LC/MS/MS Analysis of Estradiol (Underivatized) from Patient Serum on Titan C18, 5 cm Column
Materials
CONDITIONS
sample preparation
SPE (Solid Phase Extraction)
sample
A 200 μL sample of calibrator or serum was mixed with 50 μL of internal standard solution (1 nmol/L 13C4-estradiol in 30% methanol) and vortexed. (Liquid-liquid extraction was performed by addition of 1 mL of MTBE. Supernatants were dried down and reconstituted in 100 μL of 50% methanol before being transferred to the LC/MS/MS system.)
column
Titan C18, 5 cm x 2.1 mm I.D., 1.9 μm particles (577122-U)
mobile phase
[A] 0.2 mM ammonium fluoride; [B] methanol
gradient
40 to 82%B in 4.6 min; to 100%B in 0.05 min; held for 1.15 min; to 40% B in 0.05 min; held for 1.65 min
flow rate
0.4 mL/min
column temp.
40 °C
detector
MS, ESI(-), MRM transitions listed on figure
injection
20 μL
sample
patient serum sample containing estradiol, 56 pmol/L
Description
Analysis Note
A fast and robust LC/MS/MS method was developed for the measurement of estradiol in human serum using Titan C18 1.9 μm columns. Ultra high purity LC-MS solvents were used to supply low background interference and low particulate contaminants for robust, trouble-free operation. Cerilliant certified reference material grade standards provided reliable quantification.
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