Determination of sodium 3,4-diaminonaphthalene-1-sulfonate, a Congo Red derivative, in plasma and brain of hamsters by high-performance liquid chromatography after solid-phase extraction and ultraviolet absorbance

Abstract

In the search for compounds with similar or greater activity than Congo Red (CR) in protecting normal prion protein from being converted into the pathological form, we have synthesized various compounds which derive from CR. One of these is the sodium 3,4-diaminonaphthalene-1-sulfonate (RCA) which has an activity similar to CR in preliminary experiments. This study describes a method to determine RCA in plasma and in brain tissue by high-performance liquid chromatography (HPLC), using a solid-phase extraction and UV detection. RCA is an amphoteric molecule difficult to separate from biological matrices. Extraction was achieved by solid-phase extraction (ENV+ columns) together with the use of a counter ion. The resulting solid-phase extraction is efficient and rapid. RCA was separated on a Symmetry C 18 250×4.6 mm I.D. 5 μm column at 1 ml/min using a 50 m M Na 2SO 4 in 5 m M tetra- n-butylammoniumiodide (TEBA) in water–methanol (82:18, v/v) mobile phase. Retention times of RCA and I.S. were 21 and 24 min. The UV detector was set at 210 nm. The limit of quantitation was 0.5 μg/ml. The method has intra-assay and inter-assay accuracies higher than 95%, coefficients of variation ranging between 2.8 and 8.6%, and recovery rates between 74.3 and 80.1% in plasma and in brain tissue. A linear response to quantities of RCA from 0.5 to 100 μg/ml or 10 μg/g in plasma or brain was obtained. The present method allows the study of the pharmacokinetics of RCA in plasma after i.p. administration, and the distribution of the compound into the brain at the peak time.