Analyse der Peptide in Standardproben und Milchprodukten (mittels Hochleistungs-Fl�ssigchromatographie in reverser Phase (HPLC) unter Einsatz von on-line Absorptions- und Fluoreszenznachweis mit OPA

Abstract

Absorbance and post-column o-phthalaldehyde (OPA)-fluorescent detection were used to analyse standard and dairy peptides following reverse-phase HPLC. Using both detection systems on-line provides additional information on the presence of peptides in dairy products. The detection response depends on the amino acid composition of the peptide involved. Among the peptides used, glutathione, lysine-containing peptides and peptides with glycine as the N-terminal residue give the highest fluorescence after the OPA post-column reaction. Absorbance is more sensitive than fluorescence for peptides with aromatic amino acids. Different parameters, such as the flow rate of OPA, the amount of mercaptoethanol in the OPA reagent and the temperature of reaction, substantially influence the fluorescent response of peptides. The interest of using on-line absorbance and fluorescence detection is highlighted by analysing peptides from skim milk and from a tryptic hydrolysate.

The specificity of oligopeptide transport by Streptococcus thermophilus resembles that of Lactococcus lactis and not that of pathogenic streptococci

“…A 5 min isocratic phase of solvent A was followed by a linear gradient from 0 to 80 % of solvent B within 80 min. Peptides were either detected simultaneously by UV measurement (214 nm) and fluorescence measurement (excitation 340 nm and emission 425 nm) after on-line post-column derivatization by o-phthaldialdehyde (Herraiz et al, 1994), or collected after UV detection for mass spectrometry analysis.…”

Determination of reduced-form glutathione and total glutathione in blood and plasma by high performance liquid chromatography with on-column fluorescence derivatization

“…We therefore optimized the mobile phase and analytical conditions to accomplish adequate chromatographic performance, separation efficiency, and stability for long-term assays without any pretreatment of the samples. A nucleophilic reagent, such as, ~-mercaptoethanol or N-acetyl-L-cysteine is required to form stable isoindole fluorescence adducts following reaction between OPA and peptides, amino acids or amines [25,34,35]. However, GSH does not require those nucleophilic reagents to obtain fluorescence adducts, since both thiol and primary amino moieties are present in GSH [36,37].…”

“…However, most HPLC methods remain difficult because of the complex sampling procedure or troublesome equipment maintenance. Post-column derivatization methods [22][23][24][25] permit accurate determination of GSH or GSSG, but require additional equipment such as a derivatization-reagent elution pump, connector or reaction system. On the other hand, an on-column derivatization method has been developed for polyamine analysis [26,27].…”

Optimization of post-column reactor radius in capillary high performance liquid chromatography

“…It is well-known that post-column reactions can improve sensitivity and selectivity in liquid chromatographic analysis [1][2][3]. In just the past couple of years, papers using post-column reactions for a variety of bioanalytical applications have appeared, including amino acids and peptides [4,5], for fatty acid hydroperoxides in MEKC [6], for japonicus/polysaccharide [7], for glucosylated flavonoids [8], for acetyl-CoA esters [9], for phosphorylated peptides [10], for drugs (competitive binding) [11], for glucose and insulin [12], for bradykinin [13], for drugs [14], for lipoproteins [15], for tocopherols [16], for phosphorylated sugars [17], for peptides [3,[18][19][20][21]. One should include the preparation of MALDI plates as a post-column reaction [22].…”