Highly sensitive simultaneous quantitative analysis of estrone and equilin from plasma using LC/MS/MS

Significance of the Topic

Accurate measurement of trace estrogens in plasma is essential for pharmacokinetic studies, therapeutic drug monitoring and safety assessment of hormone replacement therapies. Simultaneous quantitation of estrone and equilin addresses the need to monitor endogenous and exogenous steroids with high sensitivity and specificity in complex biological matrices.

Objectives and Study Overview

This study aimed to develop and validate an ultra-sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the concurrent determination of estrone and equilin in charcoal-stripped human plasma. The goals included achieving low limits of quantitation, ensuring specificity against endogenous interferences and demonstrating robust calibration across a wide concentration range.

Methodology and Used Instrumentation

Sample Preparation:

• Plasma was treated with activated charcoal to remove endogenous estrone and interferences.
• Calibration standards and quality controls were spiked into stripped plasma at concentrations ranging from ~1 to 542 pg/mL for estrone and ~1 to 359 pg/mL for equilin.
• Samples were fortified with deuterated internal standards (estrone-D4, equilin-D4), acidified, centrifuged and subjected to solid-phase extraction (SPE) with methanol and aqueous washes.
• Extracts were evaporated under nitrogen and reconstituted in mobile phase for injection.

Instrumentation:

• UHPLC: Nexera X2 system with a 150×4.6 mm, 3 µm C18 column at 40 °C; isocratic elution (buffer/methanol) at 0.7 mL/min; injection volume 20 µL.
• Mass Spectrometry: Shimadzu LCMS-8060 triple quadrupole with heated electrospray ionization (ESI) probe; negative-mode MRM transitions: estrone (269.10>145.20), equilin (267.00>265.30+143.30) and their D4 analogues.
• Gas flows: nebulizing 3 L/min, drying 6 L/min, heating 16 L/min; interface temperature 380 °C; desolvation line 150 °C; heating block 450 °C.

Main Results and Discussion

• Limits of quantitation of 1.525 pg/mL for estrone and 1.117 pg/mL for equilin were achieved with clear baseline separation and no detectable interference in blank matrices.
• Calibration curves were linear over the tested ranges with correlation coefficients of 0.9992 (estrone) and 0.9985 (equilin).
• Intra- and inter-batch precision and accuracy met bioanalytical criteria: accuracy within 94.5–110.8% and %RSD ≤10.6% across all QC levels.
• The heated ESI probe significantly improved desolvation and ionization efficiency, lowering background noise and enhancing sensitivity.

Benefits and Practical Applications

• Enables reliable quantitation of minute estrogen levels in plasma for clinical and research settings.
• Supports pharmacokinetic profiling, therapeutic monitoring and safety evaluation of hormone therapies.
• High throughput and robustness make it suitable for routine bioanalytical laboratories.

Future Trends and Potential Uses

Advances may include multiplexed assays for broader steroid panels, integration with microflow LC for reduced solvent use, and application to other low-abundance biomarkers. Emerging ionization interfaces and high-resolution MS could further improve selectivity and throughput in clinical pharmacology.

Conclusion

The developed LC-MS/MS method on the Shimadzu LCMS-8060 platform offers ultra-sensitive, accurate and precise simultaneous measurement of estrone and equilin in plasma. It represents a comprehensive solution for bioanalysis of estrogens with potential extension to broader steroid profiling.

References

1. Yasui T, Uemura H, Tezuka M, Yamada M, Irahara M, Miura M, et al. Biological effects of hormone replacement therapy in relation to serum estradiol levels. Horm Res. 2001;56(1):38–44.
2. Giese RW. Measurement of estrogens: analytical challenges and recent advances. J Chromatogr A. 2003;1000(1–2):401–412.