Out of the 145 endogenous metabolites in the standard mix, five (1-methylnicotinamide, thiamine, choline, spermine and spermidine) were not detectable even at the highest concentration level (10 µM, approximately 1860 ng/mL). Isomers were not separated in some cases. These included 2-/3-phosphoglyceric acid, citric/isocitric acid, homoserine/threonine and isoguanosine/guanosine. The hexoses fructose, galactose, mannose, glucose and inositol were not distinguishable, as well as their phosphates (fructose-6-phosphate, glucose-1-phosphate and glucose-6-phosphate) as they were co-eluting and therefore the were not baseline separated. Pentose-phosphates, ribose-5phosphate and ribulose-5-phosphate were not baseline separated, too. For arginine and palmitic acid, no satisfactory linear regression was possible, most likely due to severe carry-over effects. The setup of the dual column approach is simple but requires two independent pumps. Thus, our dual column approach might not be transferable to another laboratory without acquiring specific equipment before. A limitation of our study was the selection of the real-life samples as both sample sets are not matched in size, demographic background and storage duration. However, these samples still have the power to showcase the unique capacities of the developed approach to detect various chemicals from endogenous and exogenous sources. Moreover, only a single reference material was analysed as experimental sample for plasma. Nonetheless, we could identify over 60 chemicals in this plasma sample showing highly convincing performance for plasma. The combination of the column effluents before introduction to the MS increases the amount of coeluting compounds, potentially causing elevated matrix effects. At the same time, the sample is diluted as the flow is doubled before the analysis. Internal standard normalization was applied for quantification. Since the set of labeled standards was limited, partially surrogate internal standards were applied reducing the reliability of the normalization for these analytes.