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 derivatize the analytes, and stable isotope-labeled compounds were used as internal standards for precise quantification. 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/mL for malic acid and oxaloacetic acid, 0.02-2 ng/mL for succinic acid and glyoxylic acid, 4-400 ng/mL for fumaric acid, 20-2000 ng/mL for 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.