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

Simultaneous Determination of Three Compounds in Rat Plasma by UHPLC-QQQ-MS/MS and Its... Hindawi Journal of Analytical Methods in Chemistry Volume 2023, Article ID 5119997, 10 pages https://doi.org/10.1155/2023/5119997 Research Article Simultaneous Determination of Three Compounds in Rat Plasma by UHPLC-QQQ-MS/MS and Its Application to Pharmacokinetics of Banxia Baizhu Tianma Tang 1,2 2 2 1,2 Zheming Ying, Guoyuan Sui, Lianqun Jia , and Guanlin Yang Te First Clinical College of Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning, China Key Laboratory of Ministry of Education for TCM Viscera-State Teory and Applications, Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning, China Correspondence should be addressed to Lianqun Jia; jlq-8@163.com and Guanlin Yang; yang_guanlin@163.com Received 20 July 2022; Revised 8 November 2022; Accepted 6 December 2022; Published 14 January 2023 Academic Editor: Mohamed Abdel-Rehim Copyright © 2023 Zheming Ying et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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. Te 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. Te detection was performed by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source in positive and negative ion modes. Te 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. Te calibration curves of the three analytes were linear with r greater than 0.994. Te intra and interday precision RSD% was less than 11.5 and 13.4. Te intra and interday precision RE% was between −10.4% and 9.33%. Te average extraction recoveries of the three analytes were no less than 86.88± 1.08%. Te developed and validated method was for the frst time applied to the pharmacokinetics of three compounds in rat plasma after intragastric administration of Banxia Baizhu Tianma Tang. in Banxia Baizhu Tianma Tang are found for treating 1. Introduction vomiting, coughing and infammation of PT [4], antitumor, Banxia Baizhu Tianma Tang originates from the book “Yixue antiviral and anti-infammatory efects of AM [5] and so on. Xinwu” of Qing Dynasty, being the classic prescription of Recently, some literature mainly concentrated on in traditional Chinese medicine, which consists of six herbs vitro and in vivo the main chemical composition analyses by including Pinellia ternata (Tunb.) Breit. (PT), Atractylodes HPLC [6] and UPLC-Q-TOF-MS [7] methods, and their macrocephala Koidz. (AM), Gastrodia elata Bl. (GE), Poria pharmacological efects of monomer compounds such as ephedrine, gastrodin, liquiritin and etc. in Banxia Baizhu cocos (Schw.) Wolf (PC), Citrus reticulata Blanco outer peel (CR), and Glycyrrhiza uralensis Fisch. (GU). Te formula has Tianma Tang [8]. To date, some pharmacokinetic studies of the function of drying dampness and resolving phlegm and the monomer compounds, such as ephedrine [9, 10], gas- calming the liver and the wind and is mainly used to treat the trodin [11], liquiritin [10, 12] in the single herbs of the wind phlegm dizziness. Modern research reported that the formula have been found, but little attention has been de- formula has the function of anti-hypertensive and vaso- voted to the pharmacokinetic studies of the chemical dilatory efects [1], and it regulates the blood pressure of compositions of Banxia Baizhu Tianma Tang. spontaneously hypertensive rats and protects the heart [2, 3]. Te six compounds including ephedrine, gastrodin, Nowadays, many pharmacological efects of the single herbs atractylenolide, pachymic acid, hesperidin, and liquiritin in 2 Journal of Analytical Methods in Chemistry OH OH O H N HO HOH C O OH H HO OH HO HO Ephedrine OH OH Liquiritin Gastrodin (a) (b) (c) OH CH OH HO CH OH O OH HO O OH HO O OH O Vitexin-2˝-O-rhamnoside (d) Figure 1: Te chemical structures of ephedrine (a), gastrodin (b), liquiritin (c), and I.S. (d). Banxia Baizhu Tianma Tang, respectively, represent the TX, USA), and formic acid (St. Louis, MO, USA) are all of main chemical markers for the quality evaluation of PT [13], HPLC grade, and other chemicals and reagents are of an- AM, GE, PC, CR, and GU [14]. Initially, the aim of this study alytical grade. was to investigate the pharmacokinetics of the six chemical compositions in Banxia Baizhu Tianma Tang in rat plasma 2.2. Chromatography and Mass Spectrometry. An UHPLC using the ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry system (1290 series, Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent 6460 Triple Quadrupole Mass (UHPLC-QQQ-MS/MS) method, but only the pharmaco- kinetic studies of ephedrine in PT, gastrodin in GE, and Spectrometer (QQQ-MS, Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ionization (ESI) liquiritin in GU were investigated at last except atractyle- source operated in positive and negative ionization modes nolide, pachymic acid, and hesperidin because only little of them were found in rat plasma. To the best our knowledge, was used to investigate the pharmacokinetic profles of ephedrine, gastrodin, and liquiritin in Banxia Baizhu this is frst report on the pharmacokinetic studies 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 Tianma Tang in rat plasma after intragastric administration in a single dose. 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 2. Materials and Methods 5–35% B from 0 to 5 min, 35%–60 B from 5 to 7 min, 60–90% 2.1. Materials and Chemicals. PT, AM, GE, PC, CR, and GU B from 7 to 9 min, and 90% B from 9 to 11 min. An aliquot of of Banxia Baizhu Tianma Tang were purchased from the 5 μL was injected, and the fow rate was 0.35 mL/min. afliated hospital of Liaoning University of Traditional Te three analytes and I.S. were directly injected into the Chinese Medicine (Shenyang, Liaoning, China) and au- MS system in order to obtain MS information. Te frag- thenticated by Prof. Rongxiang Wang, a senior expert of mentor voltage (FV) and collision energy (CE) parameters of herbal authentication at School of Pharmacy, Liaoning multiple reaction monitoring (MRM) to detect the three University of Traditional Chinese Medicine. Te reference analytes and I.S. were m/z 166.1⟶ 148.1 (FV, 90; CE8) for standards including ephedrine, gastrodin, liquiritin, and ephedrine, m/z 285.1⟶ 123.1 (FV, 100; CE, 4) for gas- vitexin-2″-O-rhamnoside [15] (internal standard, I.S.) were trodin, m/z 417.1⟶ 255.1 (FV, 180; CE, 12) for liquiritin, purchased from the National Institute of Control of Phar- and m/z 579.0⟶ 433.1 (FV200; CE, 16) for I.S. Te other maceutical and Biological Products (Beijing, China). Te parameters were as follows: drying gas (N2) fow rate, 11.0 L/ chemical structures of ephedrine, gastrodin, liquiritin, and min; drying gas temperature, 300 C; nebulizer pressure, 15 I.S. are given in Figure 1. Acetonitrile, methanol (Houston, psig; and capillary voltage, 4000 V. Journal of Analytical Methods in Chemistry 3 2.3. Calibration Standard and Quality Control Samples. 3. Method Validation Stock solutions of calibration standards including ephed- 3.1. Selectivity and Carry-Over. Selectivity was proved by rine, gastrodin, liquiritin, and I.S. were, respectively, pre- comparing MRM chromatograms of blank plasma obtained pared in methanol at concentrations of 2.40, 1.25, 5.24, and from six rats with those of corresponding standard plasma 1.88 mg/mL. Te stock solutions of three analytes and I.S. sample spiked with the three analytes and I.S., and plasma were serially diluted with methanol to be the working sample after administered to the rats via intragastric ad- standard solutions of 25.0, 10.0, 5.0, and 1.0 μg/mL in order ministration. Figure 2 shows that the MRM chromatograms to prepare calibration curves and quality control (QC) of the three analytes and I.S. were free from endogenous working standard solutions. Te fnal efective concentra- matrix interference at their respective retention times. tions of the calibration standards in rat plasma samples Te cross-talk of ephedrine, gastrodin, and liquiritin as made by spiking 10 μL the corresponding working solutions well as I.S. was evaluated by analyzing upper limit of into 100 μL blank plasma were 2.5, 5, 10, 20, 40, 100, and quantifcation (ULOQ) plasma samples without I.S. and 250 ng/mL of ephedrine, 50, 100, 200, 400, 1000, and blank plasma samples only with I.S. Te responses of the 2500 ng/mL of gastrodin, and 5, 10, 20, 40, 100, 250, and analyte and I.S. of the low limit of quantifcation (LLOQ) 500 ng/mL of liquiritin. Similarly, the low, middle, and high- were, respectively, less than 20% and 5%. Carry-over was concentration QC samples were made by the same way at assessed by injecting blank samples after injecting ULOQ. fnal concentrations of ephedrine, gastrodin, liquiritin of 5, Te results indicated that the carry-over in blank samples 40, and 200 ng/mL, 150, 600, and 2000 ng/mL, and 15, 75, following the ULOQ was not greater than 20% of the and 400 ng/mL, respectively. analyte response at the LLOQ and 5% of the response for the I.S. 2.4. Animals and Plasma Sample Preparation. Healthy male SD rats (SCXK2020-0001) (SPF grade, 300± 20 g) were 3.2. Linearity. Te calibration curves over the concentration purchased from Liaoning Changsheng Biotechnology Co. range of 2.5, 5, 10, 20, 40, 100, and 250 ng/mL of ephedrine, Ltd. (Shenyang, Liaoning, China). Te whole animal ex- 50, 100, 200, 400, 1000, and 2500 ng/mL of gastrodin, and 5, periment process was approved by the Committee of 10, 20, 40, 100, 250, and 500 ng/mL of liquiritin were de- Ethics of Animal Experimentation of Liaoning University termined using seven or six standard plasma samples via of TCM and implemented in strict accordance with the plotting the peak area ratio of the three analytes to I.S. versus guiding principles of laboratory animal protection. Rats the nominal concentration of the analyte in rat plasma. Te were kept in environmentally controlled breeding room regression equations of ephedrine, gastrodin, and liquiritin for one week, fed with standard laboratory food as well as were obtained using weighted (1/c ) least squares linear water ad libitum, and fasted overnight before the exper- regression: y � 0.1642x − 0.06984, y � 0.0795x + 0.06003, and iments. Aliquots of 100 μL of plasma sample were spiked y � 0.0051x + 0.20200, with a correlation coefcient (r ) of with 25 μL of I.S. (0.2 μg/mL) and vortex-mixed for 1 min, 0.994, 0.998, and 0.997, respectively, where y is the peak area and then 300 μL of acetonitrile was added to the sample, ratio of the analyte to I.S. and x is the spiked concentration of vortex-mixed for 1 min, and then centrifuged at 3000 rpm the analytes. 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, 3.3. Limits of Detection and Quantifcation. Te limits of vortex-mixed for 1 min, centrifuged at 15,000 rpm for detection (LOD) were determined in signal to noise ratio (S/ 3 min, and then transferred to an autosampler vial. An N) of 3, which were 0.61, 13.7, and 1.36 ng/mL for ephedrine, aliquot of 5 μL was injected into the UHPLC-QQQ-MS/MS gastrodin, liquiritin, respectively. LLOQ was defned as the system. lowest concentration on calibration curve with acceptable precision and accuracy. LLOQs of ephedrine, gastrodin, and liquiritin were 2.5, 50, and 5 ng/mL (S/N> 10) with RSDs of 2.5. Preparation of Banxia Baizhu Tianma Tang Solution. the intra and interday precisions, respectively, below 11.5% PT (9 g), AM (18 g), GE (6 g), PC (6 g), CR (6 g), and GU and 13.4% and accuracies (RE%) between −10.4% and (3 g) in Banxia Baizhu Tianma Tang were, respectively, cut 9.11%. 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 3.4. Accuracy and Precision. Precision and accuracy were combined, fltered with gauze, and concentrated to about evaluated by analyzing the QC samples at low, middle, and 10 mL under reduced pressure, and then some amount of high concentrations of the three analytes. Precision was water was added so that the total volume reached exactly expressed as relative standard deviation (RSD%), and ac- 10 mL that was stored at 4 C before use, and the solution was curacy was expressed as (mean found concentration-nom- analyzed by the UHPLC method to obtain the contents of inal concentration)/(nominal concentration) × 100%. ephedrine, gastrodin, and liquiritin which were 1.699, 0.601, Intraday precision and accuracy were determined by re- and 0.539 mg/mL, respectively. peated analysis of a set of standards on one day (n � 5), while 4 Journal of Analytical Methods in Chemistry ×10 1.03 III 1.02 IV 1.01 II 0.99 0.98 0.97 0.96 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 (a) ×10 6 1 m/z 148.1 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 II 3.9 3.85 II 3.8 3.75 m/z 123.1 3.7 3.65 3.6 3.55 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 III 5 III m/z 255.1 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 1 IV 3.5 IV 2.5 m/z 433.1 1.5 0.5 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 (b) ×10 0.8 I 0.6 m/z 148.1 0.4 0.2 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 II 0.8 II 0.6 m/z 123.1 0.4 0.2 0.5 11.5 22.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 III 0.8 III m/z 255.1 0.6 0.4 0.2 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 IV 0.8 IV 0.6 m/z 433.1 0.4 0.2 0.5 11.522.53 3.5 4 4.5 5 5.5 6 6.5 7 Counts (%) vs. Acquisition Time (min) (c) Figure 2: Typical MRM chromatograms of blank plasma (a), a blank sample spiked with the three compounds and I.S. (b), and plasma sample after intragastric administration in a single dose (c) (peak I: ephedrine; peak II: gastrodin; peak III: liquiritin; peak IV: I.S.). Journal of Analytical Methods in Chemistry 5 interday precision and accuracy were determined by re- sensitivity in MS detection. Finally, acetonitrile-water peated analysis on three consecutive days (n � 5 series per containing 0.1% formic acid, which provides better ioni- zation and higher sensitivity, was selected for the separation day). Te RSD and RE should be less than 15%, except at the LLOQ where it should not exceed 20% [15]. Te data of of the three analytes and I.S., and the excellent peak shape accuracy and precision for the three analytes from QC and mass spectral response were also obtained. Tree samples are listed in Table 1 which indicated that the present analytes and I.S. were rapidly eluted with total retention method had suitable accuracy and precision [16, 17]; at the times less than 5.0 min and the whole run time was 7.0 min, same time, the RSDs and REs of the QC samples at LLOQ of in which the retention time of ephedrine, gastrodin, I.S., and the three analytes were less than 20% which met the criteria liquiritin was 1.43, 1.64, 4.45, and 4.85 min, respectively. [17]. 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 3.5. Recovery and Matrix Efect. Te recoveries were per- dose are shown in Figure 2. formed by comparing the analytical results of extracted samples at three QC levels with corresponding extracts of 4.2. Pharmacokinetic Application. Banxia Baizhu Tianma blank spiked with the analyte after extraction that repre- Tang solution of 16.6 mL/kg approximately containing sented 100% recovery (n � 6). Te matrix efects were in- vestigated at three QC levels by comparing the peak areas of ephedrine of 28 mg, gastrodin of 10 mg, and liquiritin of 9 mg was administered to the rats via intragastric admin- post-extraction blank plasma spiked with the three analytes by directly injecting the pure standard solutions at the same istration. 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, concentrations in six replicates (n � 6). Te matrix efect 240, 360, 480 and 720 min to heparinized polythene tubes values should be in the range between 85% and 115%. Te and centrifuged at 3000 rpm for 15 min to obtain plasma recovery and matrix efect of the three analytes are listed in samples that were stored at −20 C before analysis. Te Table 1 meaning that the method had no signifcant matrix UHPLC-QQQ-MS/MS method described above was vali- efect and presented high and reproducible recovery. dated successfully and applied to the pharmacokinetic studies of ephedrine, gastrodin, and liquiritin in Banxia 3.6. Stability. Te stabilities of QC samples at three con- Baizhu Tianma Tang. Pharmacokinetic analysis was per- centrations were studied (n � 6). Short-term stability and formed using the DAS 2.0 program (Chinese Pharmacology long-term stability studies were determined at room Society, Beijing, China) to calculate the pharmacokinetic temperature for 4 h and −20 C for 1 month, respectively. parameters with both compartment and non-compartment Freeze-thaw stabilities were evaluated after three freeze approaches. Te mean plasma concentration versus time (−20 C)-thaw (room temperature) cycles. Later, the con- plots of ephedrine, gastrodin, and liquiritin are shown in centrations obtained after samples were processed and Figure 4. analyzed in this experiment were compared with the nominal values of the QC samples. Te stability results are 4.3. Incurred Sample Reanalysis (ISR). Te samples used to listed in Table 1 which demonstrated that the three ana- prepare the calibration curves and QC during method lytes were stable as no signifcant degradation of the three validation were not the actual study samples because the analytes in rat plasma occurred under various experi- diferences of protein binding, sample in homogeneity or mental conditions. concomitant medications existed and metabolites, which can afect the accuracy and precision of the analyte in such 4. Results and Discussion samples during processing and storage. Terefore, incurred 4.1. UHPLC-QQQ-MS/MS Optimization. Te standard so- samples were reanalyzed of study samples in separate runs at lutions of ephedrine, gastrodin, liquiritin, and I.S. were, diferent days to evaluate accuracy of the samples. Te respectively, injected into the mass spectrometry system to samples around the maximum concentration (C ) and max adjust the instrument setting parameters and maximize the elimination phase for ISR were selected, which could ade- responses. ESI was used in order to obtain good sensitivity quate coverage of the entire PK profle. Terefore, 12 and fragmentation. MRM was used for the quantifcation of samples (2 samples in each rat, n = 6) more than 10% of all the three analytes on account of great advantage in selec- the 66, 60 and 78 samples of ephedrine, gastrodin and tivity. Positive ion modes were used to for the quantifcation liquiritin were respectively reanalyzed for ISR. Tables 2–4 of ephedrine and I.S., negative ion modes for that of gas- indicate that the diferences between concentrations ob- trodin and liquiritin, mainly generated protonated mole- tained for the original analysis and the reanalysis were all cules m/z 166.1, 579.0, 285.1 and 417.1 as the precursor ions, within 20% of their mean, meaning that 100% ISR met the respectively. Te product ion mass spectra of positive and criteria [16]. negative ions of the three analytes and I.S. are shown in In the study, pharmacokinetic analysis was performed Figure 3. using the DAS 2.0 program to calculate the pharmaco- Acetonitrile/methanol-water (containing 0.05%, 0.1%, kinetic parameters with both compartment (a) and non- and 0.2% formic acid) being the mobile phase with gradient compartment (b) approaches, a two-compartment open elution was used to evaluate UHPLC separation and model to gastrodin, and one-compartment open model to 6 Journal of Analytical Methods in Chemistry Table 1: Precision, accuracy, recovery, and matrix efect of ephedrine, gastrodin, and liquiritin in rat plasma. Intraday (n � 5) Interday (n � 15) Stability (n � 6) Added C Extraction recovery Matrix efect (%) Analytes Found C (ng/ RSD RE Found C (ng/ RSD RE Short-term Long-term Freeze-thaw (ng/mL) (%) (n � 6) (n � 6) mL) (%) (%) mL) (%) (%) stability (%) stability (%) stability (%) 5 86.88± 1.08 100.4± 3.83 4.57± 0.52 11.5 −8.60 4.48± 0.60 13.4 −10.4 89.23± 2.63 87.43± 5.26 87.15± 3.81 Ephedrine 40 87.47± 2.35 96.40± 2.45 41.3± 2.8 6.84 3.33 37.6± 3.3 8.89 -6.0 91.40± 3.05 90.68± 2.43 90.04± 4.07 200 92.32± 3.69 96.76± 3.82 209.8± 10.3 4.93 4.90 205.6± 8.81 4.28 2.82 91.27± 5.22 91.67± 1.07 90.66± 6.42 150 89.25± 4.07 96.59± 1.26 162.7± 10.2 6.28 8.4 161.3± 18.0 11.1 −7.56 89.29± 3.99 90.11± 3.78 89.88± 2.33 Gastrodin 600 87.98± 5.20 96.72± 3.57 580.3± 20.6 5.15 −3.3 587± 35.8 6.09 −2.12 90.69± 5.33 90.85± 1.04 91.58± 1.27 2000 90.14± 3.22 95.15± 0.78 1989± 66.6 3.35 −0.53 2016± 87.2 4.32 −0.80 91.80± 4.01 93.11± 3.78 93.33± 2.63 15 88.75± 6.08 98.17± 3.69 16.1± 1.7 10.7 7.56 16.4± 1.5 9.33 9.11 89.16± 1.28 89.23± 4.65 88.29± 5.96 Liquiritin 75 90.49± 3.83 97.71± 1.54 70.7± 3.3 4.74 −5.78 76.4± 1.9 2.46 1.82 91.72± 6.47 91.87± 1.93 90.59± 4.22 400 90.81± 1.73 99.87± 7.01 390.8± 15.5 3.97 −2.3 389.5± 15.0 3.86 −2.63 92.13± 1.91 93.33± 5.74 92.70± 2.91 Journal of Analytical Methods in Chemistry 7 ×10 148.1000 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 (a) ×10 123.1000 3.5 2.5 1.5 0.5 122 123 124 125 126 127 128 129 130 131 132 (b) ×10 255.1000 1.2 0.8 0.6 0.4 0.2 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 (c) ×10 433.1000 406 408 410 412 414 416 418 420 422 424 426 428 430 432 434 436 438 440 442 444 446 (d) Figure 3: Te product ion mass spectra of positive and negative ions of ephedrine (a), gastrodin (b), liquiritin (c), and I.S. (d). ephedrine and liquiritin (weight = 1/c ) gave the best ft gastrodin. Moreover, both apparent volume of distribu- a a according to the Akaike Information Criterion (AIC) tion (V ) and clearance (CL ) of ephedrine and liquiritin values, and the main pharmacokinetic parameters of the were larger than those of gastrodin, indicating that three analytes are listed in Table 5. After intragastric ad- ephedrine and liquiritin were mainly distributed in sur- ministration, the maximum concentrations (C ) of rounding tissues leading to lower plasma concentrations. max ephedrine, gastrodin, and liquiritin in plasma were, re- In addition, the peak time (T ) of ephedrine, gastrodin, max spectively, 128.2± 28.8, 1622.7± 657.3, and and liquiritin was 0.917, 0.500, and 0.167 h, respectively, 244.7± 64.2 ng/mL, along with the corresponding values of suggesting that the three analytes were all rapidly the area under the concentration-time curves (AUC ) absorbed, in which the liquiritin was the fastest one. Te 0⟶t of the three analytes were 262.2± 42.2, 2483.5± 1194.0, terminal half-life (t ) 2.639, 1.278, and 2.176 h, indicated 1/2 and 230.3± 135.5 ng h/mL, meaning that ephedrine and that the eliminations of them were slow. Furthermore, it liquiritin had a little weak absorption compared with was noteworthy that ephedrine and liquiritin could be 8 Journal of Analytical Methods in Chemistry 160 2500 0 0 0 2841610124 01 25 3 4 Time (h) Time (h) Ephedrine Gastrodin (a) (b) 0 2468 10 12 14 Time (h) Liquiritin (c) Figure 4: Te mean plasma concentration-time curves of ephedrine (a), gastrodin (b), and liquiritin (c) (n � 6). Table 2: Incurred sample reanalysis data of ephedrine. Subject no. Sampling point (min) Original concentration (ng/mL) Incurred sample concentration (ng/mL) Mean (ng/mL) RE (%) 1 30 1.16 1.28 1.22 9.8 1 180 0.401 0.432 0.417 7.4 2 30 1.33 1.49 1.41 11.3 2 180 0.337 0.327 0.332 −3.0 3 30 2.38 2.65 2.52 10.7 3 180 0.730 0.746 0.738 2.2 4 30 1.02 1.12 1.07 9.3 4 180 0.301 0.345 0.323 13.6 5 30 1.58 1.47 1.53 −7.2 5 180 0.426 0.478 0.452 11.5 6 30 1.69 1.81 1.75 6.9 6 180 0.432 0.444 0.438 2.7 detected up to 720 min, gastrodin up to 240 min after chemical compositions can remain a longer time in body dosed, meaning that they exert a longer lasting efect in and exert a lasting therapeutic efect and increase the vivo in rats, and the advantage of TCM was that the clinical efcacy. Concentration (ng/mL) Concentration (ng/mL) Concentration (ng/mL) Journal of Analytical Methods in Chemistry 9 Table 3: Incurred sample reanalysis data of gastrodin. Subject no. Sampling point (min) Original concentration (ng/mL) Incurred sample concentration (ng/mL) Mean (ng/mL) RE (%) 1 30 1333.3 1351.6 1397.5 1.3 1 120 532.1 538.3 535.2 11.6 2 30 1159.7 1187.5 1173.6 2.4 2 120 371.8 410.9 391.4 10.0 3 30 2375 2422 2398.5 2.0 3 120 852.6 832.5 842.6 −2.4 4 30 1048.1 1008.1 528.1 7.6 4 120 412.8 419.5 416.2 1.6 5 30 1356.3 1387.0 1371.7 2.2 5 120 392.5 412.5 402.5 5.0 6 30 1275 1289 1282 1.1 6 120 367.3 371.4 369.4 1.1 Table 4: Incurred sample reanalysis data of liquiritin. Subject no. Sampling point (min) Original concentration (ng/mL) Incurred sample concentration (ng/mL) Mean (ng/mL) RE (%) 1 10 220.1 225.8 223.0 2.6 1 180 64.86 71.2 68.0 9.3 2 10 196.4 216.5 206.5 9.7 2 180 51.7 52.9 52.3 2.3 3 10 317.5 345.8 331.7 8.5 3 180 91.2 82.6 86.9 9.9 4 10 188.5 177.4 183.0 −6.1 4 180 44.39 52.2 48.3 16.2 5 10 154.7 161.9 158.3 4.5 5 180 71.9 84.7 78.3 16.3 6 10 245.4 235.3 235.4 −4.3 6 180 61.2 69.8 65.5 13.1 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, re- spectively (mean± SD, n � 6). Parameters Analytes b b b a a a a T (h) C (ng/mL) t (h) V (L/kg) CL (L h/kg) AUC (ng/mL) h AUC (ng/mL) h max max 1/2 d 0⟶t 0⟶∞ Ephedrine 0.917± 0.144 128.2± 28.8 2.639± 0.574 284.04± 52.7 106.6± 18.9 262.2± 42.2 267.81± 43.29 Gastrodin 0.5± 0.0 1622.7± 657.3 1.278± 0.48 4.904± 3.171 4.057± 2.146 2483.5± 1194.0 3012.89± 1630.6 Liquiritin 0.167± 0.0 244.7± 64.2 2.176± 0.332 3436.2± 1485.4 34.4± 14.9 230.3± 135.5 314.0± 180.8 a and b are the compartmental and non-compartmental approaches, respectively. and further studies will be conducted on other components 5. Conclusions in this formula in the future. A developed and validated UHPLC-QQQ-MS/MS method was successfully applied to the pharmacokinetic studies of Data Availability ephedrine, gastrodin, and liquiritin in rat plasma. Tree components in Banxia Baizhu Tianma Tang are simulta- Te data used to support the fndings of this study are in- neously determined in rat plasma after intragastric ad- cluded within the article. ministration for the frst time. Tis experiment provides a pharmacokinetic basis for Banxia Baizhu Tianma Tang in Conflicts of Interest clinical aspects and can make Banxia Baizhu Tianma Tang better applied in clinical treatment. Although the pharma- Te authors declare that they have no conficts of interest. cokinetic study of traditional Chinese medicine is very difcult due to its very low content of chemical components Acknowledgments in each single herb, ephedrine, gastrodin, and liquiritin which are representative of the herbs of PT, GE, and GU in Tis study was supported by the Project of the National Banxia Baizhu Tianma Tang can be determined in the study, Natural Science Foundation of China (grant no. 81974548) 10 Journal of Analytical Methods in Chemistry [14] Te Pharmacopoeia Commission of Prc, Pharmacopoeia of the Liaoning Provincial Department of Education 2020 Scien- People’s Republic of China, Chinese Medical Science and tifc Research Project (Grant L202016). Technology Press, Beijing, China, 2020. [15] X. X. Ying, X. M. Lu, X. H. Sun, X. Q. Li, and F. M. Li, References “Determination of vitexin-2-O-rhamnoside in rat plasma by ultra-performance liquid chromatography electrospray ioni- [1] C. S. Tan, Y. C. Loh, C. H. Ng et al., “Anti-hypertensive and zation tandem mass spectrometry and its application to vasodilatory efects of amended Banxia Baizhu Tianma Tang,” pharmacokinetic study,” Talanta, vol. 72, no. 4, Biomedicine & Pharmacotherapy, vol. 97, pp. 985–994, 2018. pp. 1500–1506, 2007. [2] S. Wu, Y. H. Jiang, and C. H. Yang, “Efect of Banxia Baizhu [16] Center for Drug Evaluation and Research of the U.S, “De- Tianma Tang on myocardium MAPK pathway of spontaneous partment of health and human services food and drug ad- hypertensive rats with syndrome of excessive accumulation of ministration, bioanalytical method validation guidance for phlegm-dampness,” Chinese Journal of Experimental Tradi- industry,” 2018, http://www.fda.gov/Drugs/ tional Medical Formulae, vol. 22, pp. 159–165, 2016. GuidanceComplianceRegulatoryInformation/Guidan. [3] J. Y. Jiang, X. Z. Wang, and S. S. Luo, “Efect of Banxia Baizhu [17] European Medicines Agency, “Guideline on bioanalytical Tianma decoction on the left ventricular hypertrophy of method validation,” 2011, http://www.ema.europa.eu/docs/ hypertrophied myocardium in spontaneously hypertensive en_GB/document_library/Scientifc_guideline/2011/08/ rat,” Chinese Journal of Integrated Traditional and Western WC500109686.pdf. Medicine, vol. 30, no. 10, pp. 1061–1066, 2010. [4] H. H. Xie, J. Y. Xu, T. Xie et al., “Efects of Pinellia ternata (Tunb.) Berit. on the metabolomic profles of placenta and amniotic fuid in pregnant rats,” Journal of Ethno- pharmacology, vol. 183, no. 183, pp. 38–45, 2016. [5] S. H. Gu, L. Li, H. Huang, B. Wang, and T. Zhang, “Anti- tumor, antiviral, and anti-Infammatory efcacy of essential oils from Atractylodes macrocephala Koidz. produced with diferent processing methods,” Molecules, vol. 24, no. 16, p. 2956, 2019. [6] S. L. Wang, H. M. Yang, and M. H. Li, “Fingerprint and multicomponent quantitative analysis of Banxia Baizhu Tianma decoction by HPLC,” China Modern Medicine, vol. 28, no. 8, pp. 4–8, 2021. [7] Y. Y. Fu, M. Q. Shan, M. H. Hu et al., “Chemical profling of Banxia-Baizhu-Tianma Decoction by ultra-fast liquid chro- matography with tandem mass spectrometry,” Journal of Pharmaceutical and Biomedical Analysis, vol. 174, pp. 595– 607, 2019. [8] N. Xu, S. L. Wang, and H. Y. Shi, “Research progress on chemical components and efect mechanism of Banxia Baizhu Tianma Decoction,” China Journal of Traditional Chinese Medicine and Pharmacy, vol. 36, no. 8, pp. 4802–4808, 2021. [9] J. W. Wang, M. H. Chiang, C. M. Lu, and T. H. Tsai, “De- termination the active compounds of herbal preparation by UHPLC-MS/MS and its application on the preclinical phar- macokinetics of pure ephedrine, single herbal extract of Ephedra, and amultiple herbal preparation in rats,” Journal of Chromatography B, vol. 1026, pp. 152–161, 2016. [10] J. Wang, S. J. Zheng, Y. Li et al., “Investigate the efect of honey on the absorption of seven active ingredients from Wu-Tou Decoction in rats,” Biomedical Chromatography: Biomedical Chromatography, vol. 36, no. 4, Article ID e5323, 2022. [11] Y. Zhao, X. J. Gong, X. Zhou, and Z. J. Kang, “Relative bioavailability of gastrodin and parishin from extract and powder of Gastrodiae rhizoma in rat,” Journal of Pharma- ceutical and Biomedical Analysis, vol. 100, pp. 309–315, 2014. [12] X. Chen, Y. C. Ma, M. L. Yang et al., “Pharmacokinetics and tissue distribution study of modifed Xiaochaihu granules against gastric ulcer induced by ethanol in rats by UPLC-MS/ MS,” Natural Product Communications, vol. 15, no. 7, p. 12, Article ID 1934578X2093521, 2020. [13] L. W. Fang, J. F. Xie, L. W. Lin, M. L. Tian, and K. H. Row, “Multi phase extraction of ephedrine from Pinellia ternata and herbal medicine using molecular imprinted polymer coated ionic liquid-based silica,” Phytochemical Analysis, vol. 31, no. 2, pp. 242–251, 2020. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Analytical Methods in Chemistry Hindawi Publishing Corporation

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

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Hindawi Journal of Analytical Methods in Chemistry Volume 2023, Article ID 5119997, 10 pages https://doi.org/10.1155/2023/5119997 Research Article Simultaneous Determination of Three Compounds in Rat Plasma by UHPLC-QQQ-MS/MS and Its Application to Pharmacokinetics of Banxia Baizhu Tianma Tang 1,2 2 2 1,2 Zheming Ying, Guoyuan Sui, Lianqun Jia , and Guanlin Yang Te First Clinical College of Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning, China Key Laboratory of Ministry of Education for TCM Viscera-State Teory and Applications, Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning, China Correspondence should be addressed to Lianqun Jia; jlq-8@163.com and Guanlin Yang; yang_guanlin@163.com Received 20 July 2022; Revised 8 November 2022; Accepted 6 December 2022; Published 14 January 2023 Academic Editor: Mohamed Abdel-Rehim Copyright © 2023 Zheming Ying et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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. Te 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. Te detection was performed by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source in positive and negative ion modes. Te 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. Te calibration curves of the three analytes were linear with r greater than 0.994. Te intra and interday precision RSD% was less than 11.5 and 13.4. Te intra and interday precision RE% was between −10.4% and 9.33%. Te average extraction recoveries of the three analytes were no less than 86.88± 1.08%. Te developed and validated method was for the frst time applied to the pharmacokinetics of three compounds in rat plasma after intragastric administration of Banxia Baizhu Tianma Tang. in Banxia Baizhu Tianma Tang are found for treating 1. Introduction vomiting, coughing and infammation of PT [4], antitumor, Banxia Baizhu Tianma Tang originates from the book “Yixue antiviral and anti-infammatory efects of AM [5] and so on. Xinwu” of Qing Dynasty, being the classic prescription of Recently, some literature mainly concentrated on in traditional Chinese medicine, which consists of six herbs vitro and in vivo the main chemical composition analyses by including Pinellia ternata (Tunb.) Breit. (PT), Atractylodes HPLC [6] and UPLC-Q-TOF-MS [7] methods, and their macrocephala Koidz. (AM), Gastrodia elata Bl. (GE), Poria pharmacological efects of monomer compounds such as ephedrine, gastrodin, liquiritin and etc. in Banxia Baizhu cocos (Schw.) Wolf (PC), Citrus reticulata Blanco outer peel (CR), and Glycyrrhiza uralensis Fisch. (GU). Te formula has Tianma Tang [8]. To date, some pharmacokinetic studies of the function of drying dampness and resolving phlegm and the monomer compounds, such as ephedrine [9, 10], gas- calming the liver and the wind and is mainly used to treat the trodin [11], liquiritin [10, 12] in the single herbs of the wind phlegm dizziness. Modern research reported that the formula have been found, but little attention has been de- formula has the function of anti-hypertensive and vaso- voted to the pharmacokinetic studies of the chemical dilatory efects [1], and it regulates the blood pressure of compositions of Banxia Baizhu Tianma Tang. spontaneously hypertensive rats and protects the heart [2, 3]. Te six compounds including ephedrine, gastrodin, Nowadays, many pharmacological efects of the single herbs atractylenolide, pachymic acid, hesperidin, and liquiritin in 2 Journal of Analytical Methods in Chemistry OH OH O H N HO HOH C O OH H HO OH HO HO Ephedrine OH OH Liquiritin Gastrodin (a) (b) (c) OH CH OH HO CH OH O OH HO O OH HO O OH O Vitexin-2˝-O-rhamnoside (d) Figure 1: Te chemical structures of ephedrine (a), gastrodin (b), liquiritin (c), and I.S. (d). Banxia Baizhu Tianma Tang, respectively, represent the TX, USA), and formic acid (St. Louis, MO, USA) are all of main chemical markers for the quality evaluation of PT [13], HPLC grade, and other chemicals and reagents are of an- AM, GE, PC, CR, and GU [14]. Initially, the aim of this study alytical grade. was to investigate the pharmacokinetics of the six chemical compositions in Banxia Baizhu Tianma Tang in rat plasma 2.2. Chromatography and Mass Spectrometry. An UHPLC using the ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry system (1290 series, Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent 6460 Triple Quadrupole Mass (UHPLC-QQQ-MS/MS) method, but only the pharmaco- kinetic studies of ephedrine in PT, gastrodin in GE, and Spectrometer (QQQ-MS, Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ionization (ESI) liquiritin in GU were investigated at last except atractyle- source operated in positive and negative ionization modes nolide, pachymic acid, and hesperidin because only little of them were found in rat plasma. To the best our knowledge, was used to investigate the pharmacokinetic profles of ephedrine, gastrodin, and liquiritin in Banxia Baizhu this is frst report on the pharmacokinetic studies 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 Tianma Tang in rat plasma after intragastric administration in a single dose. 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 2. Materials and Methods 5–35% B from 0 to 5 min, 35%–60 B from 5 to 7 min, 60–90% 2.1. Materials and Chemicals. PT, AM, GE, PC, CR, and GU B from 7 to 9 min, and 90% B from 9 to 11 min. An aliquot of of Banxia Baizhu Tianma Tang were purchased from the 5 μL was injected, and the fow rate was 0.35 mL/min. afliated hospital of Liaoning University of Traditional Te three analytes and I.S. were directly injected into the Chinese Medicine (Shenyang, Liaoning, China) and au- MS system in order to obtain MS information. Te frag- thenticated by Prof. Rongxiang Wang, a senior expert of mentor voltage (FV) and collision energy (CE) parameters of herbal authentication at School of Pharmacy, Liaoning multiple reaction monitoring (MRM) to detect the three University of Traditional Chinese Medicine. Te reference analytes and I.S. were m/z 166.1⟶ 148.1 (FV, 90; CE8) for standards including ephedrine, gastrodin, liquiritin, and ephedrine, m/z 285.1⟶ 123.1 (FV, 100; CE, 4) for gas- vitexin-2″-O-rhamnoside [15] (internal standard, I.S.) were trodin, m/z 417.1⟶ 255.1 (FV, 180; CE, 12) for liquiritin, purchased from the National Institute of Control of Phar- and m/z 579.0⟶ 433.1 (FV200; CE, 16) for I.S. Te other maceutical and Biological Products (Beijing, China). Te parameters were as follows: drying gas (N2) fow rate, 11.0 L/ chemical structures of ephedrine, gastrodin, liquiritin, and min; drying gas temperature, 300 C; nebulizer pressure, 15 I.S. are given in Figure 1. Acetonitrile, methanol (Houston, psig; and capillary voltage, 4000 V. Journal of Analytical Methods in Chemistry 3 2.3. Calibration Standard and Quality Control Samples. 3. Method Validation Stock solutions of calibration standards including ephed- 3.1. Selectivity and Carry-Over. Selectivity was proved by rine, gastrodin, liquiritin, and I.S. were, respectively, pre- comparing MRM chromatograms of blank plasma obtained pared in methanol at concentrations of 2.40, 1.25, 5.24, and from six rats with those of corresponding standard plasma 1.88 mg/mL. Te stock solutions of three analytes and I.S. sample spiked with the three analytes and I.S., and plasma were serially diluted with methanol to be the working sample after administered to the rats via intragastric ad- standard solutions of 25.0, 10.0, 5.0, and 1.0 μg/mL in order ministration. Figure 2 shows that the MRM chromatograms to prepare calibration curves and quality control (QC) of the three analytes and I.S. were free from endogenous working standard solutions. Te fnal efective concentra- matrix interference at their respective retention times. tions of the calibration standards in rat plasma samples Te cross-talk of ephedrine, gastrodin, and liquiritin as made by spiking 10 μL the corresponding working solutions well as I.S. was evaluated by analyzing upper limit of into 100 μL blank plasma were 2.5, 5, 10, 20, 40, 100, and quantifcation (ULOQ) plasma samples without I.S. and 250 ng/mL of ephedrine, 50, 100, 200, 400, 1000, and blank plasma samples only with I.S. Te responses of the 2500 ng/mL of gastrodin, and 5, 10, 20, 40, 100, 250, and analyte and I.S. of the low limit of quantifcation (LLOQ) 500 ng/mL of liquiritin. Similarly, the low, middle, and high- were, respectively, less than 20% and 5%. Carry-over was concentration QC samples were made by the same way at assessed by injecting blank samples after injecting ULOQ. fnal concentrations of ephedrine, gastrodin, liquiritin of 5, Te results indicated that the carry-over in blank samples 40, and 200 ng/mL, 150, 600, and 2000 ng/mL, and 15, 75, following the ULOQ was not greater than 20% of the and 400 ng/mL, respectively. analyte response at the LLOQ and 5% of the response for the I.S. 2.4. Animals and Plasma Sample Preparation. Healthy male SD rats (SCXK2020-0001) (SPF grade, 300± 20 g) were 3.2. Linearity. Te calibration curves over the concentration purchased from Liaoning Changsheng Biotechnology Co. range of 2.5, 5, 10, 20, 40, 100, and 250 ng/mL of ephedrine, Ltd. (Shenyang, Liaoning, China). Te whole animal ex- 50, 100, 200, 400, 1000, and 2500 ng/mL of gastrodin, and 5, periment process was approved by the Committee of 10, 20, 40, 100, 250, and 500 ng/mL of liquiritin were de- Ethics of Animal Experimentation of Liaoning University termined using seven or six standard plasma samples via of TCM and implemented in strict accordance with the plotting the peak area ratio of the three analytes to I.S. versus guiding principles of laboratory animal protection. Rats the nominal concentration of the analyte in rat plasma. Te were kept in environmentally controlled breeding room regression equations of ephedrine, gastrodin, and liquiritin for one week, fed with standard laboratory food as well as were obtained using weighted (1/c ) least squares linear water ad libitum, and fasted overnight before the exper- regression: y � 0.1642x − 0.06984, y � 0.0795x + 0.06003, and iments. Aliquots of 100 μL of plasma sample were spiked y � 0.0051x + 0.20200, with a correlation coefcient (r ) of with 25 μL of I.S. (0.2 μg/mL) and vortex-mixed for 1 min, 0.994, 0.998, and 0.997, respectively, where y is the peak area and then 300 μL of acetonitrile was added to the sample, ratio of the analyte to I.S. and x is the spiked concentration of vortex-mixed for 1 min, and then centrifuged at 3000 rpm the analytes. 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, 3.3. Limits of Detection and Quantifcation. Te limits of vortex-mixed for 1 min, centrifuged at 15,000 rpm for detection (LOD) were determined in signal to noise ratio (S/ 3 min, and then transferred to an autosampler vial. An N) of 3, which were 0.61, 13.7, and 1.36 ng/mL for ephedrine, aliquot of 5 μL was injected into the UHPLC-QQQ-MS/MS gastrodin, liquiritin, respectively. LLOQ was defned as the system. lowest concentration on calibration curve with acceptable precision and accuracy. LLOQs of ephedrine, gastrodin, and liquiritin were 2.5, 50, and 5 ng/mL (S/N> 10) with RSDs of 2.5. Preparation of Banxia Baizhu Tianma Tang Solution. the intra and interday precisions, respectively, below 11.5% PT (9 g), AM (18 g), GE (6 g), PC (6 g), CR (6 g), and GU and 13.4% and accuracies (RE%) between −10.4% and (3 g) in Banxia Baizhu Tianma Tang were, respectively, cut 9.11%. 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 3.4. Accuracy and Precision. Precision and accuracy were combined, fltered with gauze, and concentrated to about evaluated by analyzing the QC samples at low, middle, and 10 mL under reduced pressure, and then some amount of high concentrations of the three analytes. Precision was water was added so that the total volume reached exactly expressed as relative standard deviation (RSD%), and ac- 10 mL that was stored at 4 C before use, and the solution was curacy was expressed as (mean found concentration-nom- analyzed by the UHPLC method to obtain the contents of inal concentration)/(nominal concentration) × 100%. ephedrine, gastrodin, and liquiritin which were 1.699, 0.601, Intraday precision and accuracy were determined by re- and 0.539 mg/mL, respectively. peated analysis of a set of standards on one day (n � 5), while 4 Journal of Analytical Methods in Chemistry ×10 1.03 III 1.02 IV 1.01 II 0.99 0.98 0.97 0.96 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 (a) ×10 6 1 m/z 148.1 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 II 3.9 3.85 II 3.8 3.75 m/z 123.1 3.7 3.65 3.6 3.55 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 III 5 III m/z 255.1 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 1 IV 3.5 IV 2.5 m/z 433.1 1.5 0.5 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 (b) ×10 0.8 I 0.6 m/z 148.1 0.4 0.2 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 II 0.8 II 0.6 m/z 123.1 0.4 0.2 0.5 11.5 22.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 III 0.8 III m/z 255.1 0.6 0.4 0.2 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 ×10 IV 0.8 IV 0.6 m/z 433.1 0.4 0.2 0.5 11.522.53 3.5 4 4.5 5 5.5 6 6.5 7 Counts (%) vs. Acquisition Time (min) (c) Figure 2: Typical MRM chromatograms of blank plasma (a), a blank sample spiked with the three compounds and I.S. (b), and plasma sample after intragastric administration in a single dose (c) (peak I: ephedrine; peak II: gastrodin; peak III: liquiritin; peak IV: I.S.). Journal of Analytical Methods in Chemistry 5 interday precision and accuracy were determined by re- sensitivity in MS detection. Finally, acetonitrile-water peated analysis on three consecutive days (n � 5 series per containing 0.1% formic acid, which provides better ioni- zation and higher sensitivity, was selected for the separation day). Te RSD and RE should be less than 15%, except at the LLOQ where it should not exceed 20% [15]. Te data of of the three analytes and I.S., and the excellent peak shape accuracy and precision for the three analytes from QC and mass spectral response were also obtained. Tree samples are listed in Table 1 which indicated that the present analytes and I.S. were rapidly eluted with total retention method had suitable accuracy and precision [16, 17]; at the times less than 5.0 min and the whole run time was 7.0 min, same time, the RSDs and REs of the QC samples at LLOQ of in which the retention time of ephedrine, gastrodin, I.S., and the three analytes were less than 20% which met the criteria liquiritin was 1.43, 1.64, 4.45, and 4.85 min, respectively. [17]. 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 3.5. Recovery and Matrix Efect. Te recoveries were per- dose are shown in Figure 2. formed by comparing the analytical results of extracted samples at three QC levels with corresponding extracts of 4.2. Pharmacokinetic Application. Banxia Baizhu Tianma blank spiked with the analyte after extraction that repre- Tang solution of 16.6 mL/kg approximately containing sented 100% recovery (n � 6). Te matrix efects were in- vestigated at three QC levels by comparing the peak areas of ephedrine of 28 mg, gastrodin of 10 mg, and liquiritin of 9 mg was administered to the rats via intragastric admin- post-extraction blank plasma spiked with the three analytes by directly injecting the pure standard solutions at the same istration. 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, concentrations in six replicates (n � 6). Te matrix efect 240, 360, 480 and 720 min to heparinized polythene tubes values should be in the range between 85% and 115%. Te and centrifuged at 3000 rpm for 15 min to obtain plasma recovery and matrix efect of the three analytes are listed in samples that were stored at −20 C before analysis. Te Table 1 meaning that the method had no signifcant matrix UHPLC-QQQ-MS/MS method described above was vali- efect and presented high and reproducible recovery. dated successfully and applied to the pharmacokinetic studies of ephedrine, gastrodin, and liquiritin in Banxia 3.6. Stability. Te stabilities of QC samples at three con- Baizhu Tianma Tang. Pharmacokinetic analysis was per- centrations were studied (n � 6). Short-term stability and formed using the DAS 2.0 program (Chinese Pharmacology long-term stability studies were determined at room Society, Beijing, China) to calculate the pharmacokinetic temperature for 4 h and −20 C for 1 month, respectively. parameters with both compartment and non-compartment Freeze-thaw stabilities were evaluated after three freeze approaches. Te mean plasma concentration versus time (−20 C)-thaw (room temperature) cycles. Later, the con- plots of ephedrine, gastrodin, and liquiritin are shown in centrations obtained after samples were processed and Figure 4. analyzed in this experiment were compared with the nominal values of the QC samples. Te stability results are 4.3. Incurred Sample Reanalysis (ISR). Te samples used to listed in Table 1 which demonstrated that the three ana- prepare the calibration curves and QC during method lytes were stable as no signifcant degradation of the three validation were not the actual study samples because the analytes in rat plasma occurred under various experi- diferences of protein binding, sample in homogeneity or mental conditions. concomitant medications existed and metabolites, which can afect the accuracy and precision of the analyte in such 4. Results and Discussion samples during processing and storage. Terefore, incurred 4.1. UHPLC-QQQ-MS/MS Optimization. Te standard so- samples were reanalyzed of study samples in separate runs at lutions of ephedrine, gastrodin, liquiritin, and I.S. were, diferent days to evaluate accuracy of the samples. Te respectively, injected into the mass spectrometry system to samples around the maximum concentration (C ) and max adjust the instrument setting parameters and maximize the elimination phase for ISR were selected, which could ade- responses. ESI was used in order to obtain good sensitivity quate coverage of the entire PK profle. Terefore, 12 and fragmentation. MRM was used for the quantifcation of samples (2 samples in each rat, n = 6) more than 10% of all the three analytes on account of great advantage in selec- the 66, 60 and 78 samples of ephedrine, gastrodin and tivity. Positive ion modes were used to for the quantifcation liquiritin were respectively reanalyzed for ISR. Tables 2–4 of ephedrine and I.S., negative ion modes for that of gas- indicate that the diferences between concentrations ob- trodin and liquiritin, mainly generated protonated mole- tained for the original analysis and the reanalysis were all cules m/z 166.1, 579.0, 285.1 and 417.1 as the precursor ions, within 20% of their mean, meaning that 100% ISR met the respectively. Te product ion mass spectra of positive and criteria [16]. negative ions of the three analytes and I.S. are shown in In the study, pharmacokinetic analysis was performed Figure 3. using the DAS 2.0 program to calculate the pharmaco- Acetonitrile/methanol-water (containing 0.05%, 0.1%, kinetic parameters with both compartment (a) and non- and 0.2% formic acid) being the mobile phase with gradient compartment (b) approaches, a two-compartment open elution was used to evaluate UHPLC separation and model to gastrodin, and one-compartment open model to 6 Journal of Analytical Methods in Chemistry Table 1: Precision, accuracy, recovery, and matrix efect of ephedrine, gastrodin, and liquiritin in rat plasma. Intraday (n � 5) Interday (n � 15) Stability (n � 6) Added C Extraction recovery Matrix efect (%) Analytes Found C (ng/ RSD RE Found C (ng/ RSD RE Short-term Long-term Freeze-thaw (ng/mL) (%) (n � 6) (n � 6) mL) (%) (%) mL) (%) (%) stability (%) stability (%) stability (%) 5 86.88± 1.08 100.4± 3.83 4.57± 0.52 11.5 −8.60 4.48± 0.60 13.4 −10.4 89.23± 2.63 87.43± 5.26 87.15± 3.81 Ephedrine 40 87.47± 2.35 96.40± 2.45 41.3± 2.8 6.84 3.33 37.6± 3.3 8.89 -6.0 91.40± 3.05 90.68± 2.43 90.04± 4.07 200 92.32± 3.69 96.76± 3.82 209.8± 10.3 4.93 4.90 205.6± 8.81 4.28 2.82 91.27± 5.22 91.67± 1.07 90.66± 6.42 150 89.25± 4.07 96.59± 1.26 162.7± 10.2 6.28 8.4 161.3± 18.0 11.1 −7.56 89.29± 3.99 90.11± 3.78 89.88± 2.33 Gastrodin 600 87.98± 5.20 96.72± 3.57 580.3± 20.6 5.15 −3.3 587± 35.8 6.09 −2.12 90.69± 5.33 90.85± 1.04 91.58± 1.27 2000 90.14± 3.22 95.15± 0.78 1989± 66.6 3.35 −0.53 2016± 87.2 4.32 −0.80 91.80± 4.01 93.11± 3.78 93.33± 2.63 15 88.75± 6.08 98.17± 3.69 16.1± 1.7 10.7 7.56 16.4± 1.5 9.33 9.11 89.16± 1.28 89.23± 4.65 88.29± 5.96 Liquiritin 75 90.49± 3.83 97.71± 1.54 70.7± 3.3 4.74 −5.78 76.4± 1.9 2.46 1.82 91.72± 6.47 91.87± 1.93 90.59± 4.22 400 90.81± 1.73 99.87± 7.01 390.8± 15.5 3.97 −2.3 389.5± 15.0 3.86 −2.63 92.13± 1.91 93.33± 5.74 92.70± 2.91 Journal of Analytical Methods in Chemistry 7 ×10 148.1000 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 (a) ×10 123.1000 3.5 2.5 1.5 0.5 122 123 124 125 126 127 128 129 130 131 132 (b) ×10 255.1000 1.2 0.8 0.6 0.4 0.2 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 (c) ×10 433.1000 406 408 410 412 414 416 418 420 422 424 426 428 430 432 434 436 438 440 442 444 446 (d) Figure 3: Te product ion mass spectra of positive and negative ions of ephedrine (a), gastrodin (b), liquiritin (c), and I.S. (d). ephedrine and liquiritin (weight = 1/c ) gave the best ft gastrodin. Moreover, both apparent volume of distribu- a a according to the Akaike Information Criterion (AIC) tion (V ) and clearance (CL ) of ephedrine and liquiritin values, and the main pharmacokinetic parameters of the were larger than those of gastrodin, indicating that three analytes are listed in Table 5. After intragastric ad- ephedrine and liquiritin were mainly distributed in sur- ministration, the maximum concentrations (C ) of rounding tissues leading to lower plasma concentrations. max ephedrine, gastrodin, and liquiritin in plasma were, re- In addition, the peak time (T ) of ephedrine, gastrodin, max spectively, 128.2± 28.8, 1622.7± 657.3, and and liquiritin was 0.917, 0.500, and 0.167 h, respectively, 244.7± 64.2 ng/mL, along with the corresponding values of suggesting that the three analytes were all rapidly the area under the concentration-time curves (AUC ) absorbed, in which the liquiritin was the fastest one. Te 0⟶t of the three analytes were 262.2± 42.2, 2483.5± 1194.0, terminal half-life (t ) 2.639, 1.278, and 2.176 h, indicated 1/2 and 230.3± 135.5 ng h/mL, meaning that ephedrine and that the eliminations of them were slow. Furthermore, it liquiritin had a little weak absorption compared with was noteworthy that ephedrine and liquiritin could be 8 Journal of Analytical Methods in Chemistry 160 2500 0 0 0 2841610124 01 25 3 4 Time (h) Time (h) Ephedrine Gastrodin (a) (b) 0 2468 10 12 14 Time (h) Liquiritin (c) Figure 4: Te mean plasma concentration-time curves of ephedrine (a), gastrodin (b), and liquiritin (c) (n � 6). Table 2: Incurred sample reanalysis data of ephedrine. Subject no. Sampling point (min) Original concentration (ng/mL) Incurred sample concentration (ng/mL) Mean (ng/mL) RE (%) 1 30 1.16 1.28 1.22 9.8 1 180 0.401 0.432 0.417 7.4 2 30 1.33 1.49 1.41 11.3 2 180 0.337 0.327 0.332 −3.0 3 30 2.38 2.65 2.52 10.7 3 180 0.730 0.746 0.738 2.2 4 30 1.02 1.12 1.07 9.3 4 180 0.301 0.345 0.323 13.6 5 30 1.58 1.47 1.53 −7.2 5 180 0.426 0.478 0.452 11.5 6 30 1.69 1.81 1.75 6.9 6 180 0.432 0.444 0.438 2.7 detected up to 720 min, gastrodin up to 240 min after chemical compositions can remain a longer time in body dosed, meaning that they exert a longer lasting efect in and exert a lasting therapeutic efect and increase the vivo in rats, and the advantage of TCM was that the clinical efcacy. Concentration (ng/mL) Concentration (ng/mL) Concentration (ng/mL) Journal of Analytical Methods in Chemistry 9 Table 3: Incurred sample reanalysis data of gastrodin. Subject no. Sampling point (min) Original concentration (ng/mL) Incurred sample concentration (ng/mL) Mean (ng/mL) RE (%) 1 30 1333.3 1351.6 1397.5 1.3 1 120 532.1 538.3 535.2 11.6 2 30 1159.7 1187.5 1173.6 2.4 2 120 371.8 410.9 391.4 10.0 3 30 2375 2422 2398.5 2.0 3 120 852.6 832.5 842.6 −2.4 4 30 1048.1 1008.1 528.1 7.6 4 120 412.8 419.5 416.2 1.6 5 30 1356.3 1387.0 1371.7 2.2 5 120 392.5 412.5 402.5 5.0 6 30 1275 1289 1282 1.1 6 120 367.3 371.4 369.4 1.1 Table 4: Incurred sample reanalysis data of liquiritin. Subject no. Sampling point (min) Original concentration (ng/mL) Incurred sample concentration (ng/mL) Mean (ng/mL) RE (%) 1 10 220.1 225.8 223.0 2.6 1 180 64.86 71.2 68.0 9.3 2 10 196.4 216.5 206.5 9.7 2 180 51.7 52.9 52.3 2.3 3 10 317.5 345.8 331.7 8.5 3 180 91.2 82.6 86.9 9.9 4 10 188.5 177.4 183.0 −6.1 4 180 44.39 52.2 48.3 16.2 5 10 154.7 161.9 158.3 4.5 5 180 71.9 84.7 78.3 16.3 6 10 245.4 235.3 235.4 −4.3 6 180 61.2 69.8 65.5 13.1 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, re- spectively (mean± SD, n � 6). Parameters Analytes b b b a a a a T (h) C (ng/mL) t (h) V (L/kg) CL (L h/kg) AUC (ng/mL) h AUC (ng/mL) h max max 1/2 d 0⟶t 0⟶∞ Ephedrine 0.917± 0.144 128.2± 28.8 2.639± 0.574 284.04± 52.7 106.6± 18.9 262.2± 42.2 267.81± 43.29 Gastrodin 0.5± 0.0 1622.7± 657.3 1.278± 0.48 4.904± 3.171 4.057± 2.146 2483.5± 1194.0 3012.89± 1630.6 Liquiritin 0.167± 0.0 244.7± 64.2 2.176± 0.332 3436.2± 1485.4 34.4± 14.9 230.3± 135.5 314.0± 180.8 a and b are the compartmental and non-compartmental approaches, respectively. and further studies will be conducted on other components 5. Conclusions in this formula in the future. A developed and validated UHPLC-QQQ-MS/MS method was successfully applied to the pharmacokinetic studies of Data Availability ephedrine, gastrodin, and liquiritin in rat plasma. Tree components in Banxia Baizhu Tianma Tang are simulta- Te data used to support the fndings of this study are in- neously determined in rat plasma after intragastric ad- cluded within the article. ministration for the frst time. Tis experiment provides a pharmacokinetic basis for Banxia Baizhu Tianma Tang in Conflicts of Interest clinical aspects and can make Banxia Baizhu Tianma Tang better applied in clinical treatment. Although the pharma- Te authors declare that they have no conficts of interest. cokinetic study of traditional Chinese medicine is very difcult due to its very low content of chemical components Acknowledgments in each single herb, ephedrine, gastrodin, and liquiritin which are representative of the herbs of PT, GE, and GU in Tis study was supported by the Project of the National Banxia Baizhu Tianma Tang can be determined in the study, Natural Science Foundation of China (grant no. 81974548) 10 Journal of Analytical Methods in Chemistry [14] Te Pharmacopoeia Commission of Prc, Pharmacopoeia of the Liaoning Provincial Department of Education 2020 Scien- People’s Republic of China, Chinese Medical Science and tifc Research Project (Grant L202016). Technology Press, Beijing, China, 2020. [15] X. X. Ying, X. M. Lu, X. H. Sun, X. Q. Li, and F. M. Li, References “Determination of vitexin-2-O-rhamnoside in rat plasma by ultra-performance liquid chromatography electrospray ioni- [1] C. S. 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