Determination of radiolabelled proline and hydroxyproline in collagen hydrolysates by high-performance liquid chromatography with on-line radiometric detection.

Radiolabelled proline and hydroxyproline were separated on a C8 column (10 cm X 4.6 mm I.D.) with 10.4 mM sodium dodecyl sulphate in water-n-propanol (88:12, v/v) (pH 2.6) as the mobile phase at a flow-rate of 0.6 ml/min. The retention times of hydroxyproline and proline were 5 and 8 min, respectively. On-line radiometric detection was performed either in a homogeneous mode (liquid scintillator was added to the column effluent in the ratio 3.33:1) or in a heterogeneous mode (the detection cell was packed with a solid scintillator and 0.1 M ammonia was mixed with the column effluent in the ratio 1:6 in order to prevent adsorption of amino acids on the cell packing). Detection limits were in the range 100-900 dpm for individual isotopes and detection modes and the reproducibilities were better than 10%. The application of the method to a collagen synthesis study is reported.