Analytical Technique Optimization on the Detection of β-cyclocitral in Microcystis Species

Abstract

β-Cyclocitral, specifically produced by Microcystis, is one of the volatile organic compounds (VOCs) derived from cyanobacteria and has a lytic activity. It is postulated that β-cyclocitral is a key compound for regulating the occurrence of cyanobacteria and related microorganisms in an aquatic environment. β-Cyclocitral is sensitively detected when a high density of the cells is achieved from late summer to autumn. Moreover, it is expected to be involved in changes in the species composition of cyanobacteria in a lake. Although several analysis methods for β-cyclocitral have already been reported, β-cyclocitral could be detected using only solid phase micro-extraction (SPME), whereas it could not be found at all using the solvent extraction method in a previous study. In this study, we investigated why β-cyclocitral was detected using only SPME GC/MS. Particularly, three operations in SPME, i.e., extraction temperature, sample stirring rate, and the effect of salt, were examined for the production of β-cyclocitral. Among these, heating (60 °C) was critical for the β-cyclocitral formation. Furthermore, acidification with a 1-h storage was more effective than heating when comparing the obtained amounts. The present results indicated that β-cyclocitral did not exist as the intact form in cells, because it was formed by heating or acidification of the resulting intermediates during the analysis by SPME. The obtained results would be helpful to understand the formation and role of β-cyclocitral in an aquatic environment.

Keywords: Microcystis; SPME; analysis; volatile organic compounds.

Figure 1

Analysis of three VOCs by the solvent extraction with heating (n = 7).

Figure 2

Optimization of heating conditions for detection of β-cyclocitral by the modified solvent extraction (n = 3). (A) Heating duration, (B) Heating temperature.

Figure 3

Analysis results of β-cyclocitral, β-ionone, and β-cyclocitric acid after acidification followed by storing for 0.5, 1, 3, 6, and 24 h (n = 3).

Figure 4

Analysis results β-cyclocitral, β-ionone, and β-cyclocitric acid using the following operations (n = 3). A: Non-heat SPME, B: SPME, C: Solvent extraction after acidification, D: Solvent extraction with heating, E: Solvent extraction.