Rapid analysis of four bilirubins in domestic animal sera using high-performance liquid chromatography.

The research article presents a new method for rapid analysis of four different types of bilirubin in domestic animal blood serum using high-performance liquid chromatography.

Research Methodology

• The researchers developed a method to quickly analyze four types of bilirubin: delta-bilirubin (B delta), diconjugated bilirubin (DCB), monoconjugated bilirubin (MCB), and unconjugated bilirubin (Bu).
• High-performance liquid chromatography (HPLC), a technique used for separating the components in a mixture, was employed for this analysis. The HPLC used an internal-surface reversed-phase silica support (ISRP) column.
• The technique involved the direct injection of sera, or blood plasma without fibrinogen; it used an acetonitrile: 0.5 M Tris-HCl buffer as a simple mobile phase.
• Detection was done using a variable-wavelength detector set at 450 nm, 0.01 absorbance unit full scale (AUFS), and a recorder set at 4 mm/min. This enabled each type of bilirubin to be detected at specific times following serum injection.

Research Results

• With this method, each type of bilirubin produced a sharp peak at distinct times; B delta, DCB, MCB and Bu were detectable at approximately 4.4, 6.4, 9.2 and 14.5 minutes, respectively.
• The method provided high recovery rates; the rates for Bu and DCB in pig serum were 91.9% and 95.9%, respectively. This indicates that most of the original material was not lost a in the HPLC process.
• The method was carried out on serum samples of various domestic animals suffering from jaundice, including cows, pigs and horses. The results were found to correlate well with the bilirubin values obtained from a standard reference procedure.
• Therefore, this new method provides a rapid and efficient way to analyze bilirubin levels in animal sera, and could potentially be useful for diagnosing and monitoring jaundice in veterinary medicine.