MS method for quantification of buddlejasaponin IV in rat plasma and its application to a pharmacokinetic study

Accepted Manuscript Title: A simple and sensitive UHPLC-MS/MS method for quantification of buddlejasaponin IV in rat plasma and its application to a pharmacokinetic study Author: Yanhui Li Hui Xu Liping Chen Lei Tan PII: DOI: Reference:

S0731-7085(15)30310-1 http://dx.doi.org/doi:10.1016/j.jpba.2015.12.044 PBA 10413

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

19-9-2015 21-12-2015 22-12-2015

Please cite this article as: Yanhui Li, Hui Xu, Liping Chen, Lei Tan, A simple and sensitive UHPLC-MS/MS method for quantification of buddlejasaponin IV in rat plasma and its application to a pharmacokinetic study, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.12.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A simple and sensitive UHPLC–MS/MS method for quantification of buddlejasaponin IV in rat plasma and its application to a pharmacokinetic study Yanhui Lia, Hui Xua, Liping Chena, Lei Tanb* [email protected] a

Departments of Cardiology and Echocardiography, the First Hospital of Jilin

University, Changchun 130021, China b

Department of Orthopedic Trauma, the First Hospital of Jilin University, Changchun

130021, China *

Corresponding author at: Department of Orthopedic Trauma, the First Hospital of

Jilin University, Changchun 130021, China. Tel.: +86 043188785362; fax: +86 043188785362.

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Graphical Abstract

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Highlights An UHPLC–MS/MS method was developed for the determination of BS-IV The method is fast, simple, selective and sensitive The method was found to be linear in the range of 3.0–3000 ng/mL. Applied to pharmacokinetic and bioavailability study

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ABSTRACT Buddlejasaponin IV (BS-IV), a natural triterpene saponin isolated from several herbal plants, has drawn a lot of attention for its anti-inflammatory, antinociceptive, antihyperlipidemia, and antitumor activities. In this study, a simple and sensitive method for determination of BS-IV in rat plasma was developed for the first time, using ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Tenacissoside I was used as an internal standard (IS). Separation was achieved on an Agilent Extend-C18 column with gradient elution using methanol–water as mobile phase at a flow rate of 400 μL/min. A triple quadrupole mass spectrometer operating in the positive/negative ion-switching electrospray ionization mode with selection reaction monitoring (SRM) was used to determine BS-IV and IS transitions of 941.4 → 779.5 and 815.5 → 755.5, respectively. The lower limit of quantification was 3.00 ng/mL with a linear range of 3.0–3000 ng/mL. The intra- and inter-day precisions were both ≤10.4% for BS-IV, and the average intra- and inter-day accuracies ranged from −7.2% to 6.7%. The validated assay was successfully applied to a pharmacokinetic study of BS-IV following oral administration of 3, 6, 12 mg/kg and an intravenous administration of 0.9 mg/kg to rats.

Keywords: Buddlejasaponin IV; UHPLC–MS/MS; Rat plasma; Pharmacokinetics

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1. Introduction

Triterpene or steroid glycosides called saponins, which are found in plants, lower marine animals and some bacteria, were reported to have many biological and pharmacological properties [1–3]. Buddlejasaponin IV (BS-IV, Fig. 1) is a triterpene saponin found in plants such as Pleurospermum kamtschaticum, Physospermum verticillatum, Buddleja japonica, Bupleurum gibraltaricum, Bupleurum spinosum and Clinopodium chinense [4–8]. Many of these plants have been used in herbal medicine. Biological studies on BS-IV suggested that it possesses anti-inflammatory, antinociceptive, and cytotoxic properties against various cancer cell lines, as well as inhibitory effects on nitric oxide production in LP-induced RAW 264.7 macrophages [7, 9–12]. Moreover, it can reduce blood thiobarbituric acid-reactive substances and hydroxy radical levels, and increase superoxide dismutase activity in high cholesterol diet-induced rats; these findings suggested that BS-IV might be used to treat hypercholesterolemia or hyperlipidemia [8]. In addition, BS-IV treatment inhibits cell growth of immortalized human oral keratinocytes by inducing cell cycle arrest at the G2/M phase and apoptosis. Thus, BS-IV may be a promising chemopreventive agent for blocking the progression of human papillomavirus-induced oral carcinogenesis [13]. A number of analytical methods, such as high-performance liquid chromatography (HPLC) [14–16], thin-layer chromatography [17], and high-speed countercurrent chromatography [18], are available for the determination of BS-IV from C. chinense or products containing the herb. No bioanalytical method currently exists to determine BS-IV in plasma for the characterization of pharmacokinetic properties. Therefore, the 5

present study aimed to develop and validate a simple and highly sensitive ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for routine analysis of BS-IV in rat plasma, as well as characterize the preclinical pharmacokinetic profiles of BS-IV in rats after oral or intravenous administration. 2. Experimental 2.1. Materials and reagents BS-IV (99.5% purity) and tenacissoside I (99.1% purity) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Analytical HPLC grade methanol was purchased Tedia (Fairfield, OH, USA). Propylene glycol and carboxymethyl cellulose sodium were purchased through Sigma-Aldrich (St. Louis, Missouri, US). HPLC-grade water (> 18 mΩ) was obtained from a Milli-Q water purification system (Millipore, Milford, MA, USA). All other solvents were of analytical grades and commercially available 2.2. Instrumentation and LC–MS/MS method The UHPLC–MS/MS system consisted of an Accela 1250 UHPLC System equipped with a TSQ Quantum Ultra triple-quadrupole mass spectrometer (Thermo Scientific Inc., San Jose, CA, USA). Chromatographic separation was performed on an Agilent Extend-C18 column (4.6×50 mm, 5 μm; Agilent Technologies, Waldbronn, Germany). The mobile phase consisted of methanol (A) and water (B). The gradient elution program was as follows: 0–2.0 min, 20% A; 2.0–6.5 min, 90%A; 6.5–13.0 min, 20% A. The column 6

temperature was maintained at 35 ºC. The flow rate was 400 μL/min and the injection volume was 5 μL. After each injection, a needle wash with methanol was performed. Mass spectrometric detection was performed on a TSQ Quantum Ultra triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface. Both the analyte and IS were monitored under positive/negative ion-switching ESI conditions and quantified in selection reaction monitoring (SRM) mode with transitions of m/z 941.4 → 779.5 (negative) for BS-IV, and m/z 815.5 → 755.5 (positive) for IS. Other parameters of the mass spectrometer were as follows: sheath gas flow rate, 40 Arb; auxiliary gas flow rate, 10 Arb; spray voltage, 3250 V; vaporizer temperature, 300 ºC; and capillary temperature, 350 ºC. Argon was used as the collision gas for collision-induced dissociation (CID). 2.3. Preparation of standard solutions and samples Stock solutions of BS-IV (500 µg/mL) and IS (200 µg/mL) were prepared and dissolved in methanol. The IS working solution was diluted with methanol to 1.0 µg/mL. Standard solutions of BS-IV in rat plasma were prepared by spiking with an appropriate volume of the variously diluted stock solutions, giving final concentrations of 3.0, 10.0, 30.0, 100, 300, 1000, 3000 ng/mL for BS-IV. Quality control (QC) samples of BS-IV were prepared from blank plasma at high, medium and low concentrations of 2400, 75 and 7.5 ng/mL, respectively. All solutions were stored at –80°C prior to analysis. All the samples containing the calibration samples, QC samples and plasma samples were processed and prepared according to the procedure described following.

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The 80 μL of heparinized plasma sample, 40 μL of IS solution and 400 μL of methanol were added into a 1.5 mL polypropylene tube and vortexed for 5 min and then centrifuged at 13500 × g for 10 min. The supernatant was transferred to a clean vial and dried with nitrogen. The residue was re-dissolved with 100 μL methanol: H2O (50:50, v/v), and a 5 μL aliquot of the sample was injected into UHPLC–MS/MS for analysis. 2.4. Method validation 2.4.1. Specificity The specificity of the method was evaluated by comparing the chromatograms of blank plasma with the corresponding plasma samples spiked with BS-IV and IS, as well as real samples collected from treated rats. 2.4.2. Linearity and sensitivity Calibration curves were constructed by plotting the peak-area ratio of BS-IV to the IS versus the concentrations of the analyte. It was achieved from seven-point calibration curves covering a concentration range from 3.0 to 3000 ng/mL BS-IV in plasma samples, using a weighted least-squares linear regression (the weighting factor, 1/X2). The lower limit of quantification (LLOQ) was determined based on at least 10 times of signal-to-noise ratio at which the precision (expressed by relative standard deviation, RSD) and accuracy (calculated by relative error, RE) were lower than ±20%. 2.4.3. Accuracy and precision The intra-day accuracy and precision were investigated by determining six

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replicates of LLOQ samples and low, medium and high QC samples at concentration of 7.5, 75.0 and 2400 ng/mL on the same day. The inter-day accuracy and precision were studied by determining LLOQ and three levels of QC samples on three consecutive days. 2.4.4. Extraction recovery Extraction recoveries of BS-IV were determined by comparing the peak area of the analytes spiked and extracted from blank plasma with those of extracted blank plasma spiked with the analytes at the same concentration levels. Recoveries were determined at three QC concentrations for plasma (n=6). 2.4.5. Matrix effect and carryover Matrix effects were calculated by the mean peak area ratios of blank plasma samples spiked with BS-IV after extraction divided by the injected working solution with BS-IV at the same QC concentrations. The matrix effect of IS was evaluated at the working concentration (1.0 µg/mL) in the same manner. Carryover was evaluated by checking the responses following injection of a blank plasma sample immediately after three repeated injections of the samples of the upper limit of quantification (ULOQ, 3000 ng/mL). 2.4.6. Stability The stability was tested by analyzing six replicates of plasma samples at three QC levels under different conditions: 6 h exposure at room temperature, 24 h storage in the autosampler, three freeze/thaws cycles, and one week storage at –80 ºC. The stock solution stability of the analyte was assessed by comparing the working solutions

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stored at –80 ºC to freshly prepared solutions. Samples were considered stable if assay values were within the acceptable limits of accuracy (±15% RE) and precision (≤15% RSD). 2.4.7. Dilution integrity Six replicates of BS-IV samples with the concentration of 10 µg/mL were diluted 10-fold with blank plasma to evaluate dilution integrity. These diluted samples were analyzed along with calibration standards to calculate RE and RSD, which were required to be within ±15%. 2.5. Pharmacokinetic study 2.5.1. Animals Twenty-four male Wistar rats (weighing 240–260 g) were purchased from the Animal Center of Jilin University (Changchun, China). The rats were single-housed in plastic cages and received a standard chow and water ad libitum during the experiments. All animal experiments were performed according to the local Institutional Guidelines for Animal Care (IGAC) of Jilin University (Changchun, China). 2.5.2. Design of pharmacokinetics study The rats were randomly divided into four groups (six in each group). One group was injected with BS-IV at a dose of 0.9 mg/kg, and other three groups were treated by oral administration of BS-IV at doses of 3, 6, 12 mg/kg, respectively. The solvent used for the intravenous group was propylene glycol-5% aqueous glucose solution (3:7, v/v), and that used for the oral groups was 0.5% carboxymethyl cellulose sodium

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(CMC-Na). Blood samples (approximately 250 µL) were collected from sublingual vein into heparinized tubes at 0 (pre-dose), 5, 10, 20 and 40 min, 1, 2, 3, 4, 5, 7, 9, 12 and 18 h after dosing. Blood samples were centrifuged at 4000 × g for 10 min, then the plasma was harvested and stored at –80 ºC for analysis. 2.5.3. Sample assay The plasma samples were processed using the extraction procedure and analyzed using UHPLC–MS/MS method described above. A calibration curve was fitted by weighted linear regression (1/X2) as described in Section 2.4.2 and the concentrations in plasma were calculated. 2.5.4. Pharmacokinetic data analysis Pharmacokinetic parameters including maximum plasma concentration (Cmax) and time (Tmax), elimination half-life time (t1/2), mean residence time (MRT), systemic clearance (CL), 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-∞) were calculated using the DAS (Drug and Statistic) Software Package (version 2.0; Wenzhou Medical University, China) using a non-compartmental model. Data was expressed as mean±SD. The oral bioavailability (F) of BS-IV was calculated according to the following equation, F (%) = [dose (iv) × AUC0-∞ (oral)]/[dose(oral) × AUC0-∞ (iv)] × 100.

3. Results and discussion 3.1. Optimization of LC–MS/MS method To develop a sensitive MS/MS method for the determination of BS-IV in biological 11

fluids, the ionization of BS-IV in negative ion and positive ion modes on an ESI ion source was compared in terms of the intensity of produced ions. In the positive ion mode, only the unstable [M+Na]+ adduct ion was observed without yielding other related molecular ions. In the negative ion mode, the most abundant ion produced for BS-IV was [M–H]–, followed by a relatively low intensity of [M+Cl]–. The intensity of the most abundant quasi-molecular ion [M–H]– for BS-IV produced in the negative ion mode was much stronger than that in the positive ion mode. Therefore, the negative ion mode was used to detect BS-IV in this study. The mass spectrometer is often controlled in SRM mode to achieve higher sensitivity and specificity. In SRM mode, the precursor ion is fragmented through collision-induced dissociation to generate the corresponding production ions. The most intensive one is then chosen for monitoring. In the present study, the deprotonated quasi-molecular ion [M–H]– of BS-IV (m/z: 941.4) and its prominent fragmented ion (m/z: 779.5), produced upon losing one glucose moiety (see Fig. 2), were monitored in SRM (m/z transition: 941.4/779.5) mode to quantify BS-IV in rat plasma. The IS, tenacissoside I, yielded predominantly [M+H]+ ions at m/z 815.5 under the same MS detector conditions because of its similarity in chemical structure to BS-IV, and monitored efficiently with a m/z transition of 815.5/755.5 for SRM in positive mode. Given the short switching time, a TSQ Quantum Ultra triple-quadrupole mass spectrometer can be operated in the positive/negative ion switching modes to allow simultaneous monitoring of compounds in both the positive/negative ion modes [19]. Thus, the IS was selected throughout the

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experiment. The satisfactory resolution of BS-IV and IS was achieved with methanol and water with gradient elution at a flow rate of 400 µL/min on an Agilent Extend-C18 column (4.6×50 mm, 5 μm; Agilent Technologies). It was also noted that gradient elution dramatically narrowed the peak shape, and improved the response intensity and resolution of the analyte and IS. The LC–MS/MS method described shows good specificity and satisfactory sensitivity that meets determination requirements of BS-IV in rat plasma. 3.2. Method validation 3.2.1. Specificity Specificity was assessed by comparing the chromatograms of six different batches of blank plasma with the plasma spiked with the analyte. Typical UHPLC–MS/MS chromatograms of BS-IV and IS are presented in Fig. 3. The retention times of BS-IV and IS were 4.2 and 3.4 min, respectively. No significant interference from endogenous substances was observed at their retention times. 3.2.2. Linearity and sensitivity The standard calibration curve for BS-IV was linear over the concentration range of 3.0–3000 ng/mL by using weighted least square linear regression analysis with a weigh factor of 1/X2. The typical equation for the calibration curves for BS-IV was y = 0.02691x – 0.00612 (r = 0.9955), where y represents the peak area ratio of BS-IV to the IS and x represents the concentration of analyte in spiked plasma samples. At the LLOQ level of 3.0 ng/mL for BS-IV, the intra- and inter-day accuracy (RE) was 2.4%

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and 4.1%, and the intra- and inter-day precision (RSD) was 7.3% and 10.4%, respectively. 3.2.3. Accuracy and precision Intra- and inter-day accuracy and precision values of the LLOQ and QC samples are listed in Table 1. In this assay, the intra- and inter-day precision values were both ≤10.4% for BS-IV and the average intra- and inter-day accuracy values ranged from −7.2% to 6.7%. These results indicated an excellent accuracy and precision for the quantification of BS-IV in plasma using the current method. 3.2.4. Extraction recovery The extraction recoveries of BS-IV using six replicates of QC samples were 93.0 ± 3.5%, 86.6 ± 1.2% and 92.4 ± 3.3% (mean ± SD, n = 6) at three concentrations of 7.50, 75.0, 2400 ng/mL, with the RSDs of 3.8, 1.4, 3.6%, respectively (see Table 1). The recovery of IS was 88.7 ± 5.4%, and the RSD was 6.1%. 3.2.5. Matrix effect and carryover The matrix effects of BS-IV at the three QC concentrations above were 92.2 ± 5.4%, 95.0 ± 4.1%, 102.7 ± 3.2%, and the RSDs were 5.9, 4.3, 3.1%, respectively (see Table 1). In addition, the matrix effect of IS was 91.1 ± 1.7%, and the RSD was 1.9%. The results indicated that the protein precipitation efficiency was acceptable and no co-eluting substance could influence the ionization of BS-IV and the IS. Carryover was performed by injecting a blank sample after three repeated injections of the ULOQ samples. Results showed the absence of carryover. 3.2.6. Stability

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The stability experiment was performed under various conditions that the samples may undergo. The results demonstrated that BS-IV was stable in rat plasma at room temperature for 6 h, at 4 ºC in the autosampler for 24 h, after three freeze-thaw cycles, and after storage at –80 ºC for one week (see Table 2). It is noteworthy that no significant degradation of BS-IV was observed under the above conditions. 3.2.7. Dilution integrity Dilution integrity was performed in six replicates by a 10-fold dilution with blank plasma, and assay accuracy and precision were determined using the same sample pretreatment method. The RE was –4.0% and the RSD was 7.3%, which were within the acceptable limits of ±15%. These results demonstrated that the samples for the higher analyte concentrations (above ULOQ) could be re-analysed by an appropriate dilution. Overall, the validation of this method was highly successful for plasma, showing excellent accuracy and reproducibility, and is suitable for use in the preclinical pharmacokinetic analysis. Other HPLC quantitative methods have been described for the determination of BS-IV in herbal medicines [14–16]. However, these methods are limited by poor specificity, insufficient sensitivity, and long chromatographic run time, and none offer a chromatographic system suitable for a pharmacokinetic study of BS-IV. 3.3. Pharmacokinetics of BS-IV in rat plasma The validated assay was successfully applied to the pharmacokinetic study in rats

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following intravenous injection of 0.9 mg/kg or oral administration at three doses of 3, 6, 12 mg/kg BS-IV. The mean plasma concentration-time profiles of BS-IV in rats are illustrated in Fig. 4 and the main pharmacokinetic parameters are shown in Table 3. Results demonstrated that the plasma concentrations of BS-IV increased rapidly after oral administration of three doses and all reached the Cmax within 3.80 h, then the concentrations descended with the t1/2 values of 1.99–3.30 h and the total clearance (CL) of 4.59–5.19 L/h/kg. Linear regression analysis on the oral administration data displayed that Cmax, AUC0-t and AUC0-∞ were dose-dependent. There was no significance difference in Tmax, t1/2, CL, AUC0-t/dose, and AUC0-∞/dose (p > 0.05). The calculated oral bioavailability (F) for orally administered BS-IV was 2.06% for 3 mg/kg, 2.25% for 6 mg/kg and 2.20% for 12 mg/kg (Table 3). BS-IV showed a poor bioavailability and it might be due to the poor permeability and significant hepatic first-pass effect [20, 21]. 4. Conclusion A fast, simple, selective and sensitive quantification method of BS-IV was established to investigate pharmacokinetic study in rats. The LLOQ of this method was 3.0 ng/mL. This method was fully validated and the extraction recoveries and matrix effects were reproducible and consistent. The stability of BS-IV in stock solution and plasma samples were also proved. The developed method was successfully applied to a pharmacokinetic study of BS-IV following oral administration and intravenous administration of BS-IV to rats.

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and metabolism of

liquidchromatography/electrospray

notoginsenoside

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by J.

Figure Captions

O

(A)

HO

OH

O O

O

O HO

O OH

HO

OH OH

O

OH OH OH

OH

Exact Mass: 942.52

OH

(B)

O

O

O O

HO O

O

O

O O O

O O

Exact Mass: 814.41

Fig. 1. The chemical structures of (A) BS-IV and (B) tenacissoside I.

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DUANXUELIU_150831113615 #33RT: 0.30 AV: 1NL: 2.51E6 T:- p ESI Full ms2 941.400 [100.070-1000.000] 100

(A)

779.53

95

-H

90 85

O

617.7

80 HO

Relative Abundance

75 70 65

HO

60

O OH

HO

55

941.38

O

O

O

[M-H]-

OH

O

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779.5

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OH OH

50 OH

45

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OH

40 35 30 25

-Glc

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-Glc

15 10

119.03161.37

0

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581.43 599.27 659.31 568.90

422.93 403.62 471.29

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850

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TONGGUANTENG-I_150831114842 #37RT: 0.20 AV: 1 NL: 3.80E7 T:+ p ESI Full ms2 815.300 [100.070-1000.000] 100

755.51

95

(B)

90

+H

85 OH

80

O

715.4

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O

Relative Abundance

75 70

O

HO

65

755.5

O O

O

O

O

60 O

55

O

O 655.4

50 45

[M+H]+

40 655.41

35

815.46

30 25

715.43

20 15 633.87

10

112.31 144.35 187.16 250.75 311.25 5 0

150

200

250

300

350

673.32 400

450

500

550

600

650

m/z

Fig. 2. Product ion spectra of (A) BS-IV and (B) IS.

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700

750

800

850

RT: 0.00 - 6.50 100

5.20

(A)

Relative Abundance

90

BS-IV channel

80

4.19

70 60

0.71

0.54

1.21

1.44

2.08

2.96

2.63

4.30

3.02

3.77

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50 40 30 20 10

0 100

5.43

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IS channel

80 70

5.95

3.12 4.47

60 3.17

50 40 1.21

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0.20

30

1.44

2.08

2.70

4.98

4.30

3.06

6.04

5.66

3.64 3.38

6.46 6.17

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20 10 0

0.0

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5.0

5.5

6.0

4.17

RT: 0.00 - 6.50 100

(B)

Relative Abundance

90

BS-IV channel

80 70 60 50 0.68

40

0.75

0.94

1.78

1.87

3.04 2.12

2.73

4.29 3.33

4.02

4.76

5.27

5.33

5.42

5.90

30 20 10 0 100

3.42

90

IS channel

80 70 60 50 40 30 20 10 0

0.0

0.55 0.75 0.5

0.92 1.0

1.63 1.5

1.86 2.0

2.60 2.5

2.82

3.76 3.0 3.5 Time (min)

22

4.01 4.0

4.44 4.5

4.76

5.31 5.0

5.77 5.5

6.13 6.0

6.40

4.17

RT: 0.00 - 6.50 100

Relative Abundance

90

(C)

80

BS-IV channel

70 60 50 40 30 20 10 0.06

0 100

0.64

0.92

1.34

1.64

2.08

2.36

2.76

4.42

3.96

3.25

4.79

5.37

5.85

6.43

3.44

90

IS channel

80 70 60 50 40 30 20 10 0

0.06 0.0

0.78 0.5

1.33 1.0

1.64 1.5

2.08 2.0

2.68 2.5

3.23 3.0 3.5 Time (min)

3.82

4.38 4.62 4.5

4.0

5.15 5.0

5.65 5.5

6.30 6.0

6.5

4.17

RT: 0.00 - 6.50 100

(D)

Relative Abundance

90

BS-IV channel

80 70 60 50 40 30 20 10 0.20

0 100

0.66

1.00

1.61

2.13

2.44

2.82

3.16

4.38

3.60

5.05

5.32

5.95

6.29

5.86 6.0

6.27

3.42

90

IS channel

80 70 60 50 40 30 20 10 0

0.19 0.0

0.66 0.5

0.99 1.0

1.62 1.5

2.12 2.0

2.65 2.5

3.04 3.0 3.5 Time (min)

3.69

4.08 4.0

4.48 4.5

4.81

5.33 5.0

5.62 5.5

Fig. 3. Representative SRM chromatograms of BS-IV and IS in plasma: (A) a blank plasma sample; (B) a blank plasma sample spiked with BS-IV (final concentration 3.0 ng/mL) and IS (final concentration 1000 ng/mL); (C) a plasma sample collected at 1 h after oral administration of 6 mg/kg BS-IV; and (D) a plasma sample collected at 1 h after intravenous administration of 0.9 mg/kg BS-IV.

23

(A)

1000

Concentration (ng/mL)

3 mg/kg 6 mg/kg 12mg/kg 100

10

1

0

2

4

6

8

10

Time (h)

24

12

14

16

18

20

(B)

10000

0.9 mg/kg Concentration (ng/mL)

1000

100

10

1 0

2

4

6

8

10

12

14

16

18

20

Time (h)

Fig. 4. Mean plasma concentration–time curves of BS-IV in rats receiving (A) oral doses of 3, 6, 12 mg/kg and (B) intravenous dose of 0.9 mg/kg, respectively (n=5).

25

Tables Table 1 Accuracy, precision, extraction recovery and matrix effect of the method for BS-IV quantification in rat plasma. Spiked (ng/mL)

conc.

Accuracy (RE, %)

Precision (RSD, %)

Extraction recovery

Intra-day

Intra-day

Inter-day

Mean ± SD (%)

RSD (%)

Inter-day

Matrix effect Mean ± SD (%)

RSD (%)

3.00

2.4

4.1

7.3

10.4

–

–

–

–

7.50

–3.0

5.6

1.5

6.9

93.0 ± 3.5

3.8

92.2 ± 5.4

5.9

6.7

–0.5

1.3

1.8

86.6 ± 1.2

1.4

95.0 ± 4.1

4.3

4.6

–7.2

7.0

5.1

92.4 ± 3.3

3.6

102.7 ± 3.2

3.1

75.0 2400

26

Table 2 Stability of BS-IV under various storage conditions (n = 6). Storage conditions Concentration (ng/mL) Spiked Measured 6 h exposure at room 7.50 7.59 temperature 75.0 80.6 2400 2295

Accuracy (RE, %) 1.2 7.5 –4.4

Precision (RSD, %) 6.1 2.0 7.3

24 h storage in the autosamper

7.50 75.0 2400

6.82 77.1 2130

–9.1 2.8 –11.3

12.4 4.5 4.2

three freeze/thaws cycles

7.50 75.0 2400

8.01 71.7 2590

6.7 –4.4 7.9

5.5 6.0 9.2

one week storage at –80 ºC

7.50 75.0 2400

7.90 69.5 2304

5.3 –7.3 –4.0

1.1 3.7 8.4

27

Table 3 Pharmacokinetics parameters of BS-IV in rats (n = 5). Parameters Unit Oral (mg/kg) 3 6 AUC0-t µg h/L 583.67±146.08 1285.40±163.24 AUC0-∞ µg h/L 608.52±150.85 1325.57±172.74 Cmax ng/mL 114.96±17.93 218.33±23.39 Tmax h 2.40±0.55 3.00±1.00 t1/2 h 1.99±0.39 3.30±0.76 MRT0-t h 3.98±0.62 5.28±0.52 CL L/h/kg 5.19±1.34 4.59±0.61 F % 2.06±0.51 2.25±0.29

28

12 2548.35±351.11 2595.17±345.69 405.27±65.25 3.80±0.84 2.97±0.51 4.97±0.31 4.70±0.73 2.20±0.38

Intravenous (mg/kg) 0.9 8815.38±1111.30 8849.87±1426.08 5977.82±463.61 – 2.43±0.24 2.51±0.15 0.03±0.01 –