The measurement of homocysteine thiolactone (HTL) has received heightened attention due to its association with various cardiovascular diseases. Researchers at the University of Lodz have made significant strides by developing the first high performance liquid chromatography with ultraviolet detection (HPLC-UV) method aimed at quantifying HTL levels directly from human urine samples. This innovative method stands out not only for its effectiveness but also for its simplicity and reproducibility, providing valuable insights for both clinical diagnostics and research.
Homocysteine thiolactone, recognized as a cytotoxic metabolite of homocysteine, has been linked with detrimental effects on protein structure and function, leading to increased risk of acute myocardial infarction and ischemic strokes. Previous methods for the measurement of HTL, typically involving complex derivatization and chromatographic techniques, lacked the efficiency needed for widespread clinical use. To address these limitations, 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 derivatization step with 1-benzyl-2-chloropyridinium bromide (BBCP). After derivatization, the resulting 2-S-pyridinium derivative of homocysteine is analyzed via HPLC-UV.
Using 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 just 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 affirmed their method’s capability for precise HTL quantification down to concentrations of 100 nmol/L.
A key finding demonstrated the presence of HTL within twelve out of eighteen urine samples sourced from healthy individuals, with concentrations between 36.67 and 693.12 nmol/L. These findings not only validate the effectiveness of the new assay but also align with previous 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 not only contributes to the methodological advancements for determining HTL but emphasizes the importance of monitoring this compound within clinical settings. Future studies could explore the correlations between HTL levels and various health outcomes, potentially informing preventive strategies for cardiovascular diseases.