Supercritical fluid chromatography separates chiral synthetic cannabinoids

A study of compounds purchased online has demonstrated baseline separation of enantiomers in two racemic samples, with implications for forensic laboratories that need to characterise stereochemical composition

Researchers have demonstrated the use of subcritical or supercritical fluid chromatography to distinguish the enantiomers of selected synthetic cannabinoids. The work by a team from the Department of Physical and Macromolecular Chemistry, Charles University, Prague, Czech Republic, has addressed an analytical problem with potential forensic significance: compounds with the same molecular composition can differ in their three-dimensional structure with routine mass spectra not able to distinguish between them.

The study assessed 11 chiral synthetic cannabinoids obtained from internet vendors. High-performance liquid chromatography with optical-rotation detection indicated that two samples were racemates. The other samples did not show the same equal mixture of enantiomers, which underlined the need to characterise composition rather than assume it from a product’s stated identity.

Enantiomers are non-superimposable mirror-image forms of a chiral molecule. A racemate contains equal amounts of the two forms. Because biological receptors and enzymes depend upon three-dimensional structures they can interact differently with each enantiomer. Chemical identity alone may therefore be insufficient to describe a sample’s biological properties.

The researchers achieved simultaneous baseline enantioseparation of both racemic samples with the supercritical-fluid method. Baseline separation means that adjacent chromatographic peaks are resolved sufficiently for the signal to return to the baseline between them under the tested conditions. This provides a stronger basis for independent assessment than partially overlapping peaks, especially if the relative amounts of the enantiomers are important.

Supercritical fluid chromatography uses a compressed mobile phase, commonly based on carbon dioxide with an organic modifier, to transport analytes through a column. Temperature, pressure and composition affect its properties and the resulting separation. The term also commonly covers related subcritical operating conditions, so the precise method settings matter when one laboratory seeks to reproduce another’s results.

Chiral separation requires an environment that interacts differently with the two mirror-image forms. A suitable stationary phase can provide that distinction, which allows the enantiomers to pass through the column at different rates. The success of a separation depends on the combined behaviour of the analyte, stationary phase and mobile phase rather than on the instrument category alone.

The study also demonstrated an achiral approach with a specialised diol-bonded stationary phase. An achiral method can help separate compounds according to other chemical properties and may support an initial analytical survey. Its role is complementary to an enantiomeric method, since it does not necessarily resolve the mirror-image forms of an individual substance.

This combination is relevant to forensic workflows because laboratories often need to answer several questions about a sample. They may first need to establish which compounds are present, then determine whether a selected compound contains one enantiomer predominantly or a mixture. A single chromatographic run need not provide equally strong answers to both questions.

Optical-rotation detection adds information because chiral compounds can rotate plane-polarised light. Interpretation still requires care: the sign or magnitude of a signal cannot, without appropriate reference information, establish every aspect of absolute molecular configuration. Likewise, a small net rotation can arise from the balance between components and should be interpreted alongside the separation and the sample’s chemical identity.

Mass spectrometry remains valuable for identification but enantiomers have the same mass and can produce indistinguishable fragmentation patterns under routine conditions. A method that combines effective chiral separation with an appropriate detector can therefore supply information that mass analysis alone may lack. Compatibility of the reported separation with mass-spectrometric detection remains a further validation question.

The purchase of compounds from online vendors gave the investigation a connection with material outside a conventional reference-standard collection. However, purchased compounds are not equivalent to the full range of seized products or biological specimens.

Quantitative application would require evidence on calibration, recovery, precision, stability and the reliable detection of a minor enantiomer in the presence of a much larger peak. A visually successful separation is an essential starting point but it does not establish all those performance characteristics. Laboratories would also need to test whether extraction or storage altered the enantiomeric composition before analysis.

For further reading please visit: 10.1002/elps.70149