DETERMINATION OF DIETHYLSTILBESTROL IN HUMAN PLASMA WITH MEASUREMENT UNCERTAINTY ESTIMATION BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) using an electrospray ionization (ESI) method was developed and validated to determine the concentration of diethylstilbestrol in human plasma. Samples were extracted with methyl tert-butyl ether after alkalified by sodium tetraborate solution, using 3,5-dihydroxy-4-isopropyl toluylene as the internal standard (IS). Separation was performed on a Restek C18 column (2.1 mm × 150 mm, 5.0 µm) guarded by a Hypersil Gold pre-column (2.1 mm × 10 mm, 3 µm). The mobile phase consisted of acetonitrile–water (85:15, containing 0.2 mmol/L ammonium acetate) at a flow rate of 0.3 mL/min. Detection was performed using negative multiple reaction monitoring (MRM) mode. The mass transitions monitored were [M-H]− m/z 267.2 → 237.2 and [M-H]− m/z 253.2 → 211.0 for diethylstilbestrol and IS, respectively. The method was fully validated using total error theory, which is based on β-expectation tolerance intervals and include trueness and intermedia...