A novel hybrid (sequential) comprehensive 2D-multidimensional gas chromatography (GC × GC-MDGC) method for complex sample manipulation and separation is described. It incorporates a separation step that approximates slow modulation GC × GC, prior to microfluidic Deans switch heart-cutting of a targeted region(s) into a third analytical column. It allows discrete single or multiple components, bands or regions, or any combination of these to be selected and excised from within the 2D GC × GC separation space. The excised individual components can be further collected and studied. Alternatively, any unresolved or poorly resolved components, or regions that require further separation, can be transferred to an additional (third) column separation step. The method is applied to separation and quantitative analysis of oxygenates in a thermally stressed algae-derived biofuel oil by using flame ionization detection (FID), without any prefractionation. This permits oxygenated compounds to be fully resolved from saturated (matrix) compounds, which are completely excluded from the third column. Improved separation was obtained between target classes (aldehydes, 2-ketones, alcohols, acids). Excellent calibration linearity, and retention time and peak area reproducibility were obtained for 14 oxy-compounds present in trace amount in the complex biofuel matrix. Accuracy of microfluidic transfer to the third column, and the profile reproducibility before and after heart-cut operations, was demonstrated by extracting single components from a complex coffee volatile sample.