[Determination of hydroxyproline in collagenous proteins by high performance liquid chromatography-mass spectrometry]

A simple, rapid and accurate method for the determination of hydroxyproline in collagenous proteins by high performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI/MS) was developed. After hydrolysis of collagenous proteins with hydrochloric acid, the hydroxyproline was separated on a Spherigel C8 column using acetonitrile-0.05% trifluoroacetic acid aqueous solution (5:95, v/v) at a flow rate of 1.0 mL/min. Theanine was used as the internal standard for the quantification. Hydroxyproline was identified and quantified by electrospray ionization mass spectrometry (ESI-MS), which was operated in positive ion mode while the m/z 132 and m/z 175 ions were recorded in the selective ionization monitoring (SIM) mode. The peak area ratio of hydroxyproline to theanine versus the hydroxyproline concentration was linear over a concentration range of 11.7-117 mg/L for hydroxyproline. The correlation coefficient was 0.999 3. The reproducibility and average recovery of the method were 1.87% and 98.85% respectively. The method has the advantages of easy operation, without derivatization, less analysis time, good precision and accuracy.