Abstract
Background
Skimmin, a potential agent for treating postpartum stroke, is one of the most important coumarins extracted from the leaves of skimmia.
Objective
In this study, a specific, sensitive, and simple high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of skimmin and its metabolite umbelliferone in rat plasma was established and validated.
Method
Chromatographic separation was performed by an Inertsil ODS-3 column (50 mm × 4.6 mm, 5 μm) with a mobile phase consisting of 0.1% formic acid in distilled water–acetonitrile at a flow rate of 0.5 mL/min with gradient elution mode. All analytes were detected and quantified in negative multiple reaction monitoring (MRM).
Results
All calibration curves showed good linearity (r > 0.995) over the concentration range of 10–10 000 and 2.0–2000 ng/mL for skimmin and umbelliferone, respectively. The selectivity, sensitivity, extraction recovery, matrix effect, and stability met all requirements.
Conclusions
The analysis method was successfully applied to the pharmacokinetic study of skimmin and umbelliferone in rats following oral administration of skimmin at the doses of 10, 30, and 90 mg/kg. With the exception of AUC(0-∞) and Cmax, MRT and Cl/F of skimmin had significant statistical difference with the increasing doses. Skimmin might exhibit nonlinear pharmacokinetic characteristics in rats.
Highlights
This was the first study to investigate the pharmacokinetic characteristics of skimmin as a candidate agent for treating postpartum stroke.
Skimmia, also known as Yinyu in Chinese and Skimmia reevesiana Fort. in Latin, is a perennial shrub plant with a height of 0.4–1.0 m of the Rutaceae family that is natively distributed in China, Japan, the Philippines, and other places. In China, it is distributed mainly in southeast coastal provinces such as Hunan, Hubei, Guangxi, Guizhou, Guangdong, and Yunnan. Skimmia is an ancient Chinese herbal medicine with a medicinal history of more than 1790 years, which is used mainly for rheumatism and arthralgia, acute limb contracture, weakness of both feet, and so on (1, 2). In addition, the book Thousand Pieces of Gold Formulae, which was written by Sun Simiao in the Tang Dynasty, described “Skimmia combined with Cocculus could be used to treat postpartum stroke in women.”
Skimmin, a useful glycoside derivative of umbelliferone, is one of the most important coumarins extracted from the leaves of skimmia. Skimmin possesses many bioactive and pharmacological effects, such as anti-inflammation (3, 4), antioxidation (5), antifibrosis (6), antiamoebic activity (7), treating renal insufficiency (8), protecting diabetic cardiomyopathy (9), and improving insulin resistance (10).
There has been active interest in recent years in developing and optimizing analytical methods for determination of the active components from natural medicine in plasma samples. However, only a few reports have addressed the pharmacokinetics study of skimmin in rats. The scholars established and validated a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the estimation of skimmin in rat plasma, which was successfully applied to explore the oral and intravenous pharmacokinetics of skimmin (11). A LC-Orbitrap MS method for the simultaneous determination of skimmin, apiosylskimmin, and their metabolites 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in rat plasma was developed and validated, and the method was successfully applied to the pharmacokinetic study of Hydrangea paniculata Sieb extract in rats (12).
According to the records in the book Thousand Pieces of Gold Formulae, in our pilot study, skimmin has been proved to be beneficial for postpartum stroke due to skimmin and the major active metabolite of skimmin (the aglycone of skimmin)—umbelliferone (data not shown). Consequently, it is necessary to investigate the pharmacokinetic characteristics of skimmin and umbelliferone as a potential agent for treating postpartum stroke. As the primary aim of this study, a specific, sensitive, and simple HPLC-MS/MS method was developed and validated for the quantification of skimmin and umbelliferone in rat plasma. Then the assay was successfully applied to the preclinical pharmacokinetic study of skimmin and umbelliferone after a single oral administration of skimmin to rats at the doses of 10, 30, and 90 mg/kg. This research provides useful information for further development of skimmin as a candidate agent for treating postpartum stroke.
Experimental
Chemicals and Reagents
Skimmin and umbelliferone (both purity ≥98%; Figure 1) were obtained from Jinan Mingke Biotechnology Co., Ltd (Jinan, China). Morroniside and apigenin (both purity ≥98%), which were both used as the internal standard (IS), were purchased from Baoji Herbest Bio-Tech Co., Ltd (Baoji, China). Dimethyl-sulfoxide (DMSO) and formic acid were purchased from Damao Chemical Reagent Factory (Tianjin, China). HPLC-grade acetonitrile and methanol were obtained from Tedia Company (Fairfield, OH, USA). Distilled water was prepared using the Milli-Q Plus Ultrapure Water System (Millipore, Bedford, MA, USA). All other chemicals used were of the highest available quality.
Figure 1.
Chemical structures and product ion mass spectrum of skimmin (A), morroniside (B), umbelliferone (C), and apigenin (D).
Instrumentation and UPLC–MS/MS Conditions
Target materials (skimmin and umbelliferone) and ISs (morroniside and apigenin) in rat plasma were analyzed by using a Shimadzu Prominence UPLC (ultraperformance liquid chromatography, Shimadzu, USA) system coupled to an AB SCIEX™ 5500 Q-Trap® mass spectrometer (Applied Bio-systems, USA) equipped with an electrospray ionization interface operated in negative multiple reaction monitoring (MRM) mode. Chromatographic separation was performed by a reversed phase Inertsil ODS-3 column (50 mm × 4.6 mm, 5 μm, GL Sciences, Inc., Tokyo, Japan). The mobile phase consisted of 0.1% formic acid in distilled water–acetonitrile (ACN) at a flow rate of 0.5 mL/min with gradient elution mode. The gradient elution procedures were optimized as follows: 0–0.2 min, 20% ACN; 0.2–2.0 min, 20–35% ACN; 2.0–2.5 min, 35–60% ACN; 2.5–3.9 min, 60% ACN; 3.9–3.91 min, 60–20% ACN; and 3.91–5.0 min, 20% ACN. The column and autosampler temperatures were maintained at 40°C and 4°C, respectively. The injection volume was 10 μL. The optimized source-dependent mass parameters were set as follows: IonSpray Voltage (IS), -4500 V; temperature (TEM), 550°C; curtain gas (CUR), 35.0 psi; collision gas (CAD), medium; Gas 1, 55.0 psi; Gas 2, 55.0 psi. The MRM mode of m/z 369.1 → 160.8 [M+HCOO]- for skimmin, m/z 451.0 → 242.9 [M+HCOO]- for morroniside (IS), m/z 160.8 → 77.0 [M-H]- for umbelliferone, and m/z 269.0 → 117.1 [M-H]- for apigenin (IS) were used for quantitative analysis (Figure 1). The system control and data analysis were performed using AB SCIEX Analyst software (version 1.6.3).
Preparation of Calibration Standards and Quality Control (QC) Samples
The stock solutions of skimmin, umbelliferone, morroniside (IS), and apigenin (IS) were prepared at 10 mg/mL in DMSO. The morroniside (IS) and apigenin (IS) stock solutions were diluted to 80 μg/mL and 20 μg/mL in methanol for routine use, respectively. The appropriate volumes of the stock solutions of the two analytes were mixed and diluted with methanol to prepare a primary working solution containing 1.0 and 0.2 mg/mL of skimmin and umbelliferone, respectively. The primary working mixture was then diluted with methanol to obtain a series of working solutions at appropriate concentrations. The calibration curves were prepared by spiking 10 μL of appropriate working solutions into 990 μL drug-free rat plasma to yield a series of concentrations at 10, 25, 100, 2000, 5000, 7500, and 10 000 ng/mL for skimmin and 2.0, 5.0, 20, 400, 1000, 1500, and 2000 ng/mL for umbelliferone. The QC samples were separately prepared similarly to calibration standards to produce four different concentration levels (LLOQ, low QC, medium QC, and high QC) for skimmin (10, 20, 2500, and 8000 ng/mL) and umbelliferone (2.0, 4.0, 500, and 1600 ng/mL), respectively. The calibration standards and QC samples were freshly prepared before each analysis and treated in accordance with the biosample preparation procedure. All stock and mixed working solutions of analytes and ISs were stored at -20°C.
Biosample Preparation Procedure
A total of 50 μL of rat plasma was mixed with 200 μL of methanol, 10 μL of morroniside (IS), and 10 μL apigenin (IS) solution in a 1.5 mL polypropylene tube. All the mixtures were vortexed for 1 min and centrifuged at 13 000 rpm for 10 min at 4°C to remove precipitated proteins, and 10 µL of the supernatant was injected into the LC-MS/MS system for analysis.
Method Validation
The newly developed assay was validated in terms of US Food and Drug Administration guidance on bioanalytical method validation (US Food and Drug Administration, 2018), including selectivity and specificity, linearity, sensitivity, precision, accuracy, extraction recovery, matrix effects, and stability (13, 14).
Pharmacokinetic Study
Eighteen male Sprague-Dawley rats weighing 200 ± 20 g [certificate number: SCXK (Shandong) 2019-0003] were randomly divided into three equal groups, which were supplied by Shandong Laboratory Animal Center of Shandong Academy of Medical Sciences (Jinan, China). The rats housed in plastic cages with a 12/12 h light–dark cycle were kept under controlled temperature (23–25°C) and relative humidity (40–70%). All the animal protocols were approved by the Animal Ethics Committee at Maternal and Child Health Care Hospital of Shandong Province (Jinan, China) and strictly in accordance with the guide for the care and use of laboratory animals (National Research Council of USA, 1996) before performing the research.
For the preclinical pharmacokinetic study, the three groups of rats were administered skimmin at the doses of 10, 30, and 90 mg/kg by oral administration. Blood aliquots (150 µL) were collected in heparin-containing tubes at 0 (prior to dose), 5, 10, 20, 30, 60, 90, 120, 180, 240, 300, and 420 min after drug administration. Blood samples were centrifuged at 4500 rpm for 10 min at 4°C to separate plasma. The plasma aliquots were stored at -20°C until analysis in polypropylene tubes. The main pharmacokinetic parameters, including AUC(0-∞), MRT(0-∞), T1/2, Tmax, Cl/F, and Cmax, were estimated through a noncompartmental method by using the Drug and Statistics 2.0 (DAS 2.0) software package (Mathematical Pharmacology Professional Committee of China, Shanghai, China) (15, 16).
Data Analysis
Experimental data were shown as mean ± standard error of mean (SEM). Statistical analyses were performed with statistical software IBM SPSS Statistics 20.0 (IBM Corporation, Armonk, NY, USA). Statistical differences of pharmacokinetic parameters among different doses were evaluated by the one-way ANOVA (P < 0.05).
Results and Discussion
Method Validation
Six different batches of blank samples obtained from six different sources were prepared. The retention times of skimmin and umbelliferone, morroniside (IS), and apigenin (IS) in blank rat plasma were 1.82, 3.51, 1.61, and 4.05 min, respectively. No notable interference from enratenous substance was found in the retention region of either analytes or ISs, which suggested a good specificity of this method (Figure 2).
Figure 2.
MRM chromatograms in rat plasma. (I) Chromatographic profile of blank rat plasma; (II) chromatographic profile of rat plasma spiked with skimmin (A, 10 ng/mL), morroniside (B), umbelliferone (C, 2.0 ng/mL), and apigenin (D); (III) chromatographic profile of a plasma sample 60 min after administration of skimmin to a rat (m: other metabolites).
The calibration curves were generated by plotting the peak area ratios of the analytes to ISs against the nominal concentrations of each analyte by using a weighted (1/x2) least-squares regression mode in the freshly prepared rat plasma calibrators. Significant linearity was observed within the ranges of 10–10 000 ng/mL for skimmin and 2.0–2000 ng/mL for umbelliferone, as demonstrated by the correlation coefficients of >0.995 (r > 0.995). The typical calibration curves for skimmin and umbelliferone were y = (5.88 ± 0.61) × 10−4x + (9.33 ± 1.01) × 10−4 and y = (3.37 ± 0.46) × 10−4x + (1.06 ± 0.12) × 10−4, respectively. The LLOQs were 10 ng/mL and 2.0 ng/mL for skimmin and umbelliferone, respectively, and the obtained intra- and inter-batch precision and accuracy were within acceptable limits (±15% of the nominal concentrations, n = 6, Table 1).
Table 1.
The intra- and inter-batch accuracy and precision of skimmin and umbelliferone in rat plasma (n = 3 batches, six replicates per batch)
| Nominal concentration, ng/mL | Skimmin |
Umbelliferone |
|||
|---|---|---|---|---|---|
| Accuracy | Precision (RSD) | Accuracy | Precision (RSD) | ||
| Intra-batch, % | 10 | 108.58 | 8.76 | 100.39 | 11.23 |
| 20 | 98.25 | 7.70 | 97.58 | 12.26 | |
| 2500 | 103.47 | 11.79 | 100.87 | 8.37 | |
| 8000 | 104.19 | 6.78 | 98.45 | 7.57 | |
| Inter-batch, % | 2.0 | 109.02 | 12.42 | 99.25 | 12.71 |
| 4.0 | 109.38 | 3.27 | 107.07 | 5.40 | |
| 500 | 107.73 | 3.61 | 113.00 | 4.57 | |
| 1600 | 108.13 | 3.60 | 102.29 | 5.80 | |
The intra- batch and inter-batch precision and accuracy of skimmin and umbelliferone in rat plasma were determined by assay of replicate analyses of each QC sample (LLOQ, low QC, medium QC, and high QC; n = 6) during the three separate batches by using independently prepared calibration curves. The assay values were less than 15% for all concentrations and conformed to the accepted variable limits, which demonstrated that the method was reliable and reproducible for quantification of skimmin and umbelliferone in rat plasma. The results of precision and accuracy measurements are presented in Table 1.
The extraction recovery was determined by calculating the ratio of the responses of QC samples at three concentration levels (low QC, medium QC, and high QC; n = 3) with known amounts of skimmin and umbelliferone to the responses of skimmin and umbelliferone dissolved in post-extracted blank plasma at the same concentrations. The matrix effect was determined by the ratio of the responses of the analytes dissolved with blank plasma extract against those of neat standards. The extraction recovery of skimmin was within the range of 82.31–96.45%, while the extraction recovery of umbelliferone ranged from 87.96 to 94.41%. The mean matrix effects of skimmin and umbelliferone were in the range of 88.59–102.17% and 83.20–106.56%, respectively. The results suggested that the extraction recovery was within the acceptable range, and no significant matrix effect was observed on skimmin and umbelliferone in rat plasma.
The stability of skimmin and umbelliferone was evaluated by analysis of QC samples at two concentration levels (low QC, and high QC; n = 6) under the following conditions: stored at -20°C for 10 d, three freeze–thaw cycles, 6 h at ambient temperature, and 24 h at 4°C in an autosampler, which were aimed at confirming the effects of possible conditions that the samples might experience between collection and analysis. The measured concentrations were all within acceptable limits (±15% of the nominal concentrations) during the entire validation, which proved that the storage conditions, disposal, intermittent analysis, and analysis techniques were valid and reliable for skimmin and umbelliferone in rat plasma (Table 2).
Table 2.
Stability data of skimmin and umbelliferone in rat plasma under various storage conditions (n = 6)
| Storage conditions | Skimmin |
Umbelliferone |
||||
|---|---|---|---|---|---|---|
| Nominal concentration, ng/mL | Mean ± SEM, ng/mL | Accuracy, % | Nominal concentration, ng/mL | mean ± SEM, ng/mL | Accuracy, % | |
| Stored at -20°C for 10 d | 20 | 19.53 ± 0.86 | 97.63 | 4.0 | 4.35 ± 0.18 | 108.86 |
| 8000 | 8281.78 ± 307.37 | 103.52 | 1600 | 1723.55 ± 71.14 | 107.72 | |
| Three freeze–thaw cycles | 20 | 19.59 ± 0.87 | 97.94 | 4.0 | 4.24 ± 0.18 | 106.03 |
| 8000 | 7818.13 ± 211.82 | 97.73 | 1600 | 1629.53 ± 62.36 | 101.85 | |
| 6 h at ambient temperature | 20 | 20.50 ± 0.52 | 102.51 | 4.0 | 4.17 ± 0.10 | 104.28 |
| 8000 | 7354.49 ± 173.37 | 91.93 | 1600 | 1535.51 ± 55.14 | 95.97 | |
| 24 h at 4°C in autosampler | 20 | 20.04 ± 0.55 | 100.22 | 4.0 | 4.21 ± 0.12 | 105.16 |
| 8000 | 7586.31 ± 181.97 | 94.83 | 1600 | 1582.52 ± 58.52 | 98.91 | |
Pharmacokinetic Application
The established and validated LC-MS/MS method was successfully applied to the preclinical pharmacokinetic study of skimmin and umbelliferone in rats after a single oral administration of skimmin at the doses of 10, 30, and 90 mg/kg, respectively.
The AUC(0-∞) of skimmin were 194 561.35 ± 35 536.61 min•ng/mL, 275 907.64 ± 73 962.02 min•ng/mL, and 457 226.45 ± 145 644.12 min•ng/mL after a single oral administration of skimmin to rats at the doses of 10, 30, and 90 mg/kg; meanwhile, the Cmax were 3942.50 ± 688.16 ng/mL, 7582.50 ± 1332.70 ng/mL, and 11 182.50 ± 2339.21 ng/mL, respectively. As the main metabolite of skimmin, the AUC(0-∞) of umbelliferone was 5414.27 ± 1819.17 min•ng/mL, 13358.20 ± 4062.84 min•ng/mL, and 70 865.10 ± 25 122.76 min•ng/mL, and the Cmax of umbelliferone was 67.43 ± 24.52 ng/mL, 303.50 ± 108.09 ng/mL, and 1715.00 ± 686.76 ng/mL following a single intragastric administration of 10, 30, and 90 mg/kg skimmin, respectively. With the exception of AUC(0-∞) and Cmax, significant statistical differences were found for MRT and Cl/F of skimmin with the increasing doses of skimmin (P < 0.05). Therefore, it could be deduced that skimmin exhibited nonlinear pharmacokinetic characteristics in rats within the used dosage ranges.
The main pharmacokinetic parameters of skimmin and umbelliferone are summarized in Table 3. The mean plasma concentration-time curves of skimmin and umbelliferone in rats are shown in Figures 3 and 4, respectively.
Table 3.
Main pharmacokinetic parameters of skimmin and umbelliferone (mean ± SEM, n = 6)
| Analytes | Dose, mg/kg | 10 | 30 | 90 |
|---|---|---|---|---|
| Skimmin | AUC (0-∞), ng/mL·mina | 194 561.35 ± 14 507.76 | 275 907.64 ± 30 194.87 | 457 226.45 ± 59 458.96 |
| MRT(0-∞), minb | 54.16 ± 2.14 | 32.80 ± 2.30 | 36.61 ± 1.17 | |
| T1/2, minc | 25.52 ± 1.06 | 32.65 ± 7.98 | 50.44 ± 5.46 | |
| Tmax, mind | 27.50 ± 2.04 | 20.00 ± 0.00 | 23.71 ± 4.67 | |
| Cl/F, L/min/kge | 0.05 ± 0.004 | 0.12 ± 0.01 | 0.21 ± 0.03 | |
| Cmax, ng/mLf | 3942.50 ± 280.94 | 7582.50 ± 544.07 | 11 182.50 ± 954.98 | |
| Umbelliferone | AUC (0-∞), ng/mL·min | 5414.27 ± 742.67 | 13 358.20 ± 1658.65 | 70 865.10 ± 10 256.32 |
| MRT(0-∞), min | 134.49 ± 9.03 | 98.31 ± 4.05 | 83.71 ± 2.27 | |
| T1/2, min | 98.26 ± 15.40 | 131.49 ± 3.29 | 127.56 ± 2.20 | |
| Tmax, min | 25.00 ± 2.36 | 25.00 ± 2.36 | 30.00 ± 0.00 | |
| Cmax, ng/mL | 67.43 ± 10.01 | 303.50 ± 44.13 | 1715.00 ± 280.37 |
AUC = Area under the concentration–time curve.
MRT = Mean residence time.
T1/2 = Elimination half-life.
Tmax = Time to Cmax.
Cl/F = Clearance.
Cmax = Peak concentration.
Figure 3.
Plasma concentration–time curves of skimmin (mean ± SEM, n = 6).
Figure 4.
Plasma concentration–time curves of umbelliferone (mean ± SEM, n = 6).
Conclusions
A specific and sensitive quantitative assay based on the LC–MS/MS method combined with a simple protein precipitation procedure for simultaneous determination of skimmin and its metabolite umbelliferone in rat plasma was developed and validated. The total running time was 5.0 min, and the LLOQs were 10 and 2.0 ng/mL for skimmin and umbelliferone, respectively. Compared with the previous study, the assay presented was more suitable for routine analysis of bulk samples of skimmin and umbelliferone owing to its simple preparation procedure, high sensitivity, and short running time. The novel validated LC–MS/MS method was successfully applied to the pharmacokinetic study of skimmin and umbelliferone in rats after oral administration of skimmin at the doses of 10, 30, and 90 mg/kg, which was set with reference to the effective dose for treating postpartum stroke. This was the first study to investigate the pharmacokinetic characteristics of skimmin and umbelliferone as a therapeutic agent for treating postpartum stroke, and it should provide useful information for further development and utilization of skimmin.
Contributor Information
Yaqiong Sun, Shandong Provincial Maternal and Child Health Care Hospital Affiliated to Qingdao University, Key Laboratory of Birth Regulation and Control Technology of National Health Commission of China, Jinan 250014, China.
Yanyan Jiang, Shandong Provincial Maternal and Child Health Care Hospital Affiliated to Qingdao University, Key Laboratory of Birth Regulation and Control Technology of National Health Commission of China, Jinan 250014, China.
Ruihong Zhang, Shandong Provincial Maternal and Child Health Care Hospital Affiliated to Qingdao University, Key Laboratory of Birth Regulation and Control Technology of National Health Commission of China, Jinan 250014, China.
Jin Wang, Shandong Provincial Maternal and Child Health Care Hospital Affiliated to Qingdao University, Key Laboratory of Birth Regulation and Control Technology of National Health Commission of China, Jinan 250014, China.
Conflicts of Interest
The authors declare that there are no conflicts of interest.
Funding
This research was supported by the Program of Medical and Health Science and Technology Development of Shandong Province, China (No. 202005021512).
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