Agilent 1100 1200 series

A calibration standard was prepared using GA₃ and ABA in the range of 0.1–1.0 mg L⁻¹. The HPLC (high performance liquid chromatography, Agilent 1100/1200 series, Agilent Technologies, Santa Clara, CA, USA) was loaded with the standards as a control with each run. The separation was accomplished on C18 column (Agilent Eclipse XDB-C18, 5 µm of pore size, 4.6 × 200 mm, Santa Clara, CA, USA) kept at 30 °C. The mobile phase was prepared by using 26% (v/v) acetonitrile prepared in HPLC grade water, filtered through PVDF (polyvinylidene difluoride) membrane filter (diameter: 13 mm, filtration rating: 0.45µm, Agilent Technologies, Santa Clara, CA, USA). The flow rate was maintained at 0.8 mL min⁻¹ and 20 µL sample of extract was injected into the column. The wavelength for the determination of ABA and GA₃ were 206 and 265 nm, respectively. The retention time for standards and hormones were monitored on the chromatograph. Using the ESTD (External Standard) quantification procedure, peak area was calculated to determine each hormone’s concentration. Following the same guidelines as before, the extracted samples and the external standard were calibrated and evaluated. It was compared to those of the calibration sample in order to determine the amount in the extracted sample.

The contents of citric and malic acids were determined by the method of Espín and Samaniego [42 ]. An aliquot of the extract was passed through a 0.22 µm PVDF Millipore membrane and placed in a capped amber vial and analyzed by High Performance Liquid Chromatography (HPLC). The analysis was carried out using the Agilent 1100/1200 series (Waldbronn, Germany). The separation was carried out using an Agilent column (300 × 6.5 mm) at a flow of 0.7 mL min⁻¹ and at a temperature of 40 °C, using a solution of 0.01 N sulfuric acid in water type I as the mobile phase. For the analysis, 10 µL of the purified extract was injected into the equipment and the organic acids were monitored using a UV-visible detector at 210 nm. The identification and quantification of citric and malic acid was carried out by comparison with their respective standards. Results were expressed in g of each organic acid per 100 g⁻¹ of dry sample.