Simultaneous determination of the bilirubin oxidation end products Z-BOX A and Z-BOX B in human serum using liquid chromatography coupled to tandem mass spectrometry

• We introduce a validated quantification of bilirubin degradation end products.
• The working ranges are 2.74–163.0 pg/μL (Z-BOX A) and 2.12–162.4 pg/μL (Z-BOX B).
• Z-BOX A and Z-BOX B are endogenous serum components of healthy volunteers.
• Light exposure leads to photoisomerization of both analytes.

Bilirubin oxidation end products (BOXes) appear upon endogenous heme degradation and can be found in the cerebrospinal fluid after hemorrhagic stroke. BOXes are assumed to contribute to delayed cerebral vasospasm and secondary loss of brain tissue. Here, we present a validated LC–ESI-MS/MS method for the sensitive determination of the regio-isomers Z-BOX A and Z-BOX B in human serum. We found that Z-BOX A and Z-BOX B appear in serum of healthy volunteers. The sample preparation includes the addition of 5-bromonicotinamide as internal standard and protein precipitation with acetonitrile. Baseline-separation was achieved on a C-18 column with a binary solvent gradient of formic acid in water/acetonitrile at 1 mL/min within a total analysis time of 17 min. Using single reaction monitoring in the positive ion mode, the linear working ranges were 2.74–163 pg/μL (Z-BOX A) and 2.12–162.4 pg/μL (Z-BOX B) with R2 > 0.995. Intra- and inter-day precisions were <10%. The inherent analyte concentrations of Z-BOX A (14.4  ±   5.1 nM) and Z-BOX B (10.9 ±  3.1 nM) in pooled human serum were determined by standard addition. The photolability of both analytes was demonstrated. This method enables to monitor Z-BOX A and Z-BOX B as a prerequisite to systematically study the biological significance of higher order metabolites of heme degradation.