Bw. Patterson et Rr. Wolfe, CONCENTRATION-DEPENDENCE OF METHYL PALMITATE ISOTOPE RATIOS BY ELECTRON-IMPACT IONIZATION GAS-CHROMATOGRAPHY MASS-SPECTROMETRY, Biological mass spectrometry, 22(8), 1993, pp. 481-486
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.