Highly sensitive novel fluorescent chiral probe possessing (S)-2-methylproline structures for the determination of chiral amino compounds by ultra-performance liquid chromatography with fluorescence: An application in the saliva of healthy volunteer

• Published in: Journal of Chromatography A Vol. 1661; p. 462672
• Main Authors: Li, Xi-Ling, Han, Yu, Huang, Yuan, Sun, Xiaoqi, Xiao, Shuyun, Min, Jun Zhe
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 04.01.2022
• Subjects: Chiral amino enantiomer; Chromatography, High Pressure Liquid; Chromatography, Liquid; DBD-M-Pro; Female; Fluorescent chiral probe; Fluorescent Dyes; Healthy Volunteers; Human saliva; Humans; Male; Proline - analogs & derivatives; Saliva; Stereoisomerism; Ultrahigh-performance liquid chromatography with fluorescence; DBD-M-Pro; Chiral amino enantiomer; Fluorescent chiral probe; Human saliva; Ultrahigh-performance liquid chromatography with fluorescence
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2021.462672

•We have developed novel fluorescent derivatization reagents DBD-M-Pro.•The chiral 20 amino enantiomers resolution (Rs) values ranged from 1.32 to 24.11.•A highly sensitive and selective method was successfully applied to human saliva. We developed a novel fluorescent chiral probe, DBD-trans-2-methyl-L-proline (DBD-M-Pro), which can be used to target recognition of amino functional groups using chiral resolution. To investigate the chiral resolution efficiency, 20 chiral amino enantiomers (19 DL-amino acids and phenylethylamine) were labeled using ultra-performance liquid chromatography (UPLC) with a fluorescence (FL) system. Diastereomers were formed by the reactions of DBD-M-Pro with enantiomers of amino functional groups at 60 °C for 60 min and detected on a BEH C18 column (100 × 2.1 mm, 1.7 μm). Gradient elution of 10 mM ammonium acetate with 0.05% formic acid (FA) aqueous solution and 0.1% FA acetonitrile or 0.1% FA methanol solution was performed at an excitation wavelength (Ex) 460 nm and emission wavelength (Em) 550 nm. Each resulting derivative of D- and L- type was effectively separated. The results showed that the resolution (Rs) of 17 amino acids and phenylethylamine (PEA) in the range of 1.59–24.11, except for histidine (His) (Rs = 1.32) and serine (Ser) (Rs = 1.47), achieved completely separation. The DBD-M-Pro chiral probe has a robust chiral selectivity for D-amino acids. Furthermore, a new method for the simultaneous determination of six DL-amino acids (Pro, Val, Trp, Phe, Leu, Lys) in human saliva was developed. The proposed method showed resolution values of 1.78–16.38, and an excellent linear relationship was obtained in the range of 2.5–500 pmol (R2 ≥ 0.9990). The limit of detection (S/N = 3) ranged from 0.5 to 3.75 pmol. The intra-day and inter-day coefficient of variation (CV) were within the range of 1.75–11.73%. The average addition recoveries in saliva ranged from 95.99 to 106.97%. The methodology was used to determine the content of DL-amino acids and the D/L-amino acid ratio in the saliva of 40 healthy volunteers (15 males and 25 females), as well as evaluating the differences between men and women. Our study suggests that the DBD-M-Pro chiral probe could be an effective tool for screening potential D-amino acid biomarkers in disease.