Determination of ephedrine, pseudoephedrine, sinapine thiocyanate, tetrahydropalmatine and amygdalin by HPLC-MS/MS after oral administration of extracts of Majie cataplasm in rabbits plasma: Application to a pharmacokinetic study

This study sinapine thiocyanate, tetrahydropalmatine and amygdalin of extracts of Majie cataplasm after oral administration in rabbits plasma and describe the pharmacokinetics of this five components. The qualitative detection of the five compounds was accomplished by two methods. One method for the simultaneous determination of ephedrine, pseudoephedrine, sinapine thiocyanate and tetrahydropalmatine was developed and validated for the first time, while the other method was for amygdalin. Chromatographic separations were achieved on a C18 column using gradient elution with the mobile phase consisting of acetonitrile and water (containing 0.1% formic acid and 5mM ammonium formate) for ephedrine, pseudoephedrine, sinapine thiocyanate and tetrahydropalmatine, while acetonitrile and water for amygdalin, at a flow rate of 0.4 mL/min. The initial gradient was extended in order to isolate ephedrine and pseudoephedrine better. The detection was performed in multiple-reaction monitoring (MRM) mode using electrospray ionization (ESI). The results provided a basis for further study on the bioactivity of Majie cataplasm. Waldbronn, Germany) and an Agilent 6490 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.


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Majie cataplasm consisting of Mahuang (Herba Ephedrae), Kuxingren (Semen Armeniacae Amarum), Baijiezi (Semen Sinapis) and Yanhusuo (Rhizoma Corydalis), is a classical transdermal administration dosage form employed in treating asthma (Zhang, 2009;Kong et al., 2016). Such topical cataplasm has been widely and clinically applied in China for great effect. Millions of people used it to prevent asthma during the period of hottest days each year Liu et al., 2016;Chen, 2005).
Pharmacokinetics play a key role in the evaluation of drug action and are regarded as available means in elucidating the mechanism of traditional Chinese medicine (TCM) (Yan et al., 2015). In order to explore the possible mechanism and the time-concentration relation of absorption of Majie cataplasm, there is a need to do the pharmacokinetic analysis. In the present study, HPLC-MS/MS methods were validated for quantification of five components.
From the perspective of TCM theory, some traditional Chinese herbs, such as Semen Sinapis and Rhizoma Corydalis cannot pass into the bloodstream through skin but have the effects of local stimulation and promoting blood circulation when use externally Wang et al., 2011).
So in the present study, the rabbits were given extracting solution of Majie cataplasm by gavage to make sure that the active ingredients can pass into the bloodstream. Then the blood samples were taken from the rabbits as the research objects, and the determination method were established so that to measure the condition of the active ingredients by transdermal administration.
As we all know, complexities are the basic characters of Chinese herbal medicine. The pharmacodynamics of Majie cataplasm are generated from the synergistic effects of different herbal and components. Consequently, it is not enough to select monomeric component as analyte to conduct the pharmacokinetic study of Majie cataplasm. Thus, it is better to select several effective ingredients as the targets to investigate the pharmacokinetics of the whole prescription (Wang XQ, 2014). Ephedrine (E), pseudoephedrine (PE), Sinapine thiocyanate (ST), Tetrahydropalmatine (THP) and amygdalin (AG) (Fig. 1) are major pharmacologically active compounds and serve as the quality control standard of Ephedra Herb, Semen Sinapis and Semen Armeniacae Amarum listed in the Pharmacopeia of the People's Republic of China, respectively (China Pharmacopoeia Committee, 2010;Tang et al., 2016;Tanaka et al., 2014;Guo et al., 2013;Lee et al., 2014). In addition, as the major active components of Herba Ephedrae, Semen Armeniacae Amarum and Semen Sinapis, E, PE, ST, THP and AG have been exhibited to have antiasthmatic effects (Song et al., 2014;Song et al., 2015;Guo et al., 2013;Lin et al., 2014). As a promising antiasthmatic agent, a better understanding of its pharmacokinetics is necessary for correlation of its therapeutic effect. Therefore, in the present study, we choose E, PE, ST, THP and AG as research subjects to established HPLC-MS/MS method.
In the previous studies, several detection methods have been developed, including LC-MS/MS method for E, PE and AG in rats (Jiang et al., 2015), LC-MS/MS method for ST and THP in plasma , LC-ESI-MS/MS method for E and THP in human blood . However, to our best knowledge, these methods had limitation such as high limit of detection, which was not adequate to meet current needs. And there was no attempt to establish HPLC-MS/MS method for the determination of the five representative compounds in one study. Therefore, this study explored two HPLC-MS/MS methods for the determination of the E, PE, ST, THP and AG in rabbits plasma, and has been successfully applied to characterize oral administrations of them in pharmacokinetic study.

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).

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℃ . According to the method described above, sufficient extracts were prepared in this experiment.

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.

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).

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.

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.

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.%

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.

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.

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-∞).

Specificity
The specifiity of the assay was evaluated using blank plasma samples. Blank rat plasma was spiked with the working solutions of E, EP, THP, ST and AG. Under these conditions, the retention times of E, EP, THP, ST and AG were at 8.9, 9.5, 13.3,12.5 and 2.5 min, respectively (Fig.2). There was no interference observed from endogenous substances in the plasma at the retention time of the analytes.

Calibration curves
The calibration curves of E, EP, THP, ST and AG were obtained by weighted (1/x) linear regression analysis, and showed good linearity. The calibration curves, correlation coefficients and linear ranges of these five analytes in plasma were listed in Table 2. The calibration curves of these five analytes showed good linearity (r > 0.99) over the concentration ranges.

Extraction recovery
Across the concentration range studied, the relative recovery at the three concentrations of 1.0, 5.0 and 20 ng/mL in rabbits plasma showed in Table 3.

Reproducibility
Reproducibility was determined by analysis of the low, medium and high QCs (1, 50, and 500 ng/mL, n = 6) on three different assays. The results were showned in Table 4.

Pharmacokinetic study
The validated method was successfully applied to the pharmacokinetic study of Majie cataplasm in rabbits following oral administration (n = 6 for each administration). The mean plasma concentrations vs time profiles of E, EP, THP, ST and AG following i.g. administration of herbal aqueous extracts were shown in Fig 3. And its pharmacokinetic parameters using non compartmental analysiswere summarized in Table, the results of PK parameters include Cmax, Tmax, (AUC0-2880)and (MRT0-∞) were shown in Table 5.

Conclusions
The two HPLC-MS/MS methods for the determination of E, PE, ST, THP and AG in rabbits plasma was developed and validated. To the best of our knowledge, this is the first report of oral administration of extracts of Majie cataplasm using HPLC-MS/MS method. Compared with the analytical method reported in the literature, the methods offered superior minimum detection limit and reproducibility, and better separation of ephedrine and pseudoephedrine. We report a fully validated (LC/MS/MS) method for the simultaneous determination of E, PE, ST and THP in rabbits plasma. The method meets the requirement of high sample through-put in bioanalysis and has been successfully applied to a pharmacokinetic study of Majie cataplasm in rabbits.

Consent for publication
Not applicable.

Availability of data and materials
The data used to support the findings of this study are available from the corresponding author upon request.    Figure 1 The chemical structure of ephedrine, pseudoephedrine, sinapine thiocyanate, tetrahydropalmatine and amygdalin.