Development & validation of LC–MS/MS assay for the quantification of pegcantratinib Research Article
Figure 2. In-source collision-induced dissociation/Q1 mass spectra of pegcantratinib. (A) at declustering potential 20 V (B) at declustering potential 186 V and (C) MS/MS mass spectra of pegcantratinib.
standard solutions to tumor homogenate (90 μl), to produce final concentrations of 2.5, 5.0, 10.0, 50.0, 100 and 250 ng/ml. A blank sample (homogenate processed without IS), a zero blank sample (homog- enate processed with IS) and three concentrations of QC samples in triplicate were included alongside the standard curve. QC samples were prepared by adding 10 μl of working QC solutions to blank homogenate tumor (90 μl) to generate concentrations of 8.0, 80.0 and 160 ng/ml.
Processing samples Aliquots of homogenate (100 μl) from study samples, standards or QC samples were added with 10 μl (100 ng) of the IS working solution and 900 μl of acetonitrile and samples were vortex-mixed for 20 s. Following extraction, samples were centrifuged at 4000 × g for 6 min at 4°C. The supernatants obtained were transferred to Eppendorf polypropylene tubes, evaporated to dryness under nitrogen at 37°C, and then reconstituted in 100 μl of mobile phase (MP) A and MP B in the proportion 1:1, v/v. MP A consisted of 1% formic acid in water and MP B was 1% formic acid in acetonitrile. After vortex mixing, samples were centrifuged at 4000 × g for 6 min at 4°C, supernatants
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were transferred to autosampler glass vials and 10 μl of each sample injected onto the HPLC–MS/MS system.
Chromatography conditions The HPLC system consisted of a Series 200 autos- ampler and micropump (Perkin Elmer, Beaconsfield, Bucks, UK). Samples were separated on a Kine- tex C18 chromatographic column (2.6 μm, 50.0 × 4.6 mm) coupled with a precolumn of the same material, both thermostatically controlled at 44°C. The HPLC system was set at a constant flow rate of 0.5 ml/min and run under gradient conditions: step 1 – 50% MP A for 1 min, step 2 – 50% MP A to 15% over 4 min; step 3 – constant 15% MP A for 2 min; step 4 – 15% MP A to initial conditions over 1 min; step 5 – reconditioning for 2 min.
MS conditions Detection was obtained using an API 4000 triple quadrupole mass spectrometer SCIEX (CA, USA). Standard solutions (10 μg/ml) of pegcantratinib and IS were infused at a flow rate of 10 μl/min to optimize MS parameters, with mass spectra (MS1) and prod- uct ion spectra (MS2) generated in positive ion mode. The HPLC–MS/MS system was equipped with a
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