Spectrophotometric determination of methylene blue in biological fluids after ion-pair extraction and evidence of its adsorption on plastic polymers

Methylene blue has gained a renewed interest in medicine. It is now used in combination with visible light as a photoinactivating agent of membrane viruses such as HIV and HBV in single-donor fresh frozen plasma. MB has also been proposed for the prophylaxis and reversal of ifosfamide induced encephalopathy and in septic shock. In spite of its wide use, its pharmacokinetics in humans are little known and an improved methodology for its quantitation in biological fluids was therefore required. We report an improved spectrophotometric determination of methylene blue in whole blood, plasma and urine. Liquid-liquid ion-pair extraction of MB from the biological matrix is achieved by addition of 500 mu l (to 1.2 ml whole-blood aliquot) or 125 mu l (to 1 ml plasma or 250 mu l urine) of 5% sodium hexanesulfonate solution and subsequent extraction with 2.0 ml 1,2-dichloroethane. After centrifugation, the organic layer is assayed at 657 nm. In urine samples, the reduced form leucomethylene blue is also assayed after oxidation to methylene blue (addition of 38 mu l HCl 0.5M to 250 mu l urine sample and heating on a water bath at 70 degrees C for 1 min) prior to the ion-pairing extraction. Methylene blue can be accurately determined in spiked whole blood and plasma over the working range 0.1-9.1 mu g/ml. At higher concentrations, likely to occur in urine (up to 55 mu g/ml), the measurements deviate more than 10% from the nominal values. Analysis of spiked samples shows that methylene blue is adsorbed to some extent to polypropylene and polystyrene plastic tubes. This phenomenon is influenced by the biological matrix and is more marked with low volumes of concentrated (>3 mu g/ml) samples. At low concentrations, the adsorption is likely to occur but is more difficult to quantitate due to assay variability. This phenomenon should be kept in mind since it could potentially affect the biological activity of methylene blue. (C) 1997 Elsevier Science B.V.