Research article Received: 24 November 2014,

Revised: 30 March 2015,

Accepted: 20 April 2015

Published online in Wiley Online Library: 29 May 2015

(wileyonlinelibrary.com) DOI 10.1002/bmc.3498

Development and validation of an LC-MS/MS method for the determination of bullatine A in rat plasma: application to a pharmacokinetic study Shi-Yong Tenga, Si-Xi Zhangb, Kai Niuc, Li-Jie Zhaid and Shi-Ji Wange* ABSTRACT: Bullatine A is a diterpenoid alkaloid of Xue-Shang-Yi-Zhi-Hao (Aconitum brachypodum), which is widely used in traditional Chinese medicine for the treatment of rheumatism and pain. The plasma levels of bullatine A were measured by a rapid and sensitive LC-MS/MS method. Samples were prepared using acetonitrile precipitation and the separation of bullatine A was achieved on a Capcell Pak MG-C18 column by isocratic elution using acetonitrile (phase A) and 0.1% formic acid (phase B, pH 4.0; A:B, 30:70, v/v) as the mobile phase at a flow rate of 0.5 mL/min. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple-reaction monitoring of the transitions at m/z 344.2 → 105.2 for bullatine A and m/z 256.2 → 167.1 for the internal standard. The linearity was found to be within the concentration range of 1.32–440 ng/mL with a lower limit of quantification of 1.32 ng/mL. Only 1.3 min was needed for an each analytical run. This method was successfully applied in the determination of the active component bullatine A in rat plasma after intramuscular administration of A. brachypodum injection. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: bullatine A; pharmacokinetics; LC-MS/MS; rat plasma

Introduction

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Aconitum brachypodum, an important member of the Ranunculaceae family, is widely applied as a traditional Chinese medicine to treat rheumatism and pain (China Pharmacopoeia Committee, 1977; Li, 1990). Pharmacological research has shown that the alkaloids of this herb possess therapeutic effects, such as analgesia and anti-inflammatory (Ameri, 1998). Furthermore, the A. brachypodum extract could have anti-inflammatory functions by inhibiting the damage and apoptosis of mouse peritoneal macrophages induced by lipopolysaccharide and by decreasing reactive oxygen species and nitric oxide production (Huang et al., 2012). Along with the excellent efficacy of this herbal medicine, toxicities such as respiratory paralysis, serious ventricular arrhythmia and palpitation also occur occasionally (Huang et al., 2013; Singhuber et al., 2009). Bullatine A (see Fig. 1), which structurally belongs to the diterpenoid alkaloid family (Shen et al., 2010), is considered one of the major active constituents of A. brachypodum for the quality control (QC) of the herb (Gao et al., 2005; Zou et al., 2014). Bullatine A potently inhibits ATP-induced BV-2 cell death/apoptosis by selectively antagonizing the P2X7 receptor and may be a potential candidate to efficiently treat neurodegenerative diseases, such as arthritis (Li et al., 2013). Some analytical methods have been reported based on highperformance liquid chromatography (HPLC) with ultraviolet detection to determine bullatine A in raw materials or its preparations (Gao et al., 2005; Li et al., 2008; Geng et al., 2011; Zou et al., 2014). However, the reported HPLC assays suffer from long analytical run times, lack of specificity or inadequate lower limit of quantification (LLOQ).

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Nevertheless, high-performance capillary electrophoresis has been reported to analyze 10 alkaloids, including bullatine A, in human urine and blood with LLOQs of 40 or 50 ng/mL (Zhang et al., 2007). However, if this method is introduced to quantify bullatine A concentrations in biological fluids, the sensitivity to bullatine A should be greatly improved to meet its very low concentration in the biological matrix. Moreover, LC-MS/MS is considered a gold standard technique for drug analysis in biological samples. However, the reported method encounters several disadvantages, such as employing an external standard (no internal standard, IS, was used), time-consuming elution procedures, large sample volumes, inappropriate QC samples, incomplete validation procedures and inadequate LLOQs (Wang et al., 2013). In addition, this method * Correspondence to: Shi-Ji Wang, Department of Critical Care Medicine, the First Hospital of Jilin University, Changchun 130021, People’s Republic of China. Email: [email protected] a

Department of Anesthesiology, the First Hospital of Jilin University, Changchun, 130021, People’s Republic of China

b

Department of Pharmacy, the First Hospital of Jilin University, Changchun, 130021, People’s Republic of China

c

Department of Otorhinolaryngology Head and Neck Surgery, the First Hospital of Jilin University, Changchun, 130021, People’s Republic of China

d

Department of Medicine Management, the Second Hospital of Jilin University, Changchun, 130041, People’s Republic of China

e

Department of Critical Care Medicine, the First Hospital of Jilin University, Changchun, 130021, People’s Republic of China Abbreviations used: MRM, multiple-reaction monitoring.

Copyright © 2015 John Wiley & Sons, Ltd.

Determination of bullatine A H O H O

H

H H

N

Figure 1. The chemical structure of bullatine A.

could presumably be appropriate for therapeutic drug monitoring and does not provide any pharmacokinetic data. Therefore, development of a simple, rapid, sensitive and specific analytical method is necessary to accurately determine bullatine A concentration in biological samples. In the present study, a robust LC-MS/MS method is developed to determine bullatine A in rat plasma. The method is fully validated and then applied to a preclinical pharmacokinetic study on the active component bullatine A in rats after intramuscular administration of A. brachypodum injection.

the IS was diluted to a concentration of 50 ng/mL with acetonitrile. All the solutions were stored at 4 °C. The calibration standards of bullatine A (1.32, 4.40, 13.2, 44.0, 132 and 440 ng/mL) were prepared by adding 20 μL of the working standard solution to blank plasma. QC samples at concentrations of 3.30 (LQC), 33.0 (MQC) and 396 (HQC) ng/mL for bullatine A in plasma were prepared separately in the same fashion.

Sample preparation An aliquot of 50 μL of the IS working solution (50 ng/mL) was added to 100 μL of plasma sample followed by the addition of 200 μL acetonitrile. The sample mixtures were vortex mixed for 3 min. After centrifugation at 13,000 rpm for 5 min, the supernatant (2 μL) was injected into the LC-MS/ MS system for analysis.

Method validation The method was validated following the European Medicines Agency (2011) guidelines.

Experimental Materials and reagents Bullatine A (>98% purity, Fig. 1) and diphenhydramine hydrochloride (>98% purity) were purchased from the National Institutes for Food and Drug Control (Beijing, China). HPLC-grade acetonitrile and analytical-grade formic acid were obtained from Tedia (Fairfield, OH, USA). Fresh blank rat plasma containing sodium heparin as the anticoagulant was prepared from male Sprague–Dawley rats. Deionized water used was purified by a Milli-Q system (Millipore, MA, USA).

Preparation of A. brachypodum injection Powdered and dry roots (10 g) of A. brachypodum were extracted with 80% acidic ethanol (pH 2) by percolation (2 × 120 mL, 12 h extraction time at room temperature). After filtration, the ethanol extract was evaporated to dryness under reduced pressure to yield a syrupy residue (1.37 g) (He et al., 2009). The residue was then resolved in 100 mL of propylene glycol–physiological saline (2:8, v/v) for dosing, with the bullatine A content as 76.2 μg/mL.

Instrumentation and LC-MS/MS conditions The LC-MS/MS system comprised Agilent 1200 liquid chromatography and an Agilent 6460 triple-quadrupole mass spectrometer with an electrospray ionization source (Agilent Technologies, USA). Data were analyzed by MassHunter software (version B.01.04). Chromatography separation was performed on a Capcell Pak MG-C18 column (4.6 × 50 mm i.d., 5 μm, Shiseido, Tokyo, Japan) maintained at 30 °C using an isocratic elution with acetonitrile (phase A) and 0.1% formic acid (phase B, pH 4.0; A:B=30:70, v/v). The flow rate was set at 0.5 mL/min. Quantification was performed in multiple-reaction monitoring (MRM) mode with specific ion transitions of protonated precursor ion to product ion at m/z 344.2 → 105.2 for bullatine A and m/z 256.2 → 167.1 for the IS. The fragmentor energy was 145 V for bullatine A and 135 V for IS. The optimized collision energy was 60 eV for bullatine A and 20 eV for IS. Nitrogen was used as nebulizer and drying gas, and gas temperature and flow were adjusted to be 300 °C and 10.0 L/min, respectively. Other electrospray ionization source conditions were: capillary voltage, 4.0 kV; nebulizer pressure, 35 psi; dwell time, 250 ms; and Delta electron multiplier voltage, 200 V.

Specificity. Specificity was assessed by extracting MRM ionchromatograms for blank plasma samples spiked with the standard and IS. Blank samples from six different batches of rat plasma were prepared similarly as described in the sample preparation section. The absence of peaks interfering with the analyte or IS peak at the LLOQ was used as a criteria to examine specificity. Precision and accuracy. The intra- and inter-day precision was determined at the three QC concentrations of the analyte (n = 6) on the same day and three consecutive days, respectively. Precision was expressed in terms of percentage coefficient of variation (CV) and was calculated based on the quotient between the standard deviation and the mean for each concentration level. Accuracy was expressed in terms of percentage bias and was calculated according to the difference between the determined concentration and the spiked concentration of the samples. Linearity and LLOQ. The calibration curves ranging from 1.32 ng/mL to 2 440 ng/mL were determined through weighted (1/x ) least squares liner regression based on the analyte/IS peak area ratio. LLOQ was defined as the lowest concentration on the calibration curves that could be assayed with precision (CV) and accuracy (bias) ≤20%. Extraction recovery and matrix effect. The extraction recovery was examined by determining the ratio of the amounts of QC samples obtained against those spiked with analyte in the blank plasma (n = 6). The matrix effect was evaluated by comparing the peak areas of the post-extraction plasma spiked with analyte with those prepared in redissolved acetonitrile–0.1% formic acid solution (30:70, v/v; n = 6). Stability. The analyte stability was investigated by determining the three QC levels of plasma samples during different storage and processing procedures. Short-term stability was tested by determining the QC samples maintained at room temperature for 6 h, which exceeded the routine preparation time. Long-term stability was evaluated by assaying the QC samples at –20 °C for 15 days. Freeze–thaw stability was checked after three freezing (–20 °C) and thawing (room temperature) cycles. Post-preparation stability was investigated by analyzing the extracted QC samples maintained in the autosampler at 4 °C for 12 h. Carry-over. Carry over was evaluated following the injection of the blank plasma samples immediately after five repeats of the upper limit of quantification (ULOQ). Carry-over should not be greater than 20% of the LLOQ and 5% of the IS (European Medicines Agency, 2011).

Standard solution and QC samples

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Application to a pharmacokinetic study The applicability of the developed assay to pharmacokinetic study of bullatine A in rats was explored. Male Sprague–Dawley rats (230–250 g) were supplied by the Experimental Animal Research Center, Jilin University,

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The stock solutions of bullatine A and IS were prepared at concentrations of 133.3 and 500 μg/mL, respectively. A series of mixed working standards having 13.2–4400 ng/mL for bullatine A were obtained by gradually diluting the stock solutions with acetonitrile. In addition, the stock solution of

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+

Figure 2. The chemical structures and product ion mass spectra of the [M + H] ions of bullatine A (A) and IS (B).

China. The rats were housed on a 12 h light–dark cycle with free access to food and water for 7 days. The rats were fasted for 12 h and had free access to water before dosing. After a single intramuscular administration of the A. brachypodum injection at a dose of 5 mL/kg body weight (equivalent to 0.381 mg/kg of bullatine A), approximately 0.25 mL of blood samples were collected into heparin-contained tubes via oculi chorioideae vein at 0, 2, 5, 10, 20, 30, 40, 60 and 90 min post-dose, and immediately centrifuged at 12,000 rpm for 5 min to obtain plasma. All the samples were stored at – 20 °C until analysis.

Results and discussion Optimization of MS conditions

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When the neat bullatine A solution was infused, the precursor ion m/z 344.2 of bullatine A [M + H]+ (molecular formula: C22H34NO2+)

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was observed in the positive ionization mode. The product spectrum of the [M + H]+ ion of bullatine A highly depended on the collision energy. No fragment ion formed at the low collision energy (10–50 eV). At the higher collision energy (55–85 eV), the product ion fragments of m/z 192.3, 178.1, 165.2, 140.9, 128.1, 117.1 and 105.2 were observed (Fig. 2), with the most abundant ion at m/z 105.2 (molecular formula: C8H+9 ). Similarly, the precursor ion m/z 256.2 of the IS [M + H]+ (molecular formula: C12H22NO+) was observed in the positive ionization mode, and the product ion spectrum of the [M + H]+ ion exhibited a major fragment ion at m/z 167.1 (molecular formula: C13H+11) at CE 8–30 eV. The optimum collision energy (20 eV) was determined by observing the maximum response obtained at m/z 167.1. The precursor/product ion pairs monitored for the MRM analysis were m/z 344.2 → 105.2 (bullatine A) and m/z 256.2 → 167.1 (IS). Bullatine A and IS exhibited

Copyright © 2015 John Wiley & Sons, Ltd.

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Determination of bullatine A x101

+ESI MRM Frag=145.0V Frag=145.0V [email protected] [email protected] (344.2-> (344.2 ->105.2) 105.2)blank blank 1

6.4



1 2

A-1

6.2 6 5.8 5.6 5.4

x102 1 0.95 0.9 0.85 0.8 0.75 0.7 0.65 0.6 0.55

x102 3.5 3

A-2

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2

Counts vs. Acquisition Time (min)

1 2

2

B-1

bullatine A

x103 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2

+ESI MRM Frag=135.0V Frag=135.0V [email protected] [email protected] (256.2-> (256.2 ->167.1) 167.1)LLOQ.d… LLOQ.d… 1

1 2

2

B-2

IS 0.84

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2

Counts vs. Acquisition Time (min)

Counts vs. Acquisition Time (min)

+ESI MRM Frag=145.0V Frag=145.0V [email protected] [email protected] (344.2-> (344.2 ->105.2) 105.2)rat1-20min rat1 - …… 1 2

C-1

2 0.39

bullatine A

2.5 2 1.5 1

x103 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2



1 2

Counts vs. Acquisition Time (min)

0.39

1

+ESI MRM Frag=135.0V Frag=135.0V [email protected] [email protected] (256.2-> (256.2 ->167.1) 167.1)blank blank 1

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2

+ESI MRM Frag=145.0V Frag=145.0V [email protected] [email protected] (344.2-> (344.2 ->105.2) 105.2)LLOQ.d… LLOQ.d… 1

x102 1 0.95 0.9 0.85 0.8 0.75 0.7 0.65 0.6 0.55

…… +ESI MRM Frag=135.0V Frag=135.0V [email protected] [email protected] (256.2-> (256.2 ->167.1) 167.1)rat1-20min rat1 -20mi 1

1 2

C-2

2 0.84

IS

23

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2

Counts vs. Acquisition Time (min)

Counts vs. Acquisition Time (min)

Figure 3. Representative multiple reaction monitoring chromatograms including blank rat plasma (A); blank rat plasma spiked with 1.32 ng/mL bullatine A (LLOQ) and IS (B); and a real plasma sample at 20 min after single intramuscular administration of Aconitum brachypodum injection (C). 1, Bullatine A; 2, IS.

maximum and stable responses with mass parameters of collision and fragmentor energies. Optimization of chromatography conditions The mobile phase was optimized with various organic solvent percentages and different modifiers in water. Acetonitrile was

selected as the organic phase because sharper peak shape, better resolution and shorter chromatographic run time were obtained compared with methanol. Adding formic acid (0.1%) into the water could improve sensitivity; thus, the mobile phase selected consisted of acetonitrile and 0.1% formic acid in water. Finally, a mobile phase that consisted of acetonitrile (phase A) and 0.1% formic acid (phase B, pH 4.0; A:B, 30:70, v/v) was applied at a

Table 1. The intra- and inter-day precision and accuracy of the assay for bullatine A in rat plasma (n = 6) Intra-day

Spiked concentration (ng/mL)

Determined concentration (ng/mL)

CV (%)

Bias (%)

Determined concentration (ng/mL)

CV (%)

3.31 ± 0.15 30.81 ± 2.90 421.43 ± 40.25

4.53 9.41 9.55

0.30 –6.64 6.42

3.22 ± 0.30 32.03 ± 1.95 407.62 ± 32.13

9.32 6.09 7.88

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Copyright © 2015 John Wiley & Sons, Ltd.

Bias (%) –2.42 –2.94 2.93

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3.30 33.0 396

Inter-day

Bullatine A

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IS

3.30 33.0 396 25.0

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87.61 ± 4.03

93.03 ± 2.20

89.17 ± 1.35 87.02 ± 3.34 91.55 ± 7.51

90.55 ± 5.17 90.31 ± 0.78 95.39 ± 2.80

–2.72 –6.18 2.77 3.21 ± 0.21 30.96 ± 2.50 406.95 ± 30.19 –2.42 –1.18 2.32

6.54 8.07 7.42

CV (%) Determined concentration (ng/mL)

8.39 9.60 4.69 3.22 ± 0.27 32.61 ± 3.13 405.18 ± 19.02 2.42 6.48 –2.85 3.47 ± 0.41 31.22 ± 2.10 413.60 ± 29.09

11.82 6.73 7.03

CV (%)

5.15 –5.39 4.44

3.38 ± 0.12 35.14 ± 1.85 384.71 ± 42.62

3.55 5.26 11.08

CV (%) Determined concentration (ng/mL) Bias (%)

Long-term stability

Bias (%)

Copyright © 2015 John Wiley & Sons, Ltd.

3.30 33.0 396

Analyte Spiked concentra- Recovery Matrix effect tion (ng/mL) (mean ± SD, %) (mean ± SD, %)

Determined concentration (ng/mL)

Table 2. Recovery and matrix effect of bullatine A and IS in rat plasma (n = 6)

Freeze–thaw stability

Matrix effects. The matrix effects of bullatine A at concentrations of 3.30, 33.0 and 396 ng/mL were 93.03 ± 2.20, 90.55 ± 5.17 and 90.31 ± 0.78%, respectively. The matrix effect for IS was 95.39 ± 2.80% (Table 2). The results proved that no significant interference on the assay of the analyte from the plasma matrix was found.

Spiked concentration (ng/mL)

Extraction recovery. The extraction recoveries of bullatine A from rat plasma were 87.61 ± 4.03, 89.17 ± 1.35 and 87.02 ± 3.34% at concentration levels of 3.30, 33.0 and 396 ng/mL, respectively (n = 6). The average extraction recovery of the IS was 91.55 ± 7.51% (Table 2).

Table 3. Stability of bullatine A in rat plasma under different conditions (mean ± SD, n = 6)

Linearity and LLOQ. The calibration curves for bullatine A were constructed in the concentration range of 1.32–440 ng/mL using diphenhydramine as the IS at 50.0 ng/mL concentration, with a correlation coefficient (r2) ≥0.99 for the analyte. The peak area ratios of the analyte/IS compared with the nominal concentrations were plotted, and the calibration model was selected based on a weighted quadratic regression function (1/x2). The typical equation for the calibration curve was y = 8.909 × 10–4x + 4.174 × 10–4 with an excellent linear regression (r2 = 0.9940). The precision and accuracy at the LLOQ were determined by analyzing six replicates of the sample at the LLOQ concentration fixed at 1.32 ng/mL. The precision (CV) and accuracy (bias) were 4.20 and 1.27%, respectively, and the signal-to-noise ratio was ≥10 (Fig. 3B).

Short-term stability

Precision and accuracy. Table 1 shows the intra- and inter-day precision and accuracy. The intra-day precisions (CV) of the low, medium and high QC samples of bullatine A were 4.53, 9.41 and 9.55%, respectively. The inter-day precision (CV) of the low, medium and high QC samples of bullatine A were 9.32, 6.09 and 7.88%, respectively. The intra- and inter-day accuracies for bullatine A ranged from –6.64 to 6.42%. In conclusion, these results reveal that the developed method was precise and accurate.

Bias (%)

Specificity. Specificity was assessed in this study using independent drug-free plasma from six different batches of rats. Figure 3 shows the typical chromatograms of the blank plasma sample, blank plasma sample spiked with bullatine A and IS, and a real plasma sample. No interfering endogenous peaks were observed at the retention times of bullatine A and IS in the MRM chromatograms.

CV (%)

Method validation

Determined concentration (ng/mL)

Post-preparative stability

constant flow rate of 0.5 mL/min. The total run time was 1.3 min. Bullatine A and IS were eluted at 0.39 and 0.84 min, respectively. The retention times were short and suitable for high-throughput sample determination in pharmacokinetic study.

Bias (%)

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Determination of bullatine A (China State Drug Administration, China) was employed to calculate the pharmacokinetic parameters. The maximum concentration (Cmax) in plasma (17.59 ± 4.28 ng/ mL) for bullatine A was attained at 10.83 ± 4.92 min (Tmax). The area under the plasma concentration time curve from time zero to last measurable time point (AUC0 t) and area under the plasma concentration–time curve from time zero to infinity time point (AUC0–∞) for bullatine A were 480.12 ± 169.68 and 547.49 ± 204.20 μg min/L, respectively. The terminal half-life (t1/2) was found to be 25.30 ± 19.82 min. The pharmacokinetic data showed that bullatine A was absorbed and eliminated quickly in plasma after intramuscular administration of the A. brachypodum injection.

Plasma concentration (ng/mL)

20

15

10

5

0 0

20

40

60

80

100

Time (min) Figure 4. Mean plasma concentration–time profiles of bullatine A after single intramuscular administration of A. brachypodum injection (5 mL/kg) to Sprague–Dawley rats (n = 6).

Conclusion To our knowledge, this is the first fully validated LC-MS/MS method for the accurate quantification of bullatine A in biological samples. The method adhered to the EMA requirements for specificity, sensitivity, precision, accuracy, linearity, recovery, matrix effect and stability. This method was successfully applied in the determination of the active component bullatine A in rat plasma after intramuscular administration of A. brachypodum injection.

Table 4. Pharmacokinetic parameters of bullatine A after intramuscular administration of Aconitum brachypodum injection to rats (n = 6)

Acknowledgments

Pharmacokinetic parameters

The authors are grateful for the financial support provided by the Special Research Projects for Public Welfare of National Health and Family Planning Commission of the P.R.C. (no. 201202011).

Tmax (min) Cmax (ng/mL) t1/2 (min) AUC0–t (μg min/L) AUC0–∞ (μg min/L) MRT0–t (min) MRT0–∞ (min) Cl (L/min/kg) Vd (L/Kg)

Mean ± SD 10.83 ± 4.92 17.59 ± 4.28 25.30 ± 19.82 480.12 ± 169.68 547.49 ± 204.20 23.33 ± 6.16 35.35 ± 19.09 0.806 ± 0.369 24.86 ± 13.76

Tmax, Time to reach peak concentration in plasma; Cmax, peak concentration in plasma; t1/2, half-life; AUC, area under the plasma concentration–time curve; MRT, mean residence time; Cl, clearance; Vd, volume of distribution.

Stability. The stability was investigated under various conditions. All results are within the acceptance limit of 15% for precision and ±15% for accuracy, as shown in Table 3. Carry-over. The analyte and IS in the blank samples injected after the ULOQ samples did not exhibit any significant peak (≥20% of the LLOQ and 5% of the IS).

Application to a pharmacokinetic study

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The validated method was successfully applied to assay the plasma concentrations of bullatine A in rats following intramuscular administration of the A. brachypodum injection (5 mL/kg). The mean plasma concentration–time profiles of bullatine A are depicted in Fig. 4, and the main pharmacokinetic parameters are listed in Table 4. Noncompartmental analysis of DAS 2.0 software

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Biomed. Chromatogr. 2015; 29: 1798–1804

MS method for the determination of bullatine A in rat plasma: application to a pharmacokinetic study.

Bullatine A is a diterpenoid alkaloid of Xue-Shang-Yi-Zhi-Hao (Aconitum brachypodum), which is widely used in traditional Chinese medicine for the tre...
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