Chiral Separation and Determination of Carnitine Enantiomers in Infant Formula by Ultra-Performance Convergence Chromatography

A method for chiral separation and determination of 2 carnitine enantiomers (D-carnitine and L-carnitine) in infant formula by ultra-performance convergence chromatography (UPC²) was established. Samples were extracted by ultrasound with 0.1 mol·L^-1 hydrochloric acid solution for 5 min, and saponified with 1 mol·L^-1 potassium hydroxide solution in a water bath at 60℃ for 30 min. The supernatant obtained by centrifugation was purified on an activated Oasis MCX type mixed strong cation exchange solid phase extraction column, and eluted with methanol solution containing 4% (φ) ammonium hydroxide. The eluate was blown to near dryness with nitrogen and derivatized with acetonitrile solution containing L-alanine-β-naphthylamine in the presence of catalyst at 20℃ for 60 min. After centrifugation, concentration and filtration, the targets in the filtrate were subjected to gradient elution on an Acquity Trefoil CEL1 chiral column with mobile phases composed of supercritical carbon dioxide and methanol solution containing 1% (φ) ammonium hydroxide ammonium hydroxide at different volume ratios. The results showed that the method could effectively separate carnitine racemates, and linear relationships between mass concentrations of two carnitine enantiomers and their corresponding peak areas were kept in the range of 0.25-10.00 mg·L^-1, with the same lower limit of determination (10S/N) of 25 mg·kg^-1. Test for spiked recovery was made on the blank samples at 3 concentration levels, giving results of recovery in the range of 93.0%-110%, with RSDs (n=6) of the determined values in the range of 3.9%-5.5%. The proposed method was used for the analysis of 20 actual samples, and no D-carnitine was detected, while the determined values of L-carnitine met the requirements of GB 28050-2011 and GB 13432-2013, and there was no significant difference between the prescribed method and the spectrophotometric method of GB 29989-2013.