DNA N6-methyladenine (6mA) modification is widespread in prokaryotes and unicellular eukaryotes but relatively rare in multicellular organisms. Intriguingly, we found that the deoxynucleoside 6mA within the liquid chromatography (LC) flow path and causes overestimation in 6mA content. To identify the rare yet authentic presence of DNA 6mA in mammals and understand its biological functions, it is critical to eliminate the interference flow-path-related 6mA contamination. Here, we designed a flow path 6mA migration-retardation separation approach using two tandem LC columns. By this new design authentic 6mA released from mammalian genomic DNA can be completely resolved from flow-path-related 6mA contamination. Armed with this innovative design, DNA 6mA is detected with a limit of quantification of approximately 5 × 10⁻¹⁸ mol (signal-to-noise ratio (S/N) ≥ 10). Furthermore, the method was validated in complex biological matrices, demonstrating negligible matrix effects and high recovery. Application across 12 mammalian cell lines revealed that conventional single-column LC methods can overestimate 6mA levels by up to 30-fold, whereas our dual-column strategy effectively eliminates this systematic bias for accurate ultra-trace quantification.