Investigation on the iodination reaction of methylene blue by liquid chromatography-mass spectrometry with ionspray ionisation

Radioactive iodine (I-131 and I-123) labelled methylene blue is used for th e early diagnosis of melanoma metastases. We studied the iodination reactio n of methylene blue (using "cold" iodine) in order to characterise the iodi nation product(s) as far as number and position of iodine atoms introduced on the aromatic ring(s) is concerned. The reaction was carried out under th e same experimental conditions used for the radioactive one, that is in a l arge excess of methylene blue. The ionspray HPLC-MS analysis of the reactio n mixture showed that the iodinated methylene blue was present only in a ve ry small amount and the main iodinated product was a demethylated one, comi ng out from the iodination of an impurity azure B. We also studied the iodi nation reaction of azure B in order to better explain the reaction pathway. Commercial azure B contains impurities of methylene blue and all the possi ble demethylated derivatives. HPLC-MS analysis of the reaction mixture allo wed a complete characterisation of the iodinated and bis-iodinated products . (C) 1999 Elsevier Science B.V. All rights reserved.