Influence of extraction solvent (buffer, methanol, acetone) and time on the quantification of indoIe‐3‐acetic acid in plants

The effect of extraction solvent and time on the measured indole‐3‐acetic acid (IAA) level was investigated in plant materials having different contents of lAA‐conjugates, Tissues from pine (Pinus sylvestris L.). tobacco (Nicotiana tabacum L.), and maize (Zea mays L.) were extracted for 1–9 h with Na‐phosphate buffer (pH 7.5). 80% methanol and 70% acetone. IAA was measured by combined gas chromatography‐selected ion minitoring‐mass spectromctry (GC‐SIM‐MS) with [13C6]‐IAA as an internal standard. Extraction of maize seedlings with buffer gave a higher estimate of free IAA than did extraction with methanol or acetone, which produced similar values. The increase in free IAA after buffer extraction was paralleled by a stoichiometric decrease in lAA‐ester conjugates, indicating that free IAA was formed during buffer extraction by hydrolysis of these conjugates, which are abundant in maize seedlings. The amount of hydrolysis during a 1‐h extraction period was estimated to be ca 3% of the total lAA‐ester pool. However, in the pine extraxylary tissues and tobacco in‐ternodes which lack a significant lAA‐ester pool, buffer extraction resulted in the same IAA estimate as extraction with the organic solvents, but produced a cleaner extract. For all the plant materials investigated, a 1‐h extraction period was sufficient for equilibrating the internal standard with the endogenous IAA pool.