SIMULTANEOUS QUANTIFICATION OF MULTIFAMILY PLANT GROWTH REGULATORS IN GRAPES AND AVOCADOS BY QuEChERS-LC-MS/MS

LEONARDO BOLCATO; BERLI, FEDERICO; FONTANA, ARIEL

Barcelona

Conferencia; XXII European Conference on Analytical Chemistry (EUROANALYSIS 2025); 2025

European Chemical Society

Plant growth regulators (PGRs) are involved in several life cycle processes of plants such as cell division and differentiation, growth, sprouting, and maturity, as well as in protective responses. Every plant produces a number of PGRs, such as auxins, gibberellins, cytokinins, and abscisic acid (ABA), with levels varying according to the growth stage. In addition, a number of synthetic PGRs are also available and regularly used in agriculture including cytokinins such as 6-benzyl aminopurine (6-BA) and forchlorfenuron (CPPU). The quantification of PGRs in fruits is relevant not only to evaluate specific biochemical processes associated to their level’s variation but also because a common agricultural practice is the spraying of crops to optimize fruits growth and quality. In this work, a rapid and simple analytical method for the simultaneous quantification of 14 multifamily PGRs namely, gibberellins (gibberellic acid (GA3), gibberellin 4 (GA4), gibberellin 5 (GA5)), auxins (indol-3-acetic acid (IAA), indol-3-butyric acid (IBA)), cytokinins (zeatin (ZT), kinetin (KT) and 6-benzyladenine (6-BA)), abscisic acid (ABA), salicylic acid (SA), nicotinic acid (NA), methyl jasmonate (MJ) and CPPU within a single experiment is presented. The method is based on a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) sample preparation approach coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS). After optimization, the sample preparation method involved the extraction of fruit material (acidified with 1 % formic acid) with an acetonitrile-ethyl acetate mixture (1:1) for white and red grapes; and with acetonitrile for avocado by homogenization for 2 min by hands-shaking. The pH of the extract was enhanced by adding ammonium acetate and salting-out performed with 1 g of MgSO4, followed by homogenization and centrifugation. The supernatant extract was cleaned by dispersive solid phase extraction (d-SPE) using a combination of PSA and C18 sorbents (50 mg of each, per mL extract). To achieve high-throughput screening of tested samples, the final extract was analyzed directly by LC-MS/MS in positive and negative ion switching mode of electrospray ionization (ESI). The NA, IAA, IBA, KT, ZT, MJ, 6-BA and CPPU PGRs were monitored in positive ESI, whereas SA, ABA, GA3, GA4 and GA5 in negative ESI mode. Quantification was performed by scheduled multiple reaction monitoring (sMRM). All compounds were analyzed by a single chromatographic run of 18 min by using gradient chromatographic conditions. Significant matrix induced signal suppression for white grapes (9-28 %), red grapes (57-98 %) and avocado (12-66 %) extracts were observed when the responses for pre- and post-extraction addition of analytes were compared. This could be resolved by the use of matrix-matched calibration standards. The proposed method could be successfully applied in the analysis of natural and exogen incurred samples. Therefore, it is useful not only to determine the endogenous levels of PGRs to understand biochemical processes, but also in the evaluation of exogenous applications of PGRs on the basis of measured endogenous levels.