Development of an easy-to-set-up multiple heart-cutting achiral-chiral LC-LC method for the analysis of branched-chain amino acids in commercial tablets

Ina Varfaj; Ghaid W A Abualzulof; Simone Moretti; Anna Migni; Irene Uda; Laura Goracci; Federica Ianni; Andrea Carotti; Roccaldo Sardella

doi:10.1002/elps.202300278

Development of an easy-to-set-up multiple heart-cutting achiral-chiral LC-LC method for the analysis of branched-chain amino acids in commercial tablets

Electrophoresis. 2024 Mar 13. doi: 10.1002/elps.202300278. Online ahead of print.

Authors

Ina Varfaj¹, Ghaid W A Abualzulof¹, Simone Moretti², Anna Migni¹, Irene Uda¹, Laura Goracci³, Federica Ianni¹, Andrea Carotti¹, Roccaldo Sardella¹

Affiliations

¹ Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.
² Molecular Horizon, Bettona, Perugia, Italy.
³ Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy.

PMID: 38477690
DOI: 10.1002/elps.202300278

Abstract

In this paper, the development and application of a multiple heart-cutting achiral-chiral LC-LC method (mLC-LC) for the analysis of dansylated (Dns) branched-chain amino acids in commercial tablets are described. In the first dimension, a Waters Xbridge RP C18 achiral column was used under gradient conditions with buffered aqueous solution and acetonitrile. The elution order Dns-valine (Dns-Val) < Dns-isoleucine (Dns-Ile) < Dns-leucine (Dns-Leu) turned out with full resolution between adjacent peaks: 7.25 and 1.50 for the Val/Ile and the Ile/Leu pairs, respectively. A "research" validation study was performed, revealing high accuracy (Recovery%) and precision (RSD%) using two external set solutions, respectively, in the range 93.7%-104.1% and 0.4%-3.2%. The C18 column was connected via a two-position six-port switching valve to the quinidine-based Chiralpak quinidine-anion-exchange chiral column. A water/acetonitrile, 30/70 (v/v) with 50 mM ammonium acetate (apparent pH of 5.5) eluent allowed getting the three enantiomers' pairs resolved: R_S equal to 4.3 for Dns-Val and Dns-Ile, and 1.7 for Dns-Leu. The application of the mLC-LC method confirmed that the content of Val, Ile, and Leu in the tablets was compliant with that labeled by the producer. Only l-enantiomers were found in the food supplement, as confirmed by LC-MS/MS analysis.

Keywords: branched-chain amino acids; food supplement; multiple heart-cutting two-dimensional chromatography; quality control; quinidine-based chiral selector.