Accurate and sensitive high-performance liquid chromatographic method for geometrical and structural photoisomers of bilirubin IX alpha using the relative molar absorptivity values

It has been reported that considerable differences exist between the relati ve molar absorptivity values of the geometrical and structural photoisomers of bilirubin. We have devised an accurate HPLC method for photoisomer quan tification based on the following principle: the sum of both the integrated peak areas corrected by each factor for each photoisomer, and the integrat ed peak area of unchanged (ZZ)-bilirubin [(ZZ)-B] after an anaerobic photoi rradiation, should be constant and equal to the integrated peak area of ini tial (ZZ)-bilirubin [(ZZ)-Bi] before photoirradiation. On this basis, the f ollowing equation can be used to determine each factor. [(ZZ)-Bi] = [(ZE)-B]/alpha + [(EZ)-B]/beta + [(EZ)-CB]/gamma + [(EE)-CB]/de lta + [(ZZ)-B] alpha, beta, gamma and delta represent the factors used to correct the inte nrated peak areas of individual bilirubin photoisomers, and they are arrang ed in the order of the formula. It was demonstrated that the relative 455 n m molar absorptivity values for (ZZ)-bilirubin and all its geometrical and structural photoisomers, i.e., (ZZ)-bilirubin, (ZE)-bilirubin (EZ)-bilirubi n, (EZ)-cyclobilirubin (= lumirubin) and (EE)-cyclobilirubin in the HPLC el uent, are, respectively, 1.0, 0.81 (= alpha), 0.54 (= beta), 0.47 (= gamma) and 0.39 (= delta). (C) 1999 Elsevier Science B.V. All rights reserved.