Isotachophoretic separation of organic acids in biological fluids

The operating conditions for the isotachophoretic separation of organic acids were evaluated. At pH values ranging from 2.90 to 4.25 both relative step heights and molar flow-rates were determined experimentally for 26 anions. Comparing the observed values with simulated data, highly significant (p = 0.0001) correlation coefficients of 0.993 and 0.920, respectively, were found at pH 3.50. Whereas the concentration of the leading electrolyte did not affect the relative step heights, it increased the molar flow-rates significantly. The same applied to the detection current. The time of analysis was observed to be a function of the concentration of the leading electrolyte. However, the time elapsed between injection of the analyte and its detection depended solely on the volume and not on the amount of analyte injected. In isotachophoresis, incomplete separation of two compounds is indicated by the occurrence of a mixed zone which can hardly be distinguished from a pure zone. Thus, knowledge of the separation capacity is a prime prerequisite in optimizing the system for the analysis of biological fluids. The separability of nine equimolar pairs of anions was determined at pH values ranging from 2.90 to 4.25. Although two ionogenic constituents would separate only when their migration rates in the mixed state were different, no clear correlation was observed between separation capacity and difference in relative mobility. Separability, however, was found to increase with increasing concentration of the leading electrolyte. While the separation capacity was not influenced by the electric current, it was significantly affected by the volume injected. In subsequent analyses of serum, cerebrospinal fluid, seminal plasma and prostatic fluid, a variety of organic acids could be detected. Calibration graphs for the detected anions revealed a detection limit of 1 nmol and linearity over their biological concentration ranges. Further, the isotachophoretic results correlated well with high-performance liquid chromatographic and enzymatic analyses of citric acid and lactic acid in human seminal plasma and cerebrospinal fluid, respectively.