New liquid chromatographic assay with electrochemical detection for the measurement of amifostine and WR1065

A high-performance liquid chromatographic method (HPLC) was developed for the analysis of the radio- and chemo-protectant, amifostine and its active metabolite-WR1065 in deproteinized human whole blood and plasma. The two compounds were quantified by measuring WR1065 after two different sample pretreatment procedures. During these procedures, amifostine was quantitatively converted into WR1065, by incubating the sample at 37°C for 4 h at pH<1.0. The resulting amounts of WR1065 were determined by HPLC with coulometric detection (analytical cell: E₁ = 200 mV and E₂ = 600 mV; guard cell: E_G = 650 mV). The WR1065 standard curve ranged from 0.37 to 50.37 μM. The lower limit of quantitation of WR1065 was 0.25 μM. The within- and between-day precisions were ≤4.3% and ≤6.0% for amifostine, ≤4.4% and ≤3.8% for WR1065, respectively. The within- and between-day accuracy ranged from 95.4 to 97.7% and 95.4 to 97.8% for amifostine, and from 97.1 to 101.7% and 97.2 to 99.7% for WR1065, respectively. This method minimizes WR1065 loss during sample preparation, and allows for rapid analysis of both compounds on one system. Furthermore, the application of a coulometric electrode is more efficient and requires less maintenance than previously published methods for the two compounds.