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.

Native plasmids restrict growth of Phaeobacter inhibens DSM 17395: Energetic costs of plasmids assessed by quantitative physiological analyses

“…Amino acid derivatives were detected upon change of wavelengths 31 min after sample injection as described by Buha et al . (). Calibration curves were prepared in the range of 5–500 µM using a purchased amino acid standard (1 mM; part‐no.…”

“…Mobile phases (A: 0.1 M sodium acetate, pH 6.5; B: acetonitrile) and eluent gradients were adapted initially from Schuster (1988) and modified as follows (share of eluent B): 3% for 5.3 min, 3-6% within 0.05 min, 6-10% within 4.65 min, 10% for 1 min, 10-15% within 1 min, 15% for 13 min, 15-65% within 10 min, 65-100% within 0.1 min, and 100% for 2.9 min. Amino acid derivatives were detected upon change of wavelengths 31 min after sample injection as described by Buha et al (2011). Calibration curves were prepared in the range of 5-500 mM using a purchased amino acid standard (1 mM; part-no.…”

Anatomical and physiological studies of bigheaded carps demonstrate that the epibranchial organ functions as a pharyngeal taste organ

“…Algal filtrate (AF) was prepared from the algal food formula by centrifugation and vacuum filtered to a 6 μm size threshold. AF and well water (WW, control) were analysed using an Agilent 1200 series HPLC-DAD and Agilent 1100 series fluorescence detector (Agilent Technologies, Santa Clara, CA, USA), to determine the absolute concentration of L-amino acids (Buha et al, 2011). Stock stimuli were prepared 24 h prior to use and were stored refrigerated in 250 ml Pyrex bottles (Schott Duran).…”

Frequent amplification of ORAOV1 gene in esophageal squamous cell cancer promotes an aggressive phenotype via proline metabolism and ROS production

“…The cell pellets were lysed with 240 μL of methanol and were treated with ultrasonic. Debris was removed by centrifugation, and the proline concentrations were determined using high-performance liquid chromatography fluorescence detection and the pre-column derivatization procedure, as previously described [27]. The experiment was performed in triplicate.…”