Rapid and Sensitive Determination of Timosaponin AIII in Rat Plasma by LC-MS/MS and Its Pharmacokinetic Application
Abstract
:1. Introduction
2. Experimental Section
2.1. Chemicals and Reagents
2.2. Instruments and Analytical Conditions
2.2.1. Instruments
2.2.2. Analytical Conditions
2.3. Sample Preparation
2.4. Animals and Pharmacokinetic Study
2.5. Method Validation
2.5.1. Selectivity
2.5.2. Linearity and Lower Limit of Quantification (LLOQ)
2.5.3. Precision and Accuracy
2.5.4. Extraction Recovery
2.5.5. Analyte Stability
2.5.5.1. Post-Preparative Stabilities at Room Temperature for 24 h
2.5.5.2. Stabilities of Unpreparative Sample at Room Temperature for 24 h
2.5.5.3. Three of Freeze-Thaw Cycles
2.5.5.4. Long-Term Stability Storage at −20 °C for 50 days
2.5.6. Matrix Effect
2.6. Statistical Analysis
3. Method Validation
3.1. Selectivity
3.2. Linearity and Lower Limit of Quantification (LLOQ)
3.3. Precision and Accuracy
3.4. Extraction Recovery
3.5. Analyte Stability
3.6. Matrix Effect
4. Pharmacokinetic Analysis
5. Conclusions
Acknowledgements
References
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Time (min) | A (H2O) | D (Acetonitrile) | Flow (mL/min) |
---|---|---|---|
0 | 85% | 15% | 0.4 |
0.5 | 85% | 15% | 0.4 |
6.0 | 10% | 90% | 0.4 |
7.0 | 10% | 90% | 0.4 |
7.1 | 85% | 15% | 0.4 |
9 | 85% | 15% | 0.4 |
Sample Name | Precursor/Product Ion | Dwell (msec) | Fragmentor | CE |
---|---|---|---|---|
Timosapoin AIII | 739.2/577.2 | 200 | 260 | 30 |
Ginsenoside Re | 945.4/475.2 | 200 | 280 | 60 |
Concentrations (ng/mL) | 11.14 | 55.7 | 111.4 | 278.5 | 557 | 1114 |
---|---|---|---|---|---|---|
Peak-area ratios (n = 6) | 0.0526 | 0.2105 | 0.4075 | 0.7726 | 1.2768 | 2.4608 |
Calibration curve | y = 0.0021x + 0.1111(r = 0.9960, n = 6) |
Concentration (ng/mL) | 1 | 2 | 3 | 4 | 5 | 6 | AVE | RSD (%) |
---|---|---|---|---|---|---|---|---|
11.14 | 10.5 | 10.7 | 10.9 | 10.9 | 11.4 | 12.9 | 11.2 | 7.8 |
557 | 593.3 | 554.8 | 580.6 | 602.4 | 558.5 | 562.8 | 575.4 | 3.4 |
1114 | 1143.3 | 1137.0 | 1108.6 | 1101.0 | 1036.9 | 1065.0 | 1098.0 | 3.8 |
Concentration (ng/mL) | 1d | 2d | 3d | 4d | 5d | 6d | AVE | RSD (%) |
---|---|---|---|---|---|---|---|---|
11.14 | 11.2 | 11.1 | 11.1 | 11.1 | 11.0 | 11.9 | 11.2 | 3.0 |
557 | 575.4 | 536.5 | 571.4 | 577.9 | 561.8 | 566.9 | 566.5 | 2.7 |
1114 | 1098.0 | 1081.4 | 1085.4 | 1072.7 | 1042.3 | 1166.0 | 1092.0 | 3.5 |
Concentration (ng/mL) | RE (%) | AVE (%) | |||||
---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | ||
11.14 | 6.4 | 12.2 | 11.3 | 4.3 | 4.1 | 11.5 | 9.1 |
557 | 5.8 | 5.0 | 1.3 | 9.2 | 10.9 | 11.4 | 7.3 |
1114 | 4.8 | 11.3 | 10.4 | 0.3 | 6.6 | 5.0 | 6.4 |
Sample | Extract recovery (%) | AVE (%) | RSD (%) | |||||
---|---|---|---|---|---|---|---|---|
Concentration (ng/mL) | ||||||||
11.14 | 93.6 | 93.8 | 99.6 | 96.1 | 89.5 | 88.4 | 93.5 | 3.9 |
557 | 83.2 | 93.2 | 92.5 | 94.4 | 94.0 | 96.4 | 92.3 | 5.0 |
1114 | 95.6 | 97.5 | 97.1 | 97.2 | 96.2 | 89.6 | 95.5 | 1.2 |
Concentration (ng/mL) | 1 | 2 | 3 | 4 | 5 | 6 | AVE | RE (%) |
---|---|---|---|---|---|---|---|---|
11.14 | 10.8 | 11.4 | 11.4 | 11.1 | 11.5 | 11.2 | 11.2 | 0.8 |
557 | 600.7 | 592.4 | 603.7 | 588.7 | 621.9 | 566.9 | 595.7 | 6.9 |
1114 | 1135.0 | 1153.7 | 1076.2 | 1062.6 | 1152.3 | 1081.9 | 1110.3 | 3.3 |
Concentration (ng/mL) | 1 | 2 | 3 | 4 | 5 | 6 | AVE | RE (%) |
---|---|---|---|---|---|---|---|---|
11.14 | 10.2 | 10.6 | 11.4 | 11.7 | 11.2 | 11.7 | 11.1 | 0.2 |
557 | 582.4 | 512.3 | 582.0 | 583.8 | 559.5 | 580.7 | 566.8 | 1.7 |
1114 | 1149.1 | 1076.6 | 1066.7 | 1131.9 | 1073.3 | 1070.2 | 1094.6 | 1.7 |
Concentration (ng/mL) | 1 | 2 | 3 | 4 | 5 | 6 | AVE | RE (%) |
---|---|---|---|---|---|---|---|---|
11.14 | 10.9 | 10.6 | 11.0 | 10.6 | 10.9 | 12.1 | 11.0 | 1.0 |
557 | 560.4 | 547.0 | 547.2 | 524.5 | 553.6 | 563.7 | 549.4 | 1.4 |
1114 | 969.8 | 1091.1 | 984.6 | 1007.6 | 1018.7 | 996.3 | 1011.4 | 9.2 |
Concentration (ng/mL) | 1 day | 50 days | AVE | RE (%) |
---|---|---|---|---|
11.14 | 11.9 | 11.2 | 11.6 | 3.3 |
557 | 566.9 | 564.3 | 565.6 | 1.5 |
1114 | 1166.0 | 1096.9 | 1131.5 | 1.6 |
Sample | Matrix Effect of TA-III (%) | ||||||
---|---|---|---|---|---|---|---|
Concentration (ng/mL) | 1 | 2 | 3 | 4 | 5 | AVE (%) | RSD (%) |
11.14 | 112.8 | 106.1 | 106.0 | 110.4 | 104.0 | 107.9 | 3.4 |
557 | 110.2 | 107.8 | 92.2 | 113.6 | 115.0 | 107.8 | 8.5 |
1114 | 105.3 | 109.8 | 99.3 | 97.7 | 97.5 | 101.9 | 5.3 |
Time (h) | Rat 1 | Rat 2 | Rat 3 | Rat 4 | Rat 5 | AVE | SD |
---|---|---|---|---|---|---|---|
0.5 | 3.7 * | 0.0 | 0.4 * | 1.5 * | 12.2 | 3.5 * | 1.42 |
1 | 17.2 | 3.1 * | 6.1 * | 13.7 | 17.8 | 11.6 | 0.57 |
2 | 42.0 | 58.9 | 18.6 | 47.0 | 44.7 | 42.2 | 0.35 |
4 | 95.0 | 90.5 | 54.1 | 82.6 | 98.6 | 84.2 | 0.21 |
6 | 95.4 | 97.5 | 62.7 | 127.2 | 86.9 | 93.9 | 0.25 |
7 | 100.7 | 91.3 | 93.2 | 131.0 | 106.3 | 104.5 | 0.15 |
8 | 37.7 | 32.7 | 28.4 | 89.0 | 16.7 | 40.9 | 0.68 |
10 | 33.9 | 22.8 | 25.9 | 25.3 | 9.7 * | 23.5 | 0.37 |
12 | 18.4 | 13.1 | 42.3 | 39.4 | 11.0 * | 24.8 | 0.60 |
14 | 12.3 | 9.7 * | 47.4 | 34.5 | 16.4 | 24.1 | 0.67 |
24 | 6.6 * | 8.4 * | 31.8 | 21.7 | 11.1 * | 15.9 | 0.67 |
27 | 3.5 * | 3.9 * | 24.5 | 13.1 | 1.5 * | 9.3 * | 1.03 |
30 | 5.9 * | 3.8 * | 11.6 | 4.1 * | 0.2 * | 5.1 * | 0.81 |
36 | 1.9 * | 0.5 * | 13.0 | 11.3 * | 0.5 * | 5.4 * | 1.14 |
48 | 0.2 * | 1.4 * | 7.7 * | 1.9 * | 0.0 * | 2.2 * | 1.40 |
Time (h) | Rat 1 | Rat 2 | Rat 3 | Rat 4 | Rat 5 | AVE | SD |
---|---|---|---|---|---|---|---|
0.5 | 5.9 * | 27.6 | 17.8 | 17.1 | 12.7 | 16.2 | 0.49 |
1 | 6.6 * | 29.3 | 37.2 | 11.8 | 10.7 * | 19.1 | 0.70 |
2 | 12.6 | 63.1 | 94.5 | 85.0 | 15.2 | 54.1 | 0.71 |
4 | 29.5 | 122.2 | 129.7 | 183.8 | 53.3 | 103.7 | 0.60 |
6 | 45.7 | 129.5 | 237.4 | 148.0 | 122.3 | 136.6 | 0.50 |
7 | 71.4 | 237.4 | 340.8 | 401.1 | 131.3 | 236.4 | 0.58 |
8 | 109.6 | 285.6 | 285.5 | 381.8 | 261.3 | 264.8 | 0.37 |
10 | 234.0 | 199.9 | 226.5 | 88.0 | 181.2 | 185.9 | 0.32 |
12 | 55.0 | 44.9 | 108.2 | 104.4 | 131.6 | 88.8 | 0.42 |
14 | 69.1 | 84.7 | 87.9 | 110.4 | 107.5 | 91.9 | 0.19 |
24 | 1.0 * | 51.1 | 5.9 * | 10.9 * | 6.3 * | 15.0 | 1.36 |
27 | 11.6 | 10.6 * | 0.0 | 2.7 * | 6.2 * | 6.2 * | 0.80 |
30 | 13.6 | 11.0 * | 11.0 * | 5.8 * | 9.6 * | 10.2 * | 0.28 |
36 | 3.5 * | 1.8 * | 1.8 * | 0.0 | 3.2 * | 2.0 * | 0.68 |
48 | 0.4 * | 2.3 * | 1.7 * | 1.6 * | 0.0 | 1.2 * | 0.81 |
Time (h) | Rat1 | Rat 2 | Rat 3 | Rat 4 | Rat 5 | AVE | SD |
---|---|---|---|---|---|---|---|
0.5 | 0.0 | 26.3 | 0.0 | 0.0 | 0.0 | 5.3 * | 2.24 |
1 | 30.1 | 38.2 | 2.6 * | 9.4 * | 2.8 * | 16.6 | 0.99 |
2 | 15.1 | 38.2 | 11.6 | 42.0 | 20.2 | 25.4 | 0.54 |
4 | 54.6 | 63.2 | 8.0 | 61.1 | 51.9 | 47.8 | 0.47 |
6 | 170.1 | 71.7 | 48.9 | 274.2 | 71.9 | 127.3 | 0.74 |
7 | 304.2 | 180.0 | 104.3 | 357.5 | 414.1 | 272.1 | 0.47 |
8 | 546.1 | 298.7 | 363.0 | 607.3 | 583.3 | 479.7 | 0.29 |
10 | 300.8 | 305.0 | 196.9 | 350.9 | 247.3 | 280.2 | 0.21 |
12 | 141.6 | 156.4 | 117.1 | 181.0 | 99.1 | 139.0 | 0.23 |
14 | 121.1 | 104.9 | 121.9 | 88.9 | 60.7 | 99.5 | 0.26 |
24 | 51.8 | 67.0 | 22.1 | 59.8 | 103.5 | 60.8 | 0.48 |
27 | 37.9 | 31.5 | 19.8 | 50.5 | 22.2 | 32.4 | 0.38 |
30 | 49.2 | 142.5 | 6.4 * | 13.1 | 22.6 | 46.8 | 1.20 |
36 | 12.7 * | 12.7 | 26.9 | 16.3 | 31.5 | 20.0 | 0.43 |
48 | 5.6 * | 6.8 * | 8.8 * | 3.0 * | 12.5 | 7.3 * | 0.49 |
Parameters | Rat1 | Rat 2 | Rat 3 | Rat 4 | Rat 5 | MEAN ± SD |
---|---|---|---|---|---|---|
T (peak)(h) | 7 | 6 | 7 | 7 | 7 | 6.8 ± 0.4 |
C (max)(ng/mL) | 100.7 | 97.5 | 93.2 | 131 | 106.3 | 105.7 ± 14.9 |
Ke (1/h) | 0.352 | 0.374 | 0.051 | 0.274 | 0.419 | 0.294 ± 0.146 |
Ka (1/h) | 0.407 | 0.416 | 0.984 | 0.314 | 0.472 | 0.519 ± 0.166 |
t1/2 (ka)(h) | 1.703 | 1.665 | 0.704 | 2.209 | 1.47 | 1.55 ± 0.546 |
t1/2 (h) | 1.969 | 1.854 | 5.693 | 2.528 | 1.653 | 2.74 ± 1.68 |
MRT (0–∞)(h) | 9.35 | 9.051 | 19.842 | 13.592 | 8.421 | 12.1 ± 4.8 |
AUC (0–∞)[(ng/mL)h] | 766.1 | 729.6 | 1347.2 | 1093.9 | 672.1 | 921.8 ± 289.0 |
Parameters | Rat1 | Rat 2 | Rat 3 | Rat 4 | Rat 5 | MEAN ± SD |
---|---|---|---|---|---|---|
T (peak)(h) | 10 | 8 | 7 | 7 | 8 | 8 ± 1.2 |
C (max)(ng/mL) | 234 | 285.6 | 340.8 | 401.1 | 261.3 | 304.6 ± 66.8 |
ke (1/h) | 0.279 | 0.195 | 0.205 | 0.3 | 0.209 | 0.238 ± 0.048 |
ka (1/h) | 0.343 | 0.218 | 0.238 | 0.354 | 0.238 | 0.278 ± 0.065 |
t1/2 (ka)(h) | 2.022 | 3.175 | 2.912 | 1.958 | 2.91 | 2.60 ± 0.56 |
t1/2 (h) | 2.482 | 3.556 | 3.38 | 2.313 | 3.311 | 3.01 ± 0.57 |
MRT (0–∞)(h) | 12.226 | 11.663 | 9.877 | 10.122 | 11.304 | 11.038 ± 1.007 |
AUC (0–∞)[(ng/mL)h] | 1395.4 | 2682.6 | 3300.0 | 2694.1 | 2347.9 | 2484.0 ± 698.8 |
Parameters | Rat1 | Rat 2 | Rat 3 | Rat 4 | Rat 5 | MEAN ± SD |
---|---|---|---|---|---|---|
T (peak)(h) | 8 | 10 | 8 | 8 | 8 | 8.4 ± 0.9 |
C (max)(ng/mL) | 546.1 | 305 | 363 | 607.3 | 583.3 | 480.9 ± 137.4 |
Ke (1/h) | 0.386 | 0.088 | 0.313 | 0.364 | 0.548 | 0.340 ± 0.166 |
Ka (1/h) | 0.567 | 0.332 | 0.496 | 0.571 | 0.7 | 0.533 ± 0.134 |
t1/2 (ka)(h) | 1.222 | 2.088 | 1.396 | 1.213 | 0.991 | 1.382 ± 0.420 |
t1/2 (h) | 1.794 | 7.908 | 2.216 | 1.902 | 1.264 | 3.017 ± 2.756 |
MRT (0–∞)(h) | 14.236 | 17.46 | 19.741 | 12.753 | 16.889 | 16.216 ± 2.754 |
AUC (0–∞)[(ng/mL)h] | 2938.3 | 4081.9 | 1992.8 | 3432.8 | 2424.3 | 2974.07 ± 822.2 |
Parameters | 25 mg/kg | 50 mg/kg | 75 mg/kg |
---|---|---|---|
Ke (1/h) | 0.294 ± 0.146 | 0.238 ± 0.048 | 0.340 ± 0.166 |
Ka (1/h) | 0.519 ± 0.166 | 0.278 ± 0.065 | 0.642 ± 1.393 |
t1/2 (ka)(h) | 1.55 ± 0.546 | 2.60 ± 0.56 | 1.382 ± 0.420 |
t1/2 (h) | 2.74 ± 1.68 | 3.01 ± 0.57 | 3.02 ± 2.76 |
T (peak)(h) | 6.8 ± 0.4 | 8 ± 1.2 | 8.4 ± 0.9 |
C (max)(ng/mL) | 105.7 ± 14.9 | 304.6 ± 66.8 | 480.9 ± 137.4 |
AUC [(ng/mL)h] | 921.8 ± 289.0 | 2484.0 ± 698.8 | 2974.07 ± 822.2 |
MRT (h) | 12.1 ± 4.8 | 11.0 ± 1.0 | 16.2 ± 2.8 |
© 2013 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
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Liu, Y.; Pu, Y.; Zhang, T.; Ding, Y.; Wang, B.; Cai, Z. Rapid and Sensitive Determination of Timosaponin AIII in Rat Plasma by LC-MS/MS and Its Pharmacokinetic Application. Int. J. Mol. Sci. 2013, 14, 3656-3670. https://doi.org/10.3390/ijms14023656
Liu Y, Pu Y, Zhang T, Ding Y, Wang B, Cai Z. Rapid and Sensitive Determination of Timosaponin AIII in Rat Plasma by LC-MS/MS and Its Pharmacokinetic Application. International Journal of Molecular Sciences. 2013; 14(2):3656-3670. https://doi.org/10.3390/ijms14023656
Chicago/Turabian StyleLiu, Yanping, Yiqiong Pu, Tong Zhang, Yue Ding, Bing Wang, and Zhenzhen Cai. 2013. "Rapid and Sensitive Determination of Timosaponin AIII in Rat Plasma by LC-MS/MS and Its Pharmacokinetic Application" International Journal of Molecular Sciences 14, no. 2: 3656-3670. https://doi.org/10.3390/ijms14023656