High-Throughput LC-MS/MS Method for Direct Quantification of Glucuronidated, Sulfated, and Free Enterolactone in Human Plasma

Sulfation and glucuronidation constitute a major pathway in humans and may play an important role in biological activity of metabolites including the enterolignan, enterolactone. Because the aromatic structure of enterolactone has similarities to steroid metabolites, it was hypothesized that enterolactone may protect against hormone-dependent cancers. This led to numerous epidemiological studies. In this context, there has been a demand for rapid, sensitive, high-throughput methods to measure enterolactone in biofluids. Different methods have been developed using GC MS, HPLC, LC-MS/MS and a fluoroimmunoassay; however, most of these methods measure the total concentration of enterolactone, without any specification of its conjugation pattern. Here for the first time we present a high-throughput LC-MS/MS method to quantify enterolactone in its intact form as glucuronide, sulfate, and free enterolactone. The method has shown good accuracy and precision at low concentration and very high sensitivity, with LLOQfor enterolactone sulfate at 16 pM, enterolactone glucuronide at 26 pM, and free enterolactone at 86 pM. The short run time of 2.6 min combined with simple sample clean up and high sensitivity make this method attractive for the high-throughput of samples needed for epidemiological studies. Finally, we have adapted the new method to quantify enterolactone and its conjugates in 3956 plasma samples from an epidemiological study. We found enterolactone glucuronide to be the major conjugation form and that conjugation pattern was similar between men and women.