Liquid-chromatographic determination of indole-3-acetic acid and 5-hydroxyindole-3-acetic acid in human plasma.

Abstract

We describe a "high-performance" reversed-phase liquid-chromatographic method for determination of indole-3-acetic acid (I) and 5-hydroxyindole-3-acetic acid (II) in human plasma. I is eluted at 1.0 mL/min with a mixture of 1-pentanesulfonic acid (pH 3.1), methanol, and water. It is detected by fluorometry. A mixture of citric acid/sodium phosphate solution (pH 4.8) and methanol, at 1.5 mL/min, is used to elute II, which is detected with an electrochemical cell. Platelet-poor plasma samples were pretreated with HCl, perchloric acid, and trichloroacetic acid for protein precipitation. Best results were obtained with the last (protein precipitation is incomplete with HCl, while recoveries of I are concentration dependent with perchloric acid). Analytical recoveries were 58% (SD 3.1%, CV 5.3%, n = 12) and 79% (SD 3.3%, CV 5.3%, n = 9) for I and II, respectively. Concentrations of I and II in plasma ranged from 0.61 to 3.32 (mean 1.54, SD 0.59, n = 15) mumol/L and from 33.0 to 102.6 (mean 51.8, SD 20.1, n = 16) nmol/L, respectively.