Certification of bilirubin SRM 916a

The process used to certify Standard Reference Material (SRM) 916a Bilirubin is described. The certification involved the use of various analytical techniques to detect or quantitate impurities, as well as to characterize the SRM itself. Bilirubin (BR) is believed to exist, in human serum, as the IXα isomer. Samples prepared commercially, including this SRM, also contain the IIIα and XIIIα isomers which are believed to be formed during purification. For the SRM, the three isomers were measured by HPLC and TLC. 1H NMR was used to detect and quantitate chloroform in the BR. Biliverdine and mesobilirubin were not detected. Impurities insoluble in chloroform, the residue from the ashing of BR, and volatiles were measured, in addition to non-acidic impurities and impurities more acidic than BR. The absorptivity of BR in chloroform was measured. A pink fluorescent impurity was detected and measured by TLC. From these analyses, a best estimate of the total amount of impurities was determined, and the BR was issued as SRM 916a with a certified purity of 98.3±0.3%.