Determination of aerobic‐anaerobic metabolism‐related compounds in a Chaoborus flavicans population by infusion ion trap mass spectrometry of extracts of individual larvae

Abstract

In a daily migration, the aquatic larvae of Chaoborus flavicans (a phantom midge) alternate oxygen-saturated and anoxic lake strata. To investigate this cycle, larvae were collected at a natural environment, and acetate, propionate, pyruvate, lactate, glycerol, phosphate, maleate, succinate, glucose and citrate were determined. Each larva was homogenized with 200 microL water and deproteinized with a spin-filter; 50 microL aliquots were mixed with 50 microL of a buffer containing 80 mM propylamine, 20 mM HCl and 0.06 mM 2,4-dihydroxybenzoic acid (internal standard) in methanol. The extracts were infused in an electrospray ionization ion-trap mass spectrometer. The limits of detection for the [M-H](-) peaks ranged from 2 microM for pyruvate and lactate to 200 microM for acetate and glycerol. The MS(2) ion-trap spectra obtained at pH 7 (ammonium acetate buffer) were used to distinguish maleate (cis-2-butenedioic), which gave [M-CO(2)-H](-) (m/z 71), from fumarate (trans-2-butenedioic), which showed first a loss of water yielding an instable peak at m/z 97. The compounds involved in the aerobic-anaerobic adjustment of the metabolism were revealed by linear discriminant analysis. Acetate, citrate, glucose, maleate (which decreased during the daytime), and particularly succinate (which increased), showed the maximal discrimination power between the day- and night-time samples.