Journal of Biochemistry International

Multi-component natural formulations are increasingly being investigated as alternative matrices for therapeutic validation. However, establishing chemical compatibility and profiling secondary metabolites without chemical degradation remain analytical challenges. This study presents a comparative gas chromatography-mass spectrometry (GC-MS) investigation of individual extracts of Nigella sativa (NS) seeds, Sphagneticola calendulacea (SC) leaves, and their combined co-extract (NS-SC), prepared using a simultaneous cold-extraction process. The total ion chromatograms mapped highly distinctive molecular profiles for each matrix. The NS profile was characterised by the volatile therapeutic quinone thymoquinone (1.44% at 12.239 min), alongside a dense fatty-acid environment comprising hexadecanoic acid (20.95%) and major unsaturated octadecadienoate derivatives (>54%). Conversely, the SC extract yielded substantial phenolic fractions (11.47%) and 1-(+)-ascorbic acid 2,6-dihexadecanoate (8.19% at 29.710 min). Notably, the combined co-extract (NS-SC) preserved active parent markers, including thymoquinone (1.96% at 12.243 min) and core phenolic compounds, while exhibiting an increased relative peak area for the ascorbic acid derivative (14.18% at 29.830 min) within a stable fatty-acid matrix. Relative percentage values were determined using total peak-area normalisation, and chemical identities were putatively assigned by mass-spectral library matching. The absence of newly generated baseline fragments or destructive cross-reactivity indicates chemical compatibility under the extraction conditions used. These findings establish a baseline phytochemical profile for the NS-SC combination matrix and support its use in future biological evaluation.