Sequential hybrid three-dimensional gas chromatography with accurate mass spectrometry: a novel tool for high-resolution characterization of multicomponent samples

A novel sequential three-dimensional gas chromatography–high-resolution time-of-flight mass spectrometry (3D GC–accTOFMS) approach for profiling secondary metabolites in complex plant extracts is described. This integrated system incorporates a nonpolar first-dimension (¹D_np) separation step, prior to a microfluidic heart-cut (H/C) of a targeted region(s) to a cryogenic trapping device, directly followed by the rapid reinjection of a trapped solute into a polar second-dimension (²D_PEG) column for multidimensional separation (GC_np–GC_PEG). For additional separation, the effluent from ²D_PEG can then be modulated according to a comprehensive 2D GC process (GC×GC), using an ionic liquid phase as a third-dimension (³D_IL) column, to produce a sequential GC_np–GC_PEG×GC_IL separation. Thus, the unresolved or poorly resolved components, or regions that require further separation, can be precisely selected and rapidly transferred for additional separation on ²D or ³D columns, based on the greater separation realized by these steps. The described integrated system can be used in a number of modes, but one useful approach is to target specific classes of compounds for improved resolution. This is demonstrated through the separation and detection of the oxygenated sesquiterpenes in hop (Humulus lupulus L.) essential oil and agarwood (Aquilaria malaccensis) oleoresin. Improved resolution and peak capacity were illustrated through the progressive comparison of the tentatively identified components for GC_np–GC_PEG and GC_np–GC_PEG×GC_IL methods. Relative standard deviations of intraday retentions (¹t_R, ²t_R,, and ³t_R) and peak areas of ≤0.01, 0.07, 0.71, and 7.5% were achieved. This analytical approach comprising three GC column selectivities, hyphenated with high-resolution TOFMS detection, should be a valuable adjunct for the improved characterization of complex plant samples, particularly in the area of plant metabolomics.