High-performance liquid chromatographic separation and quantification of the four biliverdin dimethyl ester isomers of the IX series

High-performance liquid chromatography (hplc) has been used to separate and quantificate the dimethyl ester (DME) derivatives of the four biliverdin isomers of the IX series: biliverdin-IXα, -IXβ, -IXγ, and -IXδ. Samples of 0.5 to 10.0 nmol of biliverdin DME were detected quantitatively upon elution by monitoring the absorbance at 375 nm. A technique was developed in which p-bromoacetanilide (Dupont's recommended test compound for their Zorbax column) is used as a marker for biliverdin-IXα DME. To facilitate quantification of biliverdin-IXβ DME, its extinction coefficient was determined. This method has been used to study biliverdin isomers in various biological species. High-resolution NMR (360 MHz) was used to further characterize the isomers.