Separation of citric acid cycle intermediates by high-performance liquid chromatography with ion pairing

A method to separate underivatized tricarboxylic acid cycle intermediates within 20 min using the commonly available C15 high-performance liquid chromatography column has been developed. Ion pairing using tetrabutylammonium cations and isocratic conditions is used to separate the intermediates which are then detected at 210 nm. Separation was optimized by altering pH, the concentration of sodium sulfate and the pairing ion. This technique permits the detection of as little as 120 nmol of citrate to 0.5 nmol of fumarate. Physiological samples of rat liver mitochondria, human urine, and orange juice were analyzed.