QUANTITATIVE-ANALYSIS OF 2-OXOGLUTARATE IN BIOLOGICAL SAMPLES USING LIQUID-CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION

This paper describes a technique for quantitative analysis of 2-oxoglu tarate (alpha-ketoglutarate) in biological samples using liquid chroma tography with electrochemical detection (LC-EC), This method utilizes a simplified, efficient sample preparation designed to select for 2-ox oglutarate and similar compounds by derivatization with phenylhydrazin e. The response was linear over the range from 62.5 to 1000 ng/ml. The least quantifiable concentration was 62.5 ng/ml and the least detecta ble concentration was 25 ng/ml. To test the ability of the assay to me asure 2-oxoglutarate in biological samples, this method was used to qu antitate the 2-oxoglutarate content in chick osteoblast cultures and t o determine the ability of the assay to accurately measure a standard addition of 500 ng/ml 2-oxoglutarate when added to a sample of the for ementioned groups. The 2-oxoglutarate content of these cells was 6.67 +/- 1.20 ng/mu g DNA or 105 +/- 18 ng/cell layer (mean +/- 95% confide nce interval) and the assay accurately measured the standard addition. This method was also used to quantitate 2-oxoglutarate content in who le embryonic chick calvariae containing 6.40 +/- 0.95 ng/mg dry bone w eight or 37.5 +/- 5.5 ng/bone. This assay provides significantly lower detection limits than the currently available procedures and is suita ble for determination of 2-oxoglutarate in biological samples where ve ry low amounts of 2-oxoglutarate are found. This method is the first a pplication of LC-EC for quantitating 2-oxoglutarate. (C) 1994 Academic Press, Inc.