Development of a liquid chromatography-mass spectrometric method for the measurement of binding of memantine to different melanins

A sensitive and selective liquid chromatography–mass spectrometric method was validated for the determination of free memantine in melanin binding studies. The sources of melanin studied were sepia, synthetic and bovine melanin. Memantine was chromatographed on a reversed-phase column (Prodigy 5 μm, ODS(3), 100 Å, 100×4.6 mm) using gradient elution with mobile phases of 0.1% formic acid in deionised water and 0.1% formic acid in methanol at a flow-rate of 0.8 ml/min. The mode of ionisation was atmospheric pressure–electrospray and detection by single ion monitoring of the memantine ion m/z 180. Validation of the method showed that the assay was linear from 0.1 to 1200 nM and 0.5 to 1200 nM memantine in deionised water and phosphate-buffered saline (PBS), respectively. Accuracy for sample preparations in deionised water was between 80 and 108% and between 80 and 123% for PBS. For both media, intra- and inter-day precision was below 1% for retention time and below 5% for analyte peak area. At the LLOQ, the variation of peak area was less than 17%. Binding of memantine to melanin was measured indirectly by determining the unbound fraction of memantine. After incubation of melanin with memantine, the sample was centrifuged and filtered to separate the memantine–melanin complex effectively from suspension. The filtrate was then assayed for free memantine from which the extent of binding was then calculated