Stable isotope-coded quaternization for comparative quantification of estrogen metabolites by high-performance liquid chromatography-electrospray ionization mass spectrometry.
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, N-methyl-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 high-performance liquid chromatography (HPLC) mobile phase. This quaternization resulted in a highly efficient separation of 16 estrogen metabolites in 7 min at a detection level below 1 ng/mL. By using a deuterated derivatizing reagent (C1-d(3)-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.
Yang
Wen-Chu
WC
Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA.
Regnier
Fred E
FE
Sliva
Dan
D
Adamec
Jiri
J
D001943:Breast Neoplasms / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D045166:Estradiol Congeners / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D045166:Estradiol Congeners