Maximizing molecular coverage in ultrahigh-resolution orbitrap analysis of marine dissolved organic matter using two-stage design-of-experiments strategy.

Dissolved organic matter (DOM) is one of the ocean's largest reservoirs of organic carbon, yet molecular characterization by high-resolution mass spectrometry remains highly sensitive to acquisition settings. Although Orbitrap instruments are increasingly used as a more accessible alternative to FT-ICR MS for DOM study, parameter tuning is still typically done one factor at a time, which cannot capture interactions between settings and therefore often misses truly optimal conditions. Here we apply a two-stage Design of Experiments (DoE) workflow to systematically optimize negative-mode ESI acquisition parameters on an Orbitrap Fusion Lumos Tribrid 1 M mass spectrometer using the coastal marine DOM reference material TRM-0522. We first conducted a Plackett-Burman screening design and then performed response-surface optimization by means of a Doehlert design. Of the eleven parameters tested, AGC target, resolving power, methanol proportion, auxiliary gas flow rate, and number of microscans emerged as the dominant drivers of overall spectral performance. Two independent Doehlert designs, run at resolving power 500.000 (500 k) and 1.000.000 (1 M) at m/z 200, yielded second-order polynomial models with strong predictive performance (R2 = 0.985 and 0.852, respectively). At 500 k resolving power, the response surface of Doehlert design is shaped by pronounced synergistic interactions among AGC target, microscans, and methanol proportion, effects that would be missed by single-factor optimization and that are consistent with a hardware-imposed AGC ceiling limiting ion populations at 1 M resolving power. Validation experiments confirmed that the predicted optimum at 500 k produced a 447% increase in the global analytical response relative to the pre-optimization 1 M reference condition (normalized score 0.53 ± 0.08). Overall, this work establishes a transferable optimization framework that combines a composite, multi-criterion performance score with a two-stage DoE approach applied to a certified reference material. The workflow is readily applicable across Orbitrap platforms and provides a rigorous basis for interlaboratory harmonization of high-resolution DOM characterization.