OPTIMIZED HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-FLUORESCENCE DETECTION METHOD FOR THE MEASUREMENT OF GLYCINE, PROLINE, AND HYDROXYPROLINE CONCENTRATIONS IN PORCINE GELATIN
DOI:
https://doi.org/10.22159/ijap.2018.v10s1.72Keywords:
Derivatization, Fluorenylmethoxycarbonyl chloride, Glycine, High-performance liquid chromatography, Hydroxyproline, Optimization, Porcine gelatin, ProlineAbstract
Objective: The aim of this study is to develop an optimized method for glycine, proline, and hydroxyproline content quantitation in porcine skin
gelatin.
Methods: Gelatin was isolated from porcine skin by hydrolysis for 24 h in 0.5 M acetic acid, heating in distilled water at 55°C for 3 h, and drying at
60°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 28.57±0.74%, 19.24±0.48%, and 2.89±0.33% for glycine, proline, and
hydroxyproline, respectively.
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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References
Damanik A. Halal Gelatin and Non-halal Gelatin. Jakarta: Jurnal Halal
LPPOM MUI No. 36; 2001.
Schrieber R, Gareis H. Gelatin Handbook: Theory and Industrial
Practice. Weinheim: Wiley-VCH Verlag Gmbh; 2007.
Gelatin Manufacturer’s Institute of America. Standard Methods for the
Testing of Edible Gelatin. America: Gelatin Manufacturers Institute of
America, Inc.; 2012.
Azilawati MI, Hashim DM, Jamilah B, Amin I. Validation of a
reverse-phase high-performance liquid chromatography method
for the determination of amino acids in gelatins by application
of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent.
J Chromatogr A 2014;1353:49-56.
Azilawati MI, Hashim DM, Jamilah B, Amin I. RP-HPLC method using
-aminoquinolyl-N-hydroxysuccinimidyl carbamate incorporated
with normalization technique in principal component analysis to
differentiate the bovine, porcine and fish gelatins. Food Chem 2015;172:
-76.
Fabiani A, Versari A, Parpinello GP, Castellari M, Galassi S. Highperformance
liquid chromatographic analysis of free amino acids in
fruit juices using derivatization with 9-fluorenylmethyl-chloroformate.
J Chromatogr Sci 2002;40:14-8.
Coppex L. Derivatives for HPLC Analysis. Jerman: Faculty of
Chemistry and Pharmacy University of Genf; 2000.
Fakhri S, Mohammadi B, Jalili R, Hajialyani M, Bahrami G. Screening
and confirmation of different synthetic adulterants in slimming
products. Asian J Pharm Clin Res 2018;11:260-4.
Ahda M, Guntarti A, Kusbandari A. Application of high-pressure
liquid chromatography for analysis of lard in the meatball product
combined with principal component analysis. Asian J Pharm Clin Res
;9:120-3.
Bakar RA, Miran H. Validated high performance liquid chromatography
(HPLC) method for analysis of methamphetamine in human urine using
liquid-liquid extraction. Asian J Pharm Clin Res 2015;8:199-201.
Sompie M, Surtijono SE, Pontoh JH, Lontaan NN. The effects of acetic
acid concentration and extraction temperature on physical and chemical
properties of porcine skin gelatin. First Int Symp Food Agro Biodivers
;3:383-8.
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