Agilent 6120 quadrupole mass spectrometer

¹H NMR (400 MHz, DMSO-d₆) δ 7.92 (t, J = 5.6 Hz, 1 H), 7.56 (dt, J = 42.2, 6.0 Hz, 1 H), 7.38–7.03 (m, 5 H), 5.36 (dd, J = 14.9, 5.6 Hz, 1 H), 4.45 (td, J = 5.6, 2.2 Hz, 1 H), 3.72 (dd, J = 5.6, 2.6 Hz, 1 H), 3.45–2.94 (m, 4 H), 2.70 (ddt, J = 10.4, 5.8, 1.8 Hz, 2 H), 2.51 (p, J = 1.9 Hz, 1 H), 0.96 (d, J = 6.9 Hz, 3 H), 0.90–0.63 (m, 6 H); HPLC using an Agilent 1100 equipped with an Eclipse + C18 4.1 × 55 mm 1.8-micron column and a flow rate of 0.5 mL/min eluting with a 5 to 100% gradient of acetonitrile in water modified with 0.1% formic acid over 9 minutes provided a t_R = 5.93 min and 100% purity as detected at 220 and 254 nM. Mass was determined using an Agilent 6120 quadrupole mass spectrometer M + 1⁺ = 337.2.

Ba/F3 cells expressing NPM-ALK (9×10⁴ cells/200 μL) were cultured with crizotinib (0.5 μM) in combination with α-tocopherol (25 μM) for 60 or 120 min and washed with ice-cold PBS, and then lysed with 125 μL of 50% acetonitrile. Samples were centrifuged at 5,000 r.p.m at 4°C for 2 min and the supernatant was analyzed by an Agilent 6120 quadrupole mass spectrometer equipped with an electrospray interface attached to an Agilent 1200 series (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separations were performed on Agilent ZORBAX Eclipse Plus C18 column (4.6 × 100 mm, 3.5 μm) at a flow rate 0.5 mL/min. The mobile phases used were water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). At time zero the flow consisted of 90% of mobile phase A and 10% mobile phase B. One minute after injection, the proportion of B was linearly increased to 100% over 4 min followed by keeping constant at 100%B for 5 min. From this point, the mobile phase was set to initial conditions (90%A and 10%B) and the column was equilibrated for 5 min prior to the next injection. The electrospray ionization probe was set at 350°C, with the nebulizing gas pressure and electrospray gas flow set at 20 psi. and 13 L/min, respectively. Detection of crizotinib was carried out by selected ion monitoring of [M+H]⁺ ions at m/z 450 in the positive ion mode.