Validation of a radioimmunoassay for indole-3-acetic Acid using gas chromatography-selected ion monitoring-mass spectrometry.

A radioimmunoassay for indole-3-acetic acid (IAA) has been validated by comparison with a physico-chemical assay utilizing gas chromatography-selected ion monitoring-mass spectrometry and 4,5,6,7-tetradeutero-indole-3-acetic acid as an internal standard. The radioimmunoassay provided a reliable estimate of the free IAA content of etiolated corn shoots. However, base hydrolysis of extracts for determination of ester IAA released substances which interfered with the radioimmunoassay. Interference was detected by internal controls and by lack of agreement with the mass spectral method. Interfering compounds could be removed from extracts by chromatography on diethylaminoethyl- and hydroxypropylated (lipophilic) Sephadex G-25. Following such purification the radioimmunoassay estimate of the total (free + ester) IAA content of etiolated corn shoots agreed with the mass spectral method within 2% on the average.