Immunoaffinity chromatography of abscisic acid combined with electrospray liquid chromatography–mass spectrometry

Polyclonal antibodies with high specificity for C1-immobilised (+)-cis,trans-abscisic acid (ABA) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of an immunoaffinity chromatography (IAC) gel. The detection limit of the ELISA was approximately 4.6 × 10−10 mol/L. Sensitive electrospray liquid chromatography–mass spectrometry (LC–ESI-MS) methods were also developed with detection limits below 0.1 × 10−12 mol. The IAC allowed quick, single-step processing of samples prior to the analyses. The LC–ESI-MS and LC–ELISA techniques were used for comparative estimation of endogenous ABA levels in immunoaffinity purified extracts of normal and water-stressed Nicotiana tabacum L. leaves. The analytical approaches were validated using deuterium- and tritium-labelled internal standards, respectively. The IAC method was found to be highly effective, sensitive and convenient for isolating the target analyte from plant material.