Quantification of bilirubin from dry blood spots using tandem mass spectrometry

Abstract

Background: because hyperbilirubinemia is harmful and associated with many kinds of diseases, especially in neonates, it is necessary to have methods available to detect bilirubin in blood as early as possible. In this study, a new screening method for the determination of the concentration of bilirubin in blood from a dry blood spot (DBS) using tandem mass spectrometry (MS/MS) was developed. Methods: the serum bilirubin of controls and blood samples were prepared as DBSs and then extracted into a methanol solution containing ²H₃ isotope-labeled myristoyl-carnitine as the standard. The extraction was subjected to HPLC, followed by MS/MS in positive ion electric spray mode. Multiple-reaction-monitoring (MRM) of m/z 585–299 for unconjugated bilirubin (UBIL), m/z 761–585 for bilirubin monoglucuronide (BMG) and m/z 937–585 for bilirubin diglucuronide (BDG) were used to detect bilirubin. Blood concentration of UBIL was measured by MS/MS (UBIL_MS) using a known concentration of labeled external standard multiplied by the signal ratio of UBIL to the standard. Conjugated bilirubin was measured by MS/MS (CB_MS) = BMG + BDG. Total bilirubin was measured by MS/MS (TB_MS) = UBIL_MS + CB_MS. Results: the recoveries of UBIL_MS were 90–120% between 17 μmol L⁻¹ and 390 μmol L⁻¹ of UBIL, with an R² value of 0.928 after linear regression (p < 0.001). The UBIL_MS of controls were within an acceptable range, and the coefficients of variation (CV) were less than 30%. UBIL_MS and unconjugated bilirubin showed a significant relationship with a Pearson correlation coefficient of 0.63, p < 0.001. CB_MS/TB_MS and direct bilirubin/total bilirubin showed a significant relationship with a Pearson correlation coefficient of 0.5, p < 0.001. The blood UBIL_MS in neonates aged 3–7 days (68.14 ± 21.56 μmol L⁻¹) was higher than that in neonates aged 8–30 days (62.85 ± 23.91 μmol L⁻¹), and it was higher in neonates than in children aged older than 1 month (38.24 ± 7.95 μmol L⁻¹, p < 0.01). The median and 90th percentile of CB_MS/TB_MS was 0.28 and 0.42 in all the samples. Conclusion: quantification of UBIL_MS from a dry blood spot by MS/MS is reliable, and feasible for screening tests. CB_MS/TB_MS showed a good relationship with the ratio of direct to total bilirubin measured by a routine clinical biochemical test.