Stable isotope-coded quaternization for comparative quantification of estrogen metabolites by high-performance liquid chromatography–electrospray ionization mass spectrometry

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 870; no. 2; pp. 233 - 240
• Main Authors: Yang, Wen-Chu, Regnier, Fred E., Sliva, Dan, Adamec, Jiri
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.07.2008; Elsevier Science
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Breast Neoplasms - blood; Chromatography, High Pressure Liquid - methods; Estradiol Congeners - analysis; Estradiol Congeners - blood; Estradiol Congeners - chemistry; Estrogen; Fundamental and applied biological sciences. Psychology; General pharmacology; HPLC; Humans; Isotope Labeling; Mass spectrometry; Medical sciences; Pharmacology. Drug treatments; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization - methods; Mass spectrometry; HPLC; Estrogen; Edible fish; Quaternization; Metabolite; HPLC chromatography; Ovarian hormone; Electrospray; Cod; Sex steroid hormone; Isotopes; Comparative study; Quantitative analysis
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2008.06.026

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.