Optimised separation procedures for the simultaneous assay of three plant hormones in liquid biofertilisers

Introduction – The overuse of petrochemical‐based synthetic fertilisers has caused detrimental effects to soil, water supplies, foods and animal health. This, in addition to increased awareness of organic farming, has generated considerable interest in the evaluation of renewable biofertilisers. Objective – The three objectives of the current research were: (1) to evaluate and optimise a solid phase extraction procedure for extraction of three plant hormones, IAA, GA₃ and ABA from two model biofertilisers produced from coconut shells and pineapple peels; (2) to develop an HPLC analysis procedure for the simultaneous separation and quantification of three plant hormones (IAA, GA₃ and ABA); and (3) to evaluate the changes in three plant hormones levels at four different fermentation time periods and varying number of general bacteria, lactic acid bacteria and yeast. Result – An optimised procedure for sample preparation, separation and simultaneous analysis of three plant hormones [indole‐3‐acetic acid (IAA), gibberellic acid (GA₃) and abscisic acid (ABA)] produced in liquid biofertilisers was developed. This method involves sample cleanup using a Sep‐pack Oasis^®MAX cartridge containing mixed‐mode anion‐exchange and reverse‐phase sorbents that provided optimum recovery of 85.6, 91.9 and 94.3%, respectively, for the three hormones, IAA, GA₃, and ABA. Baseline separation of three hormones was achieved using mobile phase consisting of 1% acetic acid and acetonitrile (75:25, v/v) at pH 4.0. The amounts of hormones produced in liquid biofertilisers were influenced by fruit types, fermentation time and total number of general bacteria, lactic acid bacteria and yeasts. The quantities of three plant hormones produced during fermentation correlated well with the total number of microorganisms present in the liquid biofertilisers. Conclusion – A simple and rapid sample preparation procedure followed by RP‐HPLC with UV detection was optimised and developed for simultaneous quantification and identification of three plant hormones namely, IAA, GA₃ and ABA in the liquid biofertilisers. This procedure allows quantification of the three plant hormones in their natural states without any prior derivatisation step. The results presented illustrate that the contents of the three plant hormones depended on the type of fruit wastes, fermentation time and the number of microorganisms found in liquid biofertilisers. This method can be extended to determine the quantity of three hormones in other matrices. This assay procedure will aid in the development of liquid biofertilisers, a valuable alternative fertilisers to promote plant growth. This process will help farmers to reduce production cost and pollution problems. Copyright © 2009 John Wiley & Sons, Ltd.