HPLC trace of gefitinib (500 pmol injection) monitored at 254 nm by HPLC-UV-MS.
Mass spectrum (Q1 scan, positive polarity) corresponding to the UV peak at 11.29 min.
| Assay Date(s): | 02/28/16 |
| Acquisition Time: | 2:51 |
| HPLC Hardware: | Shimadzu Prominence UFLC (Shimadzu); Pumps: LC-20AD; Autosampler: SIL-20AC-HT; UV-Vis Detector: SPD-20A; Column Oven: CTO-20AC; Controller: CBM-20A; Degasser: DGU-20A3 |
| Autosampler Parameters: | Rinse Mode: Before and after aspiration; Rinsing volume: 200 µL; Cooler Temperature: 15°C |
| Column Oven Parameters: | Temperature: 40°C |
| UV-Vis Detector Parameters: | Lamp: D2; Sampling Frequency: 10 Hz; Polarity: Positive; Response: 1.0 sec; Cell Temperature: 40°C |
| MS Hardware: | QTRAP5500 (SCIEX); Ion Source: Turbo V; Probe: TurboIonSpray ESI; Gas Source: Source5000 LC/MS TriGas Generator (Parker-Balston) |
| MS Scan Parameters: | Scan Type: Q1; Polarity: Positive; Scan Mode: Profile; Q1 Resolution: Unit; Scan Rate: 200 Da/s; Scan Range: 100-1250 Da |
| MS Instrument Parameters: | CUR (N2): 40 psi; IS: 5500 V; TEM 500 °C; GS1 (Zero Air): 50 psi; GS2 (Zero Air): 50 psi; DP: 180 V, EP: 10 V |
| Processing Software: | Analyst 1.6.2 (SCIEX) |
| UV Processing Parameters: | IntelliQuan; Noise Percentage: 50%; Peak Split Factors: 5; Baseline Sub Window: 0.25 min; Minimum Height: 500 cps; Minimum Width: 0 sec |
| MS Processing Parameters: | Background Subtraction: 30 sec prior to peak base; Spectrum averaging: Peak base to base |
| Stock Solution | |
| Concentration: | 100 µM |
| Solvent: | Water (Optima LC/MS Grade) |
| LC-UV-MS Parameters | |
| Column: | Luna C18(2) 3 µm 100Å, 50×2mm (Phenomenex, 00B-4251-B0) |
| Guard Column: | C18 SecurityGuard Cartridge 4x2 mm (Phenomenex, AJ0-4286) |
| Mobile Phase: | A: 1 mM NH3COOH with 0.1% Formic acid (pH 2.75 ± 0.05); B: Acetonitrile |
| Equilibration Time: | 10 min |
| Run Time: | 20 min |
| Gradient Timetable: | 0 min = 5% B; 5 min = 5% B; 15 min = 95% B, 20 min = 95% B |
| Injection Volume: | 5 µL |
| Monitoring Wavelength: | 254 nm |
| Results | Retention Time: | 11.29 min |
| Calculated [M+H]+: | m/z 447.9 |
| Observed [M+H]+: | m/z 446.6 |
| Drug Purity by HPLC: | 99.25% |
| Related Standard Operating Procedures (SOPs) | |
| Assay SOPs: | DToxS SOP A – 10.0 HPLC-UV-MS Drug Characterization |
| Performed by: | A. J. Pickard, T. F. Bartenstein |