Stable Isotope-Coded Quaternization for Comparative Quantification of Estrogen Metabolites by High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Authors

Wen-Chu Yang, Bindley Bioscience Center, Purdue University, West Lafayette, IN
Fred E. Regnier, Department of Chemistry, Purdue University, West Lafayette, IN
Dan Silva, Cancer Research Laboratory, Methodist Research Institute, Indianapolis, IN
Jiri Adamec, University of Nebraska-LincolnFollow

Date of this Version

2008

Citation

J Chromatogr B Analyt Technol Biomed Life Sci. 2008 July 15; 870(2): 233–240

Comments

Open Access. Used by Permission.

Abstract

A fast and sensitive LC-ESI-MS method is described for the comparative quantification of 16 estrogen metabolites based on the derivatization of estrogens with a novel derivatizing reagent, Nmethyl- nicotinic acid N-hydroxysuccinimide ester (C1-NA-NHS). The process introduces a quaternary amine to the analytes, making the analytes permanently charged regardless of the pH of the HPLC mobile phase. This quaternization resulted in a highly efficient separation of 16 estrogen metabolites in 7 minutes at a detection level below 1 ng/mL. By using a deuterated derivatizing reagent (C1-d3-NA-NHS), a complete set of deuterated standards was utilized and used as internal standards in a comparative quantification and recovery study, demonstrating acceptable results over a wide concentration range. A pooled breast cancer serum sample was analyzed using the described method, and 15 estrogens were detected in the range of 80 – 530 pg/mL.