Cardiovascular risk-linked compound homocysteine thiolactone can now be detected in urine with HPLC-UV test

Homocysteine thiolactone (HTL) has been associated with various cardiovascular diseases. A high performance liquid chromatography with ultraviolet detection (HPLC-UV) method – which is aimed at measuring HTL levels directly from human urine – has been developed by researchers, Dr. Justyna Piechocka, Professor Rafał Głowacki and doctoral student Marta Gaweł, all of the University of Lodz in Poland. The novel method is both effective, simple and reproducible and provides a valuable insight for both clinical diagnostics and research.

HTL is recognised as a cytotoxic metabolite of homocysteine and has been linked with increased risk of acute myocardial infarction and ischemic strokes with its negative effects on protein structure and function. The research team aimed to create a method combining effective sample preparation with the robustness of HPLC-UV techniques.

The process begins with the liquid-liquid extraction of HTL from urine, followed by re-extraction with hydrochloric acid. This two-step extraction is necessary to separate HTL from other urine constituents before the derivatisation step with 1-benzyl-2-chloropyridinium bromide (BBCP). After derivatisation, the resulting 2-S-pyridinium derivative of homocysteine is analysed via HPLC-UV.

Using Agilent’s Zorbax SB-C18 columns, the researchers established effective chromatographic separation conditions. These include gradient elution at ambient temperatures, allowing for the analysis of samples within six minutes – a marked improvement over existing methods which require longer processing times. The eluent composition using 0.1% acetic acid and acetonitrile at controlled flow rates proved efficient for resolving HTL detection clearly against background noise.

The validation of the method showcased impressive performance metrics, including accuracy ranging from 92.56% to 114.31% and precision levels between 5.89% and 14.25%. With these metrics, the researchers confirmed their methodology for precise HTL measurement to concentrations as low as 100 nmol/L.

The study process also found the presence of HTL in twelve of eighteen urine samples taken from healthy volunteers, with concentrations between 36.67 and 693.12 nmol/L. The findings validated the assay’s effectiveness and also aligned with other studies, underlining the reliability of HTL measurements.

By integrating commonly used laboratory equipment and following straightforward procedures, this novel HPLC-UV method enhances prospects for HTL testing across laboratories worldwide. The ease of use and reduced time commitment required for sample processing compared to other analytical techniques broaden the avenues for future studies investigating the biological impact of HTL.

Conclusively, this research contributes to the methodological advances for determining HTL and also emphasises the importance of testing for this compound in a clinical setting.

For further reading please visit: https://www.nature.com/articles/s41598-025-92323-y