Isotope ratios must be measured precisely when stable isotopic tracers are used for in vivo metabolic kinetic studies since low enrichments are generally achieved above relatively high natural abundance backgrounds. We have observed that the (m + 1)/(m + 0) isotope ratio for the molecular ion of methyl palmitate (measured by electron impact ionization selected ion monitoring gas chromatography/mass spectrometry) is limited by a dependence of the isotope ratio on the quantity of sample analyzed. Since it is not practical to analyze exactly the same quantity of sample in a series of samples, this concentration dependence decreases the apparent precision of the isotope ratio measurement. The apparent natural abundance (m + 1)/(m + 0) isotope ratio increased from approximately 0.185 (0.2 nmol analyzed) to 0.20 (2.0 nmol). The concentration dependence was not altered as the quadrupole tuning resolution was varied and did not appear to arise from system non‐linearity. The major source of concentration dependence derived from hydrogen abstraction during ion–molecule collisions within the ionization chamber. The concentration dependence was decreased as the repeller voltage increased and ion residence times in the source were reduced. Furthermore, the integrated (m + 1) peak area increased with the square of the (m + 0) peak area, consistent with a bimolecular ion–molecule collision process. A simple mathematical correction for the concentration dependence afforded a ten‐fold improvement in the coefficient of variation of the isotope ratio measurement. Although hydrogen abstraction has been a well‐recognized phenomenon in electron impact ionization sources for many decades, the effects of this process on the apparent precision of isotope ratio measurements has not been generally acknowledged in applications involving in vivo metabolic tracers.