Determination of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione in plasma by direct injection into a coupled column liquid chromatographic system.

The chemical substance 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) is in clinical use for the treatment of hereditary tyrosinemia type 1. In the present study, the plasma concentration of NTBC was determined by a coupled column liquid chromatographic method. A 20-microl volume of plasma was diluted with phosphate buffer, pH 2, and injected into a small precolumn (BioTrapAcid C18) with a mobile phase containing sulfuric acid. The precolumn was based on the restricted access principle, i.e., retention of NTBC within the lipophilic pores, while polar and large endogenous compounds were eluted with the void volume. NTBC was transferred to the analytical column using a mobile phase with a high content of acetonitrile. The compound was monitored by UV detection at 278 nm. The standard curve was linear between 0.3 and 69 microM, and the between-day precision (RSD) was 3% (n=6 days) at 13.8 microM and 14% (n=6 days) at 0.3 microM NTBC in plasma. The quantitation limit was approximately 0.3 microM using 20 microl of plasma.