Submitted to: Analytical Chemist
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/20/1999
Publication Date: N/A
Citation: Hachey,D.L., Parsons,W.R., McKay,S., Haymond,M.W. 1999. Quantitation of monosaccharide isotopic enrichment in physiologic fluids by electron ionization or negative chemical ionization GC/MS using di-O-isopropylidene derivatives. Analytical Chemistry. 71(20):4734-4739. Interpretive Summary: Using a simple modification, the pentafluorobenzoyl derivative can be made. The PFBz derivatives are about 100-fold more sensitive when analyzed by NCI GC/MS than the acetate derivative making them attractive for studies in infants and other subjects from whom limited amounts of blood are available. In addition, studies of galactose, mannose, and fructose metabolism become practical. These carbohydrates are normally absent in the fasting state, and their concentration in the fed state may be more than 50-fold lower than glucose. The NCI spectrum has a dominant molecular anion, unlike many PFBz esters that produce mainly a pentafluorobenzoyl anion. Because glucose is present at about 5-10 mmol/L-1 in plasma, the EI methods are adequate for most routine isotopomer analyses.
Technical Abstract: The aldonitrile pentaacetate and other derivatives lack ions in the electron ionization (EI) spectra possessing an intact hexose structure and thus must be analyzed by chemical ionization GC/MS in order to study multiple isotopomers. We report methods for quantitation of hexose di-O-isopropylidene acetate (IPAc) or pentafluorobenzoyl (PFBz) esters. These were prepared in a two-step procedure using inexpensive reagents that do not adversely impact the isotopomer structure of the sugar. The acetate derivative possesses an abundant [M - CH3] ion in the EI spectrum which is suitable for quantitative analysis of isotopomers. The negative chemical ionization (NCI) spectrum of the corresponding pentafluorobenzoyl derivative has a dominant molecular anion. Moreover, the PFBz derivative is about 100-fold more sensitive than the acetate, which offers some advantages for analysis of minor hexoses found in plasma. Isotopic calibration curves of [U-13C]glucose are linear over the 0.1-60% tracer/tracee range tested. The useful range for isotopic tracer studies is 25-2500 pmol for EI analysis of the acetate derivative and 0.1-55 pmol for NCI analysis of PFBz derivative (sample amount injected). For most studies where sample size is not limited, EI-GC/MS analysis of the IPAc derivative is preferred. NCI-GC/MS analysis is reserved when sample size is limiting or when studies involve hexoses other than glucose that are normally present at low concentration.