Assessment of In Silico and In Vitro Selpercatinib Metabolic Stability in Human Liver Microsomes Using a Validated LC-MS/MS Method
Abstract
:1. Introduction
2. Results and Discussion
2.1. In Silico SLP Metabolic Stability
2.2. LC–MS/MS Method
2.3. Validation Parameters
2.3.1. Specificity
2.3.2. Sensitivity and Linearity
2.3.3. Precision and Accuracy
2.3.4. Matrix Effects and Extraction Recovery
2.4. Metabolic Stability
3. Material and Methods
3.1. Materials and Instruments
3.2. In Silico SLP Metabolic Stability Assessment
3.3. LC-MS/MS Analytical Methodology
3.3.1. Liquid Chromatographic
3.3.2. Mass Spectrometry
3.4. SLP Working Solutions
3.5. SLP Calibration Standards
3.6. Method Validation
3.6.1. Specificity
3.6.2. Linearity and Sensitivity
3.6.3. Accuracy and Precision
3.6.4. Matrix Effect and Extraction Recovery
3.7. SLP Metabolic Stability
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
Abbreviations
References
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SLP Nominal Concentrations (ng/mL) | Mean | SD | RSD (%) | Accuracy (%) | Recovery |
---|---|---|---|---|---|
1 (LLQC) | 0.95 | 0.02 | 2.08 | −5.08 | 94.92 |
3 (LQC) | 3.09 | 0.03 | 1.00 | 3.15 | 103.15 |
15 | 15.16 | 0.19 | 1.28 | 1.07 | 101.07 |
50 | 50.98 | 1.10 | 2.15 | 1.97 | 101.97 |
100 | 105.06 | 1.47 | 1.40 | 5.06 | 105.06 |
300 | 303.45 | 2.36 | 0.78 | 1.15 | 101.15 |
500 | 502.91 | 1.41 | 0.28 | 0.58 | 100.58 |
900 (MQC) | 898.67 | 4.33 | 0.48 | −0.15 | 99.85 |
1500 | 1492.58 | 11.37 | 0.76 | −0.49 | 99.51 |
2400 (HQC) | 2387.68 | 34.32 | 1.44 | −0.51 | 99.49 |
3000 | 3025.03 | 36.16 | 1.20 | 0.83 | 100.83 |
SLP in HLM’s Matrix (ng/mL) | Intra-Batch Assay (Twelve Replicates in the Same Day) | Inter-Batch Assay (Six Replicates in Three Consecutive Days) | ||||||
---|---|---|---|---|---|---|---|---|
1 (LLQC) | 3 (LQC) | 900 (MQC) | 2400 (HQC) | 1 (LLQC) | 3 (LQC) | 900 (MQC) | 2400 (HQC) | |
Mean | 0.95 | 3.09 | 898.67 | 2387.68 | 0.93 | 3.16 | 912.08 | 2375.59 |
SD | 0.02 | 0.03 | 4.33 | 34.32 | 0.02 | 0.10 | 15.59 | 24.66 |
Precision (%RSD) | 2.08 | 1.00 | 0.48 | 1.44 | 2.01 | 3.08 | 1.71 | 1.04 |
% Accuracy | −5.08 | 3.15 | −0.15 | −0.51 | −6.56 | 5.22 | 1.34 | −1.02 |
Recovery (%) | 94.92 | 103.15 | 99.85 | 99.49 | 93.44 | 105.22 | 101.34 | 98.98 |
Time (min) | Mean a (ng/mL) | X b | LN X | Linearity Parameters |
---|---|---|---|---|
0 | 626 | 100.00 | 4.61 | Regression equation: y = −0.0291x + 4.6221 |
2.5 | 586 | 93.61 | 4.54 | |
7.5 | 527 | 84.19 | 4.43 | R2 = 0.9971 |
15 | 418 | 66.77 | 4.20 | |
20 | 353 | 56.39 | 4.03 | Slope: −0.0291 |
30 | 264 | 42.17 | 3.74 | |
40 | 238 | 38.02 | 3.64 | t1/2: 23.82 min and |
50 | 227 | 36.26 | 3.59 | Clint: 34 mL/min/kg |
60 | 218 | 34.82 | 3.55 | |
70 | 197 | 31.47 | 3.45 |
Analytes | Methanol | ACN | Solid Phase Extraction | Protein Precipitation | C18 or C8 Column | PFP Column |
---|---|---|---|---|---|---|
SLP | 1.71 min | 2.1 min | Low recovery | High recovery | 2.65 min | 2.1 min |
Tailed | Perfect | Irreproducible | Reproducible | Tailed | Perfect | |
FLG | 1.34 min | 1.1 min | Low recovery | High recovery | 2.34 min | 1.1 min |
Overlapped | Perfect | Irreproducible | Reproducible | Perfect | Perfect |
Drug | ESI Mode | Rt | Precursor (m/z) | Qualification Traces (m/z) | Quantification Traces (m/z) | Collision Energy (CE, eV) | Cone Voltage (V) |
---|---|---|---|---|---|---|---|
SLP | +ve | 2.1 | 526.0 | 52.98 | 122.0 | 66/28 | 42 |
FLG (IS) | +ve | 1.1 | 426.0 | 223.17 | 291.11 | 24/38 | 38 |
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Attwa, M.W.; AlRabiah, H.; Mostafa, G.A.E.; Bakheit, A.H.; Kadi, A.A. Assessment of In Silico and In Vitro Selpercatinib Metabolic Stability in Human Liver Microsomes Using a Validated LC-MS/MS Method. Molecules 2023, 28, 2618. https://doi.org/10.3390/molecules28062618
Attwa MW, AlRabiah H, Mostafa GAE, Bakheit AH, Kadi AA. Assessment of In Silico and In Vitro Selpercatinib Metabolic Stability in Human Liver Microsomes Using a Validated LC-MS/MS Method. Molecules. 2023; 28(6):2618. https://doi.org/10.3390/molecules28062618
Chicago/Turabian StyleAttwa, Mohamed W., Haitham AlRabiah, Gamal A.E. Mostafa, Ahmed H. Bakheit, and Adnan A. Kadi. 2023. "Assessment of In Silico and In Vitro Selpercatinib Metabolic Stability in Human Liver Microsomes Using a Validated LC-MS/MS Method" Molecules 28, no. 6: 2618. https://doi.org/10.3390/molecules28062618