Thermo TSQ Altis high performance liquid chromatography combined with triple quadrupole mass spectrometer (Thermo Company, USA), XS105 electronic balance (Metler-Toledo Group), JA5003 electronic balance (Shanghai Shunyu Hengping Scientific Instrument Co. Ltd.), Neofuge 1600R desktop cryogenic high-speed centrifuge (Shanghai Lishen Scientific Instruments Co. Ltd.), miVac Duo vacuum centrifuge concentrator (GeneVac Company, UK), VG 3 S25 vortexer (IKA Company of Germany); KQ-100DE digital ultrasonic cleaner (Kunshan Ultrasound Instrument Co., Ltd.); Elemental 18120 Molecular Ultra Pure Water System (Shanghai Moller Scientific Instrument Co., Ltd.).
Reagents And Materials
NXTCs (Shanxi Buchang Pharmaceutical Co., Ltd., batch number: 190193) and adrenaline hydrochloride injection (Suicheng Pharmaceutical Co., Ltd., specifications: 1mL: 1 mg, batch number: 61903011) both were purchased from pharmacy of the First Affiliated Hospital of Henan University of Chinese Medicine (Zhengzhou, China). Reference standards of caffeic acid, ferulic acid, stigmatin, tanshinone II A were purchased from Nanjing Liangwei Biological Technology Co., Ltd. with purity above 98% (Nanjing, China). Internal standards including clarithromycin and propione sulphonate were purchased from National Institutes for Food and Drug Control with purity above 98% (Beijing, China). Ultra-pure water was purified by Molecular waters purification system. Acetonitrile (HPLC grade) and methanol (HPLC grade) were purchased from Merck KGaA (Darmstadt, Germany). Formic acid (HPLC grade) was purchased from Dikma Technology Co., Ltd (Tianjin, China). Ultra-pure water was purified by using a Milli-Q system (Milford, MA, USA).
Female Sprague–Dawley (SD) rats (6-8 weeks old, weighing 220 ± 20 g) were purchased by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Before the experiment, the rats were fed adaptively for one week, keeping the circadian rhythm for 12 hours and feeding and drinking freely. The temperature and humidity meet the requirements of animal feeding. Animal care and experimental protocols were performed based on ‘Detailed Rules and Regulations for Administration and Implementation of Biomedical Animal Experiments’ (No. 1998-55, Ministry of Public Health, China). The study protocol was approved by the Ethical Committee of the First Affiliated Hospital of Henan University of Chinese Medicine.
NXTC Solution Preparation
The content of NXTC was dispersed in 0.5% CMC-Na aqueous solution to produce the NXT suspension with the concentration of 0.33 g/mL.
Preparation of reference substance reserve solution, internal standard reserve solution and quality control sample
Caffeic acid, ferulic acid, anthocyanin and tanshinone II A were weighed accurately and then serially dissolved in methanol to obtain the reference stock solutions with different concentrations of 30.5, 30, 38 and 21.5 mg/mL respectively. Six series of mixed reference solutions were gained with appropriate individual reference stock solution mixed and diluted with methanol in volumetric flask: 0.5, 1, 5, 10, 50 and 100 ng/mL caffeic acid, 1, 5, 10, 50, 100 and 500 ng/mL ferulic acid, 0.05, 0.1, 0.5, 1, 5 and 10 ng/mL anthocyanin and 0.5, 1, 5, 10, 50 and 100 ng/mL tanshinone II A.
As internal standard substances, clarithromycin and probenecid were weighed precisely and dissolved with methanol to obtain the internal standard reserve solutions with concentrations of 532 and 536 mg/mL respectively. Appropriate amount of internal standard reserve solutions was diluted with methanol to provide the mixed internal standard reserve solution with clarithromycin of 106.4 ng/mL and probenecid of 107.2 ng/mL.
Quality control (QC) samples of caffeic acid with concentrations of 30, 50, 500 ng/mL, ferulic acid with concentrations of 10, 20, 200 ng/mL, anthocyanin with concentrations of 0.5, 1, 10 ng/mL and tanshinone II A with concentrations of 1, 5, 50 ng/mL were prepared with blank plasma.
Chromatography and mass spectrometry conditions
Liquid chromatographic analysis and mass spectrometry detection was performed on Thermo Scientific™ TSQ Altis™ (Thermo Company, USA). Chromatographic separation was carried out on an Acquity UPLC BEH C18 column (2.1 ´ 100 mm, 1.7 μm) maintained at 30°C. The mobile phase gradient conditions consisted of 0.1% aqueous formic acid (A) and methanol (B): 0–2 min, 40–100% B; 2–5 min, 100% B; at 5–5.1 min, 100–40% B; 5.1–8 min, 40% B. The flow rate was 0.2 mL/min. The autosampler was conditioned at 10 °C and the sample injection volume was 1 μL.
The ESI source was set in both positive and negative ionization mode. The parameters in the source were set in the following manner: positive ion scanning voltage, 3500 V; negative ion scanning voltage, 2500 V; sheath gas velocity, 25 arb; auxiliary air velocity, 7 arb; collision gas pressure, 1.5 mTorr; ion transmission tube temperature, 325 °C; ion source temperature, 400 °C. The scanning mode was set multiple reaction monitoring (MRM) mode and the selected monitor ion were m/z 178.96®135.04 for caffeic acid, m/z 192.95®134.00 for ferulic acid, m/z 267.00®251.97 for anthocyanin, m/z 295.05®277.20 for tanshinone IIA, m/z 748.35®590.29 for clarithromycin, and m/z 284.00®240.05 for propafenone. The collision energy and RF-lens were optimized for precursor/product ion pairs of each analyte and the selected values are presented in Table 1.
Modeling, drug administration and preparation of plasma samples
In this experiment, twenty rats were randomly divided into blank administration group and model administration group with 10 rats in each group respectively. After 7 days of acclimation, the rats in the model administration group were given adrenaline hydrochloride injection (0.8 mg/kg) subcutaneously. After 2 h, the rats were placed in 0°C ~ 2°C ice water to swim for 4 minutes, the first time 4 hours after the administration of epinephrine hydrochloride injection, subcutaneous injection of epinephrine hydrochloride injection 0.8 mg/kg was performed again, resulting in a model of acute blood stasis in rats. All rats fasted for 12 hours and drank freely. NXTC suspension was administrated by gavage at a dose of 5g/kg. At 0.083, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10 and 12 hours after administration, blood samples were collected from the eye orbital venous plexus of rats and placed in a heparinized centrifuge tube. Plasma was separated by 3000 r/min centrifugation for 10 min and stored at -80 °C for determination.
Plasma Sample Preparation
100 mL of plasma sample was precisely pipetted and 20 mL of 10% formic acid water, 10 mL of internal standard solution were added, the mixture was vortexed for 3 min, then 300 mL of methanol was added and vortexed for 5 min additionally. Subsequently, the sample was centrifugated at 14 000 r/min for 10 min. The supernatant was centrifugally concentrated to dryness under vacuum condition. The residue was re-dissolved in 100 mL methanol, agitated on a vortexer for 3 min and centrifugated at 14000 r/min for 10 min, then the supernatant was obtained for analysis.
The blank plasma of rats, blank plasma of rats added with reference solution and internal standard solution, and the plasma samples obtained from rats of model group were taken 100 mL respectively, the samples were processed applying the sample processing procedure in item 2.5. The ion chromatograms of the ingredients were acquired abiding by the corresponding chromatographic and mass spectrometric methods in item 2.3.
linearity, limit of detection (LOD) and lower limit of quantification (LLOQ)
Six duplicates of 100 mL blank rat plasma were added with a series of mixed reference solutions with different concentrations. The samples were processed according to the sample processing procedure in item 2.5 and conducted LC-MS/MS analysis. Taking the concentration of each substance to be measured as the abscissa (x), the ratio of the peak area of the substance against the internal standard as the ordinate (y), and the reciprocal of the concentration (1/x) as the weighting coefficient for linear regression, the regression equations of the calibration curves of each ingredient in rat plasma were established. By comparing the results of samples with known analyte concentration and those of blank samples, the signal-to-noise ratio (S/N) of 3:1 was determined to be the LOD, the S/N of 10:1 was determined to be the LLOQ, which is the lowest concentration point of the standard curve.
Precision and accuracy
Certain duplicates of 100 mL blank plasma were respectively added with different concentrations of mixed reference solution and internal standard solution to prepare the lower limit of quantification sample and quality control samples with low, medium and high concentrations. Six samples were prepared in parallel with each concentration, which were processed according to the procedure displayed in item 2.5. The measured concentration of QC sample was were calculated according to the accompanying standard curve. Measurements for 3 consecutive days were implemented to investigate the accuracy and precision of the established analysis method.
Recovery of extraction and matrix effect
Quality control samples of low, medium and high concentrations prepared from 100 mL blank plasma were processed according to the sample processing method above. The peak area of each component was recorded as A1. Other duplicates of 100 mL blank plasma were also processed according to the method above. The residues were respectively re-dissolved in reference solutions of low, medium and high concentration containing internal standard. The peak area of each component was recorded as A2. The low, medium and high concentration reference solutions containing internal standard were also respectively injected into the analysis system. The peak area of each component was recorded as A3 here. Each sample was prepared in 6 duplicates. The recovery (%) was the ratio of A1 to A3, and the matrix effect (%) was the ratio of A2 to A3.
The quality control samples with low, medium and high concentration were prepared and placed at 4 °C for 0, 2, 4, 8, 12 and 24 h for analysis. The quality control samples were also frozen at -80 °C and thawed at room temperature for 3 cycles with 6 duplicates for each concentration respectively. According to the established calibration curve, the measured concentration of QC sample was calculated, and compared with the labeled concentration to investigate the stability of each component in rat plasma.
Data processing and analysis
The data collection and sample analysis were controlled by Tracefinder 4.1 software and the data were processed with Microsoft Excel. The content of each compound was expressed as x ± s. The obtained data of content were processed with Phoenix Winnonlin (version 6.2, Pharsight Corporation, USA) pharmacokinetic software, and the pharmacokinetic parameters were calculated applying non-compartment model.