Enantioselective Separation of Mirtazapine and Its Metabolites by Capillary Electrophoresis with Acetonitrile Field-Amplified Sample Stacking and Its Application
Abstract
:1. Introduction
2. Results and Discussion
2.1. Optimization of Enantioseparation Conditions
2.1.1. CD Type and Concentration
2.1.2. Optimization of Borate-Phosphate Buffer pH and Concentration and Applied Voltage
2.1.3. Optimization of Sample Stacking Performance
2.1.3.1. Selection of Injection Modes, Time and Voltage
2.1.3.2. Selection of Sample Solvent
2.1.4. Sample Preparation
2.2. Method Validation
2.2.1. Specificity
2.2.2. Linearity of Calibration Curves and Lower Limits of Quantification
2.2.3. Accuracy, Precision and Extraction Recovery
Enantiomer | Range(ng/mL) | Regression Equations | Correlation Coefficient (R2) | SD for the Slope | SD for the Inercept |
---|---|---|---|---|---|
(−)-R-MRT | 0.5–50 | y = 0.256x + 0.0188 | 0.998 | 0.050 | 0.027 |
(+)-S-MRT | 0.5–50 | y = 0.232x + 0.0156 | 0.992 | 0.048 | 0.015 |
(−)-R-DMR | 0.5–50 | y = 0.315x − 0.0314 | 0.995 | 0.037 | 0.030 |
(+)-S-DMR | 0.5–50 | y = 0.290x − 0.0290 | 0.993 | 0.041 | 0.020 |
(−)-R-8-OHM | 0.5–50 | y = 0.145x − 0.0517 | 0.995 | 0.044 | 0.030 |
(+)-S-8-OHM | 0.5–50 | y = 0.130x − 0.0444 | 0.994 | 0.039 | 0.033 |
Enantiomer | LLOQ | Low QC | Medium QC | High QC | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
0.5 ng/mL | 1 ng/mL | 5 ng/mL | 40 ng/mL | |||||||||
Mean ± SD | RE | RSD | Mean ± SD | RE | RSD | Mean ± SD | RE | RSD | Mean ± SD | RE | RSD | |
(ng/mL) | (%) | (%) | (ng/mL) | (%) | (%) | (ng/mL) | (%) | (%) | (ng/mL) | (%) | (%) | |
(−)-R-MRT | 0.54 ± 0.041 | 8.0 | 8.2 | 1.08 ± 0.12 | 7.8 | 11.3 | 5.16 ± 0.36 | 3.3 | 7.0 | 40.34 ± 1.4 | 0.8 | 3.4 |
(+)-S-MRT | 0.46 ± 0.060 | −8.1 | 14.0 | 0.99 ± 0.10 | −1.2 | 10.0 | 4.88 ± 0.33 | −2.4 | 6.7 | 39.32 ± 1.3 | −1.7 | 3.4 |
(−)-R-DMR | 0.55 ± 0.069 | 10.4 | 13.2 | 1.03 ± 0.15 | 2.7 | 14.2 | 4.61 ± 0.27 | −7.9 | 6.0 | 39.57 ± 1.2 | −1.1 | 3.0 |
(+)-S-DMR | 0.49 ± 0.052 | −2.2 | 9.2 | 1.04 ± 0.11 | 3.7 | 10.8 | 5.08 ± 0.25 | 1.6 | 4.9 | 39.28 ± 1.5 | −1.8 | 3.9 |
(−)-R-8-OHM | 0.45 ± 0.041 | −10.8 | 7.9 | 0.97 ± 0.09 | −3.4 | 9.7 | 4.68 ± 0.30 | −6.3 | 6.3 | 43.68 ± 1.8 | 9.2 | 4.0 |
(+)-S-8-OHM | 0.54 ± 0.041 | 7.6 | 8.2 | 0.95 ± 0.08 | −4.8 | 8.8 | 5.35 ± 0.28 | 6.9 | 5.3 | 39.94 ± 0.95 | −0.2 | 2.4 |
Enantiomer | Low QC | Medium QC | High QC | ||||||
---|---|---|---|---|---|---|---|---|---|
1 ng/mL | 5 ng/mL | 40 ng/mL | |||||||
Mean ± SD | RE | RSD | Mean ± SD | RE | RSD | Mean ± SD | RE | RSD | |
(ng/mL) | (%) | (%) | (ng/mL) | (%) | (%) | (ng/mL) | (%) | (%) | |
(−)-R-MRT | 1.04 ± 0.11 | 4.3 | 10.2 | 5.10 ± 0.36 | 2.1 | 7.1 | 41.26 ± 1.8 | 3.1 | 4.3 |
(+)-S-MRT | 0.99 ± 0.10 | 1.0 | 9.5 | 4.98 ± 0.32 | −0.5 | 6.5 | 39.69 ± 1.4 | −0.8 | 3.6 |
(−)-R-DMR | 1.07 ± 0.14 | 6.8 | 12.9 | 4.86 ± 0.33 | −2.6 | 6.8 | 39.70 ± 1.3 | −0.8 | 3.2 |
(+)-S-DMR | 1.02 ± 0.10 | 1.6 | 9.6 | 5.05 ± 0.29 | 0.9 | 5.7 | 39.08 ± 1.5 | −2.2 | 3.9 |
(−)-R-8-OHM | 0.93 ± 0.10 | −6.7 | 10.8 | 4.92 ± 0.35 | −1.6 | 7.2 | 42.83 ± 1.8 | 7.1 | 4.1 |
(+)-S-8-OHM | 0.99 ± 0.09 | −0.6 | 9.0 | 5.14 ± 0.31 | 2.8 | 6.1 | 40.61 ± 1.5 | 1.5 | 3.6 |
Enantiomer | Concentration (ng/mL) | Recovery (%) (mean ± SD) | RSD (%) |
---|---|---|---|
(−)-R-MRT | 1 | 86.7 ± 8.9 | 10.3 |
5 | 89.4 ± 5.7 | 6.4 | |
40 | 90.2 ± 4.8 | 5.3 | |
(+)-S-MRT | 1 | 87.7 ± 7.3 | 8.4 |
5 | 90.1 ± 3.9 | 4.3 | |
40 | 92.0 ± 5.4 | 5.9 | |
(−)-R-DMR | 1 | 82.8 ± 8.6 | 10.4 |
5 | 87.5 ± 7.6 | 8.7 | |
40 | 87.5 ± 3.0 | 3.4 | |
(+)-S-DMR | 1 | 83.5 ± 6.8 | 8.2 |
5 | 85.8 ± 8.9 | 10.3 | |
40 | 88.9 ± 3.7 | 4.1 | |
(+)-R-8-OHM | 1 | 81.8 ± 5.9 | 7.3 |
5 | 85.1 ± 8.7 | 10.2 | |
40 | 89.5 ± 2.3 | 2.5 | |
(+)-S-8-OHM | 1 | 83.2 ± 9.6 | 11.5 |
5 | 86.1 ± 7.5 | 8.8 | |
40 | 87.8 ± 4.4 | 5.0 |
2.2.4. Stability
Enantiomer | Nominal Concentration (ng/mL) | Freeze-Thaw Stability a | 30-Day Stability b | Bench Top Stability c | Autosampler Stability d | ||||
---|---|---|---|---|---|---|---|---|---|
Measured Concentration (ng/mL) (mean ± SD) | RSD (%) | Measured Concentration (ng/mL) (mean ± SD) | RSD (%) | Measured Concentration (ng/mL) (mean ± SD) | RSD (%) | Measured Concentration (ng/mL) (mean ± SD) | RSD (%) | ||
(−)-R-MRT | 1 | 0.99 ± 0.11 | 11.5 | 1.08 ± 0.12 | 10.8 | 1.04 ± 0.12 | 11.8 | 0.99 ± 0.10 | 9.7 |
5 | 4.70 ± 0.25 | 5.4 | 4.98 ± 0.29 | 5.8 | 5.33 ± 0.29 | 5.5 | 5.13 ± 0.31 | 5.9 | |
40 | 37.61 ± 2.0 | 5.4 | 41.96 ± 1.3 | 3.0 | 41.33 ± 2.3 | 5.5 | 38.34 ± 1.4 | 3.6 | |
(+)-S-MRT | 1 | 0.95 ± 0.09 | 9.7 | 1.07 ± 0.12 | 1.6 | 1.02 ± 0.12 | 12.3 | 1.03 ± 0.12 | 11.8 |
5 | 5.06 ± 0.35 | 6.9 | 4.89 ± 0.28 | 5.8 | 5.08 ± 0.30 | 5.8 | 5.06 ± 0.30 | 5.9 | |
40 | 38.6 ± 1.8 | 4.7 | 42.5 ± 2.1 | 5.0 | 44.0 ± 1.6 | 3.7 | 36.3 ± 1.9 | 3.5 | |
(−)-R-DMR | 1 | 0.91 ± 0.079 | 8.6 | 1.08 ± 0.12 | 10.8 | 1.02 ± 0.088 | 9.1 | 1.03 ± 0.078 | 8.2 |
5 | 4.98 ± 0.25 | 5.1 | 4.63 ± 0.23 | 4.9 | 5.33 ± 0.29 | 5.5 | 4.86 ± 0.26 | 5.4 | |
40 | 39.20 ± 2.1 | 5.2 | 37.26 ± 1.8 | 4.7 | 40.55 ± 2.6 | 6.3 | 39.00 ± 2.0 | 5.0 | |
(+)-S-DMR | 1 | 1.03 ± 0.12 | 11.1 | 1.07 ± 0.12 | 11.6 | 1.00 ± 0.070 | 6.8 | 1.03 ± 0.12 | 11.7 |
5 | 4.65 ± 0.28 | 6.0 | 4.52 ± 0.26 | 5.7 | 5.40 ± 0.34 | 6.2 | 4.98 ± 0.25 | 4.9 | |
40 | 39.20 ± 2.1 | 5.2 | 39.98 ± 2.3 | 5.6 | 36.51 ± 1.4 | 3.9 | 40.61 ± 1.5 | 3.6 | |
(−)-R-8-OHM | 1 | 0.91 ± 0.081 | 8.6 | 1.02 ± 0.11 | 10.5 | 1.13 ± 0.10 | 9.2 | 0.95 ± 0.09 | 9.4 |
5 | 5.12 ± 0.22 | 4.3 | 5.07 ± 0.18 | 3.6 | 4.99 ± 0.25 | 4.9 | 5.31 ± 0.36 | 6.8 | |
40 | 39.55 ± 0.93 | 2.4 | 40.4 ± 2.0 | 4.8 | 42.9 ± 1.8 | 4.1 | 40.0 ± 2.0 | 4.9 | |
(+)-S-8-OHM | 1 | 0.97 ± 0.11 | 10.9 | 1.01 ± 0.09 | 8.5 | 1.02 ± 0.12 | 12.3 | 0.98 ± 0.11 | 10.8 |
5 | 5.10 ± 0.20 | 3.9 | 5.06 ± 0.19 | 3.7 | 5.04 ± 0.27 | 5.3 | 4.93 ± 0.34 | 6.8 | |
40 | 40.12 ± 2.0 | 5.0 | 40.71 ± 1.3 | 3.2 | 42.55 ± 1.8 | 4.1 | 38.72 ± 1.8 | 4.7 |
2.3. Application to Pharmacokinetic Studies
Enantiomer | Cmax (ng/mL) | Tmax (h) | t1/2(h) | MRT * (h) | AUC0-96 (ng h/mL) | AUC0−∞ (ng h/mL) |
---|---|---|---|---|---|---|
(−)-R-MRT | 41.81 ± 4.1 | 1.42 ± 0.66 | 23.22 ± 4.9 | 27.37 ± 5.5 | 570.94 ± 98 | 613.84 ± 103 |
(+)-S-MRT | 34.15 ± 5.9 | 1.30 ± 0.71 | 15.54 ± 4.4 | 17.43 ± 4.0 | 276.94 ± 76 | 284.14 ± 74 |
(−)-R-DMR | 12.73 ± 2.5 | 1.32 ± 0.54 | 19.80 ± 4.3 | 22.55 ± 5.7 | 133.75 ± 36 | 149.83 ± 39 |
(+)-S-DMR | 10.02 ± 1.8 | 1.29 ± 0.73 | 11.45 ± 3.3 | 17.90 ± 4.5 | 96.10 ± 34 | 109.84 ± 36 |
3. Experimental
3.1. Chemicals and Reagents
3.2. Apparatus and Capillary Electrophoretic Conditions
3.3. Preparation of Stock Solutions, Calibration Samples and Quality Control Samples
3.4. Preparation of Human Plasma Sample
3.5. Clinical Pharmacokinetic Study
4. Conclusion
Acknowledgements
Author Contributions
Conflicts of Interest
References
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Wen, J.; Zhang, W.-T.; Cao, W.-Q.; Li, J.; Gao, F.-Y.; Yang, N.; Fan, G.-R. Enantioselective Separation of Mirtazapine and Its Metabolites by Capillary Electrophoresis with Acetonitrile Field-Amplified Sample Stacking and Its Application. Molecules 2014, 19, 4907-4923. https://doi.org/10.3390/molecules19044907
Wen J, Zhang W-T, Cao W-Q, Li J, Gao F-Y, Yang N, Fan G-R. Enantioselective Separation of Mirtazapine and Its Metabolites by Capillary Electrophoresis with Acetonitrile Field-Amplified Sample Stacking and Its Application. Molecules. 2014; 19(4):4907-4923. https://doi.org/10.3390/molecules19044907
Chicago/Turabian StyleWen, Jun, Wen-Ting Zhang, Wei-Qun Cao, Ji Li, Fang-Yuan Gao, Nan Yang, and Guo-Rong Fan. 2014. "Enantioselective Separation of Mirtazapine and Its Metabolites by Capillary Electrophoresis with Acetonitrile Field-Amplified Sample Stacking and Its Application" Molecules 19, no. 4: 4907-4923. https://doi.org/10.3390/molecules19044907