New specific and sensitive HPLC-assays for ethacrynic acid and its main metabolite--the cysteine conjugate--in biological material.

A reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for the determination of the loop diuretic ethacrynic acid and its potentially active main metabolite, the ethacrynic acid-cysteine conjugate, in biological material. Simple and rapid sample preparation procedures were established using solid-phase extraction for the parent drug and direct injection after one washing step for the metabolite. HPLC separation was performed on a Spherisorb ODS II (3 microns) analytical column using isocratic elution with different mixtures of mobile phases (phosphoric acid-methanol-acetonitrile-tetrahydrofuran or triethylamine buffer-methanol, respectively). The analytes were detected by measuring the UV absorption of the eluate at 275 nm. Stability studies revealed that considerable amounts of ethacrynic acid may be released from the cysteine conjugate unless the urine samples are pH stabilized (pH 3-4). The assay provided high sensitivity with limits of quantification of 20 ng ml-1 for ethacrynic acid in plasma and urine, and 240 ng ml-1 for the cysteine conjugate in urine. All validation parameters were within the required limits. For the presented assays, the applicability to pharmacokinetic studies and routine analyses was proved.