raptor ARC-18 150 mm x 3.0 mm ID, 2.7 µm (cat.# 9314A6E)
Guard Column: Raptor ARC-18 EXP guard column cartridge 5 mm x 3.0 mm ID, 2.7 µm (cat.# 9314A0253)
Mobile phase A: Water, 6 mM ammonium formate, 0.1% formic acid
Mobile phase B: Acetonitrile, 0.1% formic acid
Column Temp: 30 °C
Sample Concentration: 50 ppm
Injection Volume: 3 µL
Detector: UV/Vis monitored at 228 nm
Flow Rate: 0.8 mL/min
  Time (min) %B
  0.00 70
  8.00 74
  8.01 100
  10.00 100
  10.01 70
  12.00 70
 

Figure 7: Chromatogram obtained for 16 cannabinoids in solvent by the method conditions outlined in Table IV.

 

Figure 8A: Chromatogram obtained from conditions outlined in Table IV for hemp oil.

Hemp oil was prepared by aliquoting 50 µL of oil and adding 950 µL of acetonitrile. After vortexing for 30 seconds, 750 µL was transferred to a vial and 250 µL of water added. The sample was vortexed, and a 1:20 dilution was performed. Analytes were spiked at 50 ppm.

Figure 8B: Chromatogram obtained from conditions outlined in Table IV for CBD hemp flower.

Flower was prepared by weighing 500 mg in a centrifuge tube and extracted with 10 mL of 80:20 methanol:water. Samples were vortexed for 15 seconds and sonicated for 5 minutes for 3 cycles. Flower was centrifuged at 4000 rpm for 5 minutes. Supernatant was diluted at 1:50 and all analytes spiked at 50 ppm, except CBDA which was measured at endogenous levels.

 

Choosing the right column dimension for your laboratory’s HPLC cannabis testing depends largely on your analytes of interest. A small panel of cannabinoids can easily be analyzed using a shorter 50 mm length column. For larger panels, a larger column might be necessary, but it is important to remember that a longer column does not always result in a longer run time. In cases where there is a need to resolve challenging compounds, there are many tools at your disposal to help achieve the desired result. Larger column dimensions can give more resolving power than smaller column dimensions, but it is also good to consider buffer concentration and organic modifier to drive selectivity changes.

References

  1. J. York, Analyzing THC concentrates? Look for isomers!, ChromaBLOGraphy, Restek Corporation, 2022.
  2. J. York, How extra-column volume affects cannabinoids analysis and LC column choice, Technical article, FFAR3688-UNV, Restek Corporation, 2022.
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