Research article Received: 24 September 2014,

Revised: 12 December 2014,

Accepted: 4 January 2015

Published online in Wiley Online Library: 10 February 2015

(wileyonlinelibrary.com) DOI 10.1002/bmc.3436

Simultaneous quantification of curdione, furanodiene and germacrone in rabbit plasma using liquid chromatography– tandem mass spectrometry and its application to a pharmacokinetic study Baolian Wang, Shuang Yang, Li Sheng* and Yan Li ABSTRACT: A simple, rapid and sensitive method was developed for the simultaneous quantification of curdione, furanodiene and germacrone in rabbit plasma using a LC-MS/MS analysis. The plasma sample preparation was a simple deproteinization by the addition of 3 vols of acetonitrile followed by centrifugation. The analytes and internal standard (IS) costunolide were separated on a Zorbax SB-C18 column (3.5 μm, 2.1 × 100 mm) with mobile phase of methanol–water (90:10, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min with an operating temperature of 25°C. Detection was carried out by atmospheric pressure chemical ionization in positive ion selected reaction monitoring mode. Linear detection responses were obtained for the three test compounds ranging from 5 to 5000 ng/mL and the lower limits of quantitation were 5-10ng/mL. The intra- and interday precisions (relative standard deviations) were within 9.4% for all analytes, while the deviation of assay accuracies was within ±10.0%. The average recoveries of analytes were >80.0%. All analytes were proved to be stable during all sample storage, preparation and analytical procedures. The method was successfully applied to the pharmacokinetic study of the three compounds after vaginal drug delivery of Baofukang suppository to rabbit. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: curdione; furanodiene; germacrone; quantification; pharmacokinetics

Introduction

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Experimental Materials Curdione (batch 20130613; purity 99.4%), furanodiene (batch 20130609; purity 98.9%), germacrone (batch 11665-201103; purity 99.8%) and Baofukang suppository (batch 20130709; 34.44 mg of Ezhu’s essential oil for each suppository) were provided by Hainan Bikai Pharmaceutical Co. Ltd. Costunolide (IS, >98.0% purity) was purchased from Shanghai Jingchun Co. Ltd. Methanol and acetonitrile were of HPLC grade (Fisher, USA). All other chemicals were of analytical reagent grade. Ultrapure

* Correspondence to: L. Sheng, Institute of Materia Medica, Chinese Academy of Medical Sciences and Perking Union Medical College, Beijing 100050, People’s Republic of China. Email: [email protected] Beijing Key Laboratory of Non-Clinical Drug Metabolism and PK/PD Study, State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Perking Union Medical College, Beijing 100050, People’s Republic of China Abbreviations used: APCI, atmospheric pressure chemical ionization; SRM, selected reaction monitoring.

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Rhizoma Curcumae (rhizome of Curcuma; Ezhu in Chinese) is a common traditional Chinese medicine. The essential oils are considered as the active part of Rhizoma Curcumae, and have been reported to possess antitumor and antiviral activities (Xia et al., 2004). Multiple agents containing Ezhu’s essential oil, like Baofukang suppository or Ezhu’s essential oil injection, have been used for treatment of cervical carcinoma, cutaneum carcinoma, colpitis and so on. According to a previous report, curdione, furanodiene and germacrone are the major components possessing active pharmacological effects in Ezhu’s essential oil (Deng et al., 2006; Manzan et al., 2003; Matsuda et al., 2001; Wang and Wang, 2001; Yang et al., 2006). Thus, it is necessary to characterize the pharmacokinetic profiles of curdione, furanodiene and germacrone for the rational use of essential oils and their preparations. There have been several analytical methods published for determination of curdione, furanodiene or germacrone in biological samples using HPLC or HPLC-MS/MS (Ma et al., 2012; Pei et al., 2012; You et al., 2005). However, these methods, which determined only one analyte in each method, are time-consuming and not sensitive enough for pharmacokinetic studies with extravascular administration. To our knowledge, there has been no analytical method reported for simultaneous quantification of curdione, furanodiene and germacrone in biological samples. We have developed a simple, sensitive and specific HPLC-MS/ MS method for the simultaneous determination of curdione,

furanodiene and germacrone in rabbit plasma with costunolide as the internal standard (see Fig. 1). The method was successfully applied to a pharmacokinetic study of the three compounds after vaginal drug delivery of Baofukang suppository to rabbit.

B. Wang et al.

Figure 1. Chemical structures of curdione (A), furanodiene (B), germacrone (C) and costunolide (D, internal standard) for plasma analysis.

water, prepared using a Milli-Q Reagent water system (Millipore, MA, USA), was used throughout the study.

Preparation of stocks, calibration standards and quality control samples The stock solution containing the three compounds was prepared in acetonitrile at the same concentration of 1 mg/mL for each single ingredient and appropriate dilutions were made with acetonitrile. A 0.5 mg/mL solution of costunolide in acetonitrile was prepared for use as the internal standard stock solution. All stock solutions were stored at –20°C prior to use. Calibrations standards samples were prepared by spiking diluted stock solutions into blank rabbit plasma, giving final concentrations of 5, 10, 20, 50, 100, 200, 500, 1000, 2000 and 5000 ng/mL for each analyte. High-, medium- and low-level quality control samples contained 10, 200 and 2000 ng/mL of the three analytes. These samples were prepared in a manner similar to that used for preparation of the calibrator samples.

Sample processing The internal standard costunolide (20 μg/mL) 20 μL and 280 μL of acetonitrile were added to plasma sample (100 μL/sample). The mixture was vortexed for 30 s followed by centrifugation at 14,000 rpm for 5 min at room temperature. A 5 μL aliquot of each supernatant was injected into the LC/MS system for the analysis.

HPLC-MS/MS HPLC-MS/MS determination was carried out using a Thermo TSQ Quantum Access mass spectrometry equipped with a series Surveyor HPLC system consisting of a quaternary pump, an automatic solvent degasser, an autosampler and an automatic thermostatic column compartment. The analytical column used was a Zorbax SB-C18 (3.5 μm, 2.1 × 100 mm, Agilent, USA) with a 0.5 online filter (Upchurch Scientific Ltd.). The mobile phase, methanol–water containing 0.1% formic acid (90:10, v/v), was run at a flow rate of 0.3 mL/min with an operating temperature of 25°C. Mass detection was carried out by atmospheric pressure chemical ionization (APCI) in positive ion selected reaction monitoring (SRM) mode. The precursor-to-product ion transitions were m/z 237.2 → 135.2 for curdione, m/z 217.2 → 149.2 for furanodiene, m/z 219.3 → 201.1 for germacrone and m/z 233.2 → 187.2 for costunolide (IS), respectively. The optimum APCI conditions for these compounds included a discharge current of 5.0 A, a vaporizer temperature of 300°C, a capillary temperature of 350°C, shealth gas pressure of 25 psi and auxiliary gas pressure of 8 psi.

Bioanalytical method validation

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Calibration curves, precision and accuracy. The linearity of the LCMS/MS method for the determination of three analytes was evaluated

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Figure 2. Mass spectrum of curdione (A), furanodiene (B), germacrone (C) and costunolide (D, internal standard) in MS/MS mode. by a calibration curve in the range of 5–5000 ng/mL. The calibration curves were obtained by plotting the peak area ratio vs the analyte concentration prepared. Least squares linear regression analysis was used to

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Simultaneous determination of curdione, furanodiene and germacrone

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Copyright © 2015 John Wiley & Sons, Ltd.

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Figure 3. Selected reaction monitoring mass chromatograms of (1) curdione (m/z 237.2 → 135.2), (2) furanodiene (m/z 217.2 → 149.2), (3) germacrone (m/z 219.3 → 201.1) and (4) the internal standard costunolide (m/z 233.2 → 187.2) during HPLC-MS/MS determination in (A) blank rabbit plasma, (B) a blank plasma spiked at 20 ng/mL of three analytes and 1μg/mL internal standard (costunolide), and (C) a rabbit plasma sample at 6 h after vaginal drug delivery of Baofukang suppository.

B. Wang et al. determine the slope, intercept and correlation coefficient. The calibra2 tion curve requires a correlation coefficient (r ) of 0.99 or better. To evaluate the precision, at least five QC samples of three different concentrations of the three substances were processed and injected on a single day (intra-day) and on different days (inter-day). The variability of three compounds determination was expressed as the coefficient of variation (CV), which should be ≤15% at all tested concentrations. Accuracy is expressed as percentage bias, which should be within limits of ±15% at all concentrations of three analytes.

prior to study. The rabbits were maintained on a 12 h light/12 h dark cycle at 22 ± 1°C and at 60% relative humidity. All animals were weighed daily and observed twice daily, in order to assess their general health. Food was prohibited for 12 h before the experiment while water was taken freely. The plasma pharmacokinetics of three compounds was studied in rabbits after vaginal drug delivery of Baofukang suppository (one suppository for each rabbit). First, we determined the concentration of the three active components in the suppository. The result showed that each Baofukang suppository (batch 20130709) contained 3.36 mg of curdione, 3.29 mg of furanodiene and 2.67 mg of germacrone. For the suppository delivery, we first introduced a lubricous polyethylene tube into the rabbit vagina with the help of lubricant (glycerol). Then, the suppository was delivered into the vagina through the tube. Blood samples were collected from the marginal ear vein into heparinized 1.5 mL polythene tubes at intervals of 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h after dosing and centrifuged at 3000g for 10 min. A 0.1 mL plasma sample was subsequently collected and stored at –20°C until analysis.

Recovery and matrix effect. The evaluation of the matrix effect was tested by comparing the MS/MS response of three compounds at a concentration of 10, 200 and 2000 ng/mL spiked in blank rabbit plasma after deproteinization to the MS/MS responses of the analytes spiked in acetonitrile–water (3:1) solutions at equivalent concentrations (n = 5). The recovery was determined by comparing the MS/MS response of three compounds at a concentration of 10, 200 and 2000 ng/mL spiked in blank rabbit plasma before deproteinization to the MS/MS responses of the analytes spiked in blank rabbit plasma after deproteinization at equivalent concentrations (n = 5).

Pharmacokinetics analysis All plasma concentration data fitting and pharmacokinetic parameter calculations were carried out using analysis software Phoenix WinNonlin (Pharsight, USA). Noncompartmental analysis was chosen for the calculation of pharmacokinetic parameters.

Stability. The effect of three freeze–thaw cycles and the stability of compound in plasma at room temperature for 48 h were determined by repeated analysis (n = 5) of QC plasma samples. Long-term stability in plasma was also tested by assaying frozen plasma samples after storage at –20°C for 1 month. In addition, the stability of plasma samples post-precipitate and left in the autosampler tray for 48 h was tested. The amount of the compounds in plasma samples was determined using a newly prepared calibration curve. The stability of the analytes was expressed as a percentage of nominal concentration.

Results and discussion The method for the simultaneous determination of curdione, furanodiene and germacrone in biological samples was successfully established for the first time in our study. Compared with the published methods for individual analyte determination, the chromatographic run time of the three compounds was shortened from 6–20 min to 5 min, while the lower limit of quantitation of curdione was reduced from 20 to 5 ng/mL. Thus, this

Pharmacokinetic experiments design All animal protocols were approved by Institute Animal Care and Welfare Committee. Japanese female rabbits (2.1–2.3 kg) were obtained from Beijing HFK Bioscience Co. Ltd. The animals were quarantined for 1 week

Table 1. Calibration curves of curdione, furanodiene and germacrone in rabbit plasma Compound

Concentration ranges (ng/mL)

Curdione Furanodiene Germacrone

5–5000 5–5000 10–5000

Regression equation

Correlation coefficient (r2)

y = 0.0109278 + 0.012798x y = 0.00130764 + 0.00476571x y = 0.00201149 + 0.00350812x

0.9989 0.9994 0.9994

y, peak area ratio (analyte/internal standard); x, concentration of compound in plasma (ng/mL).

Table 2. Intra- and inter-day accuracy and precision of HPLC-MS/MS determination of curdione, furanodiene and germacrone in rabbit plasma (n = 5) Analysts

Curdione

Furanodiene

Germacrone

Spiked concentration (ng/mL)

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10 200 2000 10 200 2000 20 200 2000

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Intra-day

Inter-day

Measured concentration Precision (ng/mL) (%RSD) 10.38 ± 0.4698 200.6 ± 6.791 2003 ± 47.87 9.215 ± 0.4944 192.8 ± 4.932 1974 ± 55.93 20.86 ± 1.867 184.4 ± 10.83 1999 ± 50.78

4.5 3.4 2.4 5.3 2.6 2.8 9.0 5.9 2.5

Accuracy (%) 103.8 100.3 100.1 92.1 96.4 98.7 104.3 92.2 99.9

Copyright © 2015 John Wiley & Sons, Ltd.

Measured concentration Precision Accuracy (ng/mL) (%RSD) (%) 10.10 ± 0.3968 204.5 ± 6.521 2101 ± 71.70 10.34 ± 0.9062 192.8 ± 11.52 2094 ± 57.41 20.65 ± 1.857 205.4 ± 19.25 2047 ± 102.1

4.0 3.2 3.4 8.8 6.0 2.7 9.0 9.4 5.0

101.0 102.3 105.0 103.4 96.4 104.7 103.3 102.7 102.3

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101.5 98.1 100.8 102.6 101.9 105.8 108.1 96.4 97.5 10.15 ± 0.1998 196.2 ± 9.724 2016 ± 66.84 10.26 ± 0.6625 203.7 ± 19.17 2116 ± 137.77 21.61 ± 1.266 192.7 ± 18.28 1950 ± 114.5 97.3 101.0 99.5 98.4 98.1 97.8 101.9 99.6 97.7 9.730 ± 0.4273 202.0 ± 5.753 1989 ± 43.37 9.841 ± 0.8215 196.1 ± 5.613 1955 ± 35.71 20.38 ± 2.397 199.1 ± 11.42 1953 ± 43.54

method, characterized by simple procedure of sample preparation, very short chromatographic run time and high sensitivity, was very suitable for the pharmacokinetic study of the three active analytes with extravascular administration.

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Accuracy (%): measured concentration/nominal concentration ×100%.

Germacrone

Furanodiene

10 200 2000 10 200 2000 20 200 2000

10.09 ± 0.3957 199.3 ± 4.499 2038 ± 37.27 9.692 ± 0.8534 192.6 ± 7.811 2005 ± 45.28 20.47 ± 1.169 198.4 ± 5.350 2051 ± 59.67

100.9 99.7 101.9 96.9 96.3 100.2 102.3 99.2 102.6

10.56 ± 0.5839 203.9 ± 3.172 1993 ± 55.73 9.154 ± 0.1571 195.3 ± 3.677 1964 ± 64.07 20.25 ± 3.078 206.6 ± 5.907 1991 ± 70.84

105.6 102.0 99.7 91.5 97.7 98.2 101.2 103.3 99.6

LC-MS/MS conditions

Curdione

Measured concentration Accuracy (ng/mL) (%) Measured concentration Accuracy (ng/mL) (%) Measured concentration Accuracy (ng/mL) (%)

Measured concentration Accuracy (ng/mL) (%)

Three freeze–thaw cycles Room temperature (48 h)

Nominal concentration (ng/mL) Analysts

Figure 4. Plasma concentration–time curves of curdione, furanodiene, germacrone after vaginal drug delivery of Baofukang suppository to rabbits. Each point represents the mean ± SD of three rabbits.

The LC-MS/MS method described in this paper has high sensitivity and specificity that enables the simultaneous determination of the three active components in rabbit plasma. The application of APCI ion source could greatly increase the MS/MS response intensity of three analytes compared with an electrospray ionization source. Formic acid was added to the mobile phase to stimulate the ionization of the analytes. The precursor-to-product ion transitions monitored were chosen according to the signal-to-noise ratio and stability of the chromatographic peaks (Fig. 2). Under the described HPLC conditions, the retention times of curdione, furanodiene, germacrone and the IS were about 1.2, 3.1, 1.7 and 1.2 min, respectively, as shown in Fig. 3. The resolution of method was satisfactory through both chromatographic separation and SRM mode mass detection. No interfering peaks were detected at these retention times in blank plasma samples from rabbit (see example in Fig. 3). The overall chromatographic

Table 4. Mean pharmacokinetic parameters of curdione, furanodiene and germacrone after vaginal drug delivery of Baofukang suppository to rabbits (n = 3) Parameter

Curdione

Furanodiene

Germacrone

t1/2 (h) Cmax (ng/mL) Tmax (h) AUClast (ng/mL h) MRTlast (h) Vz/F (mL/kg) CLz/F (mL/h/kg)

2.14 870.8 1 2,507.85 2.89 4,151.15 1,344.05

2.18 275.7 3 1,445.56 4.07 7,234.837 2,297.04

1.85 118.55 1 460.09 2.86 18,645.65 6,980.98

t1/2, elimination half-life; Cmax, maximum concentration; AUClast, area under the concentration-time curve from time zero to final sample; Vz/F, apparent volume of distribution; CLz/F, apparent oral clearance.

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Table 3. Stability of curdione, furanodiene and germacrone in rabbit plasma (n = 5)

Stored at

20°C for 1 month

Autosampler tray (48 h)

Simultaneous determination of curdione, furanodiene and germacrone

B. Wang et al. run time was finished within 5 min. The retention time was short, and thus the method is suitable for the high number of samples determined in pharmacokinetic studies. Recovery and matrix effects The recovery from each compound in rabbit plasma was 103.7–115.9% for curdione, 84.5–103.3% for furanodiene and 110.8–128.4% for germacrone at final concentrations from 10 to 2000 ng/mL (n = 5). The overall mean matrix factors were around 0.8–0.9 and the CV at each tested concentration was 0.99, indicating a good linear detector response over the dynamic range investigated. The lower limits of quantitation of curdione, furanodiene and germacrone were 5, 5 and 10 ng/mL. This sensitivity has proven useful in the analysis of pharmacokinetic data.

method are summarized in Table 4. After vaginal drug delivery of Baofukang suppository (one suppository for each rabbit), all the three active analytes could be detected at 0.25 h in the rabbit plasma, which suggested that the entrance of three compounds into plasma via vaginal mucosa was fast. The results also revealed that the distribution and elimination processes of three compounds might be quick, as indicated by the short time of maximum concentration (Tmax) (1–3 h) and mean residence time from the time of dosing to the time of the last measurable concentration (MRTlast) (2.86–4.07 h). All the data shown here indicate that the method established in this study can be used in pharmacokinetic studies of Baofukang suppository or other forms of pharmaceutics/herbal extracts containing the three active compounds. In conclusion, an LC-MS/MS method has been developed that showed excellent sensitivity, good linearity of response and high precision for the simultaneous quantification of curdione, furanodiene and germacrone in rabbit plasma and successfully applied to the preliminary pharmacokinetic study of three compounds following vaginal drug delivery of Baofukang suppository to female rabbits.

Acknowledgments We are very grateful to Hainan Bikai Pharmaceutical Company for financially support of the work and providing the purified analytes and Baofukang suppository.

Precision and accuracy of the detection method Analytical accuracy and precision data are shown in Table 2. The reproducibility of the method was defined by examining both intra- and inter-day variance. The intra-day and inter-day CVs of three analytes at low to high concentrations were 85% at the concentration range from 10 to 2000 ng/mL. Three freeze–thaw cycles of the QC samples appeared to have no effect on quantification of the analytes. Test compounds remained stable when stored at –20°C for a 1 month period with their mean recoveries from the nominal concentration >90%. The analytes in plasma samples post precipitate were also approved stable for 48 h when placed in the autosampler tray. Pharmacokinetic study of three compounds in rabbit The utility of LC-MS/MS assay for the quantitative analysis of three analytes in rabbit plasma in support of in vivo studies was demonstrated. The mean plasma concentration vs time profiles of three analytes are shown in Fig. 4 and the main pharmacokinetic parameters assessed using the noncompartmental

References Deng C, Ji J, Li N, Yu Y, Duan G and Zhang X. Fast determination of curcumol, curdione and germacrone in three species of Curcuma rhizomes by microwave- assisted extraction followed by headspace solid-phase microextraction and gas chromatography–mass spectrometry. Journal of Chromatography A 2006; 1117: 115–120. Ma J, Dong J, Lin G, Yue L, Xiang Z, Xu R, Hu L and Wang X. Determination of curdione in rabbit plasma by liquid chromatography mass spectrometry. Biomedical Chromatography 2012; 26: 655–659. Manzan AC, Toniolo FS, Bredow E and Povh NP. Extraction of essential oil and pigments from Curcuma longa [L] by steam distillation and extraction with volatile solvents. Journal of Agricultural and Food Chemistry 2003; 51: 6802–6807. Matsuda H, Morikawa T, Ninomiya K and Yoshikawa M. Hepatoprotective constituents from zedoariae rhizoma: absolute stereostructures of three new carabrane-type sesquiterpenes, curcumenolactones A, B, and C. Bioorganic and Medicinal Chemistry 2001; 9: 909–916. Pei L, Liu S, Zheng J and Chen X. A sensitive method for determination of furanodiene in rat plasma using liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study. Biomedical Chromatography 2012; 26: 826–832. Wang Y and Wang M. Study on the quality of rhizoma curcumae. Yaoxue Xuebao 2001; 36: 849–853. Xia Q, Huang Z, Li S, Zhang P, Wang J and He L. The experiment study of the anti-virus effects of zedoary oil on influenzavirus and respiratory syncytial virus. Chinese Pharmacological Bulletin 2004; 20: 357–358. Yang FQ, Wang YT and Li SP. Simultaneous determination of 11 characteristic components in three species of Curcuma rhizomes using pressurized liquid extraction and high-performance liquid chromatography. Journal of Chromatography A 2006; 1134: 226–231. You J, Cui F, Li Q, Wang Y, Han X and Yu Y. A HPLC method for the analysis of germacrone in rabbit plasma and its application to a pharmacokinetic study of germacrone after administration of zedoary turmeric oil. Journal of Chromatography B 2005; 823: 172–176.

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Biomed. Chromatogr. 2015; 29: 1393–1398

Simultaneous quantification of curdione, furanodiene and germacrone in rabbit plasma using liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study.

A simple, rapid and sensitive method was developed for the simultaneous quantification of curdione, furanodiene and germacrone in rabbit plasma using ...
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