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Chromatography 101: Resolution of Delta 8-THC and Delta 9-THC 

A blog series designed to demystify common concepts in chromatography. The Ionization Labs Chemistry Team will be posting weekly updates to this educational content series.

Introduction

The past year has seen a lot of consumers and extractors interested in a cannabinoid called delta-8-tetrahydrocannabinol, also known as d8-THC, or colloquially just “D8”. Delta 8 THC is an isomer of delta-9-tetrahydrocannabinol (d9-THC) due to a difference in the position of the endocyclic alkene [1]. This means that the compounds share the same chemical formula (C21H30O2) but are different chemical structures due the position of a carbon-carbon double bond located on the 8th or 9th carbons in Figure 1 and Figure 2. This slight difference in structure results in different chemical properties, and ultimately different legal and regulatory statuses. Therefore, accurate quantification of each isomer is required to avoid the potential for non-compliance and illegality.However, the accurate quantification of d9-THC and d8-THC is complicated by the two compounds’ chemical similarity.

Figure 1. Dibenzopyran Numbering System commonly used for naming cannabinoids. Numbering and base structure depicts the THC-type cannabinoids.

Figure 2. Differences between d8-THC and d9-THC is shown by the position of the endocyclic double bond.

The standard technology for quantification of cannabinoids is high-performance liquid chromatography (HPLC). Quantification by HPLC relies on the physical separation of compounds in a chromatography column, followed by the detection and quantification of the analytes in the HPLC detector. The response observed in the detector relates to the amount of analyte in the sample. With similar chemical structures and polarities, the two cannabinoids elute close to one another. Without a well-developed separation method, the two cannabinoids can interfere with the quantification of each other and provide incorrect results. This incorrect quantification, caused by poor separation, can lead laboratories to report inaccurate levels of d9-THC and d8-THC. It is essential to have both the D8 and D9 peaks well separated for accurate quantification.

Good Resolution

Resolution is a quantitative measure of the separation between two chromatographic peaks [2]; the larger the resolution the better the separation. The resolution, Rs, can be quantitatively measured using the equation:

Where tr is the retention time for peaks A and B, and W is the width of peaks A and B.

We can readily visualize the effect resolution has on the overall quality of the separation of 2 analytes in the example chromatograms shown in Figure 3. We see that the point where the peaks are considered fully resolved is when the resolution approaches 1.5.

Figure 3. Chromatograms showing 2 analytes with different degrees of resolution. Chromatograms were simulated using [3]

Below, Figure 4, is an example of the separation for d8-THC and d9-THC using Ionization Lab’s Cann-ID separation method. Full baseline resolution (Rs > 1.5) is observed between d8-THC and d9-THC for a 50ug/mL standard mix. Baseline resolution ensures the most accurate and reproducible quantification of d8-THC and d9-THC and limits the risk of accidentally (or negligently) mislabeling products. 

Figure 4. Example of the separation obtained by using Ionization Lab's Cann-ID separation method. Inset shows graphically how the peak statistics are calculated using Agilent’s OpenLab Chemstation software.

Bad Resolution

We were able to highlight the issues that arise from poor resolution by performing a blind inter-lab study to test a third-party lab’s ability to measure d9-THC in the presence of a predominantly d8-THC sample. We will link to that article here once the write-up is complete, but an example of the lab’s “resolution” for d8-THC and d9-THC is shown in Figure 5.

Now, It is not impossible to deconvolute coeluting peaks (peaks that are not well resolved) and achieve accurate quantitation. However, since d8-THC and d9-THC are so similar they share many physical properties like parent ion mass and UV spectra, so traditional deconvolution techniques are made considerably more complicated. The easiest and most robust route for accurate quantitation is to achieve baseline resolution (Rs>1.5) in the separation method and avoid separation methods resulting in coelutions like those shown in Figure 5.

Figure 5. Results of an inter-lab study showing the third-party lab's poor resolution between d8-THC and d9-THC.

Summary Advice

  • Always ask your third-party laboratory for your chromatographs if you are unsure about the results, especially for d8-THC products.
  • Ask for a comparison between the calibration standard and the sample chromatographs as well as the observed resolution. 
  • Avoid laboratories that are unwilling to provide supplemental information regarding your sample's test data. 
  • Another way to help labs with the quantification of these products would be having certified reference material (CRM) created with predominantly d8-THC containing some d9-THC, and vice versa. This will allow inter-lab method comparisons to determine if the lab is appropriately testing products containing appreciable amounts of d8-THC.
  • Until then, ask to see your chromatographs and utilize a trustworthy, accredited, and ethical lab. 

References