HPLC Analysis of Histidine and Related Impurities

Application description

Histidine is one of the essential amino acids which is zwitterionic in nature. It contains one carboxylic acid fragment and two basic groups, one weak basic group in the imidazole ring and a primary amine in the side chain of the ring. Histidine is a precursor for histamine, a nitrogen containing a compound necessary for implantation. Histidine can be converted to 3-methylhistidine which serves as a biomarker for muscle damage. Like any other amino acids, histidine is not retained well in reversed-phase chromatography without the use of ion-pairing reagents, which are not compatible with mass spectrometry usually required in bio and chemical studies. When histidine is retained on a traditional RP column, it produces a poor peak shape due to residual secondary interactions between basic groups of histidine and residual acidic silanols on the surface of silica gel. Histidine and related impurities are retained by a combination of weak reversed-phase and cation-exchange mechanisms. At a lower pH (below 3), the compound is more basic and retentive on this Coresep 100 mixed-mode reversed-phase cation-exchange column. The retention time for all compounds is adjusted by playing with acetonitrile, buffer pH and buffer concentration. Various buffers and acids can be used. The selection of components of the mobile phase depends on the properties of analytes and the detection technique. The Coresep 100 column can be used for the retention and separation of underivatized amino acids without ion-pairing reagent. In case of complex sample matrices like biofluids, additional sample preparation is required for developing a robust and reproducible method for analysis of underivatized amino acids

Conditions of Experiment
Column: Coresep 100
Separation Modes: reversed-phase, cation-exchange
Column Dimenstions: 4.6 x 150 mm, 2.7 um, 90A
Mobile Phase: ACN from 2% to 20%, Na2HPO4 pH 6 from 10 mM to 50 mM in 12 min
Detection: UV 210 nm
Sample: 0.3 mg/ml
Injection: 10 uL
Flow rate: 1 mL/min
Class of compounds: Amino acid, Supplement
Nature of compounds: Hydrophilic, Polar, Zwitterionic
Compounds: Alanine, Histidine, Lysine, Arginine