A Validated Liquid Chromatography–Tandem Mass Spectrometric (LC-MS/MS) Method for the Estimation of Fulvestrant in Human Plasma

A fast, reliable and sensitive high-performance liquid chromatographic / tandem mass spectrometric assay (LC-MS/MS) was developed to quantify fulvestrant in human plasma using fulvestrant-D3 as an internal standard (IS). The analyte was extracted from human plasma by liquid-liquid extraction (LLE) using methyl tertiary butyl ether (MTBE) as an extracting solvent. Chromatographic separation was achieved in a run time of less than 3.0 min on chromolith RP-18e, (100 × 4.6 mm, Ea) column. Analytes quantitation was performed by turbo-ion spray tandem mass spectrometry (TIS-MS/MS). The method involves simple isocratic chromatographic conditions at a flow rate of 1.0 mLmin-1 with approximately 50% flow splitting. Mobile phase was a mixture of 0.5% acetic acid and acetonitrile (20:80, v/v, respectively). Detection by mass spectrometry was achieved in the MRM mode by monitoring the transition pairs of m/z 605.2 to m/z 427.4 ion for fulvestrant and m/z 608.6 to m/z 430.4 for the IS, using the [M H] ̄ ions for the IS. A fully validated LC-MS/MS method was developed as per ICH guidelines. The developed liquid-liquid extraction (LLE), (LC-MS/ MS) assay method demonstrated sufficient ability to track the concentrations of fulvestrant in human plasma at a level of accuracy and sensitivity suitable for pharmacokinetic studies. Calibration plots were linear (r2>0.99) over the concentration range of 0.100 to 25.0 ng. mL-1 for fulvestrant. The overall recovery was 79.29%. Precision expressed as %RSD was ≤ 3.1% and ≤ 2.97 % for intra-day and inter-day data respectively.