Fast and Sensitive LC-APCI-MS/MS Quantitative Analysis of Estrone and Estradiol in Serum without Chemical Derivatization

Importance of the Topic

The quantitative determination of estrone (E1) and estradiol (E2) at low picogram-per-milliliter concentrations is critical for clinical research and diagnostic applications. High sensitivity and specificity are required to monitor hormone levels accurately. Immunoassays can suffer from cross-reactivity and limited sensitivity, while derivatization-based LC-MS/MS workflows increase complexity and can impact instrument performance over time.

Objectives and Study Overview

This study aimed to develop and validate a fast, simple, and sensitive LC-APCI-MS/MS method for measuring E1 and E2 in serum or plasma without chemical derivatization. Key performance parameters such as recovery, linearity, sensitivity, precision, accuracy, ion suppression, and carryover were evaluated.

Methodology and Instrumentation

• Sample Preparation: Serum or plasma samples were spiked with a deuterated E2 internal standard, extracted via liquid-liquid extraction (LLE) using methyl tert-butyl ether (MTBE), dried under nitrogen, and reconstituted in 60% methanol before centrifugation.
• Chromatography: Thermo Scientific Accela UHPLC system equipped with a Hypersil GOLD column (150 × 2.1 mm, 3 µm). Mobile phases were water and methanol; the total run time was 6 minutes at room temperature.
• Mass Spectrometry: Thermo Scientific TSQ Vantage triple quadrupole instrument with an APCI source in negative ion mode. Detection was performed in selected reaction monitoring (SRM) mode monitoring m/z 269→145 for E1 and m/z 271→183 for E2.

Main Results and Discussion

• Recovery: Absolute recoveries for E1, E2, and the internal standard ranged from 70% to 115% (n=4) using MTBE LLE.
• Linearity: Calibration curves prepared in charcoal-stripped serum delivered excellent linearity (R² > 0.998) over 5–1 000 pg/mL for both analytes.
• Sensitivity: Lower limits of quantitation (LLOQ) were established at 3.5 pg/mL for E1 and 4.4 pg/mL for E2 with accuracy between 90.5% and 112.8%.
• Precision and Accuracy: Intra- and inter-assay precision at low (12 pg/mL) and high (300 pg/mL) levels yielded CVs below 12.5%. A pooled human plasma sample spiked at 35.4 pg/mL E1 and 18.1 pg/mL E2 showed intra-batch CVs of 2.2% and 3.6%, respectively.
• Ion Suppression and Carryover: Post-column infusion experiments revealed negligible ion suppression. No carryover was detected in blank injections following high-concentration samples.

Benefits and Practical Applications

This derivatization-free assay achieves rapid throughput (6 minutes per analysis) with high sensitivity and robustness, reducing sample handling complexity and potential contamination. It is well suited for large clinical studies, therapeutic drug monitoring, and pharmaceutical quality control.

Future Trends and Potential Applications

Future developments may involve microflow or nano-LC to boost sensitivity and decrease solvent consumption. Coupling with automated sample preparation systems can increase throughput for epidemiological studies. Expanding the assay to include broader steroid panels will support endocrinology research and personalized medicine initiatives.

Conclusion

A fully validated, fast, and sensitive LC-APCI-MS/MS method for estrone and estradiol quantitation in serum and plasma has been established. It provides excellent sensitivity, a wide dynamic range, and robust performance, making it a valuable tool for clinical and research laboratories.