Analysis of phytohormones in vermicompost using a novel combinative sample preparation strategy of ultrasound-assisted extraction and solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry

Abstract

Vermicompost (VC), a widely used premium organic fertilizer, is the by-product of symbiotic interactions between earthworms and microorganisms living within them. It has been postulated that phytohormones are plausible "magic compounds" in VC that are responsible for making them such good fertilizers. Thus, a novel approach involving ultrasound-assisted extraction (UAE) and solid-phase extraction (SPE) was developed as a fast and efficient sample preparation method to screen for different classes of phytohormones in VC by liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Nine phytohormones from three different classes, including trans-zeatin (tZ), kinetin (K), N(6)-[2-isopentyl]adenine (iP), N(6)-benzyladenine (BA), N(6)-isopentenyladenosine (iPR), indole-3-acetic acid (IAA), 4-[3-indolyl]butyric acid (IBA), 1-naphthaleneacetic acid (NAA) and (+)-abscisic acid (ABA), were simultaneously screened. The extraction parameters influencing UAE efficiency were optimized to provide comparable recovery to the conventional mix-stirring (MSt) method. The optimized UAE method was subsequently applied on the analysis of phytohormones in VC, i.e. phytohormone extract was further pre-concentrated and purified using C18 and MCX SPE cartridges prior to LC-MS/MS analysis. The following phytohormones, namely iP, iPR and IAA, were detected and quantified to be 0.49, 0.53, 79.78ngg(-1), respectively; tZ was found to be below the limit of quantitation. Recoveries of 10.2%, 9.1%, 18.9% and 0.3% for tZ, iP, iPR and IAA were obtained. This is one of the few reported works for the successful detection and quantitation of cytokinins and auxins in VC, that provided the key empirical evidence to explain the growth efficacy of applying VC in promoting plant growth. Additionally, this pioneering work could potentially be applicable for the analysis of other types of organic fertilizers such as composts and activated composted materials awaiting phytohormone analyzes for quality assessment and control.

Keywords: Auxins; Cytokinins; Liquid chromatography–tandem mass spectrometry; Phytohormones; Solid-phase extraction; Ultrasound-assisted extraction; Vermicompost.