Trace determination of α-keto acid in natural waters

Numerous chromatographic methods have been developed to detect α-keto acids in physiological or sea-water samples. These methods generally involve derivatization in a strongly acidic medium with elevated temperatures, desalting, preconcentration, and liquid-liquid extraction procedures prior to chromatographic analysis. These procedures may introduce significant errors because of adsorption losses, contamination, or decomposition of the α-keto acids. To avoid these potential problems, a chemically mild method to detect α-keto acids in sea water was developed. The method is based on the reaction of α-keto acids with 2,4-dinitrophenylhydrazine in sea water to form stable hydrazone derivatives. Desalting of the reaction mixture and preconcentration of the hydrazone derivatives is accomplished by a column-switching technique. The derivatives are separated by reversed-phase, high-performance liquid chromatography and detected by absorption spectrometry. Quantification of α-keto acids in the nM to μM concentration range shows complete recovery in sea water, excellent precision at 10-20 pmol (<5% relative standard deviation), and absorbances that are linearly related to α-keto acid concentrations. The detection limit of this method is 1-5 pmol for a 2-ml injection. Applications of this method to the detection of α-keto acids in marine sediment and sea-water samples are illustrated, and the first shipboard results are presented.