Development and validation of a liquid chromatographic method for the determination of branched-chain amino acids in new dosage forms

A reversed-phase liquid chromatographic method (RP-LC) is proposed and validated for the analysis of branched-chain amino acids (L-Leucine, L-Isoleucine and L-Valine) in new pharmaceutical formulations. The pre-column derivatization reaction of these amino acids with 2,4-dinitrofluorobenzene (DNFB) has been investigated considering the matrix effect. The compound reacts at 60°C for 10 min at pH 9 with the amino function, in presence of cetyltrimethylammonium bromide (CTAB), to give adducts that have been separated on a RP Amide C16 column and detected at 360 nm. Linear responses were observed for each derivative. The intra-day precision (R.S.D.) was ≤ 2.22% and there was no significant difference between intra- and inter-day data. Recovery studies showed good results for all amino acids (98.9%-100.9%) with R.S.D. ranging from 0.1% to 0.8%. The limit of quantitation was about 20 nmol/mL