Thirty-one compounds, including delavinone, were isolated from the methanol extract of
To our knowledge, the pharmacokinetics of delavinone have not been reported. In this paper, UPLC-MS/MS method was established to determine delavinone in mouse blood, and the pharmacokinetics of delavinone after intravenous and intragastric administration were studied, and the absolute bioavailability was obtained.
Delavinone (purity >98%, Figure
Chemical structure of delavinone (a) and hapepunine (IS, (b)).
ACQUITY I-Class UPLC and XEVO TQS-micro Triple Quadrupole Mass Spectrometer (Waters Corp, Milford, MA, USA) were used. Masslynx 4.1 software (Waters Corp.) was used for data acquisition and instrument control.
The mobile phase consisted of acetonitrile and 0.1% formic acid with a gradient elution at a flow rate of 0.4 mL/min, as well as 0-0.2 min, 0.1% formic acid 90%; 0.2-1.5 min, 0.1% formic acid 90%-15%; 1.5-2.0 min, 0.1% formic acid 15%; 2.0-2.5 min, 0.1% formic acid 15%-90%; and 2.5-4.0 min, 0.1% formic acid 90%. The BEH C18 (2.1 mm × 50 mm, 1.7
The capillary voltage was set to 2.0 kV, the ion source temperature was 150°C, and the desolvation temperature was 400°C. Nitrogen was used as the desolvation gas (800 L/h) and the cone gas (50 L/h). The MRM model was quantitatively analyzed for delavinone m/z 414.4→98.1 and IS m/z 430.5→111.9 in ESI positive interface (Figure
Mass spectrum of delavinone (a) and hapepunine (IS, (b)).
A stock solution of delavinone (1.0 mg/mL) and IS (1.0 mg/mL) was prepared with methanol-water (50:50). Working solutions were prepared by diluting with a delavinone stock solution in methanol. A working solution of the IS 50 ng/mL was prepared by diluting with a IS stock solution with acetonitrile.
The blank mouse blood was spiked with an appropriate amount of standard working solution to prepare the blood standard curve of delavinone (1, 5, 20, 50, 200, and 500 ng/mL). Quality Control (QC) samples were prepared in the same manner as the standard curve (1, 3, 180, and 450 ng/mL).
20
Eighteen mice were randomly divided into three groups: one group was intravenous administration (1.5 mg/kg), and two groups were intragastric administration (2.5, 10 mg/kg), with 6 rats in each group. Then 20
DAS 2.0 software (China Pharmaceutical University) used a noncompartmental model to fit pharmacokinetic parameters.
Figure
UPLC-MS/MS of delavinone and hapepunine (IS) in mouse blood; (a) blank blood; (b) blank blood spiked delavinone and IS; (c) a mouse blood sample.
The standard curve equation of delavinone in the mouse blood was the following: Y=0.0052C+0.0040, r= 0.9982, where Y represents the ratio of the peak area of delavinone and IS and C represents the concentration of delavinone in mouse blood. The LLOQ in the mouse blood is 1.0 ng/mL; the signal-to-noise ratio is 8.
The intraday precision RSD was less than 12%, the interday precision RSD was less than 13%, the accuracy ranged from 96.8% to 104.9%, the matrix effect was between 88.8% and 103.4%, and the average recovery was better than 80.6% in Table
Accuracy, precision, matrix effect, and recovery of delavinone in the mouse blood.
Concentration | Accuracy (%) | Precision (RSD%) | Metrix effect | Recovery | ||
---|---|---|---|---|---|---|
(ng/mL) | Intraday | Interday | Intraday | Interday | (%) | (%) |
1 | 104.9 | 96.8 | 11.9 | 12.4 | 90.5±4.9 | 92.5±3.4 |
3 | 102.1 | 98.7 | 9.4 | 9.6 | 88.8 ±7.9 | 84.1±7.5 |
180 | 97.4 | 102.9 | 5.0 | 2.7 | 99.0±4.6 | 80.6±3.7 |
450 | 96.8 | 101.3 | 6.3 | 7.6 | 103.4 ±7.5 | 84.2±8.1 |
In the mouse blood at room temperature 2h, -20°C for 30 days, and freeze-thaw stability test, the variation of delavinone was within ± 12%; RSD was less than 11%, indicating that the stability of delavinone was acceptable.
The concentration-time curve of delavinone is shown in Figure
Main pharmacokinetic parameters of delavinone in mice.
Parameters | Unit | iv (1.0 mg/kg) | ig (2.5 mg/kg) | ig (10 mg/kg) |
---|---|---|---|---|
| ng/mL | 169.8±44.2 | 48.0 ±7.0 | 229.7 ±49.6 |
| ng/mL | 245.2 ±106.1 | 60.8 ±12.4 | 241.4 ±52.5 |
| h | 1.4 ±0.1 | 2.5 ±0.2 | 1.4 ±0.2 |
| h | 9.2 ±6.3 | 4.8 ±1.0 | 1.9 ±0.4 |
| h | 9.5 ±5.2 | 4.1 ±0.9 | 2.7±0.8 |
| L/h/kg | 4.7 ±1.8 | 42.6 ±8.8 | 43.0±8.6 |
| L/kg | 46.4 ±31.9 | 242.2 ±36.0 | 162.3±51.3 |
| ng/mL | 183.7 ±33.5 | 21.3 ±6.5 | 196.9±83.3 |
Bioavailability | 11.3% | 13.5% |
Time-blood concentration curve of delavinone in mouse blood after intravenous (1.0 mg/kg) and oral (2.5, 10 mg/kg) administration.
Compared with LC-MS/MS, UPLC-MS/MS is more sensitive and has obvious advantages in the study of pharmacokinetics [
The choice of positive and negative electrodes for electrospray ESI is often evaluated in methodology [
As far as possible, the internal interfering substances are separated from the retention time by HPLC; mobile phase and chromatographic column determine the chromatographic behavior [
Before UPLC-MS/MS analysis, removing protein and potential interference is a key point of method establishment [
UPLC-MS/MS has been applied to the quantitative determination of delavinone in blood, which is faster and more sensitive than traditional HPLC. Only 4 min can complete the analysis of blood samples, which can save a lot of time and solvent. In addition, the LLOQ (1ng/mL) of delavinone is relatively low, which could be used to determine a lower blood concentration at the final sampling point. A UPLC-MS/MS method was established to study the pharmacokinetics of delavinone in mice after sublingual injection or intragastric administration. Mice have less blood, so only 20
This study developed a sensitive and rapid UPLC-MS/MS method for the determination of delavinone in mouse blood with the range of 1.0-500 ng/mL. This method was successfully applied to the pharmacokinetics of delavinone in mice, and the absolute bioavailability was 12.4%.
The data used to support the findings of this study are included within the article.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Shuanghu Wang and Zhiguang Zhang contributed equally to this work.
This work was supported by start-up funding from Wenzhou Medical University (QTJ17018), National Natural Science Foundation of Zhejiang (LS18H27001 and LYY18H280003), the Ladder Plan of the People’s Hospital of Lishui (2017TC001), and medical key disciplines research and development project of Lishui (2016zdxk05).