Quantification of Elvitegravir by LC-MS/MS

A 50 µL of media or cell lysate samples were mixed with 3-volumes of cold acetonitrile containing internal standard (IS) ritonavir (50 ng/mL). The mixed solution was vortexed and centrifuged at 8000 rpm for 10 min for protein precipitation. A clear supernatant was collected and analyzed using our established LC-MS/MS method^{66 (link)}. Briefly, a Shimadzu liquid chromatographic system (Kyoto, Japan) coupled with an AB SCIEX Triple Quad 5500 tandem mass spectrometer (Framingham, MA) was used for the analysis. Chromatographic separation was performed on an Xterra® MS C18 column (125 Å, 3.5 μm, 4.6 mm × 50 mm; Waters, Milford, MA). The mobile phase used for EVG separation consisted of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid (v/v) at a flow rate of 1 mL/min. The gradient elution was as follows: 0–1.5 min, 50% B; 1.5–5.1 min, 60% B (v/v). The quantification of the validated assay of EVG was 1 to 500 ng/mL. EVG and IS were eluted separately at approximately 3.27 and 2.72 min, respectively. The multiple reactions monitoring (MRM) transitions (m/z) Q1/Q3 selected for quantitative analyses were 447.9/343.8 for EVG and 721.3/296.1 for the IS. In order to reduce matrix effects, calibration curves were prepared with blank media or cell lysate based on the sample types.