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

Document Type

Article

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.