Research Article Zangarini, Rajan, Danilenko, Berry, Traversa & Veal
Figure 3. Typical SRM chromatograms. (A) Extracted blank tumor sample, (B) extracted blank tumor sample with internal standard (zero sample), (C) extracted tumor sample with pegcantratinib at the LLOQ (2.5 ng/ml) and IS (D) extracted tumor biopsy sample from a patient treated with pegcantratinib with internal standard. Note different intensity scale in (A).
Turbo Ion Spray source operated at 750°C and voltage of 5500 V. Clinical samples were analyzed with ESI, with zero air as nebulizer gas (60 psi) and heater gas (30 psi). Nitrogen was utilized as curtain gas (20 psi) and collision gas at 4 psi. The declustering potential (DP) was optimized and set to 186 V for in-source CID of both pegcantratinib and CT340. Quantifica- tion was carried out in SRM mode with the follow- ing transition: m/z 419.0→376.4 for both pegcantra- tinib and IS, following chromatographic separation. Data were processed using the Analyst 1.6.2 software package (SCIEX).
Method validation The method validation followed the EMA and the US FDA guidance on bioanalytical method valida- tion [12,13]. The parameters validated were recovery, matrix effect, LLOQ, linearity and range, intra- and inter-day precision and accuracy, carryover effect and stability.
Recovery The percentage extraction was determined in triplicate at three QC concentrations (8.0, 80.0 and 160 ng/ml) for pegcantratinib and at 1 μg/ml for the IS in tumor
28219 Bioanalysis (2017) 9(3)
homogenate samples. Absolute recovery was deter- mined by comparing the peak area of pegcantratinib extracted from homogenate tumor samples with the peak area in absence of matrix (true concentration of the analyte in solvent) that represents 100% recovery. The coefficient of variation (CV) was required to be within 15% [13].
Matrix effect The matrix effect was assessed in four independent sources of blank matrix for pegcantratinib at the con- centration of 100 ng/ml and IS at 10 μg/ml by calcu- lating the matrix factor (MF) for each analyte, that is the ratio of the peak area of the analyte added to a pre- extracted sample to the peak area of the same amount of analyte in solvent. The IS-normalized MF was cal- culated by dividing the MF of the pegcantratinib by the MF of the IS. The CV of the IS-normalized MF should be within 15% [12].
Limit of quantification The LLOQ of the method corresponded to the con- centration of the lowest standard with precision ≤20% and accuracy within 80–120% of the nominal value, with a S/N ≥10. The LLOQ was assessed by preparing
future science group
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58
