Automated analysis of levosimendan in human plasma by on-line dialysis and liquid chromatography

A fully automated method for quantitation of levosimendan, (R)-(-)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl) phenyl]hydrazono]propanedinitrile, in human plasma is described. The method involves on-line dialysis of the samples, trace enrichment of the dialysates, and reversed-phase high-performance liquid chromatography with UV detection at 380 nm. An internal standard was used to compensate for variations in the dialysis rate caused by temperature fluctuations. The precision and accuracy of the method were good. The between-day variation (RSD) was 2.7% at a plasma concentration of 15 ng/mL and 1.7% at 450 ng/mL. The limit of quantitation was 5 ng/mL with an RSD of 4.0%. The completion time of the assay was 19 min.