2.1 Chemicals and reagents
Herba ephedrae, Semen Armeniacae Amarum, Semen Sinapis and Rhizoma Corydalis which were purchased from Guoyitang (Beijing, China). Reference-standard E, PE, ST, THP and AG (certified to contain 99.8%) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing,China). Acetonitrile and methanol (HPLC grade) were purchased from EMD Millipore Corporation (Millipore, Milford, MA). Formic acid was obtained from Dikma technologies (Shanghai, China). Purified water used throughout the study and ammonium formate were obtained from Sigma-Aldrich (St. Louis, USA). All other reagents were of analytical grade. All solutions and sample aliquots were filtered through a 0.22um nylon filter membrane manufactured by the Jinteng Corporation (Tianjin, China).
2.2 Preparation of herbal aqueous extracts
Following the extractive method of Majie cataplasm, MaHuang (2.5 g), baijiezi (2.5 g), yanhusuo (2.5 g) and kuxingren (2.5 g) were boiled twice with 80ml of 80% ethanol of 1.5 hours for each time. An aqueous solution by combine all the above mentioned extracts was obtained by ltration and concentrated under reduced pressure at 60℃(Wang et al., 2013). According to the method described above, sufficient extracts were prepared in this experiment.
2.3 Preparation of stock and working dilution and quality control samples
Master stock solutions were prepared by dissolving E, PE, ST, THP and AG in methanol at concentrations of 2 mg/ml, each stock solution was stored in tube after a brief vortex. A series of working solutions of these analytes were obtained by diluting these stocks with methanol at appropriate concentrations of 1000ng/ml, 500ng/ml, 100 ng/ml, 50 ng/ml, 10 ng/ml, 5ng/ml, 1 ng/ml, 0.5 ng/ml, 0.1 ng/ml. Calibration standard samples were prepared by spiking 90ul blank rabbits plasma with standard mixture working solutions (10ul), at final plasma concentrations of 0.01 ng/ml, 0.05 ng/ml, 0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml.
Low, medium and high level of Quality control (QC) working solutions at the desired concentrations (1, 5, 20ng/ml and 0.1, 5, 50 ng/ml ) were prepared. All samples were vortexed and stored at -80℃ until analysis.
2.4 Sample preparation
Frozen plasma samples were thawed at 25℃. To a 100 uL of rabbits plasma in 3mL tube, 350uL of methanol was added. The samples were vortexed for 30s after centrifugation at 13000 rpm for 10 min, 60ul of the upper organic layer was then transferred into another set of tube, and 140 uL of water was added and vortexed, then filtrated it with a 0.2um micro filter, the supernatant (20ul) was injected into the HPLC–MS/MS system for analysis(Huang et al., 2009).
2.5 Instrumentation and conditions
Plasma samples were analyzed by HPLC–MS/MS method. The HPLC-MS/MS system was composed of Agilent 1290 Infinity liquid chromatography instrument (Agilent, Waldbronn, Germany) and an Agilent 6490 QQQ triple-quadrupole mass spectrometer equipped with an AJS electrospray ionization source (Agilent Technologies, Inc., CA, USA). Two analytical methods were developed for analysis of these five compounds. An Agilent MassHunter Workstation Software (Agilent Technologies, USA) was used for all data acquisition and processing.
2.6 Method
2.6.1 Method 1
Chromatographic separation was achieved on a ZORBAX RRHD Eclipse Plus C18 column (3.0×100 mm, 1.8 µm) using gradient elution with the mobile phase consisting of acetonitrile and water (containing 0.1% formic acid and 5mM ammonium formate) for E, PE, ST and THP. The temperature of the analytical column was set at 40℃. The elution conditions applied for E, PE, ST, THP were: 0.0 min, 3%B; 10.0 min, 6%B; 12.0 min, 40%B; 13.0 min, 98%B; 14.0 min, 98%B; 14.1 min, 3%B. The flow rate was 0.4 mL/min and the injection volume was 5μL.
The capillary voltage and the nozzle voltage were set at 4000V and 0V for E, PE, ST and THP, respectively. The drying gas flow and temperature were set at 15.0 L/min and 200℃. The nebulizer gas pressure was set at 35.0 psi. The sheath gas flow and temperature were set at 11.0 L/min and 350℃.
2.6.2 Method 2
Chromatographic separation was achieved on a ZORBAX RRHD Eclipse Plus C18 column (3.0×100 mm, 1.8 µm) using gradient elution with the mobile phase consisting of acetonitrile and water for AG, the temperature of the analytical column was set at 40℃. The gradient elution conditions for amygdalin was: 0.0 min, 10%B; 3.0 min, 40%B; 5.0 min, 98%B; 6.0 min, 98%B; 6.1 min, 10%B. The flow rate was 0.4 mL/min and the injection volume was 5μL.
The capillary voltage and the nozzle voltage were set at -3500V and -1500V for amygdalin. The drying gas flow and temperature were set at 15.0 L/min and 200℃. The nebulizer gas pressure was set at 35.0 psi. The sheath gas flow was set at 11.0 L/min.
The tandem mass spectrometer was operated under the multiple reaction monitoring (MRM) mode using electrospray source in positive ion mode for E, PE, ST and THP, and negative ion mode for AG. The MRM parameters for the different analytes are shown in Table 1.
2.7 Method validation
2.7.1 Specificity
Specificity was assessed by analyzing blank plasma, blank plasma spiked with E, EP, THP, ST and AG and real plasma samples from rabbits after oral administration of extracting solution of Majie cataplasm.
2.7.2 Calibration curves
The calibration curves were prepared by assaying standard plasma samples at concentrations as described in the section of Preparation of stock and working dilution. Each calibration curve was constructed based on the peak-area ratios of analyte (y) vs the concentration of analyte(x) using a 1/x weighting.
2.7.3 Reproducibility
The reproducibility was determined by analysis of three replicate QC samples at low, medium and high concentration(0.1, 5, 50 ng/ml) in three independent analysis batches. Reproducibility was expressed as R.S.D.%
2.7.4 Extraction recovery
The extraction recovery at the three concentration levels(1, 5, 20ng/ml ) was determined by comparing the peak areas ratios of QC samples that had been spiked prior to extraction, to QC working solutions had been added post-extraction.
2.7.5 Pharmacokinetic study
Six rabbits were housed at Beijing Jinmuyang animal breeding center (license number: SCXK (Beijing) 2010-0001). Environmental controls for the animal room were set at 22 ± 3℃ with 50 ± 20% relative humidity. The animal studies were approved by China national legislation. The rabbits were given herbal aqueous extracts (2.725g /Kg) via intragastric administration.
Blood samples were collected at 10, 30, 60, 90, 120, 150, 180, 360, 480, 540, 720, 1440 and 2880min post administration. Blank rabbits plasma was taken from other blank rabbits. The samples were immediately transferred to tubes and centrifuged at 4000 rpm at 4℃ for 10 min, the plasma supernatant was transferred to clean tubes and stored at 20℃ until analysis.
2.7.6 Pharmacokinetic data analysis
The plasma concentrations versus time profiles were analyzed. Pharmacokinetic parameters of E, PE, ST, THP and AG were calculated using the extravascular non-compartmental analysis tool of Kinetica software (Version 5.0). The pharmacokinetic parameters include the maximum plasma concentration (Cmax), the time to reach maximum concentration (Tmax),the area under the curve from 0 to 2880min (AUC0–2880) andmean retention time from 0 to last infinity (MRT0-∞).