Materials and reagents
ST was purchased from Chinese herbal medicine market in Anguo City, Hebei Province in July 2019, and then was authenticated by Prof. Qinan Wu, a botanist of Nanjing University of Chinese Medicine. The voucher specimens were kept in the laboratory.
The reference standards of luteoloside, apigetrin, hesperidin, and quercitrin (IS) were bought from Nanjing Jin Yibai Biological Technology Co., Ltd. Their purities were determined as above 98% by HPLC method. They were stored in accordance with their instructions.
Methanol and acetonitrile were both of LC-MS grade and obtained from Merck (Merck, Darmstadt, Germany). Formic acid of HPLC grade was purchased from Anaqua Chemicals Supply (ACS, Houston, USA). Ultrapure water was purified by a Milli-Q water purification system (Millipore, Burlington, MA, USA). All other reagents were of analytical grade.
Preparation of STVO and STAE from ST
Using a set of standard apparatus including a round-bottom flask, a volatile oil extractor, a condenser pipe and a heating equipment, STVO with a yield of 1.08% was obtained by a steam distillation method recorded in the present Chinese Pharmacopoeia (2020). To remove the trace water, some ahydrous sodium sulfate was added into STVO, which was then stored in the dark glass bottle at 4 °C and filtered before use. At the same time, to remove some impurities, the decoction in the round-bottom flask was transferred into a separating funnel and added with three times the amount of 95% ethanol. After the mixture was kept at 4 °C over night, the supernatants were collected and evaporated by a vacuum-rotary unit and dried in a vacuum desiccator to prepare STAE with a yield of 10.22%.
UHPLC-MS/MS introduction
Chromatographic analysis was carried out on a Shimadzu Nexera X2 series UHPLC system (Shimadzu, Tokyo, Japan), equipped with a LC-30AD quaternary pump, a SIL-30AC autosampler, a DGU-20A on-line degasser, and a CTO-20AC thermostat. An Agilent Eclipse Plus C18 column (3.0 mm × 150 mm, 3.5 μm) was used for separation. The mobile phase consisted of with 0.2% formic acid (A) and acetonitrile (B) at a flow rate of 0.8 mL/min with the gradient elution as follows: 0.0~2.0 min, 5~15% B; 2.0~6.0 min, 15% B; 6.0~10.0 min, 15~53% B; 10.0~12.0 min, 53~95% B; 12.0~14.0 min, 95~5% B; 14.0~17.1 min, 5% B. The column temperature was maintained at 30 °C and the injection volume was 2 μL.
An AB Sciex 4500 Qtrap™ mass spectrometer (AB Sciex, Foster City, CA, USA) with electron spray ionization (ESI) in the negative mode was utilized in this study. The precursor-to-product ion transitions in multiple-reaction monitoring (MRM) mode were used for mass analysis and quantification. Some MS setting conditions were defined as follows: curtain gas pressure at 20 psi, ion spray voltage at 5000 V, turbo gas temperature at 300 °C, both nebulizer gas 1 and auxiliary gas 2 at 55 psi. Nitrogen of high purity was employed as both nebulizing gas and drying gas. All the data were obtained and analyzed by Analyst 1.6.3, a data acquisition and processing software.
Animal experiments
In the pharmacokinetic experiment, SPF male Sprague-Dawley rats (220-250 g) were supplied by Shanghai Jiesijie Experimental Animal Co., Ltd. (SCXK (Hu) 2018-0004, Shanghai, China). The rats were housed under ambient temperature of 23±2 °C with a 12 h light/dark cycle and 50% relative humidity in the laboratory. They were well looked after in the plastic cages with fresh water and free pellet food and were acclimatized for a week. Prior to the experiment, all the rats were fasted for 12 h with free access to water. To minimize the pain and suffering of the rats, the experimental procedures were performed in line with the guidelines of the Animal Care and Use Committee of Nanjing University of Chinese Medicine (Nanjing, China). The protocol was also approved by the animal experimental committee of Nanjing University of Chinese Medicine (201909A008).
Preparation of standard solutions and quality control samples
The reference standards of the three target flavonoids were separately and accurately weighed and dissolved in methanol to prepare the stock standard solutions with the concentrations of 0.238 mg/mL for luteoloside, 0.193 mg/mL for apigetrin and 0.118 mg/mL for hesperidin. Then, the calibration standard solutions were obtained by mixing and serially diluting above three stock standard solutions with methanol. Additionally, IS stock solution of quercitrin was prepared with the concentration of 200 ng/mL in methanol. All these solutions were enclosed and stored at 4 °C until analysis.
The plasma samples of calibration standards were prepared by spiking 10 μL of appropriate standard solution into 90 μL of blank plasma to obtain the final concentrations of 1.19~238 ng/mL for luteoloside, 0.965~193 ng/mL for apigetrin, 0.590~118 ng/mL for hesperidin, respectively. Quality control (QC) samples at low, middle and high concentrations (1.19, 119 and 190 ng/ml for luteoloside; 0.965, 96.5 and 154 ng/ml for apigetrin; 0.590, 59.0 and 94.4 ng/ml for hesperidin) were also prepared according to the similar procedures.
Biological sample preparation
100 µL of plasma samples and 10 µL of IS solution was added to a centrifuge tube and mixed with 500 μL acetonitrile for protein precipitation. The mixture was vortexed for 3 min and then centrifuged at 13000 rpm for 10 min at 4 °C. The supernatant was transferred to another centrifuge tube and then desiccated with an Eppendorf Concentrator Plus (Hamburg, Germany). Finally, the residue was redissolved and vortex-mixed with 120 μL of acetonitrile/water (50:50, v/v) for 5 min and centrifuged at 13000 rpm for 10 min. The supernatant was then injected into the UHPLC-MS/MS system for analysis.
Method validation
Based on the bioanalytical guidance of FDA, the method validation was carried out in terms of its specificity, linearity, sensitivity, accuracy, precision, matrix effect, recovery and stability.
Specificity
The chromatograms of some biological samples were compared to investigate the method specificity, including blank plasma samples, plasma samples spiked with luteoloside, apigetrin, hesperidin at lower limits of quantification (LLOQ) and IS, plasma samples after oral administration of STAE+STVO and STAE.
Linearity and LLOQ
The calibration curve of each analyte was obtained by plotting its peak area ratio (y) to IS versus its nominal concentration (x) with weighted (1/x2) least-squares linear regression. The method sensitivity was evaluated by LLOQ of each analyte with its signal-to-noise ratio of 10:1 in the matrix-analyte samples, which was conformed to the lowest concentration of the standard curve with an acceptable accuracy (RE≤±20%) and precision (RSD≤20%).
Precision and accuracy
Six QC samples at each level (low, medium, high) were used to in the method precision and accuracy test. The intra-day precision test was performed by repeatedly analyzing each QC sample in a day while the inter-day precision test was carried out by analyzing each QC sample on three consecutive days. The analytical precision was expressed by relative standard deviation (RSD) and relative error (RE), respectively. RE was calculated with this formula: RE (%) = (measuring value - nominal value) × 100%/nominal value. The pass criterion for RSD was required to be less than 15%, and that for RE was required to be within ±15%.
Extraction recovery and matrix effect
The extraction recovery and the matrix effect were evaluated with six replicates by the formulas as follows: (A1/A2) × 100% and (A2/A3) × 100%, respectively. A1: the peak areas obtained from the post-treated plasma samples spiked with the target components at the three QC levels; A2: the peak areas obtained from the pre-treated plasma samples spiked with the target components at the three QC levels; A3: the peak areas obtained from the standard solutions at the equivalent concentrations. The extraction recovery and matrix effect of IS were also measured by the same approach at the identical concentration.
Stability
The stability of each analyte in rat plasma was evaluated by analyzing six replicates of the QC samples at low, middle, and high concentrations in the following practical experimental conditions: storage at 4 °C for 12 h, three freeze-thaw cycles (freezing at -80 °C for 24 h and thawing at room temperature) and long-term storage at -80 °C for 2 weeks. The acceptable RE was within ±15.0% of the nominal concentration.
Pharmacokinetic study
For pharmacokinetic study, all the rats were divided into two groups (n=6 per group) randomly. The given dosages of two test extracts were equivalent to about 5g ST/kg. STAE group: the rats were administrated orally with STAE (500mg/kg), dissolved in 0.5% carboxy methyl cellulose sodium (CMC–Na) solution. STAE+STVO group: the rats were administrated orally with STAE (500mg/kg) and STVO (50mg/kg), dissolved in 0.5% CMC–Na solution. Blood samples were collected from the venous plexus of the eye socket and transferred in a heparinized centrifuge tube (1.5 mL) after oral administration (0, 5, 10, 20, 30, 45, 60, 120, 240, 480, and 720 min). Each sample was immediately centrifuged at 13000 rpm for 10 min at 4 °C to acquire the plasma. At last, the plasma was immediately stored at -80 °C until analysis.
Data analysis
The plasma concentrations of the three flavonoids during the experiment were calculated according to the daily calibration curve. With Drug And Statistics Version 3.2.8 (DAS 3.2.8) software (Mathematical Pharmacology Professional Committee of China, Shanghai, China), a non-compartmental method was employed to analyze some representative pharmacokinetic parameters, such as the time to reach maximum concentration (Tmax), elimination half-life time (T1/2), the area under the concentration curves (AUC0→t and AUC0→∞) and the maximum concentration (Cmax). Data were demonstrated as the mean ± standard deviation (SD) with triplicate. The differences of pharmacokinetic parameters from two groups were tested by Student’s t-test. Statistical analysis was operated by Graphpad Prism 7.0 software.