Icahn School of Medicine at Mount Sinai LINCS Center for Drug Toxicity Signatures

HPLC trace of nilotinib (500 pmol injection) monitored at 254 nm by HPLC-UV-MS.

Mass spectrum (Q1 scan, positive polarity) corresponding to the UV peak at 12.77 min.

Assay Date(s): 02/27/16
Acquisition Time: 22:15
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: 12.77 min
Calculated [M+H]+: m/z 530.5
Observed [M+H]+: m/z 532
Drug Purity by HPLC: 99.75%
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

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