Poliumoside is the main active constituent of Callicarpa kwangtungensis Chun (CK), a traditional Chinese medicine for management of hemostasis. In this study, a rapid and selective ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/ Q-TOF-MS) method was developed and validated to quantify poliumoside in rat plasma. The targeted analytes in rat plasma were prepared through protein-precipitation method using 10% trichloroacetic acid (TCA). The chromatographic separation was performed on a Waters BEH C 18 column (2.1 × 100 mm, 1.7 μm) by acetonitrile-water containing 0.1% formic acid. The calibration curve was linear over the range of 50 - 10,000 ng/mL (r 2 > 0.99). The intra-day or inter-day precision was less than 7.97% and accuracy was within ?7.00% - 3.36%. The developed method was successfully applied to pharmacokinetic study of poliumoside in rat plasma. Although being rapidly absorbed (T max ≤ 30 min), poliumoside was poorly bioavailable after oral administration (the absolute bioavailability was only 0.69%).
Callicarpa kwangtungensis Chun (Verbenaceae, CK), a traditional Chinese medicine, possesses various biological activities such as hemostasis and antioxidation [
Poliumoside is the major phenylethanoid glycosides (PhGs) abundantly found in CK [
As the potential pharmacological effects, it is necessary to explore the individual pharmacokinetic characteristics of poliumoside for clinical application. Sun et al. developed an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of crude extract from CK in rat plasma after oral administration [
The aim of this article was to develop a rapid and selective UPLC/Q-TOF-MS method for the determination of poliumoside in rat plasma after oral and intravenous administration. This method combines the capabilities of scanning (quadrupole) and high resolution (time of flight). And so far as we know, it has never been used for the analysis of poliumoside in plasma samples. Furthermore, the detailed pharmacokinetic parameters and bioavailability were present for the further research of poliumoside.
Poliumoside (
Chromatographic separation of the analyte was performed on Acquity UPLC system with BEHC18 column (2.1 × 100 mm, 1.7 μm, Waters, Milford, MA, USA). An aliquot of the sample (5 μL) was injected into the UPLC system for analysis. Gradient elution was performed with formic acid (0.1%) in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.4 mL/min. The gradient elution process was used as follows: 5% B at 0 to 1 min, 5 to 95% B at 1 to 3 min, then back to the initial condition within 1 min. Before the next injection, all the samples would undergo a needle wash program to minimize the pollution, the solvent for needle wash containing methanol and water (v/v, 50:50). The column temperature was maintained at 45˚C.
Mass spectrometry was performed on the Xevo G2 Q-TOF MS (Waters) equipped with the electronic spray ion (ESI) [
Stock solutions of poliumoside (1 mg/mL) were prepared in saline. Working solutions for control and calibration samples were generated by gradient dilution of the stock solution with methanol:water (60:40). The 10% TCA used as precipitating agent was prepared in purified water. The IS (caffeic acid) was prepared by dilution of the stock solution (100 μg/mL) with the same solvent to a concentration of 10 μg/mL. All of the described solutions were stored at 4˚C before use.
The calibration solutions were obtained by spiking appropriate volume of working solutions into the aliquots of 100 μL blank rat plasma, and the plots were set up at the range of 50 - 10,000 ng/mL (50, 100, 625, 1250, 2500, 5000, 10,000 ng/mL). As the same to calibration standard, quality control (QC) samples were prepared in three different plasma concentration level (200, 1000, 8000 ng/mL). All of the samples were stored at −20˚C.
The rat plasma samples, calibration standard solution and the QC samples were thawed at room temperature before use and put them on the ice for the consideration of stability. Plasma samples were extracted through protein precipitation method by 10% TCA. In brief, an aliquot of 20 μL IS working solution (10 μg/mL) was added into 100 μL plasma samples in a 1.5 mL polypropylenes tube, followed by vortexing for 30 seconds. Subsequently, 80 μL of 10% TCA was added into each tube, vortexing for another 3 min. The mixture was centrifuged under 13,000 g for 15 min at 4˚C. The supernatant was collected and transferred into a new tube. Then, 5 μL of the supernatant was injected into the UPLC/Q-TOF-MS system for the further analysis.
The selectivity was verified through the comparison of the chromatograms of blank plasma, blank plasma spiked with poliumoside and caffeic acid, and rat plasma testing samples. To investigate the specificity and make the result dependable, three different batches were tested following the same procedure.
To construct the calibration curve, the spiked calibration samples in gradient were analyzed in three consecutive days. Peak area ratios of poliumoside to IS were plotted against analyte concentrations and the date was well fitted by least-square regression using 1/x2 as the weighting factor in the concentration range of 50 - 10,000 ng/mL.
The inter-day accuracy and precision were assessed by analyzing six replicated QC samples at three concentration levels (low, middle and high level) on three separated days. The intra-day accuracy and precision were assessed by analyzing the samples on the same day. Concentrations were calculated with calibration curves obtained daily. The relative error (RE) and relative standard deviation (RSD) were used to evaluate the accuracy and precision, respectively.
The recovery of poliumoside was measured by comparing the peak area of QC samples with those of reference QC solutions reconstituted in blank plasma extracts at low, middle and high level (n = 6). The matrix effect was evaluated by calculating the peak area ratio of the response in the presence of matrix ions to the analyte peak response in the absence of matrix ions. Six different lots of blank rat plasma spiked with analyte at three levels were used for the evaluation. The recovery and matrix effect of the IS was measured in the same way.
The stability of poliumoside in rat plasma was investigated by analyzing triplicate QC samples at three concentrations level (low, middle and high), exposed to different conditions. The stability of the spiked samples were evaluated as the following aspects, 1) after three completed freeze-thaw cycles, 2) the exposure of the spiked samples at ice-water bath for 12 h, 3) room temperature for 12 h. For stability of poliumoside in processed samples, the prepared QC samples were placed in an auto-sampler at 4˚C for 3 days. This experiment can clearly determine the stability of the collected samples and the accurate analysis for determination.
Male Sprague-Dawley (SD) rats weighing 220 - 250 g were obtained from Laboratory Animal center of Jinan University (Guangdong, China). Rats were housed in a humidity-controlled room maintained on 12 h light-dark cycles for 5 days before the experiment. Standard animal food and water were provided ad libitum. Twelve rats were anaesthetized using 10% chloral hydrate and the jugular vein were cannulated for blood collection before 24 h of the experiment. Diet was prohibited for 12 h before the experiment, while water was freely taken. All procedures were approved by the Animal Care and Ethics Committee of Jinan University (Guangzhou, China).
Rats were randomly assigned to two groups (six rats per group). Poliumoside was dissolved in saline and administrated to rats by oral and intravenous injection. Approximately 0.3 mL blood samples were collected from jugular vein into 1.5 mL heparinized centrifuge tubes. Time points were set at 2, 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360 min for the intravenous group (10 mg/kg) and 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360, 480, 720 min for the oral group (200 mg/kg). Blood samples were immediately centrifuged (5000 g, 4˚C, 8 min), and 100 μL aliquot of supernatant (plasma) was collected and frozen at −80˚C before analysis.
MS parameters were optimized by directly infusing a standard solution into the ESI source. Both positive and negative ion mode were employed to confirm the accurate ions of poliumoside and IS. All analytes showed a better response under the negative mode. Ultimately, the m/z 769.2537 for poliumoside and 179.0328 for IS were used for the further analysis under negative ion mode. Other parameters have been showed in section 2.2.
To separate the interfering compounds from the poliumoside and IS, different columns and mobile phase compositions were compared for chromatographic separation. The Waters BEH C18 column showed better peak shape and appropriate retention time for the analyte than Phenomenex Kinetex C18 column (2.1 × 50 mm, 2.6 μm). Appropriate ionization and chromatographic behavior were based on the proper mobile phase [
Appropriate extraction method of the plasma is essential for the quantitative analysis. The technique of liquid-liquid extraction and precipitation of protein was tested for alternative. The method of liquid-liquid extraction showed low recovery (30%) when ethyl acetate was used as extraction solvents, and this method was both complicated and time consuming. Various protein-precipitating reagents, such as methanol, acetonitrile and 10% TCA, were tested. Strong response value, good recovery and clear supernatant for the analytes were observed by using 10% TCA as the protein-precipitating reagent. Comparing with the neutral or alkaline conditions, poliumoside was found more stable in acidic solvents [
The calibration standards of poliumoside (50, 100, 625, 1250, 2500, 5000, 10,000 ng/mL) were obtained by spiking blank plasma with appropriate amounts of working solutions to the final concentration range of 50 - 10,000 ng/mL.
Precision and accuracy at three concentration levels of poliumoside were shown in
Concentration (μg/mL) | Intra-day | Inter-day | ||||
---|---|---|---|---|---|---|
Measured conc | RSD (%) | RE (%) | Measured conc | RSD (%) | RE (%) | |
0.2 | 0.196 ± 0.010 | 5.10 | −2.00 | 0.186 ± 0.012 | 6.45 | −7.00 |
1 | 1.013 ± 0.029 | 2.86 | 1.30 | 0.953 ± 0.076 | 7.97 | −4.70 |
8 | 8.095 ± 0.288 | 3.56 | 1.19 | 8.269 ± 0.286 | 3.46 | 3.36 |
The recovery and matrix effect of poliumoside and IS were shown in
The plasma concentration-time profiles of poliumoside were shown in
A is the peak area ratio of poliumoside to IS from different samples. The mean pharmacokinetic parameters from non-compartment modeling were listed in
Compound | Spiked concentration (μg/mL) | Extraction recovery (%) | RSD (%) | Matrix effect (%) | RSD (%) |
---|---|---|---|---|---|
Poliumoside | 0.2 | 93.89 | 1.20 | 105.34 | 2.71 |
1 | 103.32 | 7.98 | 92.49 | 14.94 | |
8 | 99.92 | 6.10 | 100.27 | 12.65 | |
Caffeic acid | 10 | 93.80 | 3.65 | 94.21 | 4.51 |
Concentration (μg/mL) | Three freeze-thaw cycles | At ice-water bath for 12 h | At room temperature for 12 h | At 4˚C for 3 days | ||||
---|---|---|---|---|---|---|---|---|
RE (%) | RSD (%) | RE (%) | RSD (%) | RE (%) | RSD (%) | RE (%) | RSD (%) | |
0.2 | 13.60 | 0.67 | −5.00 | 5.35 | −44.05 | 21.78 | 5.95 | 3.51 |
1 | −2.76 | 2.13 | 7.63 | 5.69 | −40.03 | 9.36 | 1.78 | 2.74 |
8 | −0.21 | 2.85 | 1.53 | 3.80 | −21.07 | 6.52 | −0.19 | 3.05 |
Parameters | Unit | Intravenous (10 mg/kg) | Oral (200 mg/kg) | ||
---|---|---|---|---|---|
Mean | SD | Mean | SD | ||
AUC0−t | μg/mL*min | 1044.23 | 201.13 | 146 | 9.88 |
AUC0−∞ | μg/mL*min | 1131.45 | 159.52 | 325.33 | 69.73 |
MRT | h | 0.74 | 0.29 | 15.41 | 4.17 |
Tmax | min | 29.72 | 13.87 | / | / |
Cmax | ng/mL | 561.47 | 100.33 | / | / |
t1/2 | h | 0.85 | 0.19 | 10.47 | 3.25 |
AUMC0−∞ | H*h*ng/mL | 9589.94 | 1369.04 | 98857.22 | 43873.27 |
V | L/kg | 1.91 | 1.78 | 24.18 | 2.94 |
CL | L/h/kg | 0.92 | 0.3 | 1.68 | 0.41 |
Bioavailability, F | 0.69% |
Abbreviations: AUC0−t, area under the concentration-time curve calculated from zero up to the last measured concentration; AUC0−∞, area under the concentration-time curve extrapolated from zero up to infinity; MRT, mean residence time of the analysis; Tmax, time to maximum observed concentartion; Cmax, maximum plasma concentration; t1/2, elimination; AUMC0−∞, area under the first moment of the concentration-time curve from zero up to infinity; V, volume of distribution; CL, clearance.
The systematic pharmacokinetics of poliumoside in rats was explored for the first time. Furtherly, the bioavailability of poliumoside was firstly reported to be 0.69%, which is similar to the other PhGs [
In conclusion, a rapid, sensitive and selective UPLC/Q-TOF-MS method was established and applied for the determination of poliumoside in rat plasma. Only 3 minutes were needed for the analysis time, and this method truncated the detecting time and improved the accuracy to a large extent. The pharmacokinetic parameters of poliumoside in rats were successfully characterized in this study and the bioavailability was firstly reported to be 0.69%. This study provides a novel approach for pharmacokinetics of poliumoside in rat plasma and provides a basis for further pharmacology research.
HaoQian,DanyiLu,WanLi,XiaotongZhou,BaojianWu,ZhiguoMa, (2016) Validated UPLC/Q-TOF-MS Method for Determination of Poliumoside in Rat Plasma and Its Application to Pharmacokinetic Study. American Journal of Analytical Chemistry,07,266-274. doi: 10.4236/ajac.2016.73024