IMPROVED ACCURACY AND PRECISION OF GAS-CHROMATOGRAPHY MASS-SPECTROMETRY MEASUREMENTS FOR METABOLIC TRACERS

The use of stable-isotope tracer methodology to study substrate metabo lic kinetics requires accurate measurement of the tracer to tracee rat io (TTR), often by gas chromatography/mass spectrometry (GC/MS). Many approaches for measurement of the TTR by GC/MS do not use standards of known isotopic enrichment to control for variability in instrument re sponse. In addition, most GC/MS applications exhibit some degree of co ncentration dependency whereby the measured ion abundance ratio varies with the quantity of sample analyzed, thereby placing a limitation on the accuracy of isotopic enrichment standard curves unless the quanti ties of standards and samples analyzed are closely matched. We documen t the degree to which day-to-day variability can affect the instrument response for several GC/MS analyses of metabolic tracers when isotopi c enrichment standards are not used to control for variable instrument response. Furthermore, we report a new approach that incorporates con centration dependencies within a standard curve to improve the accurac y and precision of TTR measurements over a range of sample quantities analyzed. The new approach was applied to plasma samples obtained from experimental protocols performed in human subjects with three commonl y used tracers: H-2(2)-palmitate, N-15(2)-urea, and C-13-leucine. Vari ability in the day-to-day instrument response was 84% and 26% for H-2( 2)-palmitate and N-15(2)-urea, respectively; in addition, up to 10% va riability due to concentration dependency was noted for these applicat ions, The new approach virtually eliminated these sources of variabili ty. After controlling for concentration dependency, a threefold reduct ion in the standard error was noted when the enrichment of C-13-leucin e measured by electron-impact (El) ionization GC/MS was correlated aga inst negative chemical ionization (NCI) GC/MS. These data demonstrate that our new approach decreases the errors in TTR determination caused by variations in instrument response and concentration dependency. Th is approach is generically applicable, and can improve the accuracy an d precision of TTR determinations for most GC/MS analyses. Copyright ( C) 1998 by W.B. Saunders Company.