A tandem liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS) technique to separate and quantify steroid isomers 11β-methyl-19-nortestosterone and testosterone.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become a mainstay analytical technique in pharmaceutical research and development and clinical diagnosis due to several advantages including excellent selectivity, specificity, and high sensitivity. LC-MS/MS has become the method of choice for steroids analysis due to its fast analytical time and improved specificity yet has a challenge in the separation and measurement of isomers with the same product ions. Here we describe a high-sensitivity LC/LC-MS/MS method that combines chiral chromatography and reverse-phase chromatography (LC/LC) along with MS/MS to rapidly separate and quantify steroid isomers of 11ß-methyl-19-nortestosterone (11ß-MNT) and endogenous testosterone in serum.