OPTIMIZED HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-FLUORESCENCE DETECTION METHOD FOR THE MEASUREMENT OF GLYCINE, PROLINE, AND HYDROXYPROLINE CONCENTRATIONS IN PORCINE GELATIN
Keywords:Derivatization, Fluorenylmethoxycarbonyl chloride, Glycine, High-performance liquid chromatography, Hydroxyproline, Optimization, Porcine gelatin, Proline
Objective: The aim of this study is to develop an optimized method for glycine, proline, and hydroxyproline content quantitation in porcine skin
Methods: Gelatin was isolated from porcine skin by hydrolysis for 24 h in 0.5 M acetic acid, heating in distilled water at 55Â°C for 3 h, and drying at
60Â°C. The extract was evaluated by organoleptic tests, Fourier-transform infrared spectroscopy, moisture assay, ash assay, and viscosity test. Gelatin
amino acids were derivatized using 9-fluorenylmethylchloroformate-chloride and measured by high-performance liquid chromatography (HPLC)
with fluorescence detection using a C18 column after the optimization of the mobile phase composition, flow rate, and detection wavelengths.
Results: The optimized parameters for the quantitation of glycine, proline, and hydroxyproline by HPLC with fluorescence detection were as
follows: Excitation wavelength, 265 nm; emission wavelength, 320 nm; mobile phase composition acetic buffer: acetonitrile, 55:45; and flow
rate, 0.8 mL/min. The average proportional amino acid contents were 28.57Â±0.74%, 19.24Â±0.48%, and 2.89Â±0.33% for glycine, proline, and
Conclusion: This method allows for sensitive and accurate quantitation of glycine, proline, and hydroxyproline in porcine skin gelatin samples for
quality control and source determination.
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