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

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.