Tag: Culture

The Cutting-Edge Toolkit: Unravelling Complexity with Advanced Analytical Chemistry

The groundbreaking discoveries from Stellenbosch University were made possible by employing a highly sophisticated and optimised analytical methodology: Comprehensive Two-Dimensional Liquid Chromatography hyphenated to High-Resolution Mass Spectrometry (HILIC × RP-LC-HR-MS). This advanced technique is crucial for dissecting the immense chemical complexity of the cannabis plant, where hundreds of compounds, many structurally similar, coexist across a wide range of concentrations.

Here’s a detailed look at the methodology that enabled these profound insights:

1. Sample Preparation: Isolating the Targets
   To specifically target polar phenolic compounds and avoid interference from well-known apolar compounds like cannabinoids and chlorophyll, a precise sample preparation strategy was crucial:

• Freezing and Grinding: Plant samples (inflorescence and leaves) were snap-frozen with liquid nitrogen and finely ground using a mortar and pestle. This step preserves the integrity of the compounds and maximizes extraction efficiency.
• Defatting with Hexane: The ground material was defatted three times with hexane through sonication and centrifugation. This process effectively removed non-polar cannabinoids, chlorophyll, and other lipids, ensuring that the subsequent analysis focused on the more polar phenolic compounds.
• Extraction with Aqueous Acetone: After defatting, the polar phenolics were extracted using an aqueous acetone solution (30/70 v/v H2O/acetone), followed by sonication and centrifugation. The supernatant was then evaporated, freeze-dried, and re-dissolved in a dilute H2O/MeOH solution for analysis. This selective extraction method was designed to concentrate the target compounds and minimize interference.

2. Comprehensive Two-Dimensional Liquid Chromatography (LC × LC): The Ultimate Separator
   Traditional one-dimensional (1D) liquid chromatography often struggles with complex plant extracts, as many compounds co-elute (come out of the column at the same time), making individual identification nearly impossible. 2D-LC overcomes this limitation by employing two different separation mechanisms in sequence:

• First Dimension (¹D) – Hydrophilic Interaction Liquid Chromatography (HILIC):
  • Mechanism: HILIC separates compounds based on their polarity. Polar compounds interact strongly with the stationary phase (Acquity BEH Amide column, 150 × 1.0 mm, 1.7 µm) and are retained longer, while less polar compounds elute faster.
  • Purpose: This step effectively “spreads out” the highly polar phenolic compounds, providing an initial broad separation based on a property distinct from the second dimension.
  • Dilution and Modulation: The effluent from the ¹D column was diluted with a weak reversed-phase solvent and then introduced into an interface with two 80 µL loops, acting as a modulator. This process collects small fractions from the ¹D separation and rapidly injects them onto the ²D column, preventing peak distortion.
• Second Dimension (²D) – Reversed-Phase Liquid Chromatography (RP-LC):
  • Mechanism: RP-LC separates compounds based on their hydrophobicity. Less polar compounds are retained longer on the stationary phase (Zorbax Eclipse Plus C18 column, 50 × 3.0 mm, 1.8 µm), while more polar compounds elute faster.
  • Purpose: By applying a different separation mechanism, RP-LC can resolve compounds that may have co-eluted in the HILIC dimension.
  • Fast Gradient: The ²D separation uses a very fast gradient (0.45 min) and a high flow rate (3 mL/min) to ensure rapid analysis of each ¹D fraction, maintaining high resolution.
• Orthogonality and Peak Capacity: The combination of HILIC and RP-LC is highly “orthogonal” because these two modes separate compounds based on fundamentally different chemical properties. This means compounds that co-elute in one dimension are highly likely to be separated in the other, leading to vastly improved resolution. The Stellenbosch method achieved an “excellent separation performance” with a “practical peak capacity above 3000” and an average orthogonality of 75%. This level of separation is exponentially greater than what can be achieved with 1D methods, allowing for the detection of many more individual compounds.
• Method Optimization: The team used an in-house developed predictive optimization algorithm (in Matlab R2019b) [31-33]. This software systematically explored a wide range of experimental conditions to find the optimal settings for analysis time, peak capacity, and resolution, further enhancing the method’s effectiveness.

3. High-Resolution Mass Spectrometry (HR-MS) – Quadrupole Time-of-Flight (Q-TOF): Identifying the Unknowns
   As compounds exit the ²D column, they are immediately directed to a Q-TOF mass spectrometer. This instrument is essential for identifying the separated compounds:

• Accurate Mass Measurement: Q-TOF provides highly accurate mass measurements of both precursor (intact) ions and fragment ions. This precision allows researchers to determine the exact molecular formula of an unknown compound, which is a crucial first step in identification.
• MSE Fragmentation: The instrument was operated in MSE mode, which collects both low (4 eV) and high (10-30 eV ramped) collision energy data simultaneously. Low-energy data shows the intact molecular ions, while high-energy data provides characteristic fragmentation patterns. These “fingerprints” are invaluable for elucidating the structure of compounds, even those never before seen.
• Tentative Identification: By combining accurate mass data, fragmentation patterns, UV spectral data (from the DAD detector), and relative retention times in both dimensions, researchers could tentatively identify 79 compounds, including the novel flavoalkaloids and phenolic acid sulfates.

This sophisticated analytical pipeline allowed the Stellenbosch team to peer into the complex chemistry of cannabis with unprecedented clarity, leading to discoveries that would have been impossible with less advanced techniques.