High performance liquid chromatography equipped with a cathodic detector and column-switching device as a high-throughput method for a phosphatase assay with p-nitrophenyl phosphate

LC coupled to an electrochemical detector (LC-EC) operating in cathodic mode with a column-switching system realizes a high-throughput detection of p-nitrophenol (NP). The measurement-time for each NP sample was shortened to 20 s, and the successive analyses of 39 samples was completed within 13 min. In the present system, the limits of detection and quantification were 0.15 and 0.20 μM, respectively, and further, up to 25 μM, a linear calibration curve was afforded. Relative standard deviations for standard solutions of 0.20, 1.0, and 25 μM NP were 4.3, 2.0, and 1.1% (n = 5), respectively. Between-run precisions of the analysis of 5.0 and 25 μM NP over 6 days were 4.8 and 1.3%, respectively. A comparison with the commonly used Bessey-Lowry-Brock method indicates that the present LC-EC is useful for the high-throughput assay of acid and alkaline phosphatases in urine and blood samples with a p-nitrophenyl phosphate substrate.