Analysis of multicomponent mixture and simultaneous enantioresolution of proteinogenic and non-proteinogenic amino acids by reversed-phase high-performance liquid chromatography using chiral variants of Sanger's reagent

• Published in: Analytical and bioanalytical chemistry Vol. 394; no. 6; pp. 1697 - 1705
• Main Authors: Bhushan, Ravi, Kumar, Rajender
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.07.2009; Springer-Verlag; Springer
• Subjects: Acetonitrile; Amino acids; Amino Acids - analysis; Amino Acids - chemistry; Analytical Chemistry; Benzene; Biochemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatographic methods and physical methods associated with chromatography; Chromatography; Chromatography, High Pressure Liquid - methods; Dinitrofluorobenzene - chemical synthesis; Dinitrofluorobenzene - chemistry; Exact sciences and technology; Food Science; Hydrophobic and Hydrophilic Interactions; Laboratory Medicine; Liquid chromatography; Mathematical analysis; Microwaves; Molecular Structure; Monitoring/Environmental Analysis; Original Paper; Other chromatographic methods; Proteins - chemistry; Stereoisomerism; Synthesis; α-Amino acids; Liquid chromatography; Chiral variants of Sanger’s reagent; Chiral separation; Chiral variants of Sanger's reagent; Impurity; Fluorine; Multicomponent mixture; HPLC chromatography; Selectivity; Derivatization; Racemic; Reversed phase chromatography; Accuracy; Multicomponent analysis; Ultraviolet detector; Aminoacid; Benzene; Enantiomer; Detection limit; Acetonitrile; Simultaneous measurement; Microwave irradiation; Diastereomer
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-009-2854-1

Four chiral derivatizing reagents (CDR 1-4), namely, FDNP-l-Ala, FDNP-l-Val, FDNP-l-Phe, and FDNP-l-Leu, were synthesized using microwave (MW) irradiation by substituting one of the fluorine atoms in difluoro dinitro benzene (DFDNB) with l-Ala, l-Val, l-Phe, and l-Leu (CDR 1-4). The other set of CDRs, namely, FDNP-l-Phe-NH₂, FDNP-l-Val-NH₂, and FDNP-l-Leu-NH₂, was also prepared. These reagents were used for synthesis of diastereomers of 18 proteinogenic and 08 non-proteinogenic amino acids, which were resolved by reversed-phase high-performance liquid chromatography using C18 column and gradient eluting mixture of aq.TFA and acetonitrile with UV detection at 340 nm. The reagents were used for resolution of a complex mixture of 18 racemic proteinogenic amino acids in a single chromatographic run of 65 min and to determine concentration of the d-amino acid in a solution of dl-amino acid. The resolution (R S) and selectivity (α) obtained for the two sets of diastereomers were compared among themselves and among the two groups. The method was validated for accuracy, precision, limit of detection (LOD), and limit of quantification. LOD is 0.001% impurity of d-enantiomer.