Simultaneous analysis of ten low-molecular-mass organic acids in the tricarboxylic acid cycle and photorespiration pathway in Thalassiosira pseudonana at different growth stages.

Abstract

A method using high-performance liquid chromatography coupled with tandem mass spectrometry was developed for the simultaneous determination of organic acids in microalgae. o-Benzylhydroxylamine was used to derivatizethe analytes, and stable isotope-labeled compounds were used as internal standards for precisequantification. The proposed method was evaluated in terms of linearity, recovery, matrix effect, sensitivity, and precision. Linear calibration curves with correlation coefficients >0.99 were obtained over the concentration range of 0.4-40 ng/mL for glycolic acid, 0.1-10 ng/mLfor malic acid and oxaloacetic acid, 0.02-2 ng/mLfor succinic acid and glyoxylic acid, 4-400 ng/mLfor fumaric acid, 20-2000 ng/mLfor isocitric acid, 2-200 ng mL-1 for citric acid, 100-10000 ng mL-1 for cis-aconitic acid, and 1-100 ng mL-1 for α-ketoglutaric acid.Analyte recoveries were between 80.2 and 115.1%, and the matrix effect was minimal. Low limits of detection (0.003-1 ng/mL) and limits of quantification (0.01-5 ng/mL) were obtained except cis-aconitic acid. Variations in reproducibility for standard solution at three different concentrations levels were <9%. This is the first report of the simultaneous analysis of ten organic acids in microalgae, which promotes better understanding of their growth state and provides reference value for high-yield microalgae cultures.