Determination and Pharmacokinetic Study of Gentiopicroside, Geniposide, Baicalin, and Swertiamarin in Chinese Herbal Formulae after Oral Administration in Rats by LC-MS/MS
Abstract
:1. Introduction
2. Results and Discussion
2.1. Optimization of LC-MS/MS Conditions
Components | Molecular Weight | tR (min) | Mass Fragments | Collision Energy (eV) | ||
---|---|---|---|---|---|---|
Q1 Mass (amu) | Q3 Mass (amu) | |||||
A | Gentiopicroside | 356.32 | 4.0 | 357.10 [M+H]+ | 195.10 | −10.0 |
B | Geniposide | 388.36 | 4.1 | 406.10 [M+NH4]+ | 227.10 | −10.0 |
C | Baicalin | 446.36 | 5.9 | 447.00 [M+H]+ | 271.05 | −25.0 |
D | Swertiamarin | 374.12 | 3.6 | 375.10 [M+H]+ | 195.10 | −10.0 |
E | Carvedilol (IS) | 406.48 | 5.8 | 407.20 [M+H]+ | 100.15 | −35.0 |
2.2. Protein Precipitation Methods for Sample Preparation
2.3. Method Validation
2.3.1. Selectivity
2.3.2. Linearity, the Lower Limit of Determination (LLOD) and Lower Limit of Quantification (LLOQ)
2.3.3. Precision and Accuracy
Nominal conc.(ng/mL) | Intra-day | Inter-day | ||||
---|---|---|---|---|---|---|
Observed conc. (ng/mL) | Precision, RSD (%) | Accuracy, Bias (%) | Observed conc. (ng/mL) | Precision, RSD (%) | Accuracy, Bias (%) | |
Gentiopicroside | ||||||
10 | 9.82 ± 0.98 | 10.0 | −1.8 | 10.1 ± 1.45 | 14.4 | 0.9 |
25 | 23.5 ± 2.68 | 11.4 | −6.0 | 25.7 ± 2.11 | 8.2 | 2.9 |
50 | 49.9 ± 4.29 | 8.6 | −0.3 | 49.2 ± 1.26 | 2.6 | −1.6 |
100 | 98.1 ± 2.52 | 2.6 | −1.9 | 99.6 ± 2.91 | 2.9 | −0.4 |
250 | 259 ± 6.67 | 2.6 | 3.5 | 251 ± 5.72 | 2.3 | 0.3 |
500 | 494 ± 5.09 | 1.0 | −1.1 | 500 ± 2.82 | 0.6 | −0.1 |
Geniposide | ||||||
10 | 9.37 ± 0.88 | 9.4 | −6.3 | 9.77 ± 1.16 | 11.9 | −2.3 |
25 | 25.4 ± 1.19 | 4.7 | 1.5 | 26.0 ± 1.96 | 7.5 | 3.9 |
50 | 49.5 ± 1.01 | 2.0 | −0.9 | 49.8 ± 3.06 | 6.1 | −0.4 |
100 | 98.2 ± 4.04 | 4.1 | −1.8 | 100 ± 3.02 | 3.0 | 0.1 |
250 | 256 ± 5.66 | 2.2 | 2.3 | 249 ± 3.50 | 1.4 | −0.4 |
500 | 497 ± 7.04 | 1.4 | −0.5 | 500 ± 1.48 | 0.3 | 0.1 |
Baicalin | ||||||
10 | 11.0 ± 1.01 | 9.1 | 10.1 | 10.2 ± 1.51 | 14.8 | 2.1 |
25 | 24.9 ± 1.65 | 6.6 | −0.3 | 25.8 ± 2.70 | 10.5 | 3.3 |
50 | 50.9 ± 1.92 | 3.8 | 1.9 | 47.3 ± 1.12 | 2.4 | −5.4 |
100 | 98.4 ± 4.51 | 4.6 | −1.6 | 99.3 ± 3.04 | 3.1 | −0.7 |
250 | 245 ± 8.61 | 3.5 | −1.9 | 250 ± 4.71 | 1.9 | −0.1 |
500 | 491 ± 19.6 | 4.0 | −1.7 | 505 ± 9.20 | 1.8 | 1.0 |
Swertiamarin | ||||||
5 | 5.35 ± 0.33 | 6.2 | 7.0 | 4.74 ± 0.25 | 5.3 | −5.1 |
10 | 9.91 ± 0.66 | 6.7 | −0.9 | 10.1 ± 0.53 | 5.2 | 1.0 |
25 | 24.8 ± 0.37 | 1.5 | −0.9 | 24.9 ± 0.75 | 3.0 | −0.4 |
50 | 50.0 ± 1.06 | 2.1 | −0.1 | 49.8 ± 1.16 | 2.3 | −0.4 |
100 | 99.6 ± 1.28 | 1.3 | −0.4 | 101 ± 1.35 | 1.3 | 0.9 |
250 | 251 ± 2.20 | 0.9 | 0.5 | 253 ± 7.21 | 2.9 | 1.1 |
2.3.4. Matrix Effect and Recovery
Nominal conc. | Peak Area | Matrix Effect | Recovery | ||
---|---|---|---|---|---|
(ng/mL) | Set 1 | Set 2 | Set 3 | (%) a | (%) b |
Gentiopicroside | |||||
10 | 3911 ± 321 | 4001 ± 595 | 4312 ± 285 | 102.0 ± 7.4 | 108.7 ± 9.2 |
50 | 17829 ± 774 | 20372 ± 283 | 21498 ± 1749 | 114.4 ± 3.6 | 105.6 ± 9.9 |
250 | 89698 ± 3141 | 93932 ± 2505 | 104970 ± 4074 | 104.8 ± 4.1 | 111.7 ± 2.5 |
Mean ± S.D. | 107.0 ± 7.3 | 108.7 ± 7.3 | |||
Geniposide | |||||
10 | 12233 ± 190 | 14306 ± 1062 | 17028 ± 644 | 116.9 ± 6.9 | 119.4 ± 7.9 |
50 | 58115 ± 1045 | 67062 ± 1062 | 71407 ± 1764 | 115.4 ± 1.6 | 106.5 ± 1.1 |
250 | 295991 ± 3955 | 316422 ± 6847 | 346662 ± 8807 | 106.9 ± 3.7 | 109.6 ± 0.6 |
Mean ± S.D. | 113.1 ± 6.1 | 111.8 ± 7.1 | |||
Baicalin | |||||
10 | 9173 ± 232 | 7795 ± 1246 | 7825 ± 1054 | 85.0 ± 13.6 | 101.1 ± 10.7 |
50 | 45932 ± 1992 | 39518 ± 2684 | 37717 ± 460 | 86.0 ± 3.1 | 95.7 ± 5.6 |
250 | 224485 ± 4019 | 198185 ± 9541 | 186908 ± 6196 | 88.3 ± 3.2 | 94.4 ± 2.8 |
Mean ± S.D. | 86.4 ± 7.3 | 97.1 ± 6.9 | |||
Swertiamarin | |||||
10 | 16564 ± 490 | 16634 ± 397 | 17422 ± 660 | 100.5 ± 4.5 | 104.7 ± 1.7 |
50 | 76343 ± 1289 | 82585 ± 5095 | 88245 ± 4264 | 108.1 ± 4.8 | 107.0 ± 5.2 |
250 | 404293 ± 4777 | 432683 ± 3140 | 467239 ± 7470 | 107.0 ± 0.5 | 108.0 ± 1.0 |
Mean ± S.D. | 105.2 ± 4.9 | 106.6 ± 3.1 | |||
Carvedilol (IS) | |||||
10 | 76558 ± 6622 | 87040 ± 5353 | 83146 ± 3942 | 113.9 ± 2.9 | 95.7 ± 4.7 |
2.3.5. Stability
Analytes/ | Freeze-thaw Stability | Short-term Stability | Long-term Stability | Autosampler Stability | ||||
---|---|---|---|---|---|---|---|---|
Spiked Concentration (ng/mL) | Measured Concentration (ng/mL) | Accuracy (%) | Measured Concentration (ng/mL) | Accuracy (%) | Measured Concentration (ng/mL) | Accuracy (%) | Measured Concentration (ng/mL) | Accuracy (%) |
Gentiopicroside | ||||||||
10 | 11.3 ± 0.28 | 113.0 | 11.0 ± 0.40 | 110.2 | 9.82 ± 0.21 | 98.2 | 11.0 ± 1.09 | 109.8 |
50 | 53.7 ± 1.44 | 107.4 | 55.5 ± 1.68 | 111.0 | 51.1 ± 2.71 | 102.2 | 49.0 ± 2.47 | 97.9 |
250 | 265 ± 6.60 | 106.0 | 263 ± 16.2 | 105.1 | 240 ± 6.72 | 95.9 | 253 ± 10.7 | 101.1 |
Geniposide | ||||||||
10 | 10.0 ± 0.94 | 100.3 | 11.4 ± 0.80 | 114.1 | 9.10 ± 0.77 | 91.1 | 10.3 ± 0.65 | 102.8 |
50 | 56.5 ± 1.00 | 113.0 | 52.9 ± 1.12 | 105.8 | 46.4 ± 4.16 | 92.7 | 48.9 ± 1.86 | 97.7 |
250 | 265 ± 10.9 | 106.0 | 260 ± 6.85 | 103.9 | 235 ± 15.4 | 94.1 | 248 ± 5.47 | 99.4 |
Baicalin | ||||||||
10 | 10.3 ± 1.01 | 103.0 | 11.2 ± 1.14 | 111.8 | 9.54 ± 0.97 | 95.4 | 9.28 ± 0.72 | 92.8 |
50 | 49.5 ± 0.75 | 99.0 | 48.4 ± 1.18 | 96.7 | 49.7 ± 6.84 | 99.5 | 50.5 ± 4.33 | 101.0 |
250 | 231 ± 12.0 | 92.2 | 231 ± 10.8 | 92.6 | 238 ± 33.2 | 95.3 | 231 ± 1.57 | 92.4 |
Swertiamarin | ||||||||
10 | 10.6 ± 0.76 | 105.7 | 11.3 ± 0.99 | 112.7 | 9.50 ± 0.50 | 95.0 | 10.6 ± 0.78 | 106.1 |
50 | 49.8 ± 0.66 | 99.6 | 48.5 ± 0.23 | 96.9 | 45.7 ± 3.21 | 91.3 | 49.9 ± 1.08 | 99.7 |
250 | 247 ± 2.74 | 98.7 | 252 ± 8.03 | 100.9 | 224 ± 10.7 | 89.8 | 240 ± 3.52 | 96.1 |
2.4. Application of the Analytical System in Pharmacokinetic Study
Parameter | Gentiopicroside | Geniposide | Baicalin | Swertiamarin |
---|---|---|---|---|
Cmax (ng/mL) | 5767 ± 412 a | 1164 ± 144 | 2008 ± 265 | 13.0 ± 0.5 |
Tmax (min) | 60.0 ± 6.7 | 55.0 ± 12.6 | 15.8 ± 9.0 | 97.5 ± 14.4 |
t1/2 (min) | 127 ± 7.7 | 168 ± 27.4 | 314 ± 56.3 | 188 ± 27.6 |
AUC (min µg/mL) | 1417 ± 83.8 b | 302 ± 25.8 | 753 ± 86.2 | 2.5 ± 0.1 |
Cl (mL/min/kg) | 118 ± 6.5 | 784 ± 55.5 | 112 ± 20.7 | 581 ± 58.5 |
MRT (min) | 193 ± 7.9 | 213 ± 18.2 | 298 ± 11.6 | 144 ± 3.1 |
3. Experimental
3.1. Chemicals and Reagents
3.2. Herbal Preparation of Long-Dan-Xie-Gan-Tang
3.3. LC-MS/MS and Analytical Conditions
3.4. Preparation of Calibration Standards and Quality Controls
3.5. Method Validation
3.5.1. Selectivity
3.5.2. Linearity, the Lower Limits of Detection (LLOD) and Lower Limits of Quantification (LLOQ)
3.5.3. Precision and Accuracy
3.5.4. Matrix Effect and Recovery
3.5.5. Stability
3.6. Pharmacokinetic Application
3.6.1. Experimental Animals
3.6.2. Freely Moving Rat Model
3.6.3. LDXGT Administration and Sample Collection
3.6.4. Sample Preparation
3.7. Statistical Analysis
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Lu, C.-M.; Lin, L.-C.; Tsai, T.-H. Determination and Pharmacokinetic Study of Gentiopicroside, Geniposide, Baicalin, and Swertiamarin in Chinese Herbal Formulae after Oral Administration in Rats by LC-MS/MS. Molecules 2014, 19, 21560-21578. https://doi.org/10.3390/molecules191221560
Lu C-M, Lin L-C, Tsai T-H. Determination and Pharmacokinetic Study of Gentiopicroside, Geniposide, Baicalin, and Swertiamarin in Chinese Herbal Formulae after Oral Administration in Rats by LC-MS/MS. Molecules. 2014; 19(12):21560-21578. https://doi.org/10.3390/molecules191221560
Chicago/Turabian StyleLu, Chia-Ming, Lie-Chwen Lin, and Tung-Hu Tsai. 2014. "Determination and Pharmacokinetic Study of Gentiopicroside, Geniposide, Baicalin, and Swertiamarin in Chinese Herbal Formulae after Oral Administration in Rats by LC-MS/MS" Molecules 19, no. 12: 21560-21578. https://doi.org/10.3390/molecules191221560