CONCENTRATION-DEPENDENCE OF METHYL PALMITATE ISOTOPE RATIOS BY ELECTRON-IMPACT IONIZATION GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

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 backgro unds. We have observed that the (m + 1)/(m + 0) isotope ratio for the molecular ion of methyl palmitate (measured by electron impact ionizat ion selected ion monitoring ps chromatography/mass spectrometry) is li mited by a dependence of the isotope ratio on the quantity of sample a nalyzed. Since it is not practical to analyze exactly the same quantit y of sample in a series of samples, this concentration dependence decr eases the apparent precision of the isotope ratio measurement. The app arent natural abundance (m + 1)/(m + 0) isotope ratio increased from a pproximately 0.185 (0.2 nmol analyzed) to 0.20 (2.0 mmol). The concent ration 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 abstrac tion during ion-molecule collisions within the ionization chamber. The concentration dependence was decreased as the repeller voltage increa sed and ion residence times in the source were reduced. Furthermore, t he integrated (m + 1) peak area increased with the square of the (m 0) peak area, consistent with a bimolecular ion-molecule collision pro cess. A simple mathematical correction for the concentration dependenc e afforded a ten-fold improvement in the coefficient of variation of t he 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 app lications involving in vivo metabolic tracers.