Candidate reference method for determination of total bilirubin in serum: development and validation

Area of Science:

• Clinical Chemistry
• Biochemistry
• Analytical Chemistry

Background:

• Accurate measurement of total bilirubin in serum is crucial for diagnosing and monitoring various medical conditions.
• Existing methods may have limitations in accuracy or be subject to interferences.
• A standardized reference method is needed for reliable bilirubin quantification.

Purpose of the Study:

• To develop and validate a candidate Reference Method for total bilirubin measurement in serum.
• To establish normal adult reference values for total bilirubin.
• To assess potential interferences and the method's applicability in diverse patient populations.

Main Methods:

• The method is based on the Jendrassik-Gróf principle.
• Utilizes Standard Reference Material no. 916 bilirubin for standardization.
• Bilirubin standard solutions prepared in human serum or albumin solution were evaluated for molar absorptivity.

Main Results:

• The molar absorptivity of the azopigment was found to be identical in serum and albumin solutions.
• Bilirubin recovery was quantitative in adult and neonatal serum samples.
• Negligible interference was observed from hemoglobin, ascorbic acid, and zinc; L-dopa and alpha-methyldopa were identified as interferents.

Conclusions:

• The proposed Jendrassik-Gróf-based method is a valid and reliable reference method for total bilirubin determination.
• The method demonstrates minimal interference from common substances and specific drugs.
• Established normal adult reference values and assessed intraindividual variation.