Development and Validation of a Simple, Selective, and Accurate Reversed-Phase Liquid Chromatographic Method with Diode Array Detection (RP-HPLC/DAD) for the Simultaneous Analysis of 18 Free Amino Acids in Topical Formulations

Abstract

Even though there are reported methods for the quantification of free amino acids (FAAs) in biological products, no work has been done on the analysis of these substances in formulations. Moreover, further research is required as the reported methods do not fulfill analytical method requirements. The objective of this study was, therefore, to develop and validate a rapid, reliable, and appropriate RP-HPLC/DAD method for the simultaneous determination of 18 FAAs (l-Ala, l-Arg, l-Asn, l-Asp, l-Gln, l-Glu, l-Gly, l-His, l-Ile, l-Lue, l-Lys, l-Met, l-Orn, l-Phe, l-Pro, l-Ser, l-Thr, and l-Val) in topical formulations. After appropriate method development, the technique was validated for selectivity, linearity and range, limit of detection, limit of quantification, precision, and accuracy. The samples were derivatized with 9-fluorenylmethyl chloroformate (Fmoc-Cl). Chromatographic separation was performed on InfinityLab Poroshell 120 E.C 18 (3 × 50) mm, 2.7 μm column at 25 °C. The mobile phase consisting of water and acetonitrile adjusted to appropriate pH was pumped in gradient mode at a flow rate of 0.7 mL/min. Ten microliters were injected and analyte detection was conducted using a DAD. The results indicate that the method was selective for these FAAs. It was linear over the concentration range of 5–80 µM with a correlation coefficient greater than 0.995. Moreover, it was sensitive, precise, accurate, and robust. All the reported drawbacks of RP-HPLC-based analysis of FAAs were resolved, and hence, this new method can be considered appropriate for the analysis of these FAAs in topical formulations.