Quantification of free auxins in semi-hardwood plant cuttings and microshoots by dispersive liquid–liquid microextraction/microwave derivatization and GC/MS analysis

Abstract

Several studies have suggested that differences in the natural rooting ability of plant cuttings could be attributed to differences in endogenous auxin levels. Hence, during rooting experiments, it is important to be able to routinely monitor the evolution of endogenous levels of plant hormones. This work reports the development of a new method for the quantification of free auxins in auxin-treated Olea europaea (L.) explants, using dispersive liquid–liquid microextraction (DLLME) and microwave assisted derivatization (MAD) followed by gas chromatography/mass spectrometry (GC/MS) analysis. Linear ranges of 0.5–500 ng mL⁻¹ and 1–500 μg mL⁻¹ were used for the quantification of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), respectively. Determined by serial dilutions, the limits of detection (LOD) and quantification (LOQ) were 0.05 ng mL⁻¹ and 0.25 ng mL⁻¹, respectively for both compounds. When using the calibration curve for determination, the LOQ corresponded to 0.5 ng mL⁻¹ (IAA) and 0.5 μg mL⁻¹ (IBA). The proposed method proved to be substantially faster than other alternatives, and allowed free auxin quantification in real samples of semi-hardwood cuttings and microshoots of two olive cultivars. The concentrations found in the analyzed samples are in the range of 0.131–0.342 μg g⁻¹ (IAA) and 20–264 μg g⁻¹ (IBA).