Optimizing for High Throughput Analysis of Cannabinoids in a Variety of Matrices Using Ascentis^® Express C18 Column

HPLC Analysis of Cannabinoids

The final, optimized HPLC parameters are summarized in Table 2.

When developing this method, the following were considerations:

• Chromatographic resolution of all 17 compounds.
• Cycle time (i.e., run time plus equilibration) of less than 10 minutes total.
• A rugged method with consistent performance for
• >1000 injections with stable retention times, while maintaining good peak shape and response.
• Suitable for use with different matrices such as flower, chocolate, ointment, oil, concentrate, etc.

Method Calibration

Calibration for the method was from 0.01 µg/mL to 40 µg/mL. This required a high dilution for some samples in order to bring them within this analytical range. For calibration and spiking, Cerilliant^® certified reference materials (CRMs) were used. Individual cannabinoid CRMs at 1 mg/mL (with the exception of CBLA at 0.5 mg/mL) were diluted, along with the internal standard solution, directly into HPLC mobile phase component A, to prepare a 17-component stock solution at 40 µg/ mL. This stock was then diluted further into a 30:70 mixture of HPLC mobile phases A:B, for the lower concentration calibration standards.

HPLC Column: Ascentis^® Express C18

The HPLC column used for the analysis was an Ascentis^® Express C18 column, 15 cm x 2.1 mm I.D., 2 µm. Ascentis^® Express columns contain Fused-Core^® particles with a solid core and porous shell architecture, also referred to as superficially porous. This particle structure provides higher separation efficiency than fully porous particles of the same size and allows for faster analysis times with lower backpressure than approaches using smaller (< 2 µm) fully porous particles. The particle architecture of Ascentis^® Express columns allows the use of larger particles, making them suitable for both conventional and UHPLC systems. For this method, SupraRnD used a UHPLC system, although with the proper instrument optimization, successful separation can be obtained on conventional systems as well. Specifically, this would involve minimization of system dispersion. This can be done by reducing tubing length and ID at the column inlet and outlet, and for UV detectors, using a flow cell with a volume of < 5 µL.

Method Validation and Performance

Prior to choosing the Ascentis^® Express C18 for method validation, SupraRnD screened six other columns of similar chemistry from various manufacturers. They were able to achieve chromatographic resolution and a short run time with several columns but found that the Ascentis^® Express C18 was the only column that provided retention time stability – especially for the acidic cannabinoids. This is illustrated in Figure 1 which shows chromatograms of a check standard at injection #1 and injection #1140, in between which numerous sample extracts were run.

The method using the Ascentis^® Express C18 was validated in several different matrices including hop flowers (as a surrogate matrix to cannabis), hemp seed oil, CBD concentrate, and topical ointments. Recoveries from hops ranged from 85-115% over a spiking range of 0.05 to 20% by weight. A summary of this validation, as well as the other matrices, is summarized in Table 3. The method reporting limits (MRLs) achieved for the cannabinoids (except for CBDV, CBG, CBD and CBC in the concentrate) were all 0.05 wt.%. Repeatability, as % RSD, was < 4% for all matrices. Further evaluation was done using proficiency testing in which the method successfully passed for samples of cannabis flower and hemp oil.

^{*CBD recovery not quantitated due to high incurred levels
**Δ9-THC recovery not quantitated due to high incurred levels
ŦCBDV, CBG, CBD, CBC incurred in matrix led to issues preventing calculation of MRL for these compounds}

Figure 2 shows example chromatograms of hop flowers spiked at 1% w/w and at the MRL concentration of 0.05% w/w. At the much lower spiking level, where matrix interference was more apparent, all 17 cannabinoids were discernable from background peaks and could be analyzed. The specific interferences eluting next to THCV and CBL were probably due to specific terpenes present in the hop sample. These peaks were not observed in cannabis flower. In a spiked ointment sample (Figure 3), all cannabinoids were clearly detected at the MRL.

To date, > 1,550 injections have been made on a single Ascentis^® Express C18 column. SupraRnD has noted that thus far there has been no significant increase in column backpressure, or degradation in performance. Data collected on backpressure over the course of this use, showed a net increase of 2%. They also noted that retention times were stable, allowing them to identify cannabinoid peaks in samples with more confidence. An example is illustrated in Figure 4 in which two different matrices, dark chocolate and cannabis flower, are compared. Both samples contained measurable amounts of Δ9-THC, and the difference in the retention time between the two matrices were minimal.

Conclusion

After evaluating several HPLC columns, SupraRnD has successfully developed a robust and rugged method using the Ascentis^® Express C18 column for the analysis of 17 cannabinoids in a variety of matrices. Thus far, the method has been successfully applied to five different sample types including flower, ointments, chocolate, concentrates and gummies. The Ascentis^® Express C18 column was chosen for the final method based on retention time stability over repeated use, and ability to maintain chromatographic performance for the cannabinoids. In addition, the column currently in use has shown minimal increase in backpressure over the course of > 1500 injections.

* After publication of this data SupraRnD later removed the freezing step and homogenized the whole flowers at room temperature. This was done to reduce the possibility of inflating the moisture content through condensation of atmospheric water onto the cold samples.

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