Determination of free hydroxyproline and proline in serum by HPLC with electrogenerated chemiluminescence detection using tris(2,2'-bipyridine)ruthenium(II).

A simple method for the determination of free hydroxyproline (Hyp) and proline (Pro) in serum by HPLC with electrogenerated chemiluminescence detection using tris(2,2'-bipyridine) ruthenium (II) was developed. A serum sample was applied to a centrifugal filter (Microcon, YM-10, Millipore Co., Bedford, MA, U.S.A.) and was centrifuged (14000 rpm, 60 min, 15°C). The filtrate was injected to HPLC. The HPLC conditions were as follows: separation-eluent, 20 mM sodium acetate buffer (pH 4.6), 1 mM Cu2+, 10 mM 1-octanesulfonate-acetonitrile (99 : 1, v/v); flow rate, 0.5 ml/min; column, Capcell Pak C18 (4.6 × 150 mm, Shiseido Co., Ltd); detection-reagent solution, 0.3 mM tris(2,2'-bipyridine)ruthenium chloride, 10 mM sulfuric acid, flow rate: 0.3 ml/min, electrogenerated chemiluminescence detector, ECR-2000 (Comet Co., Ltd); controlled current oxidation as 80 μA. Calibration curves plotted the peak area versus Hyp and Pro concentrations. The assay was linear in the concentration range of 0.3∼10000 and 0.6∼5000 nmol/ml serum for Hyp and Pro, respectively. The recovery was 101% (10 nmol/ml) for Hyp and 103% (50 nmol/ml) for Pro. The intera-assay coefficient of variation was less than 5% for both Hyp and Pro in the linear concentration range. The method was successfully applied to the determination of aged human serum without prior derivatization.