Identification and quantification of the molecular species of bilirubin BDG, BMG and UCB by LC‒MS/MS in hyperbilirubinemic human serum

Abstract

Background and aims: Unconjugated bilirubin (UCB) is a byproduct of the heme group that indicates irregularities in the metabolism of several important biological molecules, such as hemoglobin. UCB is processed by hepatic UGT1A1, which catalyzes its conjugation to the metabolites bilirubin diglucuronide (BDG) and bilirubin monoglucuronide (BMG). The serum concentrations of BDG and BMG may indicate liver injury or dysfunction. The aim of this study was to standardize and validate a method for the identification and simultaneous quantification of BMG, BDG and UCB by LC‒MS/MS.

Methods: Liquid‒liquid extraction allows the separation of UCB, BMG and BDG from the serum of healthy subjects or patients with liver injury. Detection and quantification were performed using an LC‒MS/MS method. Compound separation was achieved with a BEH-C18 column at 40°C. The mobile phase was prepared with 5 mM ammonium acetate (pH 6) and acetonitrile, and a flow gradient was applied.

Results: This is the first study to directly quantify BMG and UCB levels in human serum; no postcalculations or correction factors are needed. However, BDG quantification requires calculations and a correction factor. We identified the molecular species with ionic transitions m/z1+ 585.4 > 299.2 for UCB, 761.3 > 475.3 for BMG, 937.3 > 299.5 for BDG and mesobilirubin 589.4 > 301.3 (IS).

Conclusion: The procedures used in this study allowed the simultaneous identification and quantification of the molecular species of bilirubin, BDG, BMG and UCB. Analysis of the serum levels in patients with hyperbilirubinemia revealed that patients with acute-on-chronic liver failure had elevated levels of these species.

Fig 1. Representative chromatogram showing the peaks resulting from the incubation of rat liver microsomes.

The cells were incubated with unconjugated bilirubin (UCB) for 20 minutes to obtain bilirubin monoglucuronide (BMG) and bilirubin diglucuronide (BDG). The transitions for each molecular species of bilirubin were as follows: BDG m/z 937.33 > 299.5, BMG 761.30 > 474.30, and UCB 585.27 > 299.21. The retention times were as follows: UCB, 5.93 min; BMG, 4.08 min; and BDG, not observed.

Fig 2. Incubation for the bilirubin glucuronidation reaction.

Panel A, 15 min; Panel B, 20 min. The production profile of bilirubin monoglucuronide, following the m/z signal, is depicted in Panel C.

Fig 3

A) Plot of substrate concentration (UCB) vs. glucuronidation (synthesis of metabolites) and remaining UCB at the end of the reaction. B) Kinetic profiles of bilirubin glucuronidation using rat liver microsomes. The microsomal or UGT1A1 protein concentration was 6 μg/mL, and the samples were incubated for 20 min. Each data point represents the mean of three replicates. Kinetic parameters were calculated by the Lineweaver‒Burk plot (r² value of 0.9942).

Fig 4. Bilirubin molecular species.

m/z¹⁺ specified for each species. Bilirubin unconjugated (UCB) was detected at 5.93 min (585.4 > 299.2), bilirubin monoglucuronide (BMG) at 4.11 min (761.3 > 475.3), bilirubin diglucuronide (BDG) at 3.98 min (937.3 > 299.5) and mesobilirubin as an IS at 6.16 min (589.4 > 301.3).

Fig 5. m/z scan showing the molecular species of bilirubin in positive mode for each type of patient with liver disease: Acute-on-chronic liver failure, hepatic encephalopathy, liver cirrhosis, and healthy subjects.

A) Integrated m/z scan of UCB from one patient for each group. B) Integrated m/z scan from BMG for each group. C) Integrated m/z scan from the BDG for each group.