Determination of Gefitinib in Plasma by Liquid Chromatography with a C12 Column and Electrospray Tandem Mass Spectrometry Detection

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Abstract
A highly sensitive liquid chromatography electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) method has been developed for the measurement of gefitinib (ZD1839) in human plasma. The method was validated over a linear range of 0.5–1000 ng/mL, using deuterated gefitinib (D8‐ZD1839) as the internal standard (IS). Compounds were extracted from 500 µL of sodium heparin plasma by 6.0 mL butyl methyl ether liquid–liquid extraction. The dried residue was reconstituted with 250 µL of 20% acetonitrile with 1.0% formic acid and 30 µL injected onto the LC‐ESI‐MS/MS system. Chromatographic separation was achieved on a Phenomenex® Synergi 4µ MAX‐RP 80 Å C12 column (75 × 2.0 mm2) with an isocratic mobile phase of acetonitrile–1.0% formic acid (30:70, v/v). The analytes were detected with a PE Sciex API 365 triple quadrupole mass spectrometer using turbo ion spray® source with positive ionization. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 447.2 (precursor ion) to m/z 127.8 (product ion) for gefitinib and m/z 455.2 (precursor ion) to m/z 136.0 (product ion) for D8‐ZD1839. The lower limit of quantitation (LLOQ) of gefitinib was 0.30 ng/mL (S/N ≥ 10), and results from a 5‐day validation study demonstrated acceptable within‐day and between‐day precision (CV% values ≤6.0% and ≤5.2%, respectively) and accuracy (range 91.0–97.7%). This method is now used to analyze plasma samples from pediatric pharmacokinetic studies of ZD1839, and the wide linear range (∼4 log) of this method provides a distinct advantage, as shown by the results of a representative patient.

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