Quantitative gas chromatographic determination of two oxidized metabolites of the diuretic mefruside in human urine, plasma and red blood cells.

A gas chromatographic method is reported for the quantitative analysis of two metabolites of mefruside, viz., 5-oxo-mefruside (mefruside lactone) and its hydroxy-carboxylic acid analogue in human body fluids. Use was made of extractive methylation as the derivatization technique, and quantitation was achieved, with a suitable internal standard, by means of a nitrogen-sensitive detector. Because the two metabolites are linked chemically through a lactone-open acid equilibrium, interconversion prior to their separation had to be avoided. A pH partitioning study was performed to find optimal separation conditions. The lactone could be extracted quantitatively at pH 7.4, without any trace of co-extracted hydroxy acid. The latter was extracted either at pH 2 directly (in the case of plasma and urine), or after conversion to the lactone at pH 7.4 (in the case of red cells or whole blood). Concentrations down to 25 ng per sample of both compounds could be analysed with a standard deviation of 5%. The two metabolites of mefruside equilibrated instantaneously between red cells and plasma in vitro. At 37 degrees, the red cell/plasma concentration ratio was 20 for the lactone, but only 0.1 for the open acid compound. 5-Oxo-mefruside was able to displace mefruside from its red blood cell binding sites in vitro.