High-Sensitivity Micro LC-MS/MS Assay for Serum Estradiol without Derivatization.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Chemistry

Background:

• Accurate measurement of low estradiol (E2) concentrations (<20 pg/mL) is critical for postmenopausal women, men, pediatric patients, and breast cancer patients.
• Existing high-sensitivity LC-MS/MS methods for E2 often require large sample volumes and complex derivatization steps.
• There is a need for a simpler, more sensitive method to measure E2 at clinically relevant low levels.

Purpose of the Study:

• To develop and validate a high-sensitivity, underivatized micro LC-MS/MS method for measuring estradiol (E2).
• To achieve a reliable quantification of E2 concentrations below 5 pg/mL using a minimal sample volume.

Main Methods:

• Estradiol (E2) was extracted from a small sample volume (290 μL) using a hexane/ethyl acetate mixture.
• Separation was performed on a micro LC system (Eksigent Ekspert™) with a flow rate of 35 μL/min over 3.5 minutes.
• Detection was achieved using a SCIEX QTRAP 6500 mass spectrometer in negative mode, utilizing specific transitions for E2 and its deuterated internal standard (E2-d4).

Main Results:

• The method demonstrated a broad linear range (3.0-820.0 pg/mL) with excellent linearity (r2 > 0.999).
• The limit of quantification (LOQ) was determined to be 3.0 pg/mL, with total precision below 15% at all quality control levels.
• The assay showed good correlation with estradiol radioimmunoassay (RIA) (r2 = 0.96) and modest correlation with an automated immunoassay (r2 = 0.86).

Conclusions:

• A routinely applicable micro LC-MS/MS method for quantifying estradiol (E2) in blood samples was successfully developed and validated.
• The method is highly sensitive, achieving an LOQ of 3.0 pg/mL without the need for derivatization.
• This underivatized approach simplifies sample preparation and reduces sample volume requirements for E2 analysis.