Development & validation of LC–MS/MS assay for the quantification of pegcantratinib Research Article
Table 4. Interday linearity, accuracy and precision of calibration curves of pegcantratinib in human tumor homogenate.
Day
1 2 3
Mean (n = 4) SD
Accuracy (%) Precision (%) NA: Not applicable.
1.00 0.97 NA NA NA NA 97
NA
2.50 2.63 2.59 2.46 2.56 0.09 102 3
5.00 4.82 4.95 5.02 4.93 0.10 99 2
Pegcantratinib concentration (ng/ml) 10.0 9.22 9.11 10.5 9.61 0.77 96 8
50.0 50.3 57.0 50.3 52.5 3.87 105 7
through the first quadrupole into the collision cell where the collision energy was optimized to obtain product ions with a high signal (Figure 2C). The characteristic product ions were monitored in the third quadrupole at m/z 376.4 (34 eV) and 348.3 (41 eV). Pegcantra- tinib and IS were quantified using the transitions m/z 419.0→376.4 and were distinguished by their different retention times of approximately 2.7 and 2.1 min for pegcantratinib and IS, respectively. Elution of the ana- lytes was rapid and selective. No interfering peaks were present at these retention times, and the peaks were completely resolved from the tumor matrix. Figure 3 presents typical SRM chromatograms: Figure 3A is an extracted blank tumor sample; Figure 3B is an extracted blank tumor sample with IS; Figure 3C is an extracted tumor sample at the LLOQ (2.5 ng/ml) with IS added; and Figure 3D represents an extracted tumor sample from a patient treated with pegcantratinib.
Recovery Recovery was evaluated in triplicate over the three QC concentrations by comparing the peak areas of spiked
100 104
92.8 97.1 98.0 5.65 98 6
250 261 267 247 258 10.3 103 4
500 481 NA NA NA NA 96
NA
homogenate tumor samples, following extraction, with those obtained from direct injection of pegcantratinib standard solutions in MP. The recovery percentage was >86% for pegcantratinib and 85% for IS. There were no significant variations in CV (<13%) as shown in Table 1.
Matrix effect Due to difficulty in obtaining different types of human tumor, the matrix effect was evaluated during the pre- validation study, when four different types of matrix were available. No matrix effect was observed in four independent
matrix sources including three different tumors and normal skin, evaluated by assessment of IS-corrected MF, with a mean ratio of 0.99 ± 0.05 and CV of 5% observed (Table 2).
Limit of quantification The LLOQ concentration in tumor homogenate was defined to be 2.5 ng/ml, with precision and accuracy of 6 and 99%, respectively. Single values are shown in Table 3.
Table 5. Intraday precision and accuracy of the method for the analysis of pegcantratinib in human tumor homogenate.
Measured concentration
Mean (n = 5) SD
Accuracy (%) Precision (%)
8.00 8.47 7.52 7.18 7.27 8.14 7.72 0.56 96.5 7.3
Nominal concentration (ng/ml) 80.0 72.7 69.6 82.1 81.9 75.4 76.3 5.56 95.4 7.3
160 154 151 160 163 164 158
5.68 99.0 3.6
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