Development & validation of LC–MS/MS assay for the quantification of pegcantratinib Research Article
Table 1. Recovery of pegcantratinib from human tumor homogenate at quality control concentrations (n = 5).
Pegcantratinib concentration (ng/ml) 8.00 80.0 160
Internal standard CV: Coefficient of variation.
five samples from a pool of tumor homogenate added with pegcantratinib at the defined concentration [12,13].
Linearity & range The calibration curve linearity was validated over three working days, while the linear range was inves- tigated by preparing samples <50% of the LLOQ and >150% of the ULOQ over one working day. For each standard concentration, the ratio of the HPLC-MS/MS peak area for pegcantratinib to IS was calculated and plotted against the nominal concentra- tion of the drug in the sample. Linearity of the stan- dard curves was assessed by regression analysis and the goodness of the fit calculated using Pearson’s determi- nation coefficient, R2
and by comparing the true and
back-calculated concentrations of the calibration stan- dards. The accuracy of back-calculated values of an individual concentration were required to be within 85–115% of the theoretical concentration (80–120% at the LLOQ), and all standards had to meet these criteria [12,13].
Intra-/interassay precision & accuracy Five replicates per QC concentration were analyzed for intraday precision and accuracy, while interday preci- sion and accuracy were determined from three separate experiments carried out on different days. The preci- sion of the method at each concentration was reported as the CV value, expressing the SD as a percentage
of the mean calculated concentration; accuracy was determined by expressing the mean calculated con- centration as a percentage of the nominal concentra- tion. In each run, the concentration measured for QC samples was required to be within 15% of the nominal value [12,13].
Carryover Carryover of pegcantratinib and IS were evaluated by placing a blank sample, without analyte or IS, imme- diately after the highest calibration standard. Response for analyte in the carryover sample was required to be ≤20% of the response at LLOQ. The response for IS in the carryover sample was required to be ≤5% of the response for the control matrix + IS [12,13].
Stability The stability of pegcantratinib in homogenate tumor was assessed by analyzing QC samples (low and high concentrations) in triplicate following storage and han- dling. Short-term stability was assessed in QC samples unextracted and extracted (autosampler stability) from the tumor matrix after 10 days stored at 4°C, the bench-top stability was determined after 4 h at room temperature. Freeze–thaw stability was assessed for three freeze–thaw cycles. We also analyzed the sta- bility of blank frozen homogenate tumor (stored at -20°C), which was used to prepare QCs on the day of the analysis. Long-term stability was assessed in QC
Table 2. Matrix effect of four different types of human tissue homogenate on pegcantratinib (100 ng/ml) and internal standard (1μg/ml).
Tissue type
Skin with anagen follicles Cylindroma and spiradenoma Cylindroma only Spiradenoma only: – Mean – SD
– CV (%) CV: Coefficient of variation; MF: Matrix factor.
Pegcantratinib (MF) 1.05 0.97 1.07 0.92 1.00 0.07 7.1
CT340 (MF) 1.05 0.96 1.05 1.01 1.02 0.04 3.9
Ratio 1.00 1.01 1.02 0.91 0.99 0.05 5.0
Recovery (%) ± SD 89.7 ± 11.9 93.7 ± 8.94 86.4 ± 10.4 84.9 ± 1.61
CV (%) 13.2 9.54 12.0 1.90
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