Improved HPLC method for determination of phytosiderophores in root washings and tissue extracts

Phytosiderophores (PS) of the mugineic acid family can be separated effectively by HPLC on resin-based anion exchange columns. Using gradient elution with aqueous NaOH, separation of 2'-deoxymugineic acid (DMA), mugineic acid (MA), 3-hydroxymugineic acid (HMA), 3-epi-hyydroxymugineic acid (epi-HMA), and nicotianamine was obtained within 16 min with a complete cycle time of 30 min. Fluorimetric detection was performed after post-column derivatization using sodium hypochlorite (NaOCl) and orthophtaldialdehyde. External standardization revealed a linear range between 0.1-2.5 nmol of each compound applied to the column, with a detection limit of approximately 0.05 nmol. Only minimal sample pre-treatment by addition of sodium hydroxide (NaOH) was required prior to HPLC-injection of natural occurring samples such as root exudates, collected from the whole root system or from single apical root zones, xylem sap, and hot water extracts of root material, obtained from iron (Fe)-deficient maize and barley plants. The method is discussed in comparison with cation exchange HPLC which is conventionally employed for the separation of phytosiderophores.