Highly sensitive and simple method for measurement of pipecolic acid using reverse-phase ion-pair high performance liquid chromatography with tris(2,2′-bipyridine)ruthenium(III) chemiluminescence detection

A new, highly sensitive chemiluminescence method for measurement of pipecolic acid in various substances such as human serum, cow's milk, beer, and apple juice has been developed. The method is based on reverse-phase ion-pair high performance liquid chromatographic separation and subsequent tris(2,2′-bipyridine)ruthenium(III) chemiluminescence detection. It was confirmed that imino acids show strong chemiluminescence upon mixing with tris(2,2′-bipyridine)ruthenium(III). A calibration graph, based on a standard pipecolic acid solution, was linear over the range 5.0 × 10−9 M to 2.0 × 10−5 M and the detection limit was 24 fmol (signal-to-noise ratio = 3). This highly sensitive and selective determination method can be applied to selected samples without purification or pre-concentration procedures. Compared to the previous HPLC methods, the proposed method is easier, more sensitive, and time-saving.