Abstract

Pre-column derivatization of UPLC assay was established for the simultaneous determination of essential amino acids (EAA) in pharmaceutical solid oral dosage formulation. The analytical method was validated by using the total error concept. Accuracy profile with Total Error (the combination of systematic and random error) approach is a decision tool to ensure the method performance. Fluorenylmethyloxycarbonyl chloride (FMOC-Cl) was used as pre-column derivatization reagent. The amino acid derivatives were separated on C18 column (2.1×100 mm, 1.6 µm) by using gradient elution with 0.1% trifluoroacetic acid (TFA) as mobile phase A and Acetonitrile: Water (90:10, v/v) as mobile phase B. The 10 EAA could be detected at 265 nm in 35 min with a flow rate of 0.25 mL/min. Linearity range of each amino acid was between 0.1-1.0 mg/mL. Accuracy profile and risk profile were found to be good throughout the range. Pre-column derivatization method proposed in this study could be used as a suitable method for the separation and quantification of essential amino acids in solid oral dosage formulation.

Key words: Essential amino acids; Total error; Pre-column derivatization; UPLC; Accuracy profile; Risk profile.