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 for citric acid, 100-10000 ng mL for cis-aconitic acid, and 1-100 ng mL 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.

Improving liquid chromatography-tandem mass spectrometry determination of polycarboxylic acids in human urine by chemical derivatization. Comparison of o-benzyl hydroxylamine and 2-picolyl amine

“…In general, transitions involving the neutral loss of 123 Da and 108 Da were selected for quantification of oBHA and 2-PA derivatives, respectively. Transitions for oBHA derivatives were similar to those previously published [25].…”

“…Furthermore, other factors such as derivatizing agents' volume, reaction temperature and derivatization time was optimized for both oBHA and 2-PA. Regarding oBHA, the addition of 50 µL of EDC 1 M and oBHA 1 M in standards and samples and the reaction time of 1 hour at room temperature previously reported for other matrices [24,25] were found to be the optimal conditions also in urine. The addition of larger volumes, larger reaction times or higher temperatures did not improve reaction yield (Supplementary Figure 2).…”

“…Tricarboxylic acids such as CA are more prone to suffer from incomplete derivatization. Although this undesirable effect has not been reported in culture cell [25], it is a potential limitation in more complex matrices like urine. Since reaction yield can differ sample to sample, it was monitored by selection of a transition for the bis-derivatized form ( Table 1).…”

“…A broad variety of derivatizing agents are used in LC-MS/MS. For instance, Girard's reagents and hydroxylamine are used for derivatizing ketones [16,17], dansyl chloride is used for aliphatic alcohols [18] phenols and amines [19], 2-picolyl amine (2-PA) and N-methyl-2phenylethanamine are used for carboxylic acids [20][21][22][23] and o-benzylhydroxyl amine (oBHA) [24,25] is used for the derivatization of both ketones and carboxylic acids.…”

“…Previous work performed by our research group demonstrated the usefulness of 2-PA for the determination of monocarboxylic acids in complex matrices such as urine [21,22] although its suitability for derivatization of polycarboxylic acids in complex matrices remains unexplored. On the other hand, the usefulness of oBHA for the derivatization of polycarboxylic acids has been demonstrated [24,25] although its suitability for the determination of these metabolites in complex matrices has not been proved. Thus, the aim of this study is to compare 2-PA and oBHA derivatizing agents for the determination of poly-carboxylic acids in human urine.…”