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 degreesC). 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 x 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 muA. Calibration curves plotted the peak area versus Hyp and Pro concentrations. The assay was linear in the concentration range of 0.3 similar to 10000 and 0.6 similar to 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.