Background: Amantadine (1-adamantylamine) is used for treatment of influenza, hepatitis C, parkinsonism, and multiple sclerosis. Current amantadine analysis by HPLC or gas chromatography (GC) requires a laborious sample pretreatment with extraction and/or derivatization steps. We established an LC-MS/MS method without protein precipitation, centrifugation, extraction and derivatization steps.
Material and methods: 50 microl sample+50 microl of 0.4 mg/l 1-(1-adamantyl)pyridinium bromide as internal standard+1000 microl water (96-well plate). Of this 25 microl+500 microl water (96-well plate; final serum dilution 1:462). LC-MS/MS: Surveyor MS pump, Autosampler, triple-quadrupole TSQ Quantum mass spectrometer (Thermo Electron). Autosampling: 2 microl of each sample. Chromatography: isocratic water/acetonitrile (60/40 v/v) with 5 g/l formic acid, flow rate 0.2 ml/min, run time 3 min, Phenomenex Luna C8(2) (100 x 2.0 mm (i.d.); 3-microm bead size) column. Mass spectrometry: electrospray atmospheric pressure ionization, positive ion and selective reaction monitoring mode, ion transitions m/z 152.0-->135.1 (at 22 eV amantadine) and 214.1-->135.1 (at 26 eV internal standard).
Results: Calibration curves were constructed with spiked serum samples (amantadine 50-1000 microg/l, r>0.99). No carry over (5000 microg/l). No ion suppression with retention times similar to those of amantadine (1.8 min) and the internal standard (2.1 min). Detection limit 20 mg/l, linearity 20-5000 mg/l, intra-assay/inter-assay CV<6%/<8%, recovery 99-101%. Method comparison: LC-MS/MS=1.23 x GC-45 (Passing-Bablok regression). No significant bias between GC and LC-MS/MS (Bland-Altman plot).
Conclusion: We consider the sample pretreatment without deproteination, derivatization and centrifugation steps and the specificity of the tandem mass spectrometry as the most important points of our amantadine analysis method.