Micro analysis of volatile metabolites in biological fluids by gas chromatography



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The work described here includes the development of two methods for the concentration and analysis of organic volatiles in biological fluids and tissues. The objectives behind the development of the new techniques are; 1) to analyze samples as small as 100 microliters of biological fluid or 100 milligrams of tissue, and 2) to analyze small samples of fluid (about 2 or 3 milliliters) at low temperature. The first method involves the extraction of a 100 microliter sample with diethyl ether, concentration of the extract on a short column of glass wool by elution of the diethyl ether, and thermal desorption of the trace constituents into a chromatographic system. The second method employs gas phase stripping analysis. Pure helium is bubbled through 2 or 3 millileters of fluid in a glass tube packed with glass beads at 55°C. At this temperature 1) the profile of volatiles is a simple one making it easy to do a comparison study, and 2) minimizing the adverse effect of temperature on the biological fluid. Volatiles in the fluid are separated from the water-saturated sweep gas by adsorption on a porous polymer, and then thermally desorbed into a chromatographic system. This system allows a large area of contact between the fluid and the carrier gas. Consequently, equilibrium is rapidly achieved resulting in greater efficiency. Using the ether extraction technique, a comparison of serum profiles obtained from about 50 healthy individuals indicates that serum profiles show less variation among individuals than do urine profiles. This suggests that the technique may be applicable to disease diagnosis and studies of metabolic disorders. Both of the described methods are highly efficient and reproducible and lend themselves to other applications such as flavor and pollution studies.