B-178 Clinical Routine Ultrasensitive LC-MS/MS Method Development for Analysis of 17β-Estradiol in Human Serum
Authors: Tokuyama P Y , Rocha V C , Zampieri G , Osorio P S , Lopes A C 
Published: September 2023

Abstract

Background
The increasing clinical demand for the measurement of circulating 17β-Estradiol at very low concentrations consolidates the need of highly sensitive and specific analytical methodologies. These assays are suitable in situations such as antiestrogen therapy monitoring (use of aromatase inhibitor in breast cancer treatment); diagnosis of suspect inherited sex-steroid metabolic disorders; diagnosis of precocious and delayed puberty in female; hormone replacement therapy in postmenopausal women; and estrogen deficiency in men. Time-consuming methods and lack of sensitivity are common in the quantification of 17β-Estradiol in serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The strategy of chemical derivatization is common for analysis of estrogens because it enhances specificity and sensitivity, but this approach becomes laborious in the sample preparation process. For the purpose of solving these analytical challenges, the aim of this work was developing a reliable, sensitive and low-cost LC-MS/MS method without derivatization for clinical routine analysis.

Methods
Isotope-labeled internal standard 17β-Estradiol-D5 were added to calibrators, quality controls (QC) and serum samples before a liquid-liquid extraction with the organic solvent Diethyl Ether. After reconstitution, the extracts were injected into a Biphenyl column 50 × 2.1 mm, 2,6 µm, chromatographic separated by solvent gradient (Ammonium Fluoride in Water – Acetonitrile) and detected in positive electrospray ionization mode. The analytical method was validated using reference material grade standards and spiked QCs in charcoal stripped pool patient serum at 4 levels by the assessment of linearity, limit of quantification (LoQ), precision inter and intra-assay, accuracy, carryover, method comparison, cross-reactivity and stability (freeze-thawing cycles, bench top, postoperative and stock solution).

Results
The method has been validated with the analytical measurement range of 2–500 pg/mL. It was successfully optimized to achieve low levels of 17β-Estradiol as proposed with limit of quantitation of 2 pg/mL. Within-run and between-run precision (coefficient of variation) study yielded 1.3%–7.2% and accuracy within 95.7%–112.1%. No carryover, matrix effect and significant cross-reactivity was observed. Estradiol was found to be stable in serum samples under 3 freeze/thawing cycles, 4 h at ambient temperature and 72 h after extraction procedure in auto-injector. The stability of stock solutions was tested in −20 °C freezer and remained stable for 1 month. Method comparison between two reference laboratories (LC-MS/MS method) was performed and the results showed acceptable data for regression statistics (slope (a) = 0.9184; intercept (b) = −0.1576), t-test statistics (bias = 8.5%) and difference plot.

Conclusion
An ultrasensitive and derivatization-free LC-MS/MS method was developed and it may be beneficial for patients with endocrine disorders associated with estradiol levels.