title: Ultrasensitive quantification of serum estrogens in postmenopausal women and older men by liquid chromatography-tandem mass spectrometry.
abstract: An ultrasensitive stable isotope dilution liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for multiplexed quantitative analysis of six unconjugated and conjugated estrogens in human serum. The quantification utilized a new derivatization procedure, which formed analytes as pre-ionized N-methyl pyridinium-3-sulfonyl (NMPS) derivatives. This method required only 0.1mL of human serum, yet was capable of simultaneously quantifying six estrogens within 20min. The lower limit of quantitation (LLOQ) for estradiol (E2), 16α-hydroxy (OH)-E2, 4-methoxy (MeO)-E2 and 2-MeO-E2 was 1fg on column, and was 10fg on column for 4-OH-E2 and 2-OH-E2. All analytes demonstrated a linear response from 0.5 to 200pg/mL (5-2000pg/mL for 4-OH-E2 and 2-OH-E2). Using this validated method, the estrogen levels in human serum samples from 20 female patients and 20 male patients were analyzed and compared. The levels found for unconjugated serum E2 from postmenopausal women (mean 2.7pg/mL) were very similar to those obtained by highly sensitive gas chromatography-mass spectrometry (GC-MS) methodology. However, the level obtained in serum from older men (mean 9.5pg/mL) was lower than has been reported previously by both GC-MS and LC-MS procedures. The total (unconjugated+conjugated) 4-MeO-E2 levels were significantly higher in female samples compared with males (p<0.05). The enhanced sensitivity offered by the present method will allow for a more specific analysis of estrogens and their metabolites. Our observations might suggest that the level of total 4-MeO-E2 could be a potential biomarker for breast cancer cases.
authors: Center of Excellence in Environmental Toxicology and Penn SRP Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States; Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States; Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors: Qingqing
authors: Q
authors: Wang
authors: Center of Excellence in Environmental Toxicology and Penn SRP Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States; Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States; NCBS, Center for Cellular and Molecular Platforms, Bangalore, India.
authors: Kannan
authors: K
authors: Rangiah
authors: Center of Excellence in Environmental Toxicology and Penn SRP Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States; Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States.
authors: Clementina
authors: C
authors: Mesaros
authors: Center of Excellence in Environmental Toxicology and Penn SRP Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States; Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States.
authors: Nathaniel W
authors: NW
authors: Snyder
authors: Center of Excellence in Environmental Toxicology and Penn SRP Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States; Division of Pulmonary, Allergy and Critical Care, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, United States.
authors: Anil
authors: A
authors: Vachani
authors: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors: Haifeng
authors: H
authors: Song
authors: Center of Excellence in Environmental Toxicology and Penn SRP Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States; Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States. Electronic address: ianblair@mail.med.upenn.edu.
authors: Ian A
authors: IA
authors: Blair
mesh_terms: D000368:Aged; D001774:Blood Chemical Analysis / Q000379:methods* / Q000592:standards; D002138:Calibration; D002853:Chromatography, Liquid; D004967:Estrogens / Q000097:blood* / Q000378:metabolism; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D017698:Postmenopause / Q000097:blood*; D012015:Reference Standards; D053719:Tandem Mass Spectrometry
chemical_list: D004967:Estrogens
keywords: 17β-estradiol; Breast cancer; Endometrial cancer; Estrogen; Prostate cancer; Stable isotope dilution
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