Determination of tricarboxylic acid cycle acids and other related substances in cultured mammalian cells by gradient ion-exchange chromatography with suppressed conductivity detection

Abstract

An ion-exchange chromatography method was established for simultaneously analyzing the tricarboxylic acid (TCA) cycle acids and other related substances in cultured mammalian cells, including citrate, cis-aconitate, isocitrate, α-ketoglutarate, succinate, malate, fumarate, oxaloacetate, trans-aconitate, phosphate, lactate and pyruvate. A Dionex 600 ion chromatograph with an ion suppressor and a conductivity detector, and an IonPac AS11-HC analytical column were employed. An NaOH gradient elution containing 13.5% methanol contributed to sufficient separation of target substances. The stability of carboxylic acids was investigated and oxaloacetate was found to be extremely unstable especially at pH 3. TCA cycle acids and other related substances in Chinese hamster ovary (CHO) cells were separated completely, and lactate, malate, phosphate, citrate and cis-aconitate were quantified due to their higher concentrations. In the quantification of the five substances, detection limits ( S/ N=3) ranged from 0.12 to 0.48 μ M, the correlation coefficients from 0.9982 to 1.0000 in their linear ranges of concentration, and the recoveries from 87 to 95%. The metabolic status of CHO cells was analyzed on the basis of the intracellular concentrations of TCA cycle acids.