MSACL 2025 Abstract

Optimized Method for LC-MS/MS Quantification of Underivatized Estrone(E1) and Estradiol (E2) in Human Serum Using Supported Liquid Extraction

Stephanie J. Marin (1), Shahana W. Huq (2), Rajashree Chakravarti (3), Paulina Michaud (4)
Phenomenex, Inc., Torrance, CA

Stephanie Marin, PhD (Presenter) Phenomenex >> POSTER (PDF)

Presenter Bio: Stephanie J. Marin is the Senior Applied Markets Global Market Development Manager at Phenomenex. She received her Ph.D. in chemistry from Arizona State University, and has expertise in sample preparation, liquid chromatography and mass spectrometry. Dr Marin has worked in marketing, product development, method development and method validation, in addition to customer facing roles in technical support, service, education and training. She has over 10 years of experience developing and validating clinical methods from her tenure at the ARUP Institute for Clinical and Experimental Pathology. She has also held positions as an Applications Chemist at Hamilton Company and Biotage, Marketing Manager at Selerity Technologies and as a Group Leader in analytical services for specialty chemicals and polymers at Rohm and Haas (now Dow). She was also a supervisor at an EPA certified laboratory. She is the author of over 30 peer reviewed publications and book chapters and over 100 abstracts presented at national and international meetings. She is a member of SOFT, ACS and ADLM, and has served as a reviewer for SOFT and ADLM national meeting abstracts. She is also a reviewer for several journals, including Journal of Chromatography B, Journal or Analytical Toxicology, JMSACL.

Relevant Financial Disclosures (within past 24 months, reported on Apr 02, 2026) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Precise measurement of estrone (E1) and estradiol (E2) is essential for assessing reproductive and endocrine function in clinical research. Due to their low circulating levels, poor ionization efficiency, and structural similarity, direct LC-MS quantification without derivatization is analytically challenging. Although recent improvements in sample preparation and mass spectrometry sensitivity have enhanced assay performance, derivatization remains a common approach to achieve the sensitivity required for reliable detection of E1 and E2 in serum. Here, we present a sensitive method for LC-MS/MS quantitation of estrone and estradiol without derivatization using supported liquid extraction (SLE).

OBJECTIVE:
To develop a robust extraction and LC-MS/MS method for quantification of underivatized E1 and E2 in human serum using supported-liquid extraction (SLE) and optimized chromatographic separation.

METHODS:
Four reversed-phase LC columns were screened for optimal separation: Kinetex C18, Kinetex Biphenyl, Luna Omega Polar C18, and Kinetex F5. The Kinetex Biphenyl column (50 × 3.0 mm, 2.6 µm) provided the best resolution (~1 min) and highest signal response for E1.
Sample preparation was SLE using Strata-SE SLE MAX 96-well plates. Human serum was pre-treated with ammonium acetate buffer and extracted with ethyl acetate/hexane (9:1). Extracts were dried under nitrogen and reconstituted in ammonium fluoride/methanol (60:40, v/v).
LC-MS/MS analysis was performed on an Agilent® 1290 Infinity system coupled with a SCIEX® 7500 Triple Quad mass spectrometer. The mobile phase consisted of 0.5 mM ammonium fluoride in water (A) and methanol (B), with a gradient elution over 5 minutes at 0.8 mL/min.

RESULTS:
The method demonstrated excellent linearity for E1 (1–500 pg/mL, R² = 0.998) and E2 (5–500 pg/mL, R² = 0.993). LOQs were 5 pg/mL for E1 and 10 pg/mL for E2, with signal-to-noise ratios of 12 and 8, respectively. Improved S/N for E2 was observed at 20 pg/mL (S/N = 19).

DISCUSSION AND CONCLUSIONS:
A sensitive and selective LC-MS/MS method was successfully developed for quantifying underivatized estrone and estradiol in human serum with acceptable signal to noise ratio for ensuring accurate quantitation at LOQs as low as 5 pg/mL for E1 and 10 pg/mL for E2. It is reported in the literature that for E1, 95th percentile values in a population including males and females of all categories fall within 40-200 pg/mL and for E2 95th percentile values in a population of all males and females range from 40-350 pg/mL (1). This method using Strata SE SLE and Kinetex Biphenyl column (50 × 3.0 mm, 2.6 µm) on a high sensitivity MS provides a solution for the majority of samples and avoids time consuming, labor intensive derivatization typically used in LC-MS/MS estrogen methods.

Strata SE SLE extraction coupled with a Kinetex Biphenyl column and a sensitive LC-MS/MS system provided optimal chromatographic performance suitable for clinical research applications. Where lower concentration levels are desired, various derivatization procedures can be followed to attain higher sensitivity.

References:
1. The Journal of Clinical Endocrinology & Metabolism, Volume 105, Issue 3, March 2020, Pages 754–768.