Isotope dilution analysis for the quantitation of labile compounds has been limited in applicability by the amount of sample necessary to determine specific activity. A method is described for the analysis of radiolabeled compounds which allows the direct determination of specific activity by gas chromatography. It requires the availability of the radiolabeled internal standard, as is customarily used in an isotope dilution assay, and also requires a chemically related radiolabeled compound to serve as a second internal standard. It is this second internal standard, added in known amounts, that permits quantitation of the gas chromatography. The method is illustrated by assaying indole-3-acetic acid in plant extracts using [14C]indole-3-acetic acid as the internal standard and adding [14C]indole-3-butyric acid as the second internal standard for quantitation of the gas chromatographic procedures. Used with a nitrogen-specific thermionic detector the method is selective and is sensitive at the nanogram level. The synthesis of [2-ring-14C]indole-3-butyric acid is also described.
Bibliographical noteFunding Information:
We thank Dr. Richard Leavitt and Ms. Irene Armock for help with the nitrogen thermionic detector and Dr. Charles C. Sweeley, Ms. Betty L. Schocpke, and Dr. Richard Chapman for assistance in obtaining mass spectral data (facility supported by PHS RR-00480 and Michigan State University). We thank Marianne La Haine for her expert assistance in manuscript preparation. This work was supported by National Science Foundation Metabolic Biology Grants PCM 76-12356 and PCM 79-04637 to Dr. R. S. Bandurski and is journal article 9631 from the Michigan Agricultural Experiment Station.