Simultaneous Determination of Three Compounds in Rat Plasma by UHPLC-QQQ-MS/MS and Its Application to Pharmacokinetics of Banxia Baizhu Tianma Tang

A rapid and highly selective and sensitive ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method was applied to simultaneously determine ephedrine, gastrodin, and liquiritin in rat plasma. The three analytes and vitexin-2″-O-rhamnoside (I.S.) were analyzed on a Waters Acquity UPLC C18 column (1.7 μm, 2.1 mm × 100 mm) at 30°C with gradient mobile phase consisting of 0.1% formic acid aqueous solution (A) and acetonitrile (B) after one-step direct protein precipitation with acetonitrile. The detection was performed by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source in positive and negative ion modes. The product ions m/z 166.1⟶148.1, 285.1⟶123.1, 417.1⟶255.1, and 579.0⟶433.1 were used for determination of ephedrine, gastrodin, liquiritin, and I.S., respectively. The calibration curves of the three analytes were linear with r2 greater than 0.994. The intra and interday precision RSD% was less than 11.5 and 13.4. The intra and interday precision RE% was between −10.4% and 9.33%. The average extraction recoveries of the three analytes were no less than 86.88 ± 1.08%. The developed and validated method was for the first time applied to the pharmacokinetics of three compounds in rat plasma after intragastric administration of Banxia Baizhu Tianma Tang.


Introduction
Banxia Baizhu Tianma Tang originates from the book "Yixue Xinwu" of Qing Dynasty, being the classic prescription of traditional Chinese medicine, which consists of six herbs including Pinellia ternata (Tunb.) Breit. (PT), Atractylodes macrocephala Koidz. (AM), Gastrodia elata Bl. (GE), Poria cocos (Schw.) Wolf (PC), Citrus reticulata Blanco outer peel (CR), and Glycyrrhiza uralensis Fisch. (GU). Te formula has the function of drying dampness and resolving phlegm and calming the liver and the wind and is mainly used to treat the wind phlegm dizziness. Modern research reported that the formula has the function of anti-hypertensive and vasodilatory efects [1], and it regulates the blood pressure of spontaneously hypertensive rats and protects the heart [2,3]. Nowadays, many pharmacological efects of the single herbs in Banxia Baizhu Tianma Tang are found for treating vomiting, coughing and infammation of PT [4], antitumor, antiviral and anti-infammatory efects of AM [5] and so on.
Recently, some literature mainly concentrated on in vitro and in vivo the main chemical composition analyses by HPLC [6] and UPLC-Q-TOF-MS [7] methods, and their pharmacological efects of monomer compounds such as ephedrine, gastrodin, liquiritin and etc. in Banxia Baizhu Tianma Tang [8]. To date, some pharmacokinetic studies of the monomer compounds, such as ephedrine [9,10], gastrodin [11], liquiritin [10,12] in the single herbs of the formula have been found, but little attention has been devoted to the pharmacokinetic studies of the chemical compositions of Banxia Baizhu Tianma Tang.
Te six compounds including ephedrine, gastrodin, atractylenolide, pachymic acid, hesperidin, and liquiritin in Banxia Baizhu Tianma Tang, respectively, represent the main chemical markers for the quality evaluation of PT [13], AM, GE, PC, CR, and GU [14]. Initially, the aim of this study was to investigate the pharmacokinetics of the six chemical compositions in Banxia Baizhu Tianma Tang in rat plasma using the ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method, but only the pharmacokinetic studies of ephedrine in PT, gastrodin in GE, and liquiritin in GU were investigated at last except atractylenolide, pachymic acid, and hesperidin because only little of them were found in rat plasma. To the best our knowledge, this is frst report on the pharmacokinetic studies of ephedrine, gastrodin, and liquiritin in Banxia Baizhu Tianma Tang in rat plasma after intragastric administration in a single dose.  [15] (internal standard, I.S.) were purchased from the National Institute of Control of Pharmaceutical and Biological Products (Beijing, China). Te chemical structures of ephedrine, gastrodin, liquiritin, and I.S. are given in Figure 1. Acetonitrile, methanol (Houston, TX, USA), and formic acid (St. Louis, MO, USA) are all of HPLC grade, and other chemicals and reagents are of analytical grade.

Chromatography and Mass
Spectrometry. An UHPLC system (1290 series, Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent 6460 Triple Quadrupole Mass Spectrometer (QQQ-MS, Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ionization (ESI) source operated in positive and negative ionization modes was used to investigate the pharmacokinetic profles of ephedrine, gastrodin, and liquiritin in Banxia Baizhu Tianma Tang. A Waters Acquity UPLC C18 column (1.7 μm, 2.1 mm × 100 mm, Waters, Milford, MA, USA) at 30°C was used for the chromatographic separations. Te mobile phase consists of 0.1% formic acid aqueous solution (A) and acetonitrile (B), and the gradient elution procedure was 5-35% B from 0 to 5 min, 35%-60 B from 5 to 7 min, 60-90% B from 7 to 9 min, and 90% B from 9 to 11 min. An aliquot of 5 μL was injected, and the fow rate was 0.35 mL/min.
Te three analytes and I.S. were directly injected into the MS system in order to obtain MS information.

Animals and Plasma Sample Preparation.
Healthy male SD rats (SCXK2020-0001) (SPF grade, 300 ± 20 g) were purchased from Liaoning Changsheng Biotechnology Co. Ltd. (Shenyang, Liaoning, China). Te whole animal experiment process was approved by the Committee of Ethics of Animal Experimentation of Liaoning University of TCM and implemented in strict accordance with the guiding principles of laboratory animal protection. Rats were kept in environmentally controlled breeding room for one week, fed with standard laboratory food as well as water ad libitum, and fasted overnight before the experiments. Aliquots of 100 μL of plasma sample were spiked with 25 μL of I.S. (0.2 μg/mL) and vortex-mixed for 1 min, and then 300 μL of acetonitrile was added to the sample, vortex-mixed for 1 min, and then centrifuged at 3000 rpm for 15 min. Te supernatant was transferred into a 1.5 mL EP tube and evaporated to dryness under 40°C of the stream of nitrogen, and 100 μL of methanol was added, vortex-mixed for 1 min, centrifuged at 15,000 rpm for 3 min, and then transferred to an autosampler vial. An aliquot of 5 μL was injected into the UHPLC-QQQ-MS/MS system.

Preparation of Banxia Baizhu Tianma Tang Solution.
PT (9 g), AM (18 g), GE (6 g), PC (6 g), CR (6 g), and GU (3 g) in Banxia Baizhu Tianma Tang were, respectively, cut into small pieces, combined and immersed in water for 20-30 min, and then extracted twice with 500 mL water for 1 h and 0.5 h, respectively. Te twice extracted solutions were combined, fltered with gauze, and concentrated to about 10 mL under reduced pressure, and then some amount of water was added so that the total volume reached exactly 10 mL that was stored at 4°C before use, and the solution was analyzed by the UHPLC method to obtain the contents of ephedrine, gastrodin, and liquiritin which were 1.699, 0.601, and 0.539 mg/mL, respectively.

Selectivity and Carry-Over.
Selectivity was proved by comparing MRM chromatograms of blank plasma obtained from six rats with those of corresponding standard plasma sample spiked with the three analytes and I.S., and plasma sample after administered to the rats via intragastric administration. Figure 2 shows that the MRM chromatograms of the three analytes and I.S. were free from endogenous matrix interference at their respective retention times. Te cross-talk of ephedrine, gastrodin, and liquiritin as well as I.S. was evaluated by analyzing upper limit of quantifcation (ULOQ) plasma samples without I.S. and blank plasma samples only with I.S. Te responses of the analyte and I.S. of the low limit of quantifcation (LLOQ) were, respectively, less than 20% and 5%. Carry-over was assessed by injecting blank samples after injecting ULOQ. Te results indicated that the carry-over in blank samples following the ULOQ was not greater than 20% of the analyte response at the LLOQ and 5% of the response for the I.S.

Linearity.
Te calibration curves over the concentration range of 2.5, 5, 10, 20, 40, 100, and 250 ng/mL of ephedrine, 50, 100, 200, 400, 1000, and 2500 ng/mL of gastrodin, and 5, 10, 20, 40, 100, 250, and 500 ng/mL of liquiritin were determined using seven or six standard plasma samples via plotting the peak area ratio of the three analytes to I.S. versus the nominal concentration of the analyte in rat plasma. Te regression equations of ephedrine, gastrodin, and liquiritin were obtained using weighted (1/c 2 ) least squares linear regression: y � 0.1642x − 0.06984, y � 0.0795x + 0.06003, and y � 0.0051x + 0.20200, with a correlation coefcient (r 2 ) of 0.994, 0.998, and 0.997, respectively, where y is the peak area ratio of the analyte to I.S. and x is the spiked concentration of the analytes.

Accuracy and Precision.
Precision and accuracy were evaluated by analyzing the QC samples at low, middle, and high concentrations of the three analytes. Precision was expressed as relative standard deviation (RSD%), and accuracy was expressed as (mean found concentration-nominal concentration)/(nominal concentration) × 100%. Intraday precision and accuracy were determined by repeated analysis of a set of standards on one day (n � 5), while   Journal of Analytical Methods in Chemistry interday precision and accuracy were determined by repeated analysis on three consecutive days (n � 5 series per day). Te RSD and RE should be less than 15%, except at the LLOQ where it should not exceed 20% [15]. Te data of accuracy and precision for the three analytes from QC samples are listed in Table 1 which indicated that the present method had suitable accuracy and precision [16,17]; at the same time, the RSDs and REs of the QC samples at LLOQ of the three analytes were less than 20% which met the criteria [17].

Recovery and Matrix Efect.
Te recoveries were performed by comparing the analytical results of extracted samples at three QC levels with corresponding extracts of blank spiked with the analyte after extraction that represented 100% recovery (n � 6). Te matrix efects were investigated at three QC levels by comparing the peak areas of post-extraction blank plasma spiked with the three analytes by directly injecting the pure standard solutions at the same concentrations in six replicates (n � 6). Te matrix efect values should be in the range between 85% and 115%. Te recovery and matrix efect of the three analytes are listed in Table 1 meaning that the method had no signifcant matrix efect and presented high and reproducible recovery.
3.6. Stability. Te stabilities of QC samples at three concentrations were studied (n � 6). Short-term stability and long-term stability studies were determined at room temperature for 4 h and −20°C for 1 month, respectively. Freeze-thaw stabilities were evaluated after three freeze (−20°C)-thaw (room temperature) cycles. Later, the concentrations obtained after samples were processed and analyzed in this experiment were compared with the nominal values of the QC samples. Te stability results are listed in Table 1 which demonstrated that the three analytes were stable as no signifcant degradation of the three analytes in rat plasma occurred under various experimental conditions.

UHPLC-QQQ-MS/MS Optimization.
Te standard solutions of ephedrine, gastrodin, liquiritin, and I.S. were, respectively, injected into the mass spectrometry system to adjust the instrument setting parameters and maximize the responses. ESI was used in order to obtain good sensitivity and fragmentation. MRM was used for the quantifcation of the three analytes on account of great advantage in selectivity. Positive ion modes were used to for the quantifcation of ephedrine and I.S., negative ion modes for that of gastrodin and liquiritin, mainly generated protonated molecules m/z 166.1, 579.0, 285.1 and 417.1 as the precursor ions, respectively. Te product ion mass spectra of positive and negative ions of the three analytes and I.S. are shown in Figure 3. Acetonitrile/methanol-water (containing 0.05%, 0.1%, and 0.2% formic acid) being the mobile phase with gradient elution was used to evaluate UHPLC separation and sensitivity in MS detection. Finally, acetonitrile-water containing 0.1% formic acid, which provides better ionization and higher sensitivity, was selected for the separation of the three analytes and I.S., and the excellent peak shape and mass spectral response were also obtained. Tree analytes and I.S. were rapidly eluted with total retention times less than 5.0 min and the whole run time was 7.0 min, in which the retention time of ephedrine, gastrodin, I.S., and liquiritin was 1.43, 1.64, 4.45, and 4.85 min, respectively. Typical MRM chromatograms of a blank sample, a blank sample spiked with three analytes and I.S., and a plasma sample at 30 min after intragastric administration in a single dose are shown in Figure 2.

Pharmacokinetic Application. Banxia Baizhu Tianma
Tang solution of 16.6 mL/kg approximately containing ephedrine of 28 mg, gastrodin of 10 mg, and liquiritin of 9 mg was administered to the rats via intragastric administration. A blood sample of 0.25 mL was withdrawn from orbital venous at times of 0, 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360, 480 and 720 min to heparinized polythene tubes and centrifuged at 3000 rpm for 15 min to obtain plasma samples that were stored at −20°C before analysis. Te UHPLC-QQQ-MS/MS method described above was validated successfully and applied to the pharmacokinetic studies of ephedrine, gastrodin, and liquiritin in Banxia Baizhu Tianma Tang. Pharmacokinetic analysis was performed using the DAS 2.0 program (Chinese Pharmacology Society, Beijing, China) to calculate the pharmacokinetic parameters with both compartment and non-compartment approaches. Te mean plasma concentration versus time plots of ephedrine, gastrodin, and liquiritin are shown in Figure 4.

Incurred Sample Reanalysis (ISR).
Te samples used to prepare the calibration curves and QC during method validation were not the actual study samples because the diferences of protein binding, sample in homogeneity or concomitant medications existed and metabolites, which can afect the accuracy and precision of the analyte in such samples during processing and storage. Terefore, incurred samples were reanalyzed of study samples in separate runs at diferent days to evaluate accuracy of the samples. Te samples around the maximum concentration (C max ) and elimination phase for ISR were selected, which could adequate coverage of the entire PK profle. Terefore, 12 samples (2 samples in each rat, n = 6) more than 10% of all the 66, 60 and 78 samples of ephedrine, gastrodin and liquiritin were respectively reanalyzed for ISR. Tables 2-4 indicate that the diferences between concentrations obtained for the original analysis and the reanalysis were all within 20% of their mean, meaning that 100% ISR met the criteria [16].
In the study, pharmacokinetic analysis was performed using the DAS 2.0 program to calculate the pharmacokinetic parameters with both compartment (a) and noncompartment (b) approaches, a two-compartment open model to gastrodin, and one-compartment open model to Table  1: Precision, accuracy, recovery, and matrix efect of ephedrine, gastrodin, and liquiritin in rat plasma.

6)
Found C (ng/ mL)  Journal of Analytical Methods in Chemistry detected up to 720 min, gastrodin up to 240 min after dosed, meaning that they exert a longer lasting efect in vivo in rats, and the advantage of TCM was that the chemical compositions can remain a longer time in body and exert a lasting therapeutic efect and increase the clinical efcacy.  and further studies will be conducted on other components in this formula in the future.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.   Table 5: Mean pharmacokinetic parameters of ephedrine, gastrodin, and liquiritin in rat plasma after intragastric administration of Banxia Baizhu Tianma Tang solution of 16.6 mL/kg, equivalent to 28 mg/kg of ephedrine, 10 mg/kg of gastrodin, and 9 mg/kg of liquiritin, respectively (mean ± SD, n � 6).