Extending Calibration Anchors Resolves Low- <i>m</i> / <i>z</i> Shifts in Negative-Mode Orbitrap Workflows

Reliable mass accuracy is essential for the confident identification of diagnostic fragment ions in high-resolution mass spectrometry. During the analysis of perfluoroalkyl sulfonic acids (PFSAs) with a Q Exactive Orbitrap, we consistently observed the characteristic O₃Ṡ^- fragment, expected at a mass-to-charge ratio (<i>m</i>/<i>z</i>) of 79.9574, reported at <i>m</i>/<i>z</i> 79.9568, a systematic deviation of approximately -6 parts per million (ppm). Similar errors affected other ions below <i>m</i>/<i>z</i> 100, while precursors and higher-<i>m</i>/<i>z</i> fragments remained within the specification. Comparison with an Exploris 120 instrument, calibrated with anchors down to <i>m</i>/<i>z</i> 59, confirmed that the deviation is not intrinsic to the ion but originates from the limited calibration range of the Q Exactive standard calibration solution. We demonstrate that extending calibration to include additional low-<i>m</i>/<i>z</i> anchors generated <i>in situ</i> by in-source fragmentation fully corrects the error without affecting the accuracy at a higher <i>m</i>/<i>z</i>. This adjustment resolves systematic deviations for ions below <i>m</i>/<i>z</i> 100 in the Q Exactive instruments.