Rapid spectrophotometric determination of fosfestrol following on-line hydrolysis by alkaline phosphatase using flow injection and chasing zones Original Research Article

A new flow injection (FI) method is reported for the spectrophotometric determination of fosfestrol (diethyl-stilbestrol (DES) diphosphate) in pharmaceutical formulations. The proposed method is based on the on-line hydrolysis of the analyte by alkaline phosphatase (Al-Pase) using a “chasing zones” FI manifold. The orthophosphate ions, thus, generated are determined spectrophotometrically (λmax=690 nm) using the molybdenum blue approach. The chemical and FI variables affecting the enzymatic reaction were investigated. The proposed method is very precise (sr=1.1% at 1×10−4 mol l−1 fosfestrol, n=12), fast (allowing up to 40 samples h−1 to be analyzed) and has a determination range of 2×10−5 to 2×10−4 mol l−1, with a satisfactory 3σ detection limit of 5×10−6 mol l−1. The method was shown to provide accurate determinations of the fosfestrol concentration in a pharmaceutical formulation, giving relative errors, er, of +0.6 and −0.5% compared to the value stated by the supplier (Asta Medica Inc.) and the concentration derived using a method recommended by the United States Pharmacopoeia XXI, respectively. In addition, the average recoveries of known amounts of the analyte ranged between 99.2 and 101.2%.