High-performance liquid chromatographic determination of 3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648) and its deacetyl metabolite in plasma, whole blood, urine and tissue samples in rats

A reversed-phase high-performance liquid chromatographic method was developed for the determination of 3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648, I), a new rifamycin derivative, and its 25-deacetyl metabolite (KRM-1671, II) in plasma, whole blood, tissues and urine from rats. I and II were coextracted with an internal standard from each sample matrix by solid-phase extraction (Bond Elut). Plasma and urine were directly loaded onto Bond Elut, while whole blood and tissues were homogenized and extracted with methanol or dichloromethane—chloroform prior to Bond Elut extraction. The extracts were chromatographed on Shim-pack CLC-ODS(M) using acetonitrile—0.02 M citrate buffer containing 0.1 M sodium perchlorate (2:1, v/v), and peaks were detected at 643 nm. The validation data showed that the assays for I and II in plasma, whole blood, tissues and urine were selective, accurate and reproducible.