Determination of d,l-Amino Acids in Collagen from Pig and Cod Skins by UPLC Using Pre-column Fluorescent Derivatization

Abstract

A simple and sensitive analytical method for the quantification of d,l-amino acids by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection is described. The reaction of the R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS] with d,l-hydroxyproline (Hyp), glycine (Gly), d,l-aspartic acid (Asp), and d,l-proline (Pro) effectively proceeds at 55 °C for 20 min in the presence of 3% TEA to produce the corresponding fluorescent diastereomers (excitation at 460 nm, emission at 550 nm). The mixture was simultaneously separated within 20 min on an ACQUITY UPLCTM BEH C18 (1.7 μm, 100 mm × 2.1 mm i.d.) by gradient elutions using water–acetonitrile containing 0.2% formic acid as the mobile phase. Peak resolution was in the range of 1.62 (d,l-Asp), 2.99 (d,l-Pro), and 6.74 (d,l-Hyp). A good linearity was achieved from the calibration curves (r2 > 0.9984) in the range of 1.0~100 pmol; the limit of detection (S/N = 3) was 42.0–250 fmol, the inter-day and intra-day assay precisions were all less than 6.23%, and the mean recoveries (%) of the d,l-amino acids spiked in the collagen from pig and dried cod skins were 87.58–107.41%. The derivatives of the free d,l-amino acids in the collagen were successfully identified by the proposed procedure. To the best of our knowledge, for the first time, these three kinds of d-amino acids, which were d-Asp, d-Pro, and d-Hyp, were detected in the collagen samples.