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Simultaneous liquid chromatographic assay of amantadine and its four related compounds in phosphate‐buffered saline using 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole as a fluorescent derivatization reagent
Author(s) -
Higashi Yasuhiko,
Nakamura Shota,
Matsumura Hiroki,
Fujii Youichi
Publication year - 2006
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.577
Subject(s) - chemistry , rimantadine , chromatography , derivatization , amantadine , high performance liquid chromatography , hydrochloride , reagent , organic chemistry , pharmacology , medicine , virus , virology , influenza a virus , biology
Simultaneous HPLC assay of 1‐adamantanamine hydrochloride (amantadine) and its four related compounds [2‐adamantanamine hydrochloride (2‐ADA), 1‐adamantanmethylamine (ADAMA), 1‐(1‐adamantyl)ethylamine hydrochloride (rimantadine) and 3,5‐dimethyl‐1‐adamantanamine hydrochloride (memantine)] in phosphate‐buffered saline (pH 7.4) after pre‐column derivatization with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F) was developed. Phosphate‐buffered saline samples were mixed with borate buffer and NBD‐F solution in acetonitrile at 60°C for 5 min and injected into HPLC. Five derivatives were well separated from each other. The lower limits of detection of amantadine, 2‐ADA, ADAMA, rimantadine and memantine were 0.008, 0.001, 0.0008, 0.0015 and 0.01 µg/mL, respectively. The coefficients of variation for intra‐ and inter‐day assay were less than 6.4 and 8.2%, respectively. The method presented was applied to a binding study of these compounds to human α 1 ‐acid glycoprotein. While affinity constants and capacities for ADAMA, rimantadine and memantine were calculated by means of Scatchard plots, those for the others were not determined. ADAMA, rimantadine and memantine were bound with different affinities and capacities. These results indicate that NBD‐F is a good candidate as a fluorescent reagent to simultaneously determine amantadine and its four related compounds by HPLC after pre‐column derivatization. Our method can be applied to binding studies for protein. Copyright © 2005 John Wiley & Sons, Ltd.