Quantitative analysis of 2-oxoglutarate in biological samples using liquid chromatography with electrochemical...

This paper describes a technique for quantitative analysis of 2-oxoglutarate (alpha-ketoglutarate) in biological samples using liquid chromatography with electrochemical detection (LC-EC). This method utilizes a simplified, efficient sample preparation designed to select for 2-oxoglutarate and similar compounds by derivatization with phenylhydrazine. 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 detectable concentration was 25 ng/ml. To test the ability of the assay to measure 2-oxoglutarate in biological samples, this method was used to quantitate the 2-oxoglutarate content in chick osteoblast cultures and to 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 forementioned groups. The 2-oxoglutarate content of these cells was 6.67 +/- 1.20 ng/micrograms DNA or 105 +/- 18 ng/cell layer (mean +/- 95% confidence interval) and the assay accurately measured the standard addition. This method was also used to quantitate 2-oxoglutarate content in whole embryonic chick calvariae containing 6.40 +/- 0.95 ng/mg dry bone weight or 37.5 +/- 5.5 ng/bone. This assay provides significantly lower detection limits than the currently available procedures and is suitable for determination of 2-oxoglutarate in biological samples where very low amounts of 2-oxoglutarate are found. This method is the first application of LC-EC for quantitating 2-oxoglutarate.