title: HPLC-FLD for the simultaneous determination of primary and secondary amino acids from complex biological sample by pre-column derivatization.
abstract: A rapid, sensitive, and reproducible pre-column derivatisation procedure has been established for the simultaneous determination of 20 amino acids by high-performance liquid chromatography using fluorescence detection. The amino acids were derivatized using o-phthalaldehyde and 9-fluorenylmethyl-chloroformate reagents. The optimal conditions for simultaneous separation and detection of both primary and secondary amino acids were investigated. The developed method has several advantages, namely automated pre-column derivatization, short analysis time with optimal separation, a simple and economical mobile phase, high level of precision for peak area and retention time, and higher sensitivity with more reliability of peak identification. The biological media development is the key parameter for macromolecule drug discovery. Biological media amino acids in three consecutive discovery batches were determined and the results showed a good agreement with hypothetical value. The method appears suitable for application to measure biological media amino acids at various stages of macromolecule drug discovery.
authors.affiliation: Department of Biopharmaceutics, Zydus Research Centre, Cadila Healthcare Ltd., Ahmedabad-382210, Gujarat-India. sms_buha@yahoo.com
authors.forename: Shailesh M
authors.initials: SM
authors.lastname: Buha
authors.forename: Ashwin
authors.initials: A
authors.lastname: Panchal
authors.forename: Haresh
authors.initials: H
authors.lastname: Panchal
authors.forename: Ravindra
authors.initials: R
authors.lastname: Chambhare
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Kumar
authors.forename: Mukul
authors.initials: M
authors.lastname: Jain
authors.forename: Pankaj R
authors.initials: PR
authors.lastname: Patel
mesh_terms: D000596:Amino Acids / Q000032:analysis / Q000737:chemistry*; D001709:Biotechnology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019346:Sodium Acetate; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature
chemical_list: D000596:Amino Acids; D019346:Sodium Acetate