Journal of Chromatography B, 967 (2014) 255–263

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Simultaneous determination of seven bufadienolides in rat plasma after oral administration of Shexiang Baoxin Pill by liquid chromatography–electrospray ionization–tandem mass spectrometry: Application to a pharmacokinetic study Shuping Wang a,b,1 , Chengcheng Peng c,1 , Peng Jiang a , Peng Fu d , Jianfei Tao e , Lin Han a , Huimei Huang a , Wanlin Chang a , Le Li f , Weidong Zhang a,c,∗ , Runhui Liu a,∗ a

School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China Department of Pharmacy, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200001, PR China c School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China d Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai 200433, PR China e Pharmacy Department, Shanghai Yangsi Hosipital, Shanghai 200126, PR China f School of Chemistry and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, PR China b

a r t i c l e

i n f o

Article history: Received 20 April 2014 Accepted 24 July 2014 Available online 4 August 2014 Keywords: Bufadienolides Shexiang Baoxin Pill LC–ESI–MS/MS Pharmacokinetic study

a b s t r a c t A liquid chromatography–electrospray ionization–tandem mass spectrometry method was described for the simultaneous determination of resibufogenin, bufalin, gamabufotalin, telibufagin, arenobufagin, cinobufagin and bufotalin in rat plasma. Plasma samples were pretreated by liquid–liquid extraction with ethyl acetate. Chromatographic separation was carried out on an ACQUITY HSS T3 column with gradient elution using mobile phase consisting of acetonitrile–0.1% formic acid in water at a flow rate of 0.3 mL/min. All analytes showed good linearity over a wide concentration range (r > 0.99). The lower limit of quantification was in the range of 0.5–10 ng/mL for seven bufadienolides. The mean recovery of the analytes ranged from 94.36 to 104.18%. The intra- and inter-day precisions were in the range of 1.74–13.78% and the accuracies were between 89.37 and 101.38%. The validated method was successfully applied to a pharmacokinetic (PK) study of the seven bufadienolides in rat plasma after oral administration of Shexiang Baoxin Pill (SBP). The selected PK marker compounds with typical efficacy/toxicity may provide a practical solution for marker compound selection and dosage design for the therapeutic drug monitoring and PK study of SBP in its clinical applications. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Generally, pharmacokinetic (PK) study does provide scientific evidence on the efficacy and safety of traditional Chinese medicine (TCM). Because of the unavoidable limitations such as complexity of chemical composition, uncharacterized active constituents, differential disposition process and complex workings of this ancient medical system, the marker compounds selection and dosage

∗ Corresponding authors at: School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433, PR China. Tel.: +86 21 81871244; fax: +86 21 81871245. E-mail addresses: [email protected] (W. Zhang), [email protected] (R. Liu). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.jchromb.2014.07.038 1570-0232/© 2014 Elsevier B.V. All rights reserved.

regimens explanation for PK evaluation of TCM are still challenging task. Shexiang Baoxin Pill (SBP), an important and widely used Chinese patent medicine in treating coronary heart disease (CHD), consists of seven medicinal materials, including Moschus, Radix Ginseng, Calculus Bovis, Cortex cinnamomi, Styrax, Venenum Bufonis and Borneolum Syntheticum. It has been proved to be very safe and effective in the treatment of CHD with less harmful side effects [1–3]. Previous pharmacological studies demonstrate that SBP can regulate blood lipid, protect endothelial cell, inhibit the inflammation of vascular wall, stabilize the vulnerable plaques and promote angiogenesis [4–9]. Although considerable progress has been made in demonstrating the clinical effect of this prescription over the past decades, the PK evaluation of SBP was rare. The real-life challenge is not only the complexity and diversity of the chemical components, but also the trace concentration of them in vivo.

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Therefore, simultaneous determination of a broad range of components with main efficacy and toxicity would provide a simplified solution for elaborating the PK property of complex TCM in a rational manner to rapidly achieve optimal efficacy and safety of medication. As the most characteristic components of SBP, Venenum Bufonis performs as a cardiotonic, bloodpressure-stimulating, diuretic and anodyne agent in the complex prescription [10–14]. It is known that the main component exhibiting cardiotonic activity is bufadienolides, a class of cardioactive C-24 steroids with a characteristic ␣-pyrone ring at C-17, which exhibit the main efficacy and toxicity with their narrow toxic/therapeutic margins [15–17]. Therefore, an integral investigation into the PK evaluation of the bufadienolides in SBP could not only link data from pharmacological assays to clinical efficacy/toxicity, but also help exploring rational dosage regimens. Various analytical methods based on liquid chromatography technology have been developed for the determination of the bufadienolides in biological fluids [18–21]. However, high limit of quantification, long analysis time, lack of commercial assay kit, the costliness of the instrument and strict requirement for sample pretreatment or could not fulfill oral PK study due to the insufficient sensitivity and specificity, though pronounced. Recently, Xu et al. developed liquid chromatography–tandem mass spectrometry method for simultaneous determination five bufadienolides in plasma after Venenum Bufonis orally administrated to rats [22]. Although it was sensitive enough, the PK results of these components was derived from the single medicinal material, while investigation on the single-entity drugs could not represent the principle of TCM prescription. On the basis of Chinese medical philosophy and practice, the raw medicinal materials are prescribed combination to obtain synergistic effects or diminish potential adverse reactions. Considering the efficacy/toxicity and the discipline of compatibility of TCM prescription, to profile the PK properties of the active ingredients of Venenum Bufonis in SBP would substantially aid clarifying the active mechanism of the whole prescription. In this paper, a liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) method was developed and validated for the simultaneous determination of seven bufadienolides including resibufogenin, bufalin, cinobufagin, gamabufotalin, telibufagin, arenobufagin and bufotalin in rat plasma. The marker compound selection and dosage design for PK evaluation of SBP were investigated as well.

2. Experimental

2.2. Determination of seven bufadienolides in SBP The contents of resibufogenin, bufalin, cinobufagin, gamabufotalin, telibufagin, arenobufagin and bufotalin in SBP were quantitatively determined to calculate the administration dosage to rats. SBP were powdered and sieved through a No. 40-mesh sieve to get a homogeneous size. Ten milligrams was accurately weighed into a 10 mL volumetric flask. A mixed solution of methanol–dichloromethane–water (8:4:1, v/v) was added and ultrasonic extracted for 20 min, the lost volume was compensated by adding the same solvent when cooled at room temperature, and then centrifuged at 13,500 × g for 10 min, the supernatant was filtered through a syringe filter (0.22 ␮m). Five microliters of filtrate was injected into LC–MS/MS for quantitative analysis with linear calibration curve model. The LC–MS/MS conditions of this part were the same conditions applied for determination of the seven compounds in rat plasma addressed in part 2.4 and part 2.5. The contents of resibufogenin, bufalin, gamabufotalin, telibufagin, arenobufagin, cinobufagin, and bufotalin were determined as 1529.17, 2240.69, 272.44, 44.88, 277.65, 277.55, and 102.83 ␮g/g, respectively. 2.3. Calibration standards and quality control samples Stock solution of resibufogenin, bufalin, gamabufotalin, telibufagin, arenobufagin, cinobufagin, and bufotalin was separately prepared in methanol at a high concentration (200 ␮g/mL). Appropriate volume of the above stock solution was mixed and then diluted with methanol to obtain a series of working standard solutions. Calibration work solutions were prepared by adding the diluted working standard solutions into blank rat plasma (5/95, v/v), final concentration series ranged from 5 to 2500, 10 to 5000, 5 to 2500, 0.5 to 250, 2 to 1000, 0.5 to 250, and 0.5 to 250 ng/mL for resibufogenin, bufalin, gamabufotalin, telibufagin, arenobufagin, cinobufagin, and bufotalin were obtained, respectively. The IS stock solution was prepared in methanol with a concentration of 1 ␮g/mL, and the working solution (10 ng/mL) was obtained by diluting the stock solution with ethyl acetate at a ratio of 1:100 (v/v). Quality control (QC) samples include low QC (LQC), middle QC (MQC), and high QC (HQC). 10, 100, and 1000 ng/mL for resibufogenin and gamabufotalin, 20, 200, and 2000 ng/mL for bufalin, 1, 10, and 100 ng/mL for telibufagin, cinobufagin, and bufotalin, 5, 50, and 500 ng/mL for arenobufagin were also prepared in the same manner. Calibration work solutions and QC samples were stored at 4 ◦ C until LC–ESI–MS/MS analysis.

2.1. Materials and reagents 2.4. LC–ESI–MS/MS analysis Reference standards of bufalin (molecular weight, hereinafter MW, MW: 386.5), cinobufagin (MW: 442.5), resibufogenin (MW: 384.5) and tinidazole (internal standard, IS, MW: 247.3) were purchased from the National Institute for Food and Drug Control (Beijing, China); arenobufagin (MW: 416.5), bufotalin (MW: 444.6), gamabufotalin (MW: 402.5) and telibufagin (MW: 402.5) were obtained from Shanghai Sunny Biotech Co., Ltd (Shanghai, China). Their chemical structures are shown in Fig. 1. SBP samples (batch NO.: 120716) were kindly offered by Shanghai Hutchison Pharmaceuticals Company (Shanghai, China). Acetonitrile was of HPLC grade from Merck (Darmstadt, Germany); formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA); aqueous solutions were prepared with ultrapure water produced from a Milli-Q50 SP Reagent Water System (Bedford, MA, USA). Other reagents were of analytical grade or higher if not otherwise stated.

LC–ESI–MS/MS analysis was carried out on an Agilent 1200 Series liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent 6410B triple quadrupole mass spectrometer (USA) with an electrospray ionization (ESI) source. Chromatographic separations were achieved on an ACQUITY HSS T3 column (100 mm × 2.1 mm, 1.8 ␮m, Waters, MA, Ireland). Analytical column was maintained at 35 ◦ C and eluted with a mobile phase consisting of (A) acetonitrile and (B) water containing 0.1% formic acid using the following gradient program: 20% A→50% A at 0–1.0 min; 50% A at 1.0–2.5 min; 50% A→80% A at 2.5–4.0 min; 80% A at 4.0–6.5 min; 80% A→20% A at 6.5–7.0 min; 20% A at 7.0–12.0 min at a flow rate of 0.3 mL/min. The total run time was 12 min with a 2 min equilibration time. The effluent from the LC column was directed from the waste to the mass spectrometer source after the first 1.0 min of each run.

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Fig. 1. Chemical structures of the analytes and IS.

Analytical signal was acquired in positive ion multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The conditions of MS analysis were designed as follows: capillary voltage, 3500 V; nebulizer pressure, 40 psi; drying gas (N2 ),10 L/min with a temperature of 350 ◦ C; collision gas (N2 ); dwell time, 100 ms; mass analyzers Q1 and Q3 operated at unit mass resolution were used for each MRM transition. Compounddependent parameters are listed in Table 1. Data collection, peak integration, and calculations were performed using Masshunter Workstation Software from Agilent Technologies (version B.03.01) (Palo Alto, CA, USA). 2.5. Sample preparation One hundred microliters aliquot of the plasma sample was extracted with 1 mL ethyl acetate containing the IS (10 ng/mL, w/v) by vortex-mixing for 10.0 min. Then the mixture was centrifuged at 13,500 × g for 10 min, the upper organic layer was transferred to another tube and evaporated to dryness at 37 ◦ C under a gentle stream of nitrogen. The residue was reconstituted in 100 ␮L methanol–water (50:50, v/v) followed by vortex-mixing for 10 min and centrifugation at 13,500 × g for 10 min. Then, 10 ␮L aliquot of supernatant was injected to the LC–MS/MS system.

2.6. Method validation The method was validated for selectivity, linearity, accuracy and precision, extraction recovery, matrix effect, and stability following the industrial guidelines for bioanalytical method validation from the U.S. Food and Drug Administration (FDA) [23]. 2.6.1. Selectivity The chromatographic interference from endogenous plasma matrix components was estimated by comparing the response of blank plasma from six sources with blank plasma spiked with the response of analytes at the lower limit of quantification (LLOQ), and the interfering peak area should be less than 20% of the peak area for the analytes at LLOQ in rat plasma. 2.6.2. Linearity and LLOQ The linearity of the method was generated by analysis of calibration curves containing eight non-zero concentrations in duplicate in three separate runs. Each calibration curve was analyzed individually by fitting the area ratio response for analyte/IS as a function of standard concentration, using least-square weighted (1/x2 ) linear regression, and excluding the point of origin. The LLOQ was defined as the lowest concentration producing a signal-to-noise (S/N) ratio

Table 1 MS/MS transitions and parameters for the detection of the analytes and IS. Analyte

Rt (min)

MRM transitions (m/z)

Collision energy (V)

Fragmentor (V)

Dwell time (ms)

12 20

110

100

Tinidazole(IS)

2.66

248.0–121.0 248.0–128.0

Resibufogenin

7.29

385.2–107.0 385.2–105.0

36 50

140

100

Bufalin

6.17

387.3–107.0 387.3–105.0

36 50

150

100

Gamabufotalin

4.67

403.3–107.1 403.3–145.0

40 36

150

100

Telibufagin

5.97

403.4–105.0 403.4–349.2

50 20

150

100

Arenobufagin

5.22

417.2–399.4 417.2–105.0

24 50

180

100

Cinobufagin

7.32

443.2–105.0 443.2–187.0

50 24

150

100

Bufotalin

6.15

445.3–349.3 445.3–367.3

16 12

150

100

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larger than 5 with a relative standard deviation (RSD) below 20% and deviations from the nominal concentration within ±20% by six replicate analysis. 2.6.3. Accuracy and precision The intra-day accuracy and precision were determined by analysis of QC samples at low, middle, and high concentrations levels from five replicates on the same day, while the inter-day accuracy and precision were evaluated on three independent days. Accuracy was determined by calculating the percentage bias from the nominal concentration. Precision was assessed by calculating the RSD for each replicates. Values below ±15% and at LLOQ below ±20% were accepted.

2.8. Statistical analysis Pharmacokinetic parameters including observed peak concentration (Cmax ), time to reach peak concentration (Tmax ) obtained directly, elimination half-life (T1/2 ), mean residence time (MRT), area under the plasma concentration–time curve to the last measureable plasma concentration (AUC0–t ), and area under the plasma concentration–time curve to time infinity (AUC0–INF ) were calculated by WinNonlin 5.2 from Pharsight Corporation (Sunnyvale, CA, USA) based on non-compartmental analysis of plasma concentration versus time data. All data were presented as mean ±SD. 3. Results and discussion

2.6.4. Extraction recovery and matrix effect The extraction recovery and matrix effects were conducted by analyzing three sets of samples [24]: Set 1 (neat standards), Set 2 (pre-extracted matrix samples), and Set 3 (post-extracted matrix samples), three QC levels with five replicates for each set. Extraction recovery was calculated by comparing the mean area response of Set 2 to that of Set 3 at each QC level. As per the acceptance criteria, recovery should be consistent and reproducible. Matrix effect was determined by comparing the mean area response of Set 3 with mean area of Set 1. The value of matrix effect less than 85% represented ionization suppression, while more than 115% represented ionization enhancement. It was considered negligible if no more than a 15% difference was observed. 2.6.5. Stability The stability of the analytes in rat plasma was evaluated by analysis of five replicate QC samples at three concentration levels for short-term stability, long-term stability, auto-sampler stability, and freeze–thaw stability. The independent unprocessed QC samples were, respectively, subjected to placing at room temperature for 4 h for short-term stability, stored at -20 ◦ C for 4 weeks for long-term stability and three cycles of freezing at -20 ◦ C and thawing for freeze and thaw stability. The auto-sampler stability was assessed by analyzing the pretreated samples after being left in the auto-sampler at room temperature for 12 h. The measured concentrations subjected to four treatments were compared with those of freshly prepared QC samples and the percentage concentration deviation was calculated to evaluate stability. 2.7. PK study in rats Male Sprague–Dawley (SD) rats (body weight: 180–220 g) were supplied by the Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). The animals were maintained in controlled conditions (temperature 20–25 ◦ C, relative humidity 55–60%, and 12 h dark–light cycle) with free access to standard laboratory food and water for 5 days acclimation, and then fasted the night before (12 h prior to the experiment), and the time of recovery of feeding was 4 h post-dose. Six rats were intragastric administered SBP suspended in 0.5% carboxymethyl cellulose sodium aqueous solution at 4 g/kg. Blood samples (about 250 ␮L) were collected in heparin pretreated polypropylene tubes via the postorbital venous plexus veins from each rat before dosing (0 min) and at 5 min, 10 min, 20 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after dosing. All blood samples were immediately centrifuged and obtained plasma stored frozen in a freezer set to maintain −20 ◦ C until bioanalysis. The plasma collected from six vehicle-administered rats served as the blank. Animal experiments were carried out according to the Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of the Second Military Medical University.

3.1. Method development To develop a sensitive and reliable LC–ESI–MS/MS method, it was important to optimize the chromatographic and mass spectrometric conditions, as well as to obtain an efficient and simple extraction procedure for all the analytes and IS. 3.1.1. Selection of IS In this study, tinidazole was chosen as the IS. Tinidazole is a stable chemical raw material that is widely used and commercially available, which displayed a similar solubility to solvent extraction and adsorbent function to the column, compared with those of the analytes. Although tinidazole does not contain the similar chemical structure with bufadienolides, it exhibited a good response and good peak to compensate for variable extraction yields and to reduce possible effects of ion suppression/enhancement in the MS source. Moreover, tinidazole was stable during the period of pretreatment and assaying of the plasma samples and absent in SBP. 3.1.2. Optimization of mass spectrometric conditions In order to achieve the maximum response of each analyte, the mass spectrometric conditions were fine optimized by infusing 200 ng/mL standard solutions, respectively, from the following aspects: acquisition ion modes, the precursor ions, fragmentor energy, product ions, and collision energy. In the full-scan analytical signal, the ionization of all the analytes and IS gave more relative intense signals in positive ion mode than in negative ion mode. The MS/MS product ion spectra of the analytes and IS are shown in Fig. 2. To get the richest relative abundance of precursor and product ions, the parameters for fragmentor energy and collision energy were further optimized. Table 1 shows the optimized MS/MS transitions and energy parameters of all the analytes. Meanwhile, maximum abundance of the molecular ions of the analytes, acquisition parameters (source temperature and ions spray voltage) were investigated and the highest ion intensity for molecular ion of the analytes were achieved when the source temperature, the nebulizing gas (N2 ) pressure, the drying gas flow were set at 350 ◦ C, 40 psi and 10 L/min, respectively. 3.1.3. Optimization of liquid chromatographic conditions The optimization of liquid chromatographic conditions was mainly conducted from the following aspects: stationary phase, the composition and pH value of mobile phase, column temperature, and flow rate, which were considered to be major factors on the separation. Considering the physicochemical property of compounds investigated, two types of reversed-phase chromatographic columns including Zorbax Eclipse Plus C18 column (100 mm × 3.0 mm I.D., 1.8 ␮m, Agilent) and ACQUITY HSS T3 column (100 × 2.1 mm I.D., 1.8 ␮m, Waters) were investigated. ACQUITY HSS T3 column was chosen for its higher efficiency

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259

Fig. 2. Product ion mass spectra of the analytes and IS.

and improving the peak symmetry in high column temperature, especially for the separation of isomeric compounds such as gamabufotalin and telibufagin, which could not be baseline separated by conventional reverse-phase C18 column. After the optimization of the gradient of mobile phase, baseline separation was ultimately achieved, and they were attributed unambiguously by comparison of the retention behavior of the reference standards. Different mobile phase (methanol–water, acetonitrile–water, methanol–acid aqueous solution and acetonitrile–acid aqueous solution) was examined to obtain efficient chromatography and appropriate ionization. Acetonitrile–water gave the most satisfactory separation. Besides, addition of small amount of acid, such as 0.1% (v/v) formic acid in mobile phase could dramatically enhance the abundance of [M + H]+ ions and the resolution of analytes. Other factors affecting the separation were also investigated, such as column temperature and flow rate. At last, the best separation was obtained when the column temperature was kept at 35 ◦ C using a flow rate of 0.3 mL/min. 3.1.4. Optimization of sample pre-treatment Several sample preparation methods were evaluated in the present study, including protein precipitation (PPT) with acetonitrile, PPT with methanol, liquid–liquid extraction (LLE) with ethyl acetate, and solid-phase extraction (SPE) with Waters Oasis HLB cartridges (Milford, MA, USA). SPE was proved to be tedious and limited extraction efficiency due to the complex manual pre-treatment steps. Although PPT showed consistent extraction recovery, the significant matrix effect of this method led to too much deviation of quantitative results. LLE gave all the analytes the suitable recovery and little endogenous interference, which was in consistent with previous publications [19,21,22]. Therefore, taking into consideration of matrix effect and extraction recovery

together, LLE with ethyl acetate was selected as a promising and feasible approach for the extraction of bufadienolides from rat plasma. 3.2. Method validation 3.2.1. Selectivity In the present study, the chromatograms of the blank plasma, spiked plasma sample with all analytes and the IS, and the plasma samples of oral administrated rat are shown in Fig. 3. The mass transition ion-pair was followed as 385.2→105.0 for resibufogenin, m/z 387.3→107.0 for bufalin, m/z 403.3→107.1 for gamabufotalin, m/z 403.4→349.2 for telibufagin, m/z 417.2→399.4 for arenobufagin, m/z 443.2→105.0 for cinobufagin, m/z 445.3→349.3 for bufotalin, and m/z 248.0→121.0 for IS, while the retention time was 7.29, 6.17, 4.67, 5.97, 5.22, 7.32, 6.15, and 2.66 min for each number, respectively. Blank plasma samples from six sources were screened and found to be free of interference from endogenous components or other sources at the same mass transitions and retention times as the analytes and IS, which indicated that the developed method was selective. 3.2.2. Linearity and LLOQ The linear ranges, regression equations, correlation coefficients, and LLOQs of all analytes are listed in Table 2. All the calibration curves exhibited good linearity with correlation coefficients above 0.99. The LLOQ was 5 ng/mL for resibufogenin and gamabufotalin, 10 ng/mL for bufalin, 0.5 ng/mL for telibufagin, cinobufagin, and bufotalin, and 2 ng/mL for arenobufagin, allowing sufficient for PK study of the analytes following oral administration of SBP to rats.

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Fig. 3. Representative MRM chromatograms of the analytes and IS: (A) blank plasma sample, (B) blank plasma sample spiked with the analytes at LLOQ and IS, and (C) plasma sample of oral administrated rat at time point of 1 h post-dose.

3.2.3. Precision and accuracy The intra-day and inter-day precisions and accuracies of the QC samples are presented in Table 3. The intra- and inter-day precisions were in the range of 1.74–13.78% and the accuracies were between 89.37 and 101.38%. All the assay values were within the acceptable criteria, demonstrating an accurate, reliable, and reproducible method was established for the determination of all the analytes in rat plasma. 3.2.4. Extraction recovery and matrix effect As shown in Table 4, the recovery ranged from 94.36 to 104.18%, which indicated that the overall extraction recovery of LLE with ethyl acetate was efficient, consistent, and reproducible. The matrix effect of the assay was no less than 90.55% at the three QC levels. No significant matrix effect for all the analytes was observed,

which indicated that no co-eluting substance could influence the ionization of the analytes. 3.2.5. Stability The stability of all the analytes under various conditions is presented in Table 5. The consistent results indicated that these analytes were all stable in plasma at room temperature for 4 h, after three freeze–thaw cycles, at −20 ◦ C for 4 weeks, and in the autosampler at ambient temperature for 12 h after LLE with the values in the range of 85.09–101.86% (RSD was less than 15%). 3.3. Sample analysis and PK studies The plasma concentrations of resibufogenin, bufalin, gamabufotalin, telibufagin, arenobufagin, cinobufagin, and bufotalin

Table 2 Regression data and LLOQ of the analytes. Analyte

Regression equation (n = 6)

r (n = 6)

Linear range (ng/mL)

LLOQ (ng/mL)

Resibufogenin Bufalin Gamabufotalin Telibufagin Arenobufagin Cinobufagin Bufotalin

y = 0.5089x + 1.6753 y = 0.5946x + 1.7911 y = 0.1686x + 0.5155 y = 1.9893x + 0.6458 y = 0.1196x + 0.0665 y = 1.3450x + 0.2207 y = 0.9304x + 0.1117

0.9952 0.9984 0.9975 0.9968 0.9991 0.9992 0.9996

5–2500 10–5000 5–2500 0.5–250 2–1000 0.5–250 0.5–250

5 10 5 0.5 2 0.5 0.5

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Table 3 Precision and accuracy for the analytes in rat plasma (n = 15, 5 replicates per day for 3 days). Analyte

Nominal conc. (ng/mL)

Intra-day

Inter-day

Conc. found (ng/mL)

Accuracy (%)

RSD (%)

Conc. found (ng/mL)

Accuracy (%)

RSD (%)

Mean

SD

Mean

SD

Resibufogenin

10 100 1000

9.62 93.66 959.67

0.45 1.63 30.34

96.24 93.66 95.97

4.68 1.74 3.16

9.19 96.69 1000.58

0.31 5.02 41.83

91.90 96.69 100.06

3.37 5.19 4.18

Bufalin

20 200 2000

19.88 193.66 1914.89

1.11 7.65 33.72

99.40 96.83 95.74

5.58 3.95 1.76

19.44 190.29 1913.10

1.31 10.81 81.81

97.20 95.15 95.66

6.74 5.68 4.28

Gamabufotalin

10 100 1000

9.44 95.42 976.10

0.34 1.86 33.87

94.40 95.42 97.61

3.60 1.95 3.47

9.33 94.58 961.25

0.45 2.14 47.97

93.30 94.58 96.13

4.82 2.26 4.99

Telibufagin

1 10 100

0.95 9.40 93.38

0.06 0.23 5.39

95.00 94.00 93.38

6.32 2.45 5.77

0.96 9.28 93.62

0.04 0.48 3.76

96.00 92.80 93.62

4.17 5.17 4.02

Arenobufagin

5 50 500

4.70 48.72 477.53

0.25 2.52 19.09

94.00 97.44 95.51

5.32 5.17 4.00

4.65 47.78 471.32

0.22 2.36 20.12

93.00 95.56 94.26

4.73 4.94 4.27

Cinobufagin

1 10 100

0.93 8.94 93.50

0.06 0.71 4.13

93.00 89.40 93.50

6.45 7.94 4.42

0.94 9.14 90.50

0.09 0.60 3.26

94.00 91.40 90.50

9.57 6.56 3.60

Bufotalin

1 10 100

1.01 9.87 100.58

0.14 0.91 7.77

101.00 98.70 100.58

13.86 9.22 7.73

0.98 10.10 97.64

0.12 0.86 4.45

98.00 101.00 97.64

12.24 8.51 4.56

were determined by LC–ESI–MS/MS method after oral administration of SBP extract (4 g/kg) (resibufogenin 6.12 mg/kg, bufalin 8.96 mg/kg, gamabufotalin 1.09 mg/kg, telibufagin 0.18 mg/kg, arenobufagin 1.11 mg/kg, cinobufagin 1.11 mg/kg, and bufotalin 0.41 mg/kg) to rats. PK parameters were estimated by using the non-compartmental model. The mean plasma concentration–time profiles of all analytes are presented in Fig. 4, and the PK parameters of them are listed in Table 6. The mean maximum concentrations (Cmax ) for the seven bufadienolides ranged from 1.08 to 155.42 ng/mL, and all observed range from approximately 0.42 to 1.33 h post-dose (Tmax ). The results indicated that the absorption of arenobufagin, cinobufagin, and bufotalin might be rapid (Tmax less

than 1 h), which was in accordance with previously published data [19,20,22]. 3.4. Marker compound selection and dosage design Marker compound selection has always been the bottleneck in PK study due to the diversity of the components in TCMs and the complexity of their action of mechanism. Ideally, the rational marker compound for PK study of TCMs should be strongly correlated to their safety and efficacy. As suggested by the European Medicines Agency and related references, some categories of constituents are defined as marker compounds including

Table 4 Matrix effect and extraction recovery for the analytes in rat plasma (n = 5). Analyte

Nominal conc. (ng/mL)

Matrix effect

Extraction recovery

Mean

SD

RSD (%)

Resibufogenin

10 100 1000

95.65 99.61 101.84

3.40 6.51 4.25

Mean

3.55 6.54 4.17

100.33 104.18 102.97

Bufalin

20 200 2000

96.79 97.14 99.71

10.52 3.79 4.96

Gamabufotalin

10 100 1000

95.71 103.33 98.48

Telibufagin

1 10 100

Arenobufagin

SD 2.32 4.97 4.48

RSD (%) 2.31 4.77 4.35

10.87 3.90 4.97

99.08 97.47 97.46

10.13 6.53 4.15

10.22 6.70 4.26

3.37 4.02 5.19

3.52 3.89 5.27

98.24 96.28 95.98

0.90 2.30 1.97

0.92 2.39 2.05

98.24 102.26 98.37

4.61 2.94 3.64

4.69 2.88 3.70

100.24 94.88 101.15

2.06 6.65 7.18

2.06 7.01 7.10

5 50 500

98.76 97.43 102.44

1.43 4.37 7.40

1.45 4.49 7.22

96.97 97.00 98.35

1.77 1.31 5.17

1.83 1.35 5.26

Cinobufagin

1 10 100

95.77 92.89 100.49

2.48 5.40 3.85

2.59 5.81 3.83

99.69 101.98 98.08

4.40 5.12 4.08

4.41 5.02 4.16

Bufotalin

1 10 100

101.87 100.87 90.55

9.63 11.87 5.52

9.45 11.77 6.10

103.22 94.36 98.57

5.35 4.43 8.37

5.18 4.69 8.49

262

S. Wang et al. / J. Chromatogr. B 967 (2014) 255–263

Table 5 Stability of the analytes in rat plasma (n = 5). Analyte

Nominal conc. (ng/mL)

Short-term stability

Long-term stability

Conc. found (%)

Conc. found (%)

RSD (%)

Mean

RSD (%)

SD

Autosampler stability

Freeze–thaw stability

Conc. found (%)

Conc. found (%)

Mean

RSD (%)

SD

Mean

RSD (%)

Mean

SD

Resibufogenin

10 100 1000

93.78 97.61 86.25

6.14 5.45 6.22

6.55 5.58 7.21

96.11 98.76 85.09

7.64 4.55 4.29

7.95 4.61 5.04

93.92 94.75 87.34

4.43 8.15 2.24

4.72 8.60 2.56

94.51 95.71 87.01

SD 7.15 5.98 1.81

7.57 6.25 2.08

Bufalin

20 200 2000

100.09 99.69 91.68

5.97 6.55 3.76

5.96 6.57 4.10

100.32 101.70 101.86

7.79 4.12 3.18

7.77 4.05 3.12

101.47 93.23 94.63

5.14 3.78 3.94

5.07 4.05 4.16

95.75 100.62 97.20

9.29 2.22 1.18

9.70 2.21 1.21

Gamabufotalin

10 100 1000

91.54 99.48 91.97

5.45 3.69 4.18

5.95 3.71 4.54

91.71 97.60 98.61

3.18 1.75 5.35

3.47 1.79 5.43

89.81 98.48 97.65

2.08 2.74 6.35

2.32 2.78 6.50

88.85 93.48 94.33

3.38 4.53 5.92

3.80 4.85 6.28

Telibufagin

1 10 100

96.24 93.32 88.07

5.95 6.01 6.25

6.18 6.44 7.10

98.52 95.55 90.26

4.80 5.27 5.36

4.87 5.52 5.94

98.92 92.46 87.72

2.83 6.21 9.21

2.86 6.72 10.50

92.56 93.41 85.92

3.05 5.98 4.63

3.30 6.40 5.39

Arenobufagin

5 50 500

98.16 93.71 95.73

6.95 5.76 2.96

7.08 6.15 3.09

98.95 95.43 94.50

5.74 6.08 4.01

5.80 6.37 4.24

98.18 94.27 97.29

6.20 4.26 6.94

6.31 4.52 7.13

91.26 93.68 87.61

5.40 6.61 5.63

5.92 7.06 6.43

Cinobufagin

1 10 100

87.19 88.39 89.18

7.92 6.53 3.53

9.08 7.39 3.96

98.60 92.31 93.74

11.41 6.22 5.21

11.57 6.74 5.56

95.77 91.28 88.80

9.20 7.65 7.24

9.61 8.38 8.15

86.81 91.57 87.03

5.16 3.34 1.42

5.94 3.65 1.63

Bufotalin

1 10 100

91.30 86.35 92.52

8.98 7.02 4.45

9.84 8.13 4.81

97.43 86.63 90.14

12.01 6.32 3.75

12.33 7.30 4.16

86.49 87.93 92.92

8.07 7.07 7.58

9.33 8.04 8.16

88.54 88.28 89.73

11.95 5.62 2.55

13.50 6.37 2.84

Fig. 4. Plasma concentration–time profiles of the analytes following single oral administration of SBP to rats (n = 6).

principle (constituents that have known clinical activities), active (constituents that have some known pharmacological activities), negative (constituents that may have allergenic or toxic properties), and analytical (constituents that are chosen as markers for

identification and quantitative determination) markers [25,26]. However, the conventional method for discovery of marker compounds such as systematic chemical separation followed by pharmacological activity assay or bioassay guided chemical

Table 6 PK parameters of the analytes following single oral administration of SBP to rats (n = 6). Parameters

Dose (mg/kg) Cmax (ng/mL) Tmax (h) AUC0–t (ng/mL h) AUC0–INF (ng/mL h) T1/2 (h) MRT (h)

Resibufogenin

Bufalin

Mean

SD

Mean

6.12 54.70 1.08 91.73 140.15 1.60 2.86

0 18.44 0.20 24.35 66.40 1.30 1.78

8.96 155.42 1.05 309.94 469.55 1.46 2.60

Gamabufotalin

Telibufagin

Arenobufagin

Cinobufagin

Bufotalin

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

0 28.67 0.27 152.78 384.22 1.52 2.19

1.09 68.38 1.33 130.16 158.61 1.35 2.64

0 24.96 0.26 55.15 53.69 0.54 0.84

0.18 13.33 1.08 25.54 35.48 1.62 2.96

0 3.09 0.34 9.20 13.47 0.93 1.36

1.11 67.06 0.92 240.40 270.88 2.67 4.21

0 11.50 0.34 48.76 67.06 1.35 1.44

1.11 4.49 0.88 43.11 57.13 11.35 16.53

0 0.45 0.14 4.14 11.98 3.14 4.74

0.41 1.08 0.42 0.81 1.22 0.74 1.22

0 0.59 0.14 0.26 0.41 0.22 0.28

S. Wang et al. / J. Chromatogr. B 967 (2014) 255–263

separation is not only proved to be time-consuming, laborintensive, error-prone, or costly, but also cannot explain the synergistic action of multiple components of TCMs. In our previous study, the potential bioactive components of SBP and Venenum Bufonis were screened by a simplified methodology with LC-DAD coupled with electrospray tandem mass spectrometry and plasma pharmacochemistry-based approach [27,28]. As follow-up work, the selected seven bufadienolides in this paper were not merely the achievements of plasma pharmacochemistry study and bioactivity screening of Venenum Bufonis, but the compounds with these characters, including therapeutic, bioactive, characteristic, main, synergistic, correlative, toxic, and analytical. The success of PK study depends not only on selecting the proper marker compounds, but also on tailoring the appropriate dosage of the drugs. However, it is still rare in clinical practice due to the extreme complexity and diversity of the co-existing components in TCMs. Also the foundation of treatment based on syndrome differentiation including age, gender, weight, and genetic variations of an individual have significant impact on the drug disposition that is closely related to the efficacy and safety of medications. Therefore, a rational dosage design, which not only within the toxic/therapeutic margins, but also let the marker compounds determined in vivo, is a plausible solution for the PK study of TCMs. The oral dosage of SBP was set at 4 g/kg, an acceptable value not only within the window of therapeutic dosage originated from the pharmacodynamic study on rats, but also can give typical plasma concentration–time profiles of the seven compounds. 4. Conclusions A sensitive and reliable LC–ESI–MS/MS method has been developed and validated for the simultaneous determination of resibufogenin, bufalin, gamabufotalin, telibufagin, arenobufagin, cinobufagin, and bufotalin in rat plasma. The analysis was carried out in a single run of 12 min by easy-to-use and highthroughput method with LLE sample pretreatment. No interference from endogenous materials or other source was found at the same retention time as the analytes. The PK property of arenobufagin, cinobufagin, and bufotalin was characterized as rapid oral absorption. The selected PK marker compounds with typical efficacy/toxicity may provide a practical solution for marker compound selection and dosage design for the therapeutic drug monitoring and PK study of SBP in its clinical applications. Acknowledgements The work was supported by program NCET Foundation, NSFC (81230090), partially supported by Global Research Network for Medicinal Plants (GRNMP), and King Saud University, Shanghai Leading Academic Discipline Project (B906), Key Laboratory of Drug Research for Special Environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300), and the Scientific Foundation of Shanghai China (12401900801, 09DZ1975700, 09DZ1971500, 10DZ1971700, 13ZR1408500). National Major Project of China (2011ZX09307-002-03), National Key Technology R&D Program of China (2012BAI29B06, 2008BAI51B03). References [1] L.J. Wang, X.P. Luo, Y. Wang, Evaluation on tolerability and safety of long-term administration with Shexiang Baoxin Pill in patients with coronary heart disease of stable angina pectoris, Chin. J. Integr. Tradit. West. Med. 28 (2008) 399–401.

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