Feng Bai (Presenter)
LABioMed at Harbor-UCLA Medical Center
Bio: Bioanalytical Method Developer, especially in LC-MS/MS method development, validation, application, and assay troubleshooting under GLP and CLIA environments
Authorship: Feng Bai, Maria LaJoie, Andrew Leung, Ronald Swerdloff, and Christina Wang
Division of Endocrinology, Departments of Medicine, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute,
| Short Abstract We developed and validated a highly sensitive LC-MS/MS method for the simultaneous measurement of serum E1, E2, and E3 for clinical and research purposes in both women and men. The three estrogens were separated on a selected column within 3 minutes and detected on API5000 with ESI source at negative mode. The assay sensitivity reached 2 pg/mL without using sample derivatization and other sensitivity enhancing techniques. The assay precision (%CV) were 2.6 to 5.6 for E1, 4.3 to 5.2 for E2, and 4.1 to 8.7 for E3. The accuracy (%) were from 95.6 to 99.0 for E1, from 96.8 to 98.4 for E2, and from 94.7 to 97.3 for E3 respectively at spanning different estrogen concentrations. |
Long Abstract
Background: Estrone (E1), estradiol (E2), and estriol (E3) are three natural bioactive estrogens produced by women throughout their reproductive years. Estradiol is the principal active estrogen produced by women and men.
Objective: We developed and validated a highly sensitive LC-MS/MS method for the simultaneous measurement of serum E1, E2, and E3 levels for clinical and research purposes in both women and men.
Method: Internal standards (IS), deuterium-labeled d4-E1, d4-E2, and d3-E3, were obtained from Cambridge Isotope Laboratories (Andover, MA). 300ƒÊL of serum were extracted with ethyl acetate/hexane (v/v 3/2) and the organic phase applied to a Shimadzu high-performance LC 20 series system (Columbia, Maryland) connected to an Applied Biosystems API 5000 using ESI source (Foster City, California). The three estrogens were separated on a Raptor Biphenyl column (100~2.0mm, 2.7ƒÊm) (Restek, Bellefonte, PA) within 3 minutes, with a gradient profile (from 63% to 100% methanol) at a flow rate of 0.4mL/min and a mobile phase of methanol (B) and water (A). The triple quadrupole mass spectrometer operated in the negative mode using multiple reaction monitoring (MRM) with transitions m/z: 269.1/145.0 for E1, m/z 273.0/147.0 for d4-E1; 271.3/145.0 for E2, 275.2/147.0 for d4-E2; 287.3/171.0 for E3, and 290.2/174.0 for d3-E3. The calibration curve was linear over a concentration range of 2 to 2000 pg/ml for both E1 and E2, and 50 to 5000 pg/ml for E3. The lower limit of quantification was 2.0pg/ml for both E1 and E2, and 50 pg/ml for E3. The intra-assay and inter-assay precision expressed as coefficient of variation were 2.6 to 5.6 and 3.9 to 4.6 for E1; 4.3 to 5.0 and 4.6 to 5.2 for E2; 4.1 to 5.7 and 5.2 to 8.7% for E3. The accuracy was from 95.6% to 99.0% for E1, from 96.8% to 98.4% for E2, and from 94.7 to 97.3% for E3 respectively spanning different estrogen concentrations.
Results: The three natural estrogens were separated within 3 minutes and the concentrations simultaneously measured using this method. The assay sensitivity reached 2 pg/mL without using sample derivatization and other sensitivity enhancing techniques. The method was efficient, simple, and easy to translate to clinical application. We used this validated method in a study of the three estrogens in women in the first trimester of pregnancy and correlated the levels with Bisphenol A. We are currently using this method in clinical research in adult men.
Conclusion: We have developed and validated a highly sensitive and accurate LC-MS/MS assay for the natural estrogens E1, E2, and E3 in serum.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |