Capillary electrophoresis for the detection of bilirubin in rat serum.
BACKGROUND
The rat is used often to assess the toxicity of new chemical entities in preclinical drug development. Bilirubin concentration in rat serum is routinely determined by colorimetric methods, but false positive results due to hemolyzed serum or direct interferences by test compounds may occur. Capillary electrophoresis (CE) is an automated method that requires small sample volume and facilitates the direct detection of bilirubin.


OBJECTIVE
The purpose of this study was to evaluate a CE method for detecting bilirubin and albumin-bound bilirubin in rat serum and to measure potential interference by hemolysis and specific test compounds.


METHODS
Serum samples from male Sprague Dawley rats (n=20) were used in the study. Results obtained on a Beckman P/ACE MDQ CE instrument equipped with a UV-detector were compared with those obtained using a colorimetric method on a Hitachi 912 analyzer. Bilirubin standards were used to evaluate the detection and stability of bilirubin in rat serum, and vials with ultrafiltration membranes were used to separate albumin-bound bilirubin. Intraday and interday coefficients of variation (CV), linearity, and the effects of added hemoglobin and a test compound on CE results were determined.


RESULTS
The CE method was capable of detecting bilirubin and albumin-bound bilirubin in rat serum samples with reproducible results and good accuracy. CVs were <3% and linearity of the CE assay was high (R(2)=0.9951). Abnormally high bilirubin peaks due to the presence of hemoglobin or the test compound were easily distinguished by means of CE.


CONCLUSIONS
CE is a good alternative to the colorimetric methods currently used for the determination of bilirubin in rat serum.
Crivellente
Federica
F
Safety Assessment Department, GlaxoSmithKline Research Center, Verona, Italy. federica.a.crivellente@gsk.com
Bocchini
Nicola
N
Vandin
Luca
L
Cristofori
Patrizia
P
D000818:Animals; D001663:Bilirubin / Q000032:analysis / Q000097:blood*; D019075:Electrophoresis, Capillary / Q000379:methods*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity
D001663:Bilirubin