VALIDATION METHODS FOR THE ANALYSIS OF HYDROXYPROLINE FROM COLLAGEN UNDENATURED TYPE II COLLAGEN USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY FLUORESCENCE
Objective: This study aimed to validate an high-performance liquid chromatography method for analyzing undenatured Type II collagen preparations
using a fluorescence detector.
Methods: Based on the optimum analysis conditions, the compound was detected at an excitation wavelength of 255 nm and an emission wavelength
of 320 nm. The optimum mobile phase was determined to be acetate (pH 4.2) and acetonitrile (60:40) with a flow rate of 1.0 ml/min. Hydroxyproline
is a compound that does not have chromophore moiety; thus, it has to be derivatized first using 9-fluorenylmetoxycarbonyl-chloride.
Results: The developed method was validated with linearity and an equation of y=3,249,704 x+141,945,072, with a value of r=0.9994. The detected
range of hydroxyproline was 4â€“15 ppm. The limit of detection was determined to be 0.49, with an limit of quantitation of 1.64.
Conclusion: Our results indicated that the average level of hydroxyproline was 98.66%, 99.12%, and 99.85%.
Kempka AP, et al. Collagen extraction process. Int Food Res J
2. Engelking LR. Textbook of Veterinary Physiological Chemistry. Vol. 3.
St. Louis, MO: Elsevier; 2015. p. 397-405.
3. Marshall WJ, Bangert SK. Clinical Biochemistry: Metabolic and
Clinical Aspects. Churchill Livingstone/Elsevier. Available from:
4. Health Research and Development Agency of the Ministry of Health of
the Republic of Indonesia. Basic Health Research (Riskesdas). Jakarta:
Indonesian Ministry of Health; 2013.
5. Crowley DC, Lau FC, Sharma P, Evans M, Guthrie N, Bagchi M, et al.
Safety and efficacy of undenatured Type II collagen in the treatment of
osteoarthritis of the knee: A clinical trial. Int J Med Sci 2009;6:312-21.
6. Lugo JP, Saiyed ZM, Lau FC, Molina JP, Pakdaman MN, Shamie AN,
et al. Undenatured Type II collagen (UC-IIÂ®) for joint support:
A randomized, double-blind, placebo-controlled study in healthy
volunteers. J Int Soc Sports Nutr 2013;10:48.
7. Aditya A. Isolation, Purification, and Collagen Characterization
from Pig Skin, and Glisin, Prolin, and Hydroxyproline Analysis in
Characterization of High Performance Liquid Chromatography-
Fluorescence. Depok: Thesis. Faculty of Pharmacy, Universitas
8. Rediatning W, Kartini N Analisis asam amino dengan kromatografi
cairan kinerja tinggi secara derivatisasi prakolom dan pascakolom.
Proceedings Institut Teknologi Bandung 1987; 20:41-59.
9. Harmita I. Phytochemicals Analysis. Depok: Faculty of Pharmacy,
Universitas Indonesia; 2012.
10. Kupiec T. Quality-control analytical methods: High-performance liquid
chromatography. Int J Pharm Compd 2004;8:223-7.
11. Chakravarthy VA, Sailaja BB, Praveen KA. Stability indicating
reverse phase high-performance liquid chromatographic method for
simultaneous estimation of labetalol and its degradation products in
tablet dosage forms. Asian J Pharm Clin Res 2016;9:242-9.
12. Siddiraju S, Kavitha R, Sudhakar M. Reverse phase high performance
liquid chromatography method development and validation for
the simultaneous estimation of gatifloxacin and flurbiprofen in
pharmaceutical dosage form. Asian J Pharm Clin Res 2015;8:242-6.
13. Senthilraja M, Giriraj P. Reverse phase HPLC method for the
simultaneous estimation of terbutanile sulphate, bromhexine HCL and
guaifenesin in cough syrup. Asian J Pharm Clin Res 2011;4:13-5.