Sample multiplexing for increasing throughput for quantification of estrogens in serum by LC-MS/MS

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Estrogens play crucial roles in numerous physiological processes.
• Accurate and rapid estrogen quantification is vital for diagnosing and managing estrogen-related diseases.
• High-volume assay methods are needed to meet clinical demands.

Purpose of the Study:

• To develop and validate a sample-multiplexing liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of serum estrogens.
• To enhance assay throughput for estrone (E1), estradiol (E2), and estriol (E3).

Main Methods:

• Developed a sample-multiplexing LC-MS/MS method using 100 μL serum samples.
• Employed ethyl acetate extraction, dansyl chloride or pyridine-3-sulfonyl chloride derivatization, and a second liquid-liquid extraction with hexane.
• Validated the method according to FDA and CLSI guidelines.

Main Results:

• The method achieved simultaneous analysis of E1, E2, and E3 in 2 serum samples within a 7-minute run.
• Demonstrated excellent analytical performance: accuracy (87.7-110.3%), linearity (R² > 0.99), and precision (CVs 1.7-9.4%).
• Showed negligible interference, carry-over, and acceptable matrix effects.

Conclusions:

• The developed sample-multiplexing LC-MS/MS method significantly increases assay throughput for serum estrogens.
• This method provides accurate and reliable quantification of E1, E2, and E3 without compromising analytical performance.
• Offers a valuable tool for high-volume estrogen analysis in clinical diagnostics.