Journal of Ethnopharmacology 169 (2015) 305–313
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Simultaneous determination of seven anthraquinones in rat plasma by Ultra High Performance Liquid Chromatography–tandem Mass Spectrometry and pharmacokinetic study after oral administration of Semen Cassiae extract Chunjuan Yang 1, Shuhong Wang 1, Xiaowei Guo, Jiahui Sun, Lu Liu, Lijun Wu n College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang Distrct., Harbin 150081, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 September 2014 Received in revised form 17 March 2015 Accepted 10 April 2015 Available online 20 April 2015
Ethnopharmacological relevance: Semen Cassiae, called Juemingzi in China, is the seed of the annual Cassia obtusifolia L., of the leguminosae family. It has been used as healthy drinks to alleviate constipation and improve eyesight for many years in China. Aim of the study: A simple sensitive UHPLC–MS/MS method has been developed and validated for the simultaneous determination and pharmacokinetic study of chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantio-obtusin in rat plasma. Materials and methods: Chromatographic separation was accomplished on a C18 column with a 5 min gradient elution. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source and operating in the negative ionization mode. The samples were prepared by LLE with ethyl acetate after being spiked with an internal standard (butylparaben). Results: The current UHPLC–MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability. The method was linear for all analytes over investigated range with all correlation coefficients greater than 0.9900. The lower limit of quantification (LLOQ) of each analyte was lower than 5 ng/mL. Intra-day and inter-day precisions were less than 14.99%. The relative errors of accuracies were in the range of 14.60% to 5.11%. The mean recoveries and matrix effects of anthraquinones were higher than 65.54% and 93.26%, respectively. After oral administration 1.25 g/kg of Semen Cassiae extract, the maximum plasma concentration (Cmax) was 1189.25 7 333.40 ng/mL for chrysophanol, 38.48 7 3.15 ng/mL for emodin, 79.20 7 34.76 ng/mL for aloe-emodin, 152.707 23.91 ng/ mL for rhein, 461.857 266.77 ng/mL for physcion, 243.59 722.71 ng/mL for obtusifolin and 1950.44 7638.86 ng/mL for aurantio-obtusin, respectively. The time to reach the maximum plasma concentration (Tmax) was 0.333 7 0.071 h for chrysophanol, 0.333 7 0.059 h for emodin, 0.333 7 0.009 h for aloe-emodin, 0.333 70.09 h for rhein, 0.16770.002 h for physcion, 0.5 70.074 h for obtusifolin and 0.333 70.06 h for aurantio-obtusin, respectively. Conclusion: The proposed method was further applied to investigate the pharmacokinetics of seven anthraquinones after oral administration of Semen Cassia extract to rats. & 2015 Elsevier Ireland Ltd. All rights reserved.
Chemical compounds studied in this article: Chrysophanol (PubChem CID:10208) emodin (PubChem CID:3220) aloe-emodin (PubChem CID:10207) rhein (PubChem CID:10168) physcion (PubChem CID:10639) obtusifolin (PubChem CID:3083575) aurantio-obtusin (PubChem CID:155011) Keywords: UHPLC–MS/MS Semen Cassiae Anthraquinones Pharmacokinetics Rat plasma
1. Introduction Traditional Chinese Medicine (TCM) and its preparations have been widely used in China for thousands years due to its special efficacy in clinical treatment of disease. Semen Cassiae, called Juemingzi in China,
Abbreviations: (UHPLC–MS/MS), Ultra High Performance Liquid Chromatography–tandem Mass Spectrometry; (LLE), liquid—liquid extraction n Corresponding author. Tel.: þ 86 451 86681103; fax: þ 86 451 86614073. E-mail address: [email protected] (L. Wu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jep.2015.04.008 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
is the seed of the annual Cassia obtusifolia L., of the leguminosae family. It is easily grown, widely cultivated in China and Korea, and commonly drunk as a kind of roasted tea. The seeds are reported to have the effects of alleviating constipation and improving eyesight, lowering hypertension and hyperlipidemia which are anchoring and nourishing the liver (Hao et al., 2001; Chen and Chen, 2001; Jiangsu New Medical College, 1975). Semen Cassiae also exhibits antidiabetic effects and neuroprotective effects in the brain disease models (Cho et al., 2005; Guan and Zhao, 1995; Kim et al., 2007). It has been reported that a number of constituents of this plant were isolated, including naph thopyrones, anthraquinones, anthrones, flavonoids, and triterpenoids
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(Kitanaka et al., 1985; Takido, 1958; Zhang et al., 2009).The anthraquinone derivatives, anthronic, dianthronic, and anthraquinone glycosides of Cassia are responsible for the purgative action. In our previous investigation, seven anthraquinones, chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantio-obtusin, have been isolated and identified from the seed of Semen Cassiae. All of them are active compounds of the herbal preparation with pharmacological effects. Aloe-emodin and chrysophanol were found to have antimicrobacterial activity (Smolarz et al., 2013). Weber (2013) discovered that emodin, aloe-emodin and rhein possessed antitumor activities by targeting the apoptosis pathway in cancer, and these compounds possessed antiinflammatory and antiviral effects. Furthermore, emodin and aloe-emodin suppress breast cancer cell proliferation through ER α inhibition (Huang et al., 2013) and rhein also have antitumor, anticancer and hemostatic properties (You et al., 2013). Jang et al., 2007 demonstrated that emodin and obtusifolin exhibited a significant inhibitory activity on glycation end products formation, and aurantio-obtusin, and emodin showed a significant inhibitory activity on rat lens aldose reductase. In addition, physcion will be a potential candidate in the field of anticancer drug discovery against human cervical cancer (Wijesekara et al., 2014). Various analytical methods including High Performance Liquid Chromatography (Huang et al., 2010; Xu et al., 2012), Ultraviolet Spectrophotometry (Zhang et al., 2007), Capillary Electrophoresis (Koyama et al., 2003) and Ultra High Performance Liquid Chromatography coupled with Mass Spectrometry (Zhang et al., 2012) have been used in the qualitative or quantitative analysis of anthraquinones. Although many studies have reported the role of Semen Cassiae in disease prevention, and many pharmacokinetic researches about the anthraquinones from other plants or decoctions have been presented, few papers have reported integrated data on its pharmacokinetic properties in Semen Cassiae (Fang et al., 2011; Zhang et al., 2013; Yan et al., 2008; Li et al., 2013; Gong et al., 2011; Zhu et al., 2005; Shia et al., 2011). Feng et al. (2014) presented the pharmacokinetics of five rhubarb anthraquinones in dog plasma by HPLC after orally administration the rhubarb extract. Zhang et al. (2014) established a HPLC–ESI–MS/MS method to determined four active components of Semen Cassiae extract (aurantio-obtusin, chrysoobtusin, obtusin and 1-desmethylobtusin) in rat plasma after oral administration and provided their pharmacokinetic characteristics. So it is important to develop a highly sensitive, rapid and reproducible analytical method for quantifying these compounds in plasma to characterize the precise pharmacokinetic properties of anthraquinone derivatives. In this study, a rather sensitive and selective UHPLC–MS/MS method was first developed and validated for simultaneously determining chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantio-obtusin in rat plasma. The advantage of this paper is the separation of the up to seven active compounds with similar chemical structure in only five minutes. The method was applied to pharmacokinetics after oral administration of Semen Cassiae extract to rats. It was expected that the results of this study would provide some references to the further pharmacological study of Semen Cassiae.
2. Experimental 2.1. Materials and reagents Reference standards of chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantio-obtusin (Fig. 1.) were isolated from Semen Cassiae and their structures were elucidated by comparison of spectral data (UV, MS, 1H NMR and 13C NMR) with the literature values. Butylparaben was purchased from
Guangfu Fine Chemical Research Institute (Tianjin, PR China) and used as an internal standard (I.S.). HPLC grade methanol and acetonitrile were purchased from J&KMEDICAL (Beijing, China). Formic acid of HPLC grade was obtained from DIKMA (Lake Forest, USA). Ammonium acetate of HPLC grade was bought from Kermel (Tianjin, China). All other reagents were of analytical grade. Ultrapure water was prepared by using a Milli-Q water purification system (Millipore, Molsheim, France). Parched Semen Cassiae was purchased from Beijing Tongrentang (Beijing, china) and authenticated by Prof. Zhenyue Wang in Heilongjiang University of Chinese Medicine. A voucher specimen (Art. NO. 01216800) was deposited in college of pharmacy, Harbin Medical University, China. 2.2. Instruments and analytical conditions The UHPLC system was Agilent technology 1290 series equipped with an automatic degasser, a quaternary pump, and an autosampler. Chromatographic separation was achieved on a C18 column (ACQUITY UPLCs HSS T3 1.8 μm 2.1 100 mm) at 40 1C. Mobile phase composed of (A) acetonitrile and (B) aqueous ammonium acetate (5 mM) with a gradient elution as shown in Table SI. 1 was used. The flow rate was 0.4 mL/min and the analysis time was 5.0 min for each injection. A 5 μL aliquot of sample solution was injected, with a needle wash process which is used to wash the outer wall of sample needle after each injection. The mass spectrometric detection was performed on a 6430 triple-quadrupole mass spectrometer (Agilent, US) with electrospray ionization (ESI) interface set in negative ionization mode. An Agilent Mass Hunter workstation (Agilent, US) was used to control the equipment and for data acquisition and analysis. The quantification was obtained using multiple reaction monitoring (MRM) of precursor-product ion transition at m/z 253.2-225.1 for chrysophanol, m/z 269.0-225.1 for emodin, m/z 269.0-240.1 for aloe-emodin, m/z 283.0-239.0 for rhein, m/z 283.2-240.0 for physcion, m/z 283.1-268.1 for obusifolin, m/z 329.1-298.9 for aurantio-obtusin and m/z 193.0-92.0 for butylparaben (I.S.), respectively (Fig. 2). The parameters of each compound were listed in Table SI. 2 . Other parameters of the mass spectrometer were set as follows: a drying gas flow of 11 L/min; a drying gas temperature of 300 1C; a nebulizer pressure of 15 psi and a capillary voltage of 4000 V. 2.3. Preparation of extract of Semen Cassiae For the extract preparation, dried powder (200 g) of Semen Cassiae was extracted under reflux with 75% ethanol at a rate of material and liquid 1:6 for three times, 1 h each time, and then filtrated. The combined filtrate was evaporated to dryness, and the residue was reconstituted in water to get a concentration equivalent to 0.1 g/mL of the Semen Cassiae extract. 2.4. Preparation of calibration standards and quality control (QC) samples The stock solutions of chysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin, aurantio-obtusin and the I.S. (butylparaben) were individually prepared in methanol. The stock solutions of the standards were further diluted in methanol to produce combined standard working solutions at a series of concentrations. The I.S. solution (2000 ng/mL) was obtained by diluting the stock solution in methanol. The samples for standard calibration curves were prepared by spiking appropriate amount of the standard solutions in 200 μL blank plasma to yield calibration concentrations of 4.74–4854 ng/mL for chysophanol; 4.97–5090 ng/mL for emodin; 2.29–2346.5 ng/mL for aloe-emodin; 4.71–4826 ng/mL for rhein; 1.82–1868 ng/mL for
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physcion; 2.49–2550 ng/mL for obtusifolin and 2.52–2580 ng/mL for aurantio-obtusin, respectively. All the stock and working solutions were stored at 4 1C and brought to room temperature before use. The QC samples were prepared at concentrations of 9.71, 485, 3883 ng/ mL for chysophanol; 10.2, 509, 4072 ng/mL for emodin; 4.69, 235, 1877 ng/mL for aloe-emodin; 9.65, 483, 3861 ng/mL for rhein; 3.74, 187, 1494 ng/mL for physcion; 5.10, 255, 2040 ng/mL for obtusifolin and 5.16, 258, 2064 ng/mL for aurantio-obtusin in drug-free plasma. The spiked samples were processed according to the following section.
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2.5. Animal experiments Male Sprague–Dawley rats (body weight 220 750 g) were purchased and adapted under 65% RH, 25 1C. The animal handling procedures were approved by the Institutional ethical committee and conformed to the principles of the International Guide for the Care and Use of Laboratory Animals. The SD rats were fasted for 12 h before experiment, had free access to water even during the experiment. The blood (0.4 mL) was collected from the orbital venous plexus before administration and at 0.083, 0.167, 0.333, 0.5,
Fig. 2. MS/MS fragmentation patterns of (A) chrysophanol; (B) emodin; (C) aloe-emodin; (D) rhein; (E) physcion; (F) obtusifolin; (G) aurantio-obtusin; (H) I.S.
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1, 2, 3, 4, 6, 8 and 12 h after oral administration of Semen Cassiae extract at a dose of 1.25 g/kg body weight. The plasma was immediately acquired by centrifugation and stored frozen at 20 1C until analysis. 2.6. Plasma sample preparation After thawing the plasma samples at room temperature for 15 min, liquid–liquid extraction was applied for the sample preparation. I.S. 50 μL (2000 ng/mL), 100 μL methanol and 50 μL glacial acetic acid were added to rat plasma sample (200 μL). After vortexing for 60 s, a 3 mL aliquot of ethyl acetate was added. The mixture was vortexed for 2 min and then centrifuged for 5 min at 3000 r/min. The upper organic layer was removed and evaporated to dryness at 40 1C under a stream of nitrogen. The residue was then reconstituted in 100 μL acetonitrile, vortexed for 30 s and filtered by a 0.22 μm membrane. A 5 μL aliquot of the solution was injected into the UHPLC–MS/MS system for analysis. 2.7. Method validation The current UHPLC–MS/MS assay was validated for selectivity, linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability in accordance to the requirements for the analysis of biological samples. The validation runs were conducted on three consecutive days. Each validation run consisted of two sets of calibration standards and six replicates of QC samples at three concentrations. The peak area ratios of the seven analytes to I.S. of the QC samples interpolated from the calibration curve on the same day were used to calculate the concentrations of the seven analytes. The results from QC samples in the three runs were used to evaluate the intra-day and inter-day precision and accuracy of the method. 2.7.1. Specificity and selectivity Selectivity is the ability of an analytical method to differentiate and quantify the analytes in the presence of other components in the sample. In this paper, the selectivity was ascertained by comparatively analyzing blank plasma samples from six individual rats, corresponding blank plasma spiked with the seven analytes and I.S. and the plasma samples from the rats after oral administration of the Semen Cassiae extract. 2.7.2. Linearity and lower limits of quantification (LLOQ) Calibration curves were prepared by assaying standard plasma samples at seven concentration levels. The linearity of each calibration curve was determined by plotting the peak area ratio (y) of analytes to I.S. versus the nominal concentration (x) of analytes with a weighted (1/x2) least square linear regression. The LLOQ was defined as the lowest analytical concentration of the calibration curve with at which the measured precision expressed as relative standard deviation (R.S.D.) was within 720% and the accuracy expressed as relative error (R.E.) was within 7 20%. Six replicate samples were used for evaluation. 2.7.3. Precision and accuracy Three validation batches, each containing six replicates of QC samples at low, medium and high concentration levels were assayed to assess the precision and accuracy of the method at three different days. The intra-day and inter-day precisions were defined as relative standard deviation with criteria of less than 15%; the accuracy was assessed by comparing the measured concentration with its nominal value with a criterion of within 715% for all QC samples.
2.7.4. Extraction recovery and matrix effect The extraction efficiency of the seven analytes was determined by analyzing six replicates of plasma samples at LQC, MQC and HQC levels. To investigate recovery and matrix effect, extracted samples, unextracted samples (pure standard solution), and postextracted spiked samples at three QC levels were analyzed in the same assay. The recovery was obtained by comparing peak areas of analytes in extracted samples with those in post-extracted spiked samples. The matrix effect was evaluated by determining the peak area ratios of the analytes in post-extracted spiked samples to those acquired from unextracted samples. 2.7.5. Stability experiments The stability of the seven analytes in samples including freeze– thaw stability (three freeze at 20 1C and thaw cycles), long-term stability (storage for 2 weeks at 20 1C), room temperature stability (storage for 4 h at ambient temperature), postpreparation stability (storage for 12 h after sample preparation at 4 1C) was tested at LQC, MQC, HQC levels with six replicates at each level. All stability testing QC samples were determined by using the calibration curve of freshly prepared standard samples. 2.8. Application to pharmacokinetic study The validated method was applied to pharmacokinetics after oral administration of Semen Cassiae extract to rats. The maximum plasma concentration (Cmax) and the time of maximum plasma concentration (Tmax) were observed directly from the measured data. The elimination rate constant (Ke) was calculated by linear regression of the terminal points in a semi-log plot of the plasma concentration against time. The elimination half-life (t1/2) was calculated using the formula t1/2 ¼0.693/ Ke. The area under plasma concentration–time curve (AUC0–t) to the last measurable plasma concentration (Ct) was estimated by using the linear trapezoidal rule. The area under the plasma concentration–time curve to time infinity (AUC0–1) was calculated as AUC0–1 ¼ AUC0–t þCt/Ke. 3. Results and discussion 3.1. Optimization of chromatographic mass spectrometric The first step in the method development was to select precursor ions and product ions of the analytes and I.S., meanwhile, their precursor ions and product ions were ascertained for use in MRM. The anthraquinones are polyhydroxy compounds, easily falling off the proton, and the response signal is higher in negative ionization mode than positive ionization mode in theory. We found that these compounds were corresponding well in the negative ionization mode and we did not detect the molecular ion peaks in the positive ionization mode. Thus, negative ionization mode was finally employed. Fig. 2 shows the ion pairs of the seven analytes and I.S. The parameters for fragmentor energy and collision energy were optimized in order to get the richest relative abundance of precursor and product ions. Table SI. 2 shows the MS/MS transitions and energy parameters of all the compounds. Chromatographic conditions were optimized to improve peak shape, increase sensitivity and shorted run time for simultaneous analysis of seven components. Several mobile phase systems were tried in order to choose the optimal mobile phase that produces the best response, sensitivity and separation efficiency. Mobile phase systems such as methanol–water and acetonitrile–water were tested, and different buffers including ammonium acetate (2 and 5 mM), formic acid (0.1%) and acetic acid (0.2%) were added to the system. Finally, acetonitrile and 5 mM ammonium acetate in water were the best solvent composition. Satisfactory separation
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Fig. 3. Chromatograms of the seven components and I.S. in plasma: (A) blank plasma; (B) blank plasma spiked with the seven analytes and I.S. (LQC); (C) plasma sample obtained at 0.5 h from a rat after oral administration of Semen Cassiae extract: channel 1 for I.S.; channel 2 for chrysophanol; channel 3 for emodin; channel 4 for aloeemodin; channel 5 for rhein; channel 6 for physcion; channel 7 for obtusifolin and channel 8 for aurantio-obtusin.
was achieved in 5 min by gradient elution using a C18 column (ACQUITY UPLCs HSS T3 1.8 μm 2.1 100 mm) at 40 1C with a flow rate of 0.4 mL/min. As shown in Fig. 3, there were no
endogenous plasma components interfering with the analytes when the above-mentioned mobile phase was used. Compared the elution profiles of blank plasma spiked with the seven analytes
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and I.S. with that of real plasma sample, it can be seen that the satellite and addition peaks in the chromatograms of the real plasma sample, such as compounds 1, 3, 4 and 7 in Fig. 3B and C. Table 1 The regression equations, linear ranges and LLOQs for the determination of the analytes in rat plasma. Compounds
Regression equation
R2
Linear range (ng/mL)
LLOQ (ng/mL)
Chrysophanol Emodin Aloe-emodin Rhein Physcion Obtusifolin Aurantioobtusin
Y ¼ 0.4621X þ0.0021 Y ¼ 1.361X þ 0.0078 Y ¼ 0.4974X 1.154 10 4 Y ¼ 0.1358X þ5.826 10 4 Y ¼ 17.63X þ 0.0083 Y ¼ 4.859X þ0.0154 Y ¼ 0.2517Xþ 5.460 10 4
0.9932 0.9939 0.9946 0.9900 0.9949 0.9971 0.9906
4.74–4854 4.97–5090 2.29–2346 4.71–4826 1.82–1868 2.49–2550 2.52–2580
4.74 4.97 2.29 4.71 1.82 2.49 2.52
This phenomenon may be caused by the other isomers or compounds absorbed into blood, and they have the same molecular mass in precursor and product ions after one or more cleavage. So we optimized the liquid phase conditions to adjust the retention time so as to obtain the suitable resolution of the seven analytes. 3.2. Sample preparation In our experiment, liquid–liquid extraction (LLE) and protein precipitation were compared during sample preparation. Initially, we used the protein precipitation method, 3 mL methanol (or acetonitrile) was added for protein precipitation, and it was not selected because of its high MS/MS noise level. For LLE, organic extracting solvent including ethyl acetate, diethyl ether and dichloromethane was used. It was observed that LLE with ethyl acetate could obtain clean, simple, reproducible extraction and
Table 2 Precision and accuracy of the determination of seven anthraquinones in rat plasma (n¼18, 6 replicates per day for 3 days). Compounds
Spiked concentration (ng/mL)
Measured concentration (ng/mL)
Accuracy (%)
Intra-day precision (%)
Inter-day precision (%)
Chrysophanol
9.71 485 3883 10.2 509 4072 4.69 235 1877 9.65 483 3861 3.74 187 1494 5.10 255 2040 5.16 258 2064
9.81 71.37 415 750.0 3719 7503 9.40 71.34 491 733.7 3974 7400 4.49 70.63 218 728.6 1614 7139 8.69 71.20 422 753.3 3397 7271 3.65 70.50 181 718.7 1349 7187 4.98 70.68 230 722.8 1917 7271 4.88 70.56 257 737.9 2169 7270
1.08 14.60 4.22 7.68 3.50 2.40 4.31 7.08 14.00 9.97 12.47 12.02 2.25 2.94 9.72 2.32 9.90 6.02 5.52 0.35 5.11
14.06 12.82 14.09 14.45 7.15 10.65 14.38 13.53 9.17 14.47 13.27 8.33 14.36 10.26 14.72 14.37 10.37 14.93 12.13 14.99 12.64
12.85 1.33 8.25 11.57 4.05 3.20 9.46 1.12 6.45 6.45 5.81 4.79 6.85 10.54 3.73 5.34 5.68 6.06 3.51 12.74 11.02
Emodin
Aloe-emodin
Rhein
Physcion
Obtusifolin
Aurantio-obtusin
Table 3 Matrix effects and extraction recovery for the analytes in rat plasma (n¼ 6). Compounds
Chrysophanol
Emodin
Aloe-emodin
Rhein
Physcion
Obtusifolin
Aurantio-obtusin
I.S.
Spiked concentration (ng/mL)
9.71 485 3883 10.2 509 4072 4.69 235 1877 9.65 483 3861 3.74 187 1494 5.10 255 2040 5.16 258 2064 2000
Matrix effect
Extraction recovery
Mean(%)
RSD (%)
Mean (%)
RSD (%)
97.97 96.06 100.8 102.5 95.21 101.6 96.91 93.26 99.60 102.7 95.30 98.42 100.1 86.84 97.97 97.42 101.2 97.35 101.2 101.6 105.3 96.12
7.194 1.725 5.083 4.026 2.537 1.193 7.529 12.34 6.232 2.830 11.70 10.88 4.932 2.811 9.111 3.286 12.01 2.672 6.657 5.710 4.545 3.756
74.51 78.44 77.98 78.80 72.18 76.60 73.33 83.74 80.37 68.31 65.54 72.43 81.48 84.63 80.44 76.99 82.36 84.41 74.42 79.28 81.33 80.49
13.65 2.500 10.82 2.922 3.398 4.181 7.404 5.666 9.627 6.255 9.919 3.330 5.495 5.791 11.40 3.928 3.162 1.900 8.358 6.669 1.982 3.741
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Table 4 Stabilities of the analytes in rat plasma (n¼ 6). Analytes
Chrysophanol
Emodin
aloe emodin
rhein
physcion
Obtusifolin
Aurantio-obtusin
a
Spiked concentration (ng/mL)
9.71 485 3883 10.2 509 4072 4.69 235 1877 9.65 483 3861 3.74 187 1494 5.10 255 2040 5.16 258 2064
Stability (% REa) Three freeze–thaw
Short-term
Long-term
Post-preparative
8.20 11.10 8.51 1.35 4.39 0.86 4.31 2.11 12.32 0.96 0.83 9.47 10.15 1.87 8.46 8.27 7.32 8.62 12.49 6.34 14.17
6.99 1.11 8.56 5.70 2.02 1.73 7.71 1.39 6.72 4.03 9.14 5.25 3.40 7.14 0.94 5.29 6.68 0.53 6.84 6.41 7.66
9.41 13.39 13.63 7.70 6.86 2.81 10.77 3.43 5.71 0.16 6.35 7.64 5.40 10.68 1.75 0.90 5.45 2.10 3.64 5.64 0.63
8.07 4.75 5.70 10.25 8.39 0.89 2.34 7.65 9.29 7.62 2.99 12.23 4.53 1.43 3.62 3.86 3.37 0.26 8.53 3.39 9.76
RE is expressed as (measured concentration/freshly prepared concentration 1) 100%.
good recovery for the seven analytes. During the study, glacial acetic acid was considered as a critical factor for the recovery of the analytes. Different amounts (20, 50 and 100 μL) of glacial acetic acid were tested in the experiment. At last, 50 μL of glacial acetic acid was used to improve the extract efficiency. 3.3. Method validation 3.3.1. Specificity and selectivity The selectivity of the method towards endogenous plasma matrix was evaluated with plasma from six rats. Eight channels were used for recording and the retention times of chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantioobtusin were 3.8, 3.0, 2.8, 1.3, 4.0, 3.1 and 2.4 min, respectively. Typical chromatograms obtained from a blank, a spiked plasma sample with the analytes and I.S. (LQC), and 0.5 h plasma sample after an oral administration of Semen Cassiae extract are shown in Fig. 3. All the peaks of the analytes and I.S. were detected with excellent resolution as well as peak shapes, and no interference from the endogenous substances was observed at the retention time of the analytes and I.S. The analytes could be easily differentiated from the rat plasma matrix and quantitatively determined at the LLOQ level. 3.3.2. Linearity and lower limits of quantification The regression equation, correlation coefficients and linearity ranges for seven analytes are shown in Table 1. All correlation coefficients were higher than 0.9900. They all exhibited good linearity. The LLOQ of chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin, and aurantio-obtusin were 4.74, 4.97, 2.29, 4.71, 1.82, 2.49 and 2.52 ng/mL, respectively. 3.3.3. Precision and accuracy In this assay, the intra-day and inter-day precisions and accuracy were determined. Six replicates of QC samples at three concentration levels (LQC, MQC, HQC) of seven analytes on the same day and on three different days were tested. The results are listed in Table 2.
Fig. 4. Mean plasma concentration–time profiles of rhein and obtusifolin (A), chrysophanol, aurantio-obtusin and physcion (B), emodin and aloe-emodin (C) in rats after oral administration of Semen Cassiae extract. Values are expressed as means 7SD (n¼ 6).
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Table 5 Pharmacokinetic parameters of the seven constituents in rats after oral administration of Semen Cassiae extract (mean 7 S.D., n ¼6). Compounds
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUC0-t (ng h/mL)
AUC0-1 (ng h/mL)
Chrysophanol Emodin Aloe-emodin Rhein Physcion Obtusifolin Aurantio-obtusin
1189.25 7 333.4 38.487 3.15 79.20 7 34.76 152.707 23.91 461.85 7 266.77 243.597 22.71 1950.447 638.86
0.3337 0.071 0.3337 0.059 0.3337 0.009 0.3337 0.09 0.1677 0.002 0.5 7 0.074 0.3337 0.06
6.12 72.37 6.40 73.31 24.62 77.27 10.30 75.02 10.46 71.46 7.21 73.66 13.78 73.03
642.21 7119.51 39.75 72.30 80.84 750.76 199.81 722.47 218.42 715.08 235.90713.11 874.50 7103.70
650.58 7 126.26 42.83 7 6.38 106.797 71.88 208.63 7 26.50 224.417 16.84 238.94 7 15.84 991.34 7 114.65
At each QC level, the inter- and intra-day precisions (R.S.D) of seven analytes were 1.12–14.99%, the accuracy was 14.60–5.11%. These results indicated that the developed method was precise and accurate. 3.3.4. Extraction recovery and matrix effect The data of extraction recovery and matrix effect of the seven analytes are summarized in Table 3. The extraction solvent used in the experiment showed good extraction efficiency. Mean absolute recoveries of chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantio-obtusin were 74.51–78.44%, 72.18– 78.80%, 73.33–83.7%, 65.54–72.43%, 80.4–84.6%, 76.99–84.4% and 74.42–81.3% at three QC levels. These results demonstrated that the values were all in the acceptable ranges. The matrix effects derived from QC samples were between 93.2% and 105.3%, and I.S. was 96.1 73.756%. These results confirmed that the evaluated method was free from any matrix effect. 3.3.5. Stability experiments The stability of all the analytes was assessed under various conditions. The results presented in Table 4 indicate that the seven analytes were all stable in plasma after three freeze–thaw cycles, at room temperature for 4 h. Post-preparative stability of the analytes also showed that no significant degradation occurred when the extracted samples were kept at 4 1C for 12 h. Moreover, all the investigated compounds were stable for 2 weeks when kept frozen at 20 1C.
from 6.12 h to 24.62 h, which suggests that the seven components were rapidly absorbed in the stomach and eliminated in rat plasma after oral administration of Semen Cassiae extracts. In other assays (Kitanaka et al. 1985), a double-peak phenomenon appeared on the mean plasma concentration–time profiles of some of the seven compounds, which indicated that enterohepatic circulation could be involved in their absorption process or may be induced by metabolic transformation through the demethylation pathway between these compounds. In this study, only rhein showed minor double-peak in the mean plasma concentration– time curve. These results should be useful for further studies on the pharmacokinetics, pharmacy and toxicity of the TCM Semen Cassiae and should support studies seeking to determine the efficacy of this TCM in clinical therapeutic research.
4. Conclusion This validated UHPLC–MS/MS method was sensitive, accurate and fast, and met all requirements in bioanalysis. This is the first paper to simultaneously determinate of seven anthraquinones in rat plasma after oral administration of Semen Cassiae extract. In addition, this method has been successfully applied to pharmacokinetic study of seven components following oral administration of Semen Cassiae extract to rats and it is the first pharmacokinetic study of obtusifolin. The results might be helpful for investigating the bioactivity mechanism and clinical application of Semen Cassiae.
3.3.6. Application to the pharmacokinetic study The developed UHPLC–MS/MS method was applied to investigate the pharmacokinetics of the seven constituents in Semen Cassiae after a single oral administration of 1.25 g/kg (equivalent to 0.66 mg/g for chrysophanol, 0.02 mg/g for emodin, 0.04 mg/g for aloe-emodin, 0.08 mg/g for rhein, 0.3 mg/g for physcion, 5.01 mg/g for obtusifolin and 0.95 mg/g for aurantio-obtusin) to six rats. We chose an oral dosage for the rats corresponding to the human dosage in Chinese Pharmacopoeia (China Pharmacopoeia Committee, part I, 2010). The human dosage of Semen Cassiae in Chinese Pharmacopoeia is 15 g/day, so we picked the final oral dosage as 1.25 g/kg in rats based on the Meeh-Rubner method. The mean plasma concentration–time curves (n ¼6) of the analytes are shown in Fig. 4. The pharmacokinetic parameters including halftime (t1/2), maximum plasma concentration (Cmax), time to reach the maximum concentrations (Tmax), area under concentration– time curve (AUC0Ut and AUC0U1) calculated by the noncompartment model are presented in Table 5. As shown in Table 5, the range of Cmax of these seven anthraquinones was from 38.48 73.15 to 1950.447 638.86 ng/mL because of the great differences of the contents of the seven compounds in the Semen Cassiae extract. The range of AUC0Ut was from 39.75 72.30 to 874.50 7103.70 ng/mL/h. All the seven analytes achieved the maximum plasma concentration within 0–0.5 h after oral administration, meanwhile, their half life of elimination (T1/2) ranged
Acknowledgment Our work was supported by the Scientific Research Project of National Natural Science Foundation of China (No. 81202875) and Heilongjiang Province Education Department (No. 12521201).
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Feng, S.X., Li, J.S., Wang, S.M., Yang, R., Zhou, T.Q., Li, X.Y., Qu, L.B., 2014. Simultaneous determination and pharmacokinetics of five rhubarb anthraquinones in dog plasma by HPLC after orally administration the rhubarb extract. Pak. J. Pharm. Sci. 27, 847–854. Gong, H., Tang, W., Wang, H., Xia, Q., Huang, X., 2011. Effects of food and gender on the pharmacokinetics of rhein and emodin in rats after oral dosing with DaCheng-Qi decoction. Phytotherapyresearch 25, 74–80. Hao, Y.J., Sang, Y.L., Zhao, Y.Q, 2001. The advancement of the studies on the seeds of Cassia obtusifolia. Chin. Tradit. Herb. Drugs 32, 858–859. Huang, P.H., Huang, C.Y., Chen, M.C., Lee, Y.T., Yue, C.H., Wang, H.Y., Lin, H., 2013. Emodin and aloe-emodin suppress breast cancer cell proliferation through ER alpha Inhibition. Evid.-based Complement. Altern. Med.:eCAM 2013, 376123. Huang, X., Zhong, G., Zhang, X., Zhang, Y., 2010. Determination of aurantio-obtusin and chrysophanol in cassiae semen by HPLC. China J. Chin. Mater. 35, 2065–2067. Jang, D.S., Lee, G.Y., Kim, Y.S., Lee, Y.M., Kim, C.S., Yoo, J.L., Kim, J.S., 2007. Anthraquinones from the seeds of Cassia tora with inhibitory activity on protein glycation and aldose reductase. Biol. Pharm. Bull. 30, 2207–2210. Jiangsu New Medical College, 1975. Encyclopedia of Chinese MateriaMedica, I. Shanghai Science and Technology Press, Shanghai, China p. 949. Kim, D.H., Yoon, B.H., Kim, Y.W., Lee, S., Shin, B.Y., Jung, J.W., Kim, H.J., Lee, Y.S., Choi, J.S., Kim, S.Y., Lee, K.T., Ryu, J.H., 2007. The seed extract of Cassia obtusifolia ameliorates learning and memory impairments induced by scopolamine or transient cerebral hypoperfusion in mice. J. Pharm. Sci. 105, 82–93. Kitanaka, S., Kimura, F., Takido, M, 1985. Studies on contituents of the seeds of cassia obtusifolia LINN. The structures of two new anthtaquinone glycosides. Chem. Pharm. Bull. 33, 1274–1276. Koyama, J., Morita, I., Kawanishi, K., Tagahara, K., Kobayashi, N., 2003. Capillary electrophoresis for simultaneous determination of emodin, chrysophanol, and their 8-β-D-Glucosides. Chem. Pharm. Bull. 51 (4), 418–420. Li, H., Guo, H., Wu, L., Zhang, Y., Chen, J., Liu, X., Cai, H., Zhang, K., Cai, B., 2013. Comparative pharmacokinetics study of three anthraquinones in rat plasma after oral administration of Radix et Rhei-Rhizoma extract and DahuangFuzi Tang by high performance liquid chromatography–mass spectrometry. J. Pharm. Biomed. Anal. 76, 215–218. Shia, C.S., Tsai, S.Y., Lin, J.C., Li, M.L., Ko, M.H., Chao, P.D., Huang, Y.C., Hou, Y.C., 2011. Steady-state pharmacokinetics and tissue distribution of anthraquinones of Rhei-Rhizoma in rats. J. Ethnopharmacol. 137, 1388–1394.
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Smolarz, H.D., Swatko-Ossor, M., Ginalska, G., Medynska, E., 2013. Antimycobacterial effect of extract and its components from Rheum rhaponticum. J. AOAC Int. 96, 155–160. Takido, M., 1958. Studies on the constituents of the seeds of cassia obtisifolia L.I. The structure of obtusifolin. Chem. Pharm. Bull. 6, 397–400. Weber, M.L., 2013. Targeting apoptosis pathways in cancer by Chinese medicine. Cancer Lett. 332, 304–312. Wijesekara, I., Zhang, C., Van Ta, Q., Vo, T.S., Li, Y.X., Kim, S.K., 2014. Physcion from marine-derived fungus microsporum sp. induces apoptosis in human cervical carcinoma HeLa cells. Microbiol. Res. 169, 255–261. Xu, L., Chan, C.O., Lau, C.C., Yu, Z., Mok, D.K., Chen, S., 2012. Simultaneous determination of eight anthraquinones in Semen Cassiae by HPLC-DAD. Phytochem. Anal. 23, 110–116. Yan, D.M., Ma, Y.M., Shi, R., Xu, D.S., Zhang, N., 2008. Anthraquinone pharmacokinetics in Xiexin decoction and the different combinations of its constituent herbs. Eur. J. Drug Metab. Pharmacokinet. 33, 69–75. You, X., Feng, S., Luo, S., Cong, D., Yu, Z., Yang, Z., Zhang, J., 2013. Studies on a rheinproducing endophytic fungus isolated from Rheum palmatum L. Fitoterapia 85, 161–168. Zhang, W.D., Wang, P.Y., Wang, Y., Wang, Q., Gu, Y., Cao, J., Wang, S.Q., Wang, X.J., 2014. Simultaneous determination of aurantio-obtusin, chrysoobtusin, obtusin and 1-desmethylobtusin in rat plasma by UHPLC–MS/MS. Biomed. Chromatogr. 28, 369–374. Zhang, W.D., Wang, Y., Wang, Q., Yang, W.J., Gu, Y., Wang, R., Song, X.M., Wang, X.J., 2012. Quality evaluation of Semen Cassiae (Cassia obtusifolia L.) by using ultrahigh performance liquid chromatography coupled with mass spectrometry. J. Sep. Sci. 35, 2054–2062. Zhang, X.M., Yang, R.P., Li, N., et al., 2007. Determination of the content of anthraquinones in Semen Cassiae by ultraviolet spectrophotometry. Lishizhen Med. Mater. Res. 18 (1), 97–98. Zhang, Y.X., Li, J.S., Peng, W.W., Liu, X., Yang, G.M., Chen, L.H., Cai, B.C., 2013. Comparative pharmacokinetics of aloe-emodin, rhein and emodin determined by liquid chromatography–mass spectrometry after oral administration of a rhubarb peony decoction and rhubarb extract to rats. Die Pharm. 68, 333–339. Zhang, C., Li, G.L., Xiao, Y.Q., Li, L., Pang, Z., 2009. Two new glycosides from the seeds of Cassia obtusifolia. Chin. Chem. Lett. 20, 1097–1099. Zhu, W., Zhang, L., Wang, X.M., Wang, B.X., Li, X.Y., 2005. The pharmacokinetics of rhein in 12 healthy volunteers after oral administration of rhubarb extract. China J. Chin. Mater. 30, 1458–1461.
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Simultaneous determination of seven anthraquinones in rat plasma by Ultra High Performance Liquid Chromatography–tandem Mass Spectrometry and pharmacokinetic study after oral administration of Semen Cassiae extract Chunjuan Yang 1, Shuhong Wang 1, Xiaowei Guo, Jiahui Sun, Lu Liu, Lijun Wu n College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang Distrct., Harbin 150081, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 September 2014 Received in revised form 17 March 2015 Accepted 10 April 2015 Available online 20 April 2015
Ethnopharmacological relevance: Semen Cassiae, called Juemingzi in China, is the seed of the annual Cassia obtusifolia L., of the leguminosae family. It has been used as healthy drinks to alleviate constipation and improve eyesight for many years in China. Aim of the study: A simple sensitive UHPLC–MS/MS method has been developed and validated for the simultaneous determination and pharmacokinetic study of chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantio-obtusin in rat plasma. Materials and methods: Chromatographic separation was accomplished on a C18 column with a 5 min gradient elution. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source and operating in the negative ionization mode. The samples were prepared by LLE with ethyl acetate after being spiked with an internal standard (butylparaben). Results: The current UHPLC–MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability. The method was linear for all analytes over investigated range with all correlation coefficients greater than 0.9900. The lower limit of quantification (LLOQ) of each analyte was lower than 5 ng/mL. Intra-day and inter-day precisions were less than 14.99%. The relative errors of accuracies were in the range of 14.60% to 5.11%. The mean recoveries and matrix effects of anthraquinones were higher than 65.54% and 93.26%, respectively. After oral administration 1.25 g/kg of Semen Cassiae extract, the maximum plasma concentration (Cmax) was 1189.25 7 333.40 ng/mL for chrysophanol, 38.48 7 3.15 ng/mL for emodin, 79.20 7 34.76 ng/mL for aloe-emodin, 152.707 23.91 ng/ mL for rhein, 461.857 266.77 ng/mL for physcion, 243.59 722.71 ng/mL for obtusifolin and 1950.44 7638.86 ng/mL for aurantio-obtusin, respectively. The time to reach the maximum plasma concentration (Tmax) was 0.333 7 0.071 h for chrysophanol, 0.333 7 0.059 h for emodin, 0.333 7 0.009 h for aloe-emodin, 0.333 70.09 h for rhein, 0.16770.002 h for physcion, 0.5 70.074 h for obtusifolin and 0.333 70.06 h for aurantio-obtusin, respectively. Conclusion: The proposed method was further applied to investigate the pharmacokinetics of seven anthraquinones after oral administration of Semen Cassia extract to rats. & 2015 Elsevier Ireland Ltd. All rights reserved.
Chemical compounds studied in this article: Chrysophanol (PubChem CID:10208) emodin (PubChem CID:3220) aloe-emodin (PubChem CID:10207) rhein (PubChem CID:10168) physcion (PubChem CID:10639) obtusifolin (PubChem CID:3083575) aurantio-obtusin (PubChem CID:155011) Keywords: UHPLC–MS/MS Semen Cassiae Anthraquinones Pharmacokinetics Rat plasma
1. Introduction Traditional Chinese Medicine (TCM) and its preparations have been widely used in China for thousands years due to its special efficacy in clinical treatment of disease. Semen Cassiae, called Juemingzi in China,
Abbreviations: (UHPLC–MS/MS), Ultra High Performance Liquid Chromatography–tandem Mass Spectrometry; (LLE), liquid—liquid extraction n Corresponding author. Tel.: þ 86 451 86681103; fax: þ 86 451 86614073. E-mail address: [email protected] (L. Wu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jep.2015.04.008 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
is the seed of the annual Cassia obtusifolia L., of the leguminosae family. It is easily grown, widely cultivated in China and Korea, and commonly drunk as a kind of roasted tea. The seeds are reported to have the effects of alleviating constipation and improving eyesight, lowering hypertension and hyperlipidemia which are anchoring and nourishing the liver (Hao et al., 2001; Chen and Chen, 2001; Jiangsu New Medical College, 1975). Semen Cassiae also exhibits antidiabetic effects and neuroprotective effects in the brain disease models (Cho et al., 2005; Guan and Zhao, 1995; Kim et al., 2007). It has been reported that a number of constituents of this plant were isolated, including naph thopyrones, anthraquinones, anthrones, flavonoids, and triterpenoids
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(Kitanaka et al., 1985; Takido, 1958; Zhang et al., 2009).The anthraquinone derivatives, anthronic, dianthronic, and anthraquinone glycosides of Cassia are responsible for the purgative action. In our previous investigation, seven anthraquinones, chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantio-obtusin, have been isolated and identified from the seed of Semen Cassiae. All of them are active compounds of the herbal preparation with pharmacological effects. Aloe-emodin and chrysophanol were found to have antimicrobacterial activity (Smolarz et al., 2013). Weber (2013) discovered that emodin, aloe-emodin and rhein possessed antitumor activities by targeting the apoptosis pathway in cancer, and these compounds possessed antiinflammatory and antiviral effects. Furthermore, emodin and aloe-emodin suppress breast cancer cell proliferation through ER α inhibition (Huang et al., 2013) and rhein also have antitumor, anticancer and hemostatic properties (You et al., 2013). Jang et al., 2007 demonstrated that emodin and obtusifolin exhibited a significant inhibitory activity on glycation end products formation, and aurantio-obtusin, and emodin showed a significant inhibitory activity on rat lens aldose reductase. In addition, physcion will be a potential candidate in the field of anticancer drug discovery against human cervical cancer (Wijesekara et al., 2014). Various analytical methods including High Performance Liquid Chromatography (Huang et al., 2010; Xu et al., 2012), Ultraviolet Spectrophotometry (Zhang et al., 2007), Capillary Electrophoresis (Koyama et al., 2003) and Ultra High Performance Liquid Chromatography coupled with Mass Spectrometry (Zhang et al., 2012) have been used in the qualitative or quantitative analysis of anthraquinones. Although many studies have reported the role of Semen Cassiae in disease prevention, and many pharmacokinetic researches about the anthraquinones from other plants or decoctions have been presented, few papers have reported integrated data on its pharmacokinetic properties in Semen Cassiae (Fang et al., 2011; Zhang et al., 2013; Yan et al., 2008; Li et al., 2013; Gong et al., 2011; Zhu et al., 2005; Shia et al., 2011). Feng et al. (2014) presented the pharmacokinetics of five rhubarb anthraquinones in dog plasma by HPLC after orally administration the rhubarb extract. Zhang et al. (2014) established a HPLC–ESI–MS/MS method to determined four active components of Semen Cassiae extract (aurantio-obtusin, chrysoobtusin, obtusin and 1-desmethylobtusin) in rat plasma after oral administration and provided their pharmacokinetic characteristics. So it is important to develop a highly sensitive, rapid and reproducible analytical method for quantifying these compounds in plasma to characterize the precise pharmacokinetic properties of anthraquinone derivatives. In this study, a rather sensitive and selective UHPLC–MS/MS method was first developed and validated for simultaneously determining chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantio-obtusin in rat plasma. The advantage of this paper is the separation of the up to seven active compounds with similar chemical structure in only five minutes. The method was applied to pharmacokinetics after oral administration of Semen Cassiae extract to rats. It was expected that the results of this study would provide some references to the further pharmacological study of Semen Cassiae.
2. Experimental 2.1. Materials and reagents Reference standards of chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantio-obtusin (Fig. 1.) were isolated from Semen Cassiae and their structures were elucidated by comparison of spectral data (UV, MS, 1H NMR and 13C NMR) with the literature values. Butylparaben was purchased from
Guangfu Fine Chemical Research Institute (Tianjin, PR China) and used as an internal standard (I.S.). HPLC grade methanol and acetonitrile were purchased from J&KMEDICAL (Beijing, China). Formic acid of HPLC grade was obtained from DIKMA (Lake Forest, USA). Ammonium acetate of HPLC grade was bought from Kermel (Tianjin, China). All other reagents were of analytical grade. Ultrapure water was prepared by using a Milli-Q water purification system (Millipore, Molsheim, France). Parched Semen Cassiae was purchased from Beijing Tongrentang (Beijing, china) and authenticated by Prof. Zhenyue Wang in Heilongjiang University of Chinese Medicine. A voucher specimen (Art. NO. 01216800) was deposited in college of pharmacy, Harbin Medical University, China. 2.2. Instruments and analytical conditions The UHPLC system was Agilent technology 1290 series equipped with an automatic degasser, a quaternary pump, and an autosampler. Chromatographic separation was achieved on a C18 column (ACQUITY UPLCs HSS T3 1.8 μm 2.1 100 mm) at 40 1C. Mobile phase composed of (A) acetonitrile and (B) aqueous ammonium acetate (5 mM) with a gradient elution as shown in Table SI. 1 was used. The flow rate was 0.4 mL/min and the analysis time was 5.0 min for each injection. A 5 μL aliquot of sample solution was injected, with a needle wash process which is used to wash the outer wall of sample needle after each injection. The mass spectrometric detection was performed on a 6430 triple-quadrupole mass spectrometer (Agilent, US) with electrospray ionization (ESI) interface set in negative ionization mode. An Agilent Mass Hunter workstation (Agilent, US) was used to control the equipment and for data acquisition and analysis. The quantification was obtained using multiple reaction monitoring (MRM) of precursor-product ion transition at m/z 253.2-225.1 for chrysophanol, m/z 269.0-225.1 for emodin, m/z 269.0-240.1 for aloe-emodin, m/z 283.0-239.0 for rhein, m/z 283.2-240.0 for physcion, m/z 283.1-268.1 for obusifolin, m/z 329.1-298.9 for aurantio-obtusin and m/z 193.0-92.0 for butylparaben (I.S.), respectively (Fig. 2). The parameters of each compound were listed in Table SI. 2 . Other parameters of the mass spectrometer were set as follows: a drying gas flow of 11 L/min; a drying gas temperature of 300 1C; a nebulizer pressure of 15 psi and a capillary voltage of 4000 V. 2.3. Preparation of extract of Semen Cassiae For the extract preparation, dried powder (200 g) of Semen Cassiae was extracted under reflux with 75% ethanol at a rate of material and liquid 1:6 for three times, 1 h each time, and then filtrated. The combined filtrate was evaporated to dryness, and the residue was reconstituted in water to get a concentration equivalent to 0.1 g/mL of the Semen Cassiae extract. 2.4. Preparation of calibration standards and quality control (QC) samples The stock solutions of chysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin, aurantio-obtusin and the I.S. (butylparaben) were individually prepared in methanol. The stock solutions of the standards were further diluted in methanol to produce combined standard working solutions at a series of concentrations. The I.S. solution (2000 ng/mL) was obtained by diluting the stock solution in methanol. The samples for standard calibration curves were prepared by spiking appropriate amount of the standard solutions in 200 μL blank plasma to yield calibration concentrations of 4.74–4854 ng/mL for chysophanol; 4.97–5090 ng/mL for emodin; 2.29–2346.5 ng/mL for aloe-emodin; 4.71–4826 ng/mL for rhein; 1.82–1868 ng/mL for
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physcion; 2.49–2550 ng/mL for obtusifolin and 2.52–2580 ng/mL for aurantio-obtusin, respectively. All the stock and working solutions were stored at 4 1C and brought to room temperature before use. The QC samples were prepared at concentrations of 9.71, 485, 3883 ng/ mL for chysophanol; 10.2, 509, 4072 ng/mL for emodin; 4.69, 235, 1877 ng/mL for aloe-emodin; 9.65, 483, 3861 ng/mL for rhein; 3.74, 187, 1494 ng/mL for physcion; 5.10, 255, 2040 ng/mL for obtusifolin and 5.16, 258, 2064 ng/mL for aurantio-obtusin in drug-free plasma. The spiked samples were processed according to the following section.
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2.5. Animal experiments Male Sprague–Dawley rats (body weight 220 750 g) were purchased and adapted under 65% RH, 25 1C. The animal handling procedures were approved by the Institutional ethical committee and conformed to the principles of the International Guide for the Care and Use of Laboratory Animals. The SD rats were fasted for 12 h before experiment, had free access to water even during the experiment. The blood (0.4 mL) was collected from the orbital venous plexus before administration and at 0.083, 0.167, 0.333, 0.5,
Fig. 2. MS/MS fragmentation patterns of (A) chrysophanol; (B) emodin; (C) aloe-emodin; (D) rhein; (E) physcion; (F) obtusifolin; (G) aurantio-obtusin; (H) I.S.
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1, 2, 3, 4, 6, 8 and 12 h after oral administration of Semen Cassiae extract at a dose of 1.25 g/kg body weight. The plasma was immediately acquired by centrifugation and stored frozen at 20 1C until analysis. 2.6. Plasma sample preparation After thawing the plasma samples at room temperature for 15 min, liquid–liquid extraction was applied for the sample preparation. I.S. 50 μL (2000 ng/mL), 100 μL methanol and 50 μL glacial acetic acid were added to rat plasma sample (200 μL). After vortexing for 60 s, a 3 mL aliquot of ethyl acetate was added. The mixture was vortexed for 2 min and then centrifuged for 5 min at 3000 r/min. The upper organic layer was removed and evaporated to dryness at 40 1C under a stream of nitrogen. The residue was then reconstituted in 100 μL acetonitrile, vortexed for 30 s and filtered by a 0.22 μm membrane. A 5 μL aliquot of the solution was injected into the UHPLC–MS/MS system for analysis. 2.7. Method validation The current UHPLC–MS/MS assay was validated for selectivity, linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability in accordance to the requirements for the analysis of biological samples. The validation runs were conducted on three consecutive days. Each validation run consisted of two sets of calibration standards and six replicates of QC samples at three concentrations. The peak area ratios of the seven analytes to I.S. of the QC samples interpolated from the calibration curve on the same day were used to calculate the concentrations of the seven analytes. The results from QC samples in the three runs were used to evaluate the intra-day and inter-day precision and accuracy of the method. 2.7.1. Specificity and selectivity Selectivity is the ability of an analytical method to differentiate and quantify the analytes in the presence of other components in the sample. In this paper, the selectivity was ascertained by comparatively analyzing blank plasma samples from six individual rats, corresponding blank plasma spiked with the seven analytes and I.S. and the plasma samples from the rats after oral administration of the Semen Cassiae extract. 2.7.2. Linearity and lower limits of quantification (LLOQ) Calibration curves were prepared by assaying standard plasma samples at seven concentration levels. The linearity of each calibration curve was determined by plotting the peak area ratio (y) of analytes to I.S. versus the nominal concentration (x) of analytes with a weighted (1/x2) least square linear regression. The LLOQ was defined as the lowest analytical concentration of the calibration curve with at which the measured precision expressed as relative standard deviation (R.S.D.) was within 720% and the accuracy expressed as relative error (R.E.) was within 7 20%. Six replicate samples were used for evaluation. 2.7.3. Precision and accuracy Three validation batches, each containing six replicates of QC samples at low, medium and high concentration levels were assayed to assess the precision and accuracy of the method at three different days. The intra-day and inter-day precisions were defined as relative standard deviation with criteria of less than 15%; the accuracy was assessed by comparing the measured concentration with its nominal value with a criterion of within 715% for all QC samples.
2.7.4. Extraction recovery and matrix effect The extraction efficiency of the seven analytes was determined by analyzing six replicates of plasma samples at LQC, MQC and HQC levels. To investigate recovery and matrix effect, extracted samples, unextracted samples (pure standard solution), and postextracted spiked samples at three QC levels were analyzed in the same assay. The recovery was obtained by comparing peak areas of analytes in extracted samples with those in post-extracted spiked samples. The matrix effect was evaluated by determining the peak area ratios of the analytes in post-extracted spiked samples to those acquired from unextracted samples. 2.7.5. Stability experiments The stability of the seven analytes in samples including freeze– thaw stability (three freeze at 20 1C and thaw cycles), long-term stability (storage for 2 weeks at 20 1C), room temperature stability (storage for 4 h at ambient temperature), postpreparation stability (storage for 12 h after sample preparation at 4 1C) was tested at LQC, MQC, HQC levels with six replicates at each level. All stability testing QC samples were determined by using the calibration curve of freshly prepared standard samples. 2.8. Application to pharmacokinetic study The validated method was applied to pharmacokinetics after oral administration of Semen Cassiae extract to rats. The maximum plasma concentration (Cmax) and the time of maximum plasma concentration (Tmax) were observed directly from the measured data. The elimination rate constant (Ke) was calculated by linear regression of the terminal points in a semi-log plot of the plasma concentration against time. The elimination half-life (t1/2) was calculated using the formula t1/2 ¼0.693/ Ke. The area under plasma concentration–time curve (AUC0–t) to the last measurable plasma concentration (Ct) was estimated by using the linear trapezoidal rule. The area under the plasma concentration–time curve to time infinity (AUC0–1) was calculated as AUC0–1 ¼ AUC0–t þCt/Ke. 3. Results and discussion 3.1. Optimization of chromatographic mass spectrometric The first step in the method development was to select precursor ions and product ions of the analytes and I.S., meanwhile, their precursor ions and product ions were ascertained for use in MRM. The anthraquinones are polyhydroxy compounds, easily falling off the proton, and the response signal is higher in negative ionization mode than positive ionization mode in theory. We found that these compounds were corresponding well in the negative ionization mode and we did not detect the molecular ion peaks in the positive ionization mode. Thus, negative ionization mode was finally employed. Fig. 2 shows the ion pairs of the seven analytes and I.S. The parameters for fragmentor energy and collision energy were optimized in order to get the richest relative abundance of precursor and product ions. Table SI. 2 shows the MS/MS transitions and energy parameters of all the compounds. Chromatographic conditions were optimized to improve peak shape, increase sensitivity and shorted run time for simultaneous analysis of seven components. Several mobile phase systems were tried in order to choose the optimal mobile phase that produces the best response, sensitivity and separation efficiency. Mobile phase systems such as methanol–water and acetonitrile–water were tested, and different buffers including ammonium acetate (2 and 5 mM), formic acid (0.1%) and acetic acid (0.2%) were added to the system. Finally, acetonitrile and 5 mM ammonium acetate in water were the best solvent composition. Satisfactory separation
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Fig. 3. Chromatograms of the seven components and I.S. in plasma: (A) blank plasma; (B) blank plasma spiked with the seven analytes and I.S. (LQC); (C) plasma sample obtained at 0.5 h from a rat after oral administration of Semen Cassiae extract: channel 1 for I.S.; channel 2 for chrysophanol; channel 3 for emodin; channel 4 for aloeemodin; channel 5 for rhein; channel 6 for physcion; channel 7 for obtusifolin and channel 8 for aurantio-obtusin.
was achieved in 5 min by gradient elution using a C18 column (ACQUITY UPLCs HSS T3 1.8 μm 2.1 100 mm) at 40 1C with a flow rate of 0.4 mL/min. As shown in Fig. 3, there were no
endogenous plasma components interfering with the analytes when the above-mentioned mobile phase was used. Compared the elution profiles of blank plasma spiked with the seven analytes
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and I.S. with that of real plasma sample, it can be seen that the satellite and addition peaks in the chromatograms of the real plasma sample, such as compounds 1, 3, 4 and 7 in Fig. 3B and C. Table 1 The regression equations, linear ranges and LLOQs for the determination of the analytes in rat plasma. Compounds
Regression equation
R2
Linear range (ng/mL)
LLOQ (ng/mL)
Chrysophanol Emodin Aloe-emodin Rhein Physcion Obtusifolin Aurantioobtusin
Y ¼ 0.4621X þ0.0021 Y ¼ 1.361X þ 0.0078 Y ¼ 0.4974X 1.154 10 4 Y ¼ 0.1358X þ5.826 10 4 Y ¼ 17.63X þ 0.0083 Y ¼ 4.859X þ0.0154 Y ¼ 0.2517Xþ 5.460 10 4
0.9932 0.9939 0.9946 0.9900 0.9949 0.9971 0.9906
4.74–4854 4.97–5090 2.29–2346 4.71–4826 1.82–1868 2.49–2550 2.52–2580
4.74 4.97 2.29 4.71 1.82 2.49 2.52
This phenomenon may be caused by the other isomers or compounds absorbed into blood, and they have the same molecular mass in precursor and product ions after one or more cleavage. So we optimized the liquid phase conditions to adjust the retention time so as to obtain the suitable resolution of the seven analytes. 3.2. Sample preparation In our experiment, liquid–liquid extraction (LLE) and protein precipitation were compared during sample preparation. Initially, we used the protein precipitation method, 3 mL methanol (or acetonitrile) was added for protein precipitation, and it was not selected because of its high MS/MS noise level. For LLE, organic extracting solvent including ethyl acetate, diethyl ether and dichloromethane was used. It was observed that LLE with ethyl acetate could obtain clean, simple, reproducible extraction and
Table 2 Precision and accuracy of the determination of seven anthraquinones in rat plasma (n¼18, 6 replicates per day for 3 days). Compounds
Spiked concentration (ng/mL)
Measured concentration (ng/mL)
Accuracy (%)
Intra-day precision (%)
Inter-day precision (%)
Chrysophanol
9.71 485 3883 10.2 509 4072 4.69 235 1877 9.65 483 3861 3.74 187 1494 5.10 255 2040 5.16 258 2064
9.81 71.37 415 750.0 3719 7503 9.40 71.34 491 733.7 3974 7400 4.49 70.63 218 728.6 1614 7139 8.69 71.20 422 753.3 3397 7271 3.65 70.50 181 718.7 1349 7187 4.98 70.68 230 722.8 1917 7271 4.88 70.56 257 737.9 2169 7270
1.08 14.60 4.22 7.68 3.50 2.40 4.31 7.08 14.00 9.97 12.47 12.02 2.25 2.94 9.72 2.32 9.90 6.02 5.52 0.35 5.11
14.06 12.82 14.09 14.45 7.15 10.65 14.38 13.53 9.17 14.47 13.27 8.33 14.36 10.26 14.72 14.37 10.37 14.93 12.13 14.99 12.64
12.85 1.33 8.25 11.57 4.05 3.20 9.46 1.12 6.45 6.45 5.81 4.79 6.85 10.54 3.73 5.34 5.68 6.06 3.51 12.74 11.02
Emodin
Aloe-emodin
Rhein
Physcion
Obtusifolin
Aurantio-obtusin
Table 3 Matrix effects and extraction recovery for the analytes in rat plasma (n¼ 6). Compounds
Chrysophanol
Emodin
Aloe-emodin
Rhein
Physcion
Obtusifolin
Aurantio-obtusin
I.S.
Spiked concentration (ng/mL)
9.71 485 3883 10.2 509 4072 4.69 235 1877 9.65 483 3861 3.74 187 1494 5.10 255 2040 5.16 258 2064 2000
Matrix effect
Extraction recovery
Mean(%)
RSD (%)
Mean (%)
RSD (%)
97.97 96.06 100.8 102.5 95.21 101.6 96.91 93.26 99.60 102.7 95.30 98.42 100.1 86.84 97.97 97.42 101.2 97.35 101.2 101.6 105.3 96.12
7.194 1.725 5.083 4.026 2.537 1.193 7.529 12.34 6.232 2.830 11.70 10.88 4.932 2.811 9.111 3.286 12.01 2.672 6.657 5.710 4.545 3.756
74.51 78.44 77.98 78.80 72.18 76.60 73.33 83.74 80.37 68.31 65.54 72.43 81.48 84.63 80.44 76.99 82.36 84.41 74.42 79.28 81.33 80.49
13.65 2.500 10.82 2.922 3.398 4.181 7.404 5.666 9.627 6.255 9.919 3.330 5.495 5.791 11.40 3.928 3.162 1.900 8.358 6.669 1.982 3.741
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Table 4 Stabilities of the analytes in rat plasma (n¼ 6). Analytes
Chrysophanol
Emodin
aloe emodin
rhein
physcion
Obtusifolin
Aurantio-obtusin
a
Spiked concentration (ng/mL)
9.71 485 3883 10.2 509 4072 4.69 235 1877 9.65 483 3861 3.74 187 1494 5.10 255 2040 5.16 258 2064
Stability (% REa) Three freeze–thaw
Short-term
Long-term
Post-preparative
8.20 11.10 8.51 1.35 4.39 0.86 4.31 2.11 12.32 0.96 0.83 9.47 10.15 1.87 8.46 8.27 7.32 8.62 12.49 6.34 14.17
6.99 1.11 8.56 5.70 2.02 1.73 7.71 1.39 6.72 4.03 9.14 5.25 3.40 7.14 0.94 5.29 6.68 0.53 6.84 6.41 7.66
9.41 13.39 13.63 7.70 6.86 2.81 10.77 3.43 5.71 0.16 6.35 7.64 5.40 10.68 1.75 0.90 5.45 2.10 3.64 5.64 0.63
8.07 4.75 5.70 10.25 8.39 0.89 2.34 7.65 9.29 7.62 2.99 12.23 4.53 1.43 3.62 3.86 3.37 0.26 8.53 3.39 9.76
RE is expressed as (measured concentration/freshly prepared concentration 1) 100%.
good recovery for the seven analytes. During the study, glacial acetic acid was considered as a critical factor for the recovery of the analytes. Different amounts (20, 50 and 100 μL) of glacial acetic acid were tested in the experiment. At last, 50 μL of glacial acetic acid was used to improve the extract efficiency. 3.3. Method validation 3.3.1. Specificity and selectivity The selectivity of the method towards endogenous plasma matrix was evaluated with plasma from six rats. Eight channels were used for recording and the retention times of chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantioobtusin were 3.8, 3.0, 2.8, 1.3, 4.0, 3.1 and 2.4 min, respectively. Typical chromatograms obtained from a blank, a spiked plasma sample with the analytes and I.S. (LQC), and 0.5 h plasma sample after an oral administration of Semen Cassiae extract are shown in Fig. 3. All the peaks of the analytes and I.S. were detected with excellent resolution as well as peak shapes, and no interference from the endogenous substances was observed at the retention time of the analytes and I.S. The analytes could be easily differentiated from the rat plasma matrix and quantitatively determined at the LLOQ level. 3.3.2. Linearity and lower limits of quantification The regression equation, correlation coefficients and linearity ranges for seven analytes are shown in Table 1. All correlation coefficients were higher than 0.9900. They all exhibited good linearity. The LLOQ of chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin, and aurantio-obtusin were 4.74, 4.97, 2.29, 4.71, 1.82, 2.49 and 2.52 ng/mL, respectively. 3.3.3. Precision and accuracy In this assay, the intra-day and inter-day precisions and accuracy were determined. Six replicates of QC samples at three concentration levels (LQC, MQC, HQC) of seven analytes on the same day and on three different days were tested. The results are listed in Table 2.
Fig. 4. Mean plasma concentration–time profiles of rhein and obtusifolin (A), chrysophanol, aurantio-obtusin and physcion (B), emodin and aloe-emodin (C) in rats after oral administration of Semen Cassiae extract. Values are expressed as means 7SD (n¼ 6).
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Table 5 Pharmacokinetic parameters of the seven constituents in rats after oral administration of Semen Cassiae extract (mean 7 S.D., n ¼6). Compounds
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUC0-t (ng h/mL)
AUC0-1 (ng h/mL)
Chrysophanol Emodin Aloe-emodin Rhein Physcion Obtusifolin Aurantio-obtusin
1189.25 7 333.4 38.487 3.15 79.20 7 34.76 152.707 23.91 461.85 7 266.77 243.597 22.71 1950.447 638.86
0.3337 0.071 0.3337 0.059 0.3337 0.009 0.3337 0.09 0.1677 0.002 0.5 7 0.074 0.3337 0.06
6.12 72.37 6.40 73.31 24.62 77.27 10.30 75.02 10.46 71.46 7.21 73.66 13.78 73.03
642.21 7119.51 39.75 72.30 80.84 750.76 199.81 722.47 218.42 715.08 235.90713.11 874.50 7103.70
650.58 7 126.26 42.83 7 6.38 106.797 71.88 208.63 7 26.50 224.417 16.84 238.94 7 15.84 991.34 7 114.65
At each QC level, the inter- and intra-day precisions (R.S.D) of seven analytes were 1.12–14.99%, the accuracy was 14.60–5.11%. These results indicated that the developed method was precise and accurate. 3.3.4. Extraction recovery and matrix effect The data of extraction recovery and matrix effect of the seven analytes are summarized in Table 3. The extraction solvent used in the experiment showed good extraction efficiency. Mean absolute recoveries of chrysophanol, emodin, aloe-emodin, rhein, physcion, obtusifolin and aurantio-obtusin were 74.51–78.44%, 72.18– 78.80%, 73.33–83.7%, 65.54–72.43%, 80.4–84.6%, 76.99–84.4% and 74.42–81.3% at three QC levels. These results demonstrated that the values were all in the acceptable ranges. The matrix effects derived from QC samples were between 93.2% and 105.3%, and I.S. was 96.1 73.756%. These results confirmed that the evaluated method was free from any matrix effect. 3.3.5. Stability experiments The stability of all the analytes was assessed under various conditions. The results presented in Table 4 indicate that the seven analytes were all stable in plasma after three freeze–thaw cycles, at room temperature for 4 h. Post-preparative stability of the analytes also showed that no significant degradation occurred when the extracted samples were kept at 4 1C for 12 h. Moreover, all the investigated compounds were stable for 2 weeks when kept frozen at 20 1C.
from 6.12 h to 24.62 h, which suggests that the seven components were rapidly absorbed in the stomach and eliminated in rat plasma after oral administration of Semen Cassiae extracts. In other assays (Kitanaka et al. 1985), a double-peak phenomenon appeared on the mean plasma concentration–time profiles of some of the seven compounds, which indicated that enterohepatic circulation could be involved in their absorption process or may be induced by metabolic transformation through the demethylation pathway between these compounds. In this study, only rhein showed minor double-peak in the mean plasma concentration– time curve. These results should be useful for further studies on the pharmacokinetics, pharmacy and toxicity of the TCM Semen Cassiae and should support studies seeking to determine the efficacy of this TCM in clinical therapeutic research.
4. Conclusion This validated UHPLC–MS/MS method was sensitive, accurate and fast, and met all requirements in bioanalysis. This is the first paper to simultaneously determinate of seven anthraquinones in rat plasma after oral administration of Semen Cassiae extract. In addition, this method has been successfully applied to pharmacokinetic study of seven components following oral administration of Semen Cassiae extract to rats and it is the first pharmacokinetic study of obtusifolin. The results might be helpful for investigating the bioactivity mechanism and clinical application of Semen Cassiae.
3.3.6. Application to the pharmacokinetic study The developed UHPLC–MS/MS method was applied to investigate the pharmacokinetics of the seven constituents in Semen Cassiae after a single oral administration of 1.25 g/kg (equivalent to 0.66 mg/g for chrysophanol, 0.02 mg/g for emodin, 0.04 mg/g for aloe-emodin, 0.08 mg/g for rhein, 0.3 mg/g for physcion, 5.01 mg/g for obtusifolin and 0.95 mg/g for aurantio-obtusin) to six rats. We chose an oral dosage for the rats corresponding to the human dosage in Chinese Pharmacopoeia (China Pharmacopoeia Committee, part I, 2010). The human dosage of Semen Cassiae in Chinese Pharmacopoeia is 15 g/day, so we picked the final oral dosage as 1.25 g/kg in rats based on the Meeh-Rubner method. The mean plasma concentration–time curves (n ¼6) of the analytes are shown in Fig. 4. The pharmacokinetic parameters including halftime (t1/2), maximum plasma concentration (Cmax), time to reach the maximum concentrations (Tmax), area under concentration– time curve (AUC0Ut and AUC0U1) calculated by the noncompartment model are presented in Table 5. As shown in Table 5, the range of Cmax of these seven anthraquinones was from 38.48 73.15 to 1950.447 638.86 ng/mL because of the great differences of the contents of the seven compounds in the Semen Cassiae extract. The range of AUC0Ut was from 39.75 72.30 to 874.50 7103.70 ng/mL/h. All the seven analytes achieved the maximum plasma concentration within 0–0.5 h after oral administration, meanwhile, their half life of elimination (T1/2) ranged
Acknowledgment Our work was supported by the Scientific Research Project of National Natural Science Foundation of China (No. 81202875) and Heilongjiang Province Education Department (No. 12521201).
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