Quantitation of pyrrole-imidazole polyamide in rat plasma by high-performance liquid chromatography coupled with UV detection.

A simple and robust method using high-performance liquid chromatography with UV detection was developed and validated for the determination of six pyrrole-imidazole (PI) polyamides (HN.49, TGF-β1f, TGF-β1t, HN.50f, HN.50t, and LOX-1) in rat plasma. After the plasma proteins were precipitated with methanol containing phenacetin as an internal standard, the analytes were separated on a Luna C18 (2) (5 μm, 4.6 × 150 mm). Calibration curves were linear over the range of 0.5 to 200 μg/mL for HN.49, 0.25 to 200 μg/mL for TGF-β1f, TGF-β1t, HN.50t, and LOX-1, 1 to 200 μg/mL for HN.50f in rat plasma. The inter- and intraday precision were below 15%, and the accuracy was within 15% at the quality controls. The validated method was successfully applied to sample analysis for the pharmacokinetic study.