Opposite to untargeted metabolomics, targeted metabolomics approach can be applied when the biomarker is known. Enterolactone is a biomarker of healthy lifestyle and therefore used in epidemiological studies in revers association to lifestyle diseases such as type 2 diabetes, cardiovascular diseases and some forms of cancer. However, the analytical techniques to measure enterolactone in plasma developed so far are based on the hydrolysis of enterolactone with enzymes β-glucuronidase/sulfatese prior to the measurements and therefore are time consuming. Our purpose was to develop method that was rapid, reproducible, sensitive and easy to perform. Moreover, we wanted to develop a method to quantify enterolactone in its intact form as glucuronide, sulfate and free enterolactone. Tandem mass spectrometry (LC-MS/MS) was the method of choice due to its superior selectivity and sensitivity. Using the authentic standards of enterolacrone, enterolactone glucuronide, and enterolactone sulfate we developed the method that has shown good accuracy and precision at low concentration and high sensitivity, with LLOQ for enterolactone sulfate at 16 pM, enterolactone glucuronide at 26 pM and free enterolactone at 86 pM. The method was validated with blank human plasma and applied to measure 3956 plasma samples from an epidemiological study. The results of Principal Component Analyses (PCA) indicated that the total concentration of enterolactone and the concentration of enterolactone glucuronide and sulfate negatively correlated to BMI, age, ratio, cancer type, smoking status and alcohol intake but positively to sport, fruits-, vegetables- and whole-grain intake. Moreover, we found enterolactone glucuronide to be the major conjugation form and that there was no difference between men and women.

The importance of this novel, targeted LC-MS/MS method is two-fold. Firstly, we hope that measuring enterolactone in its intact forms will contribute with new knowledge on the role of enterolactone in human health. Secondly, since the method is rapid and easy to perform it can be used for high-throughput of samples and therefore will be the method of choice for future epidemiological investigations and clinical diagnosis.