Rapid and quantitative analysis of bilirubin in equines by high-performance liquid chromatography.

This research article describes the development of a quick and precise method for measuring bilirubin levels in horses using high-performance liquid chromatography. Both unconjugated and conjugated bilirubin types were detected, providing valuable diagnostic information for assessing the health status of equines, particularly in distinguishing healthy from jaundiced animals.

High-Performance Liquid Chromatography (HPLC) Method

• The study utilized high-performance liquid chromatography (HPLC) with ultraviolet detection to determine the levels of bilirubin in horses. This is a technique used for separating the components of a mixture and is widely implemented in various fields such as biochemistry and analytical chemistry.
• The mobile phase, which is the liquid that carries the components of the mixture through the chromatograph, was a blend of methanol and 0.5 M Tris-HCl buffer. This combination successfully generated clear HPLC peaks for both unconjugated bilirubin (UCBL) and conjugated bilirubin (CBL), making their detection straightforward and accurate.

UV Detection and Peak Analysis

• The wavelength detector was set to 450 nm, a frequency that gives high detection sensitivity for bilirubin. The retention time for UCBL and CBL was 7.1 minutes and 4.9 minutes respectively, providing rapid results.
• The lower limits of detection for these forms of bilirubin ranged between 0.16 to 0.78 micrograms per ml, indicating the designed technique’s sensitivity and precision.
• Orange II, with a retention time of 3.6 minutes, served as an internal standard to enable accurate measurements by adjusting for any potential variations during analysis.

Application to Healthy and Jaundiced Equine Samples

• Analysis of samples from healthy horses and those suspected of jaundice (due to a yellowish discoloration in their carcasses) revealed only UCBL peaks. This suggests that the technique might be particularly useful for identifying this form of bilirubin and thus assisting in the diagnosis of jaundice.
• No bilirubin was detected in the aqueous humor (a liquid found in the eye) of either healthy or clinically ill horses. This finding indicates the test’s specificity for serum, and possibly other bodily fluids, but not for aqueous humour.

Correlation with Other Test Methods

• There was a strong correlation between the UCBL levels determined by this HPLC method and the total bilirubin value obtained from a standard total bilirubin assay kit in serum. This suggests that this method reliably estimates bilirubin levels within this biological fluid.
• The study also found a correlation between bilirubin levels in serum samples and those in pericardial fluid (the fluid around the heart), extending the potential application of this method.