Analysis of cholesterol and phospholipid hydroperoxides by high-performance liquid chromatography with mercury drop...

High-performance liquid chromatography (HPLC) with reductive mode electrochemical detection on a mercury drop has been employed for the separation and determination of lipid hydroperoxides. Under the conditions used, baseline separation is achieved for three cholesterol hydroperoxide (ChOOH) standards, not only from one another, but also from two different phosphatidylcholine hydroperoxide (PCOOH) standards. Applying this method to a test system, photodynamically treated murine leukemia cells, we have identified and quantified a major family of overlapping PCOOHs and three ChOOHs, two of which are characteristic singlet oxygen adducts. In a typical separation, the detection limit is < 0.5 pmol for ChOOHs and < 50 pmol for more slowly eluting PCOOHs. In this respect, mercury drop detection outperforms all previously described electrochemical detection methods for lipid hydroperoxides and compares favorably with other HPLC-based approaches. However, in terms of equipment cost, relative simplicity of operation, and fewer potential artifacts, this method has a clear advantage over all other existing high-sensitivity methods.