A Validated LC–MS/MS Assay for the Simultaneous Quantification of the FDA-Approved Anticancer Mixture (Encorafenib and Binimetinib): Metabolic Stability Estimation
Abstract
:1. Introduction
2. Results and Discussion
2.1. Results of In Silico ENF and BNB Metabolic Stability
2.2. Results of In Silico BNB and ENF Structural Alert Sites and Toxicity Prediction
2.3. HPLC–MS/MS Methodology
2.4. LC–MS/MS Method Validation
2.4.1. Specificity
2.4.2. Sensitivity and Linearity
2.4.3. Precision and Accuracy
2.4.4. Matrix Effects and Extraction Recovery
2.4.5. Stability
2.5. Metabolic Stability
3. Materials and Methods
3.1. Chemicals and Reagents
3.2. In Silico Prediction of ENF and BNB Metabolic Vulnerability and Toxicity Using StarDrop WhichP450 and DEREK Modules
3.3. LC–MS/MS Methodology
3.4. Standard Solutions of ENF and BNB
3.5. Preparation of Calibration Standards
3.6. BNB and ENF Extraction from HLM Matrix
3.7. Method Validation
3.7.1. Specificity, Linearity, and Sensitivity
3.7.2. Accuracy, Precision, and Stability
3.7.3. Matrix Effect and Extraction Recovery
3.8. Metabolic Stability Assessment of BNB and ENF
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Concentration in ng/mL | BNB | ENF | ||||||
---|---|---|---|---|---|---|---|---|
Mean a | SD | RSD % | Accuracy % | Mean a | SD | RSD % | Accuracy % | |
5 | 5.72 | 0.13 | 2.27 | 114.35 | 5.70 | 0.04 | 0.63 | 113.94 |
10 | 10.09 | 0.36 | 3.58 | 100.93 | 10.56 | 0.06 | 0.57 | 105.57 |
15 (LQC) | 14.98 | 0.24 | 1.60 | 99.84 | 15.24 | 0.14 | 0.94 | 101.63 |
20 | 19.46 | 0.36 | 1.86 | 97.29 | 20.17 | 0.35 | 1.75 | 100.86 |
30 | 29.62 | 0.32 | 1.08 | 98.75 | 30.80 | 0.18 | 0.57 | 102.66 |
50 | 49.48 | 1.41 | 2.85 | 98.96 | 49.87 | 0.76 | 1.52 | 99.75 |
80 | 82.15 | 2.40 | 2.92 | 102.69 | 79.06 | 1.30 | 1.65 | 98.83 |
100 | 98.21 | 1.36 | 1.39 | 98.21 | 97.41 | 0.77 | 0.79 | 97.41 |
150 (MQC) | 148.71 | 1.51 | 1.02 | 99.14 | 149.30 | 0.83 | 0.55 | 99.53 |
200 | 200.18 | 1.42 | 0.71 | 100.09 | 199.68 | 1.47 | 0.74 | 99.84 |
300 | 303.91 | 5.47 | 1.80 | 101.30 | 302.84 | 1.00 | 0.33 | 100.95 |
400 (HQC) | 396.97 | 2.22 | 0.56 | 99.24 | 400.59 | 3.46 | 0.86 | 100.15 |
500 | 500.44 | 2.89 | 0.58 | 100.09 | 498.75 | 2.08 | 0.42 | 99.75 |
% Recovery | 100.84 | 101.61 | ||||||
SD | 4.29 | 4.20 |
HLM Matrix | Mean | SD | % RSD | % Accuracy | |||
---|---|---|---|---|---|---|---|
BNB | Conc. in ng/mL | 15.00 (LQ) | Intra-day assay * | 14.98 | 0.20 | 1.31 | 99.8 |
Inter-day assay ** | 15.02 | 0.37 | 2.48 | 104 | |||
150.00 (MQ) | Intra-day assay | 148.71 | 1.24 | 0.83 | 99.1 | ||
Inter-day assay | 148.68 | 1.37 | 0.92 | 100 | |||
400.00 (HQ) | Intra-day assay | 396.97 | 1.81 | 0.46 | 99.2 | ||
Inter-day assay | 397.78 | 3.52 | 0.88 | 101 | |||
ENF | 15.00 (LQ) | Intra-day assay | 15.24 | 0.12 | 0.77 | 101 | |
Inter-day assay | 15.16 | 0.39 | 2.60 | 96.1 | |||
150.00 (MQ) | Intra-day assay * | 149.30 | 0.67 | 0.45 | 99.5 | ||
Inter-day assay | 148.20 | 1.88 | 1.27 | 96.7 | |||
400.00 (HQ) | Intra-day assay | 400.59 | 2.83 | 0.71 | 100 | ||
Inter-day assay | 399.80 | 2.92 | 0.73 | 100 |
Concentration (ng/mL) | HLM Matrix | |||||
---|---|---|---|---|---|---|
BNB | ENF | |||||
15 | 150 | 400 | 15 | 150 | 400 | |
Mean a | 15.09 | 148.56 | 398.35 | 15.15 | 148.40 | 400.35 |
Recovery (%) | 100 | 99.1 | 99.6 | 101 | 98.9 | 100 |
SD | 0.38 | 1.30 | 3.55 | 0.36 | 1.79 | 3.03 |
Precision (RSD %) | 2.51 | 0.87 | 0.89 | 2.38 | 1.21 | 0.76 |
Nominal Concentration (ng/mL) | Mean (ng/mL) | Recovery % | Precision (RSD %) | |
---|---|---|---|---|
BNB | Room temperature for 8 h | 14.89 ± 0.2 | 99.25 | 3.07 |
15 | ||||
150 | 147.38 ± 2.74 | 98.25 | 1.70 | |
400 | 395.31 ± 4.14 | 98.85 | 1.23 | |
Three freeze–thaw cycles | 14.75 ± 0.27 | 98.32 | 0.46 | |
15 | ||||
150 | 145.18 ± 2.04 | 96.79 | 2.52 | |
400 | 389.91 ± 3.19 | 97.48 | 4.87 | |
Stored at 4 °C for 24 h | 15.01 ± 0.42 | 100.05 | 2.80 | |
15 | ||||
150 | 146.38 ± 3.99 | 97.59 | 2.73 | |
400 | 397.31 ± 4.89 | 99.33 | 1.23 | |
Stored at −20 °C for 30 days | ||||
15 | 14.51 ± 0.67 | 96.72 | 4.59 | |
150 | 146.58 ± 2.39 | 97.72 | 1.63 | |
400 | 395.11 ± 4.57 | 98.78 | 1.16 | |
ENF | Room temperature for 8 h | |||
15 | 15.25 ± 0.3 | 101.64 | 2.00 | |
150 | 150.83 ± 4.17 | 100.55 | 2.76 | |
400 | 405.84 ± 4.45 | 101.46 | 1.10 | |
Three freeze–thaw cycles | ||||
15 | 14.61 ± 0.26 | 97.37 | 1.80 | |
150 | 145.83 ± 3.93 | 97.22 | 2.70 | |
400 | 398.64 ± 3.95 | 99.66 | 0.99 | |
Stored at 4 °C for 24 h | ||||
15 | 14.51 ± 0.35 | 97.37 | 2.37 | |
150 | 143.43 ± 2.85 | 96.95 | 1.96 | |
400 | 392.84 ± 3.16 | 98.21 | 0.81 | |
Stored at −20 °C for 30 days | ||||
15 | 15.03 ± 0.28 | 100.18 | 1.84 | |
150 | 144.83 ± 3.90 | 96.55 | 2.69 | |
400 | 393.64 ± 3.05 | 98.41 | 0.77 |
Parameters | ENF | BNB | ||
---|---|---|---|---|
ENF Alone | ENF with BNB | BNB Alone | BNB with ENF | |
Regression equation a | y = −0.0161x + 4.6059 | y = −0.0151x + 4.6042 | y = −0.0115x + 4.6096 | y = −0.0119x + 4.605 |
Slope | 0.0161 | 0.0151 | 0.0115 | 0.0119 |
t1/2 b | 43.1 min | 45.9 min | 60.3 min | 58.2 min |
CLint c | 16.09 µL/min/mg | 15.09 µL/min/mg | 11.49 µL/min/mg | 11.89 µL/min/mg |
R2 d | 0.9972 | 0.9906 | 0.9757 | 0.9817 |
Agilent 1200 HPLC | Triple Quadrupole 6410 QqQ | ||
---|---|---|---|
Isocratic mobile phase | ACN (38%) | ESI source | Positive mode |
10 mM ammonium formate in H2O (62%) adjusted with formic acid to pH 3.8 | Drying gas: N2 gas Pressure (60 psi) Flow rate (12 L/min) | ||
Injection volume: 2 μL | |||
Flow rate: 0.2 mL/min | |||
Agilent Hypersil BDS-C18 | Length 125 mm, fully porous particle size (3.0 μm) and internal diameter (2.0 mm) | Source temperature: 350 °C | |
Capillary voltage: 4000 V | |||
Mode | MRM mode | ||
Collision cell gas | Nitrogen with high purity | ||
Analytes | Binimetinib (BNB) | BNB MRM transitions | m/z 441 → m/z 379, FVa: 140 V, CEb: of 22 eV |
m/z 441 → m/z 165, FV: 140 V, CE: of 20 eV | |||
Encorafenib (ENF) | ENF MRM Transitions | m/z 540 → m/z 508, FV: 135 V, CE: 18 eV | |
m/z 540 → m/z 359, FV: 140 V, CE: 20 eV | |||
IS | Avitinib (AVB) | AVB MRM transitions | m/z 488 → m/z 433, FV: 145 V, CE: of 15 eV |
m/z 488 → m/z 403, FV: 145 V, CE: of 16 eV |
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Attwa, M.W.; Darwish, H.W.; Al-Shakliah, N.S.; Kadi, A.A. A Validated LC–MS/MS Assay for the Simultaneous Quantification of the FDA-Approved Anticancer Mixture (Encorafenib and Binimetinib): Metabolic Stability Estimation. Molecules 2021, 26, 2717. https://doi.org/10.3390/molecules26092717
Attwa MW, Darwish HW, Al-Shakliah NS, Kadi AA. A Validated LC–MS/MS Assay for the Simultaneous Quantification of the FDA-Approved Anticancer Mixture (Encorafenib and Binimetinib): Metabolic Stability Estimation. Molecules. 2021; 26(9):2717. https://doi.org/10.3390/molecules26092717
Chicago/Turabian StyleAttwa, Mohamed W., Hany W. Darwish, Nasser S. Al-Shakliah, and Adnan A. Kadi. 2021. "A Validated LC–MS/MS Assay for the Simultaneous Quantification of the FDA-Approved Anticancer Mixture (Encorafenib and Binimetinib): Metabolic Stability Estimation" Molecules 26, no. 9: 2717. https://doi.org/10.3390/molecules26092717