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acid hydrolysis in rat plasma after oral administration of Verbena officinalis L. extract
Journal of Ethnopharmacology 135 (2011) 201–208
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Review
LC–MS/MS determination and pharmacokinetic study of five flavone components after solvent extraction/acid hydrolysis in rat plasma after oral administration of Verbena officinalis L. extract Kunfeng Duan, Zhifang Yuan, Wei Guo, Yan Meng, Yang Cui, Dezhi Kong, Lantong Zhang ∗ , Na Wang Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang 050017, China
a r t i c l e
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Article history: Received 21 April 2010 Received in revised form 18 December 2010 Accepted 5 January 2011 Available online 8 January 2011 Keywords: Verbena officinalis L. extract HPLC–MS/MS Pharmacokinetics
a b s t r a c t Ethnopharmacological relevance: Traditional Chinese medicine (TCM) has been used in clinical practice for several thousand years. TCM has played an indispensable role in the prevention and treatment of diseases, especially the complicated and chronic ones. Pharmacokinetic study on active constituents in herbal preparations is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of TCM. Aim of the study: A selective and sensitive HPLC–MS/MS method was first developed and validated for the determination of luteolin, kaempferol, apigenin, quercetol, and isorhamnetin in rat plasma. Materials and methods: The LC system consisted of an Agilent Technologies Series 1200 system (Agilent, USA) equipped with an automatic degasser, a quaternary pump, and an autosampler. Chromatographic separations were performed on a Waters SunFireTM C18 column (150 mm × 4.6 mm, 5 m), and the column temperature was maintained at 25 ◦ C and the sample injection volume was 20 L. The current LC–MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability. Results: The validated method was successfully applied to monitoring the concentrations and pharmacokinetic studies of five flavone compounds in rat plasma after a single oral administration of Verbena officinalis L. extract with a dosage of 8.0 mL/kg. The time to reach the maximum plasma concentration (Tmax1 ) was 0.48 ± 2.14 h for luteolin, 0.25 ± 0.13 h for kaempferol, 0.97 ± 1.08 h for apigenin, 1.04 ± 4.25 h for quercetol and 0.25 ± 0.16 h for isorhamnetin, and the maximum plasma concentration (Tmax2 ) was 3.97 ± 1.48 h, 4.05 ± 0.46 h, 4.33 ± 0.58 h, 2.99 ± 0.48 h and 4.02 ± 0.34 h. The elimination half-time (t1/2 ) of luteolin, kaempferol, apigenin, quercetol and isorhamnetin was 4.02 ± 0.81, 7.65 ± 0.71, 3.30 ± 0.83, 4.55 ± 0.49 and 5.56 ± 1.32 h, respectively. Conclusions: This paper described a simple, sensitive and validated LC–MS/MS method for simultaneous determination of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma after oral administration of V. officinalis L. extract, and investigated on their pharmacokinetic studies as well, with a short run time of 5 min. © 2011 Published by Elsevier Ireland Ltd.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chemicals and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Instrumentation and analytical conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Preparation of V. officinalis L. extract in the administration solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Preparation of calibration standards and quality control (QC) samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Application to pharmacokinetic study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author. Tel.: +86 311 86266419; fax: +86 311 86266419. E-mail address: [email protected] (L. Zhang). 0378-8741/$ – see front matter © 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2011.01.002
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K. Duan et al. / Journal of Ethnopharmacology 135 (2011) 201–208
2.7. Method validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Mass spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Liquid chromatographic conditions and matrix effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Selection of IS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Method validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1. Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2. Linearity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.3. Precision, accuracy and extraction recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.4. Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Application in pharmacokinetic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Traditional Chinese medicine (TCM) has been used in clinical practice for several thousand years. TCM has played an indispensable role in the prevention and treatment of diseases, especially the complicated and chronic ones. Pharmacokinetic study on active constituents in herbal preparations is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of TCM. Verbena officinalis L., which is known as Mabiancao mostly grows in South China, is a famous traditional Chinese medicine (TCM) and widely used for clearing away heat and detoxicating, promoting blood circulation and removing blood stasis, inducing diuresis and excreting dampness based on the Chinese medical theory (National Commission Chinese Pharmacopoeia of the People’s Republic of China, 2005). It also can be used in folk medicine as a diuretic, expectorant and anti-rheumatic (Wichtl and Bisset, 1994). In Navarra, Spain, it is used extensively in traditional medicine mainly because of its anti-inflammatory topical applications (Fernandez, 1981; Deepak and Handa, 2000; Calvo, 2006). In 1985, it was the first time to report V. officinalis L. had antitussive effect which was associated with the components including verbenalin by Gui Chenghui in China (Gui and Tang, 1985). However, as we know, one or several categories of ingredients contribute to the pharmacodyamic material basis in most traditional Chinese medicine. Up to now, the total extract of V. officinalis L. and its ethyl acetate and n-butyl alcohol parts had significant antitussive and anti-inflammatory effects (Deng and Zhou, 2005; Jin and Zhang, 2007). Phytochemical investigation found that V. officinalis L. contained many constituents such as flavonoids, iridoid glycosides, phenylpropanoid glycoside, sterols, triterpenes and glycoconjugate (Rimpler et al., 1979; Makboul, 1986; Calvo et al., 1997; Liu et al., 2002; Muller et al., 2004). It has been reported that flavonoids and iridoid glycosides are important active constituents most contributing to the pharmacological efficacy of V. officinalis L. (Zhang et al., 2002; Xin et al., 2008). Pharmacological tests have revealed that the major active compounds of anti-inflammatory are flavonoids mainly including luteolin, kaempferol, apigenin, quercetol and isorhamnetin (Tian et al., 2005; Chen et al., 2006a,b). Their chemical structures are shown in Fig. 1. To date, there has many preliminary researches on the quantitative analysis of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in plant material, natural products and preparations using HPLC, HPLC-MS and MEKC (Zhang et al., 2002; Yuan et al., 2009). In contrast, only few papers have been reported on HPLC or HPLC–MS methods for the pharmacokinetic study of luteolin in rabbits and apigenin in rats, independently (Li et al., 2005). As luteolin, kaempferol, apigenin, quercetol and isorhamnetin are the major active components of V. officinalis L., it is essential to develop a method to determine all
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five of them in plasma samples for the purpose of studying the mechanism of action. In the present study we developed and validated a sensitive, specific and accurate method for the simultaneous determination of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma. The method was successfully applied to evaluate the pharmacokinetics of luteolin, kaempferol, apigenin, quercetol and isorhamnetin after oral administration of V. officinalis L. extract to rats. 2. Experimental 2.1. Chemicals and reagents Five standards, luteolin; kaempferol; apigenin; quercetol and isorhamnetin (Fig. 1), were purchased from the National Institute for Control of Biological and Pharmaceutical Products (Beijing, China). SMZ (Sulfamethoxazolum) as internal standard was identified and supplied by Hebei Medical University (Hebei, China). Methanol (Tedia, Fairfield, USA), acetonitrile (Tedia, Fairfield, USA) and formic acid (Kermel, Tianjin, China) were of HPLC grade. The distilled water was prepared from demineralized water and used throughout the experiments. All other chemical solvents were of analytical grade from Beijing Chemical Factory (Beijing, China). V. officinalis L. was purchased from a traditional Chinese medicinal store in Shijiazhuang, China and authenticated by Professor Ding Zhao (Key Laboratory of Modern Chinese Medicines, School of Pharmacy, Hebei Medical University, Shijiazhuang, China). 2.2. Instrumentation and analytical conditions The LC system consisted of an Agilent Technologies Series 1200 system (Agilent, USA) equipped with an automatic degasser, a quaternary pump, and an autosampler. Chromatographic separations were performed on a Waters SunFireTM C18 column (150 mm × 4.6 mm, 5 m), and the column temperature was maintained at 25 ◦ C and the sample injection volume was 20 L. The mobile phase consisted of acetonitrile and 0.1% aqueous formic acid using gradient elution (0–2 min, 45–80% acetonitrile; 2–5 min, 80% acetonitrile; 5–5.1 min 80–45% acetonitrile) and was delivered at a flow rate of 0.8 mL/min. Determination was performed with 3200 QTRAPTM system from Applied Biosystems/MDS Sciex (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion trap mass spectrometer equipped with Turbo V sources and TurboIonspray interface. The analytes were determined in negative ionization mode by monitoring the precursor–product combination in multiple reaction monitoring (MRM) mode. The ion source temperature was held at 650 ◦ C. Target ions were monitored at m/z 285.0
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Fig. 1. Chemical structures of luteolin (A), kaempferol (B), apigenin (C), quercetol (D), isorhamnetin (E) and IS (F).
for luteolin, 285.0 for kaempferol, 269.0 for apigenin, 301.1 for quercetol, 315.1 for isorhamnetin and 252.0 for IS. The data acquisition and peak integration were performed using analyst software (Version 1.4.2). 2.3. Preparation of V. officinalis L. extract in the administration solution The dried powder of V. officinalis L. (100 g) was extracted twice by refluxing with 80% ethanol (1:8 and then 1:6, w/v) for 3 h and the extraction solutions were combined to be filtered, concentrated to 100 mL under reduced pressure and then precipitated with the same volume water. The precipitate was discarded and the ethanol was removed under the condition of waterbath. The solution was then extracted with same volume acetoacetate for 3 times. The acetoacetate was removed under reduced pressure and the residue was dissolved in water. The administration solution was obtained and stored at 4 ◦ C before use. To calculate the administered dose, the contents of five flavones in administration solution were quantitatively determined by external standardization using the same chromatography conditions as described above. The contents of luteolin, kaempferol, apigenin, quercetol and isorhamnetin were 0.206, 0.288, 0.332, 0.162 and 0.094 mg/mL in extract, respectively. 2.4. Preparation of calibration standards and quality control (QC) samples Stock solution of luteolin, kaempferol, apigenin, quercetol, isorhamnetin and IS was prepared in methanol at the concentration of 530 g/mL, 220 g/mL, 941 g/mL, 196 g/mL, 520 g/mL and 360 g/mL. A series of standard mixture working solutions with concentrations 0.005–2.50 g/mL for luteolin, 0.005–2.50 g/mL for kaempferol, 0.020–10.0 g/mL for apigenin, 0.025–12.5 g/mL for quercetol and 0.020–10.0 g/mL for isorhamnetin were obtained by diluting the mixture of the stock standard solutions with methanol. The working solution of IS (3.60 g/mL) was prepared by diluting SMZ. All the working solutions were kept at 4 ◦ C. Then assay standard samples were prepared by spiking 100 L blank rat plasma with standard mixture working solutions (20 L) and IS working solution (20 L), in which the concentrations were 0.1, 0.25, 1.0, 5.0, 20.0, and 50.0 g/mL for luteolin, 0.1, 0.25,
1.0, 5.0, 20.0, and 50.0 g/mL for kaempferol, 0.4, 1.0, 4.0, 20.0, 80.0, and 200.0 g/mL for apigenin, 0.5, 1.25, 5.0, 25.0, 100.0, and 250.0 g/mL for quercetol and 0.4, 1.0, 4.0, 20.0, 80.0, and 200.0 g/mL for isorhamnetin. Quality control (QC) samples (luteolin 0.0125, 0.250, and 2.0 g/mL, kaempferol 0.0125, 0.250, and 2.00 g/mL, apigenin 0.050, 1.00, and 8.00 g/mL, quercetol 0.0625, 1.25, and 10.0 g/mL, isorhamnetin 0.050, 1.00, and 8.00 g/mL) were independently prepared in the same manner. The working and assay standard solutions were prepared everyday. 2.5. Sample preparation Plasma samples (100 L each) were treated with 10 mmol/L ascorbic acid before storage at −70 ◦ C. For clean-up, the thawed plasma samples were vortex-mixed with 20 L of IS and 280 L of methanol–hydrochloric acid (25%) (4:1, v/v). The solution was centrifuged at 12,000 rpm for 10 min to separate the protein from the organic phase, then the upper supernatant was treated at 90 ◦ C for 30 min, and then the supernatant was collected and centrifuged at 12,000 rpm for another 10 min. At last 20 L of the resulting supernatant was applied to LC–MS/MS analysis. 2.6. Application to pharmacokinetic study Male Sprague–Dawley rats (250 ± 20 g) were supplied by Lab Animal Center of Heibei Medical University (Shijiazhuang, China) and kept in an environmentally controlled breeding room (temperature: 24 ± 2 ◦ C, humidity: 60 ± 5%, 12 h dark–light cycle) for at least 5 days before starting the experiments. They were fed with food and water ad libitum and fasted overnight prior to the test. Blood samples of about 0.3 mL were collected into heparinized centrifuge tubes from the fossa orbitalis vein at 0, 5, 15, 30, 60, 90, 120, 150, 180, 240, 360, 480, and 720 min in 6 healthy rats after single oral administration of V. officinalis L. extract (10 mL/kg). Then the samples were centrifuged at 4000 rpm for 10 min and the separated plasma samples were frozen in polypropylene tubes at −20 ◦ C until analysis. PK parameters including area under concentration–time curve (AUC), maximum plasma concentration (Cmax ), time to reach the maximum concentrations (Tmax ), the plasma half-life (T1/2 ), the area under the plasma concentration–time curve from zero to the time of the final measurable sample (AUC0−t ), the area under the
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plasma concentration–time curve from zero to infinity (AUC0−∞ ) and mean residence time (MRT) were estimated using PKSlover 1.0 (Zhang et al., 2007).
and 252.0/155.9 for IS. The product ion scan spectra were shown in Fig. 2. 3.2. Liquid chromatographic conditions and matrix effect
2.7. Method validation
3. Results and discussion
The chromatographic condition was optimized to improve peak shape, increase signal response of analytes and shorten run time. In the previous study, acetonitrile was chosen as the organic solvent because it provided a higher sensitivity and lower background noise than methanol. Comparative studies showed that the use of a mobile phase containing an extremely low concentration of formic acid dramatically improved the electrospray ionization efficiency of the analytes in the negative ion mode. We tested different buffers including formic acid (0.01%, 0.05%, and 0.1%), ammonium acetate (0.2, 1, and 2 mmol/L) and acetic acid (0.01%, 0.05%, and 0.1%) to identify the optimal mobile phase that produced the best sensitivity and efficiency. The results indicated that the intensity of the five analytes peak strength increased dramatically when 0.01% formic acid was added into mobile phase. Therefore acetonitrile–0.01% formic acid was used as the mobile phase with an acceptable run time of 5.0 min. The matrix effect (ME) was examined by comparing the mean peak areas of the analytes between two different sets of samples. In set 1, reference standards were dissolved in water (100 L), methanol–hydrochloric acid (25%) (4:1, v/v) (260 L) and five replicates were analyzed. In set 2, blank plasma samples (100 L) were extracted and then spiked with the same concentrations of analytes. Samples of both sets were prepared at three QC levels for the analytes (n = 6). The ME was defined as following: ME% = 100 × set 2/set1. Matrix effect results were shown in Table 4. The average matrix effect values were 96.8%, 101.7% and 92.4% for luteolin, 94.8%, 97.5% and 98.4% for kaempferol, 96.9%, 97.8% and 87.8% for apigenin, 107.6%, 102.9% and 96.2% for quercetol and 104.6%, 94.8% and 101.5% for isorhamnetin at the three QC concentration levels. Results, which were described as percentage in concentration of diluted samples to working standard solutions, were no less than 85% or more than 115% for all the six analytes, and showed that there was no significant difference between the peak areas of samples prepared from rat plasma and from water which indicated that no co-eluting unseen compounds significantly influenced the ionization of analytes and IS.
3.1. Mass spectra
3.3. Sample preparation
Luteolin, kaempferol, apigenin, quercetol, and isorhamnetin were at first characterized according to their mass spectra from syringe pump infusion analysis, respectively. Meanwhile, their precursor ions and product ions were ascertained for use in MRM. The electrospray interface (ESI) was employed for good sensitivity and fragmentation were obtained. In our experiment, we discovered that the negative ion mode was more sensitive for flavonol analysis. Positive mode also tested, but the sensitivity obtained was not satisfactory for all of the six analytes and IS. Thus, negative ion mode was finally employed. The optimum declustering potential were −70 V for luteolin, −67 V for kaempferol, −58 V for apigenin, −50 V for quercetol, −52 V for isorhamnetin and −30 V for IS. The molecular ions of luteolin, kaempferol, apigenin, quercetol, isorhamnetin and SMZ IS were fragmented at collision energy of −47 eV, −83 eV, −50 eV, −30 eV, −30 eV and −20 eV using nitrogen as collision gas. In the full scan mass spectra, the protonated molecular ions [M−H]− were stable and exhibited higher abundance, thus [M−H]− were chosen as the precursor ions for MS/MS fragmentation analysis of the analytes. The optimized mass transition ion-pairs (m/z) for quantitation were 285.0/133.0 for luteolin, 285.0/92.9 for kaempferol, 269.0/117.0 for apigenin, 301.1/151.0 for quercetol, 315.1/300.0 for isorhamnetin,
Commonly, the used extraction techniques mainly including liquid–liquid extraction (LLE), protein precipitation (PPT) and solid phase extraction (SPE). The method of SPE has good extraction recovery and reproducibility, however, this extraction method is complicated and costly so that it is not suitable for the processing of multiple samples in a limited amount of time for bioequivalence and pharmacokinetic studies. In the course of our method development, we found that liquid–liquid extraction technique had a low recovery when ethyl acetate, dichloromethane, n-butanol or their mixture in different combinations and ratios used as extraction solvents. Therefore, plasma samples were subjected to a simple protein precipitation procedure. Finally, methanol–25% hydrochloric acid (4:1, v/v) appeared to be optimal, especially for five analytes and IS. The determined concentration included the free flavones and that after acid hydrolysis. During the experiment, it was found that the five flavones were hardly to be detected after plasma precipitation only by organic solvents. The extract was a complex system, may be some compounds result in the induction of metabolic enzymes, the determined flavones rapidly transformed to their metabolites. Therefore, the total concentration of the five flavones was determined after solvent extraction/acid hydrolysis. So hydrolysis was added. At last a simple one-step protein
The current LC–MS/MS assay was validated for linearity, intraday and inter-day precisions, accuracy, extraction recovery and stability. The calibration plasma samples were prepared by adding 20 L of working solutions, 20 L of IS solution to 100 L of blank plasma and extracted as described in Section 2.5. Calibration curves were prepared by assaying standard plasma samples at six concentrations. Weighted (1/x) linear least-squares regression method was used to determine the slope, intercept and correlation coefficient. The limits of detection (LOD) and limits of quantification (LOQ) were determined at a signal-to-noise ratio of about 3 and 10 by analyzing the diluted standard solution. Three validation batches, each containing six replicates of QC samples at low, medium and high concentration levels (0.0125, 0.250, and 2.00 g/mL for luteolin, 0.0125, 0.250, and 2.00 g/mL for kaempferol, 0.050, 1.00, and 8.00 g/mL for apigenin, 0.0625, 1.25, and 10.0 g/mL for quercetol and 0.050, 1.00, and 8.00 g/mL for isorhamnetin), were assayed to assess the precision and accuracy of the method on three different days. The intra-day and inter-day precisions were defined as relative standard deviation (R.S.D.) and the accuracy was assessed by comparing the measured concentration with its true value. The extraction recovery was determined in sets of six replicate quality control samples by measuring the amount of each compound recovered after extraction and calculated by comparing the peak areas of the extracted samples with that of the unextracted standard solutions (the solvent was changed to water) containing an equivalent amount of the analytes. The stability of analytes in rat plasma was evaluated by analyzing QC samples at three concentrations exposed to different conditions (room temperature, 24 h; −20 ◦ C, 15 days; freeze-thaw cycles). Stability was assessed by comparing the mean concentration of the stored QC samples.
K. Duan et al. / Journal of Ethnopharmacology 135 (2011) 201–208
1.0e5
132.9
4.6e6
205
92.9
B
A
285.0
285.0 100
200
117.0
4.6e6
100
300
150.9
1.7e6
C
200
300
D
269.0
100
200
300.9
300.0
1.35e6
100
300
252.0
315.1 100
200
300
300
F
155.9
2.0e7
E
200
400
100
200
300
Fig. 2. MS and product ion spectra of the compounds. Luteolin (A), kaempferol (B), apigenin (C), quercetol (D), isorhamnetin (E) and IS (F).
precipitation was executed, and high recovery and good precision and accuracy were obtained. 3.4. Selection of IS An internal standard should be used when performing pharmacokinetic study. An appropriate internal standard will control for extraction, HPLC injection and ionization variability. We investigated several compounds served as suitable IS, such as paeoniflorin, puerarin, and orientin. As these compounds are also flavone, their physicochemical features and ionization characteristics are similar to those of analytes. The result showed that SMZ was more appropriate for IS, because it provided clean chromatography and shorter retention time and no significant direct interference was observed. Moreover, SMZ is not a component of biological samples after oral administration of V. officinalis L. extract. 3.5. Method validation 3.5.1. Specificity The typical chromatograms of plasma sample from a rat after oral administration of V. officinalis L. extract are shown in Fig. 3. Four channels were used for recording and the retention times of luteolin, kaempferol, apigenin, quercetol, isorhamnetin and IS were
3.22, 3.80, 3.70, 3.30, 3.85 and 3.18 min, respectively. All the peaks of the analytes and IS were detected with excellent resolution as well as peak shapes, and no interfering peaks were observed at the retention times of the analytes. At the same time, due to the high selectivity of MRM mode, there was no endogenous interference and matrix effect on ionization. 3.5.2. Linearity The regression equations, correlation coefficients, test ranges, LODs and LOQs are shown in Table 1. The results showed that there was excellent correlation between the ratio of peak area and concentration for each compound within the test ranges. The LODs were 2.0, 8.0, 2.5, 7.5 and 9.0 ng/mL for luteolin, kaempferol, apigenin, quercetol and isorhamnetin, and the LOQs were less than 25 ng/mL, indicating that this method is sensitive for the quantitative evaluation of the three compounds. 3.5.3. Precision, accuracy and extraction recovery The assay precision and accuracy results are shown in Table 2. The intra- and inter-day precisions and accuracy were determined by the replicate analyses of QC samples (n = 6) at three level concentrations (low, middle, and high). The intra- and inter-day precisions (R.S.D.) of these analytes were all less than 9.1% and 10.8%. The results of extraction recovery are shown in Table 4. The mean
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Fig. 3. Typical MRM chromatograms of analytes (A–E) and IS (F) in rat plasma sample colleted at 30 min after oral administration of Verbena officinalis L. extract. Luteolin (A), kaempferol (B), apigenin (C), quercetol (D), isorhamnetin (E) and IS (F).
extraction recoveries of luteolin, kaempferol, apigenin, quercetol and isorhamnetin ranged from 95.8% to 106.2%, from 83.9% to 105.7%, from 102.1% to 104.5%, from 81.5% to 102.8% and from 95.6% to 108.1%, respectively. The results demonstrated that the values were all within the acceptable range and the method was accurate and precise.
3.5.4. Stability Stability data are summarized in Table 3 and indicated that the three analytes in plasma were stable for 24 h in autosampler condition after preparation, at least three freeze-thaw cycles. Moreover, the results of the stability showed that all the investigated compounds were stable for at least 4 weeks when kept frozen at −20 ◦ C (Table 4).
3.6. Application in pharmacokinetic studies The validated method was successfully applied to monitoring the concentrations and pharmacokinetic studies of five flavone compounds after solvent extraction/acid hydrolysis in rat plasma after a single oral administration of V. officinalis L. extract with a dosage of 8.0 mL/kg. The mean plasma concentration–time curves (n = 6) of luteolin, kaempferol, apigenin, quercetol and isorhamnetin are shown in Fig. 4. The pharmacokinetic parameters are listed in Table 5. These five analytes exhibited consistent tendency in plasma concentration–time profiles. The time to reach the maximum plasma concentration (Tmax1 ) was 0.48 ± 2.14 h for luteolin, 0.25 ± 0.13 h for kaempferol, 0.97 ± 1.08 h for apigenin, 1.04 ± 4.25 h for quercetol and 0.25 ± 0.16 h for isorhamnetin, and the maximum plasma
Table 1 Calibration curves, LODs and LLOQs of luteolin, kaempferol, apigenin, quercetol and isorhamnetin. Components
Regression equation
Luteolin Kaempferol Apigenin Quercetol Isorhamnetin
Y = 2.1764X − 0.0695 Y = 0.7365X − 0.1575 Y = 3.5734X − 0.0593 Y = 0.1291X + 0.0172 Y = 0.1728X + 0.0034
Test range (g/mL) 0.005–2.50 0.005–2.50 0.020–10.0 0.025–12.5 0.020–10.0
r2
LOD (ng/mL)
LOQ (ng/mL)
0.9975 0.9974 0.9931 0.9987 0.9958
2 8 2.5 7.5 9
5 5 20 25 20
K. Duan et al. / Journal of Ethnopharmacology 135 (2011) 201–208
207
Table 2 Precision and accuracy for the determination of luteolin, kaempferol, apigenin, quercetol and isorhamnetin. Components
Spiked concentration (g/mL)
Intra-day (n = 6)
Inter-day (n = 6)
Measured concentration (g/mL, mean ± S.D.) Luteolin
Kaempferol
Apigenin
Quercetol
Isorhamnetin
0.0125 0.250 2.00 0.0125 0.250 2.00 0.050 1.00 8.00 0.0625 1.25 10.0 0.050 1.00 8.00
0.0116 0.255 1.95 0.0105 0.237 2.08.0 0.0521 1.03 8.11 0.0626 1.27 10.1 0.0520 0.972 8.09
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.8 17.0 52.3 0.5 19.8 29.3 0.6 5.7 13.4 1.8 3.4 18.6 0.3 4.9 25.7
R.S.D. (%)
RE (%)
Measured concentration (g/mL, mean ± S.D.)
8.5 3.2 4.0 2.8 3.0 1.8 2.3 6.7 1.9 3.6 7.3 4.2 1.7 2.4 9.1
−1.7 1.8 −1.4 −4.6 3.9 −1.6 −3.0 5.0 −3.1 −2.8 6.9 2.0 −1.3 1.2 −0.8
0.0116 0.264 2.08.6 0.0099 0.271 2.12 0.0522 1.04 8.13 0.0621 1.24 10.1 0.0517 0.925 8.13
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.0 21.5 34.6 0.7 22.1 13.5 0.4 5.8 18.3 1.3 2.8 13.2 0.5 9.4 18.4
R.S.D. (%)
RE (%)
10.8 4.7 3.5 7.5 3.9 2.3 4.9 6.8 2.5 3.8 7.2 2.9 3.0 8.4 5.7
−2.3 1.1 −2.6 −4.1 2.7 2.0 −3.5 4.6 1.8 −3.7 4.3 −8.3 −6.8 1.9 3.2
Table 3 Stability of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma (n = 3). Components
Accuracy (%, mean ± S.D.)
Concentration added (g/mL)
24 h at room temperature
Kaempferol
Apigenin
Quercetol
Isorhamnetin
0.0125 0.250 2.00 0.0125 0.250 2.00 0.050 1.00 8.00 0.0625 1.25 10.0 0.050 1.00 8.00
97.42 102.69 97.35 95.48 102.47 98.25 98.27 104.34 97.36 101.92 98.17 103.44 99.45 97.24 101.58
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.1 4.1 3.7 2.7 4.4 5.1 1.8 2.0 8.3 5.0 2.8 5.3 7.6 7.3 8.4
concentration (Tmax2 ) was 3.97 ± 1.48 h, 4.05 ± 0.46 h, 4.33 ± 0.58 h, 2.99 ± 0.48 h and 4.02 ± 0.34 h. The elimination half-time (T1/2 ) of luteolin, kaempferol, apigenin, quercetol and isorhamnetin was 4.02 ± 0.81, 7.65 ± 0.71, 3.30 ± 0.83, 4.55 ± 0.49 and 5.56 ± 1.32 h, respectively. Following oral administration of V. officinalis L. extract, double peaks were observed in mean plasma concentration curves
Table 4 The mean recovery and matrix effect of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma (n = 6). Components
Luteolin
Kaempferol
Apigenin
Quercetol
Isorhamnetin
Concentration (g/mL)
Recovery (%, mean ± S.D.)
0.0125 0.250 2.00 0.0125 0.250 2.00 0.050 1.00 8.00 0.0625 1.25 10.0 0.050 1.00 8.00
95.80 106.2 104.8 83.9 96.3 105.7 102.1 104.6 104.5 81.5 102.8 93.4 108.1 105.7 95.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.2 6.3 1.6 8.7 12.1 1.7 5.3 2.8 0.8 8.5 5.6 5.1 1.8 2.6 4.8
98.46 103.4 96.84 97.26 101.52 95.17 99.15 101.71 97.14 98.37 99.09 104.82 97.65 98.37 102.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.5 2.2 1.8 2.8 3.1 3.8 2.4 1.4 2.6 4.3 9.2 2.8 3.4 1.9 6.7
4.3 5.4 5.9 6.7 5.3 2.0 5.2 1.7 1.8 2.8 1.9 4.7 5.1 1.6 2.8
103.83 106.01 99.37 98.15 97.63 105.48 98.69 102.47 98.35 97.06 104.85 97.54 98.23 103.58 104.19
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.6 3.9 2.8 1.5 6.8 4.7 4.6 3.9 5.4 6.1 6.5 8.0 2.9 4.5 6.4
10000
Matrix effect (%, mean ± S.D.) 96.80 101.7 92.4 94.8 97.5 98.4 96.9 97.8 87.8 107.6 102.9 96.2 104.6 94.8 101.5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
of all five individual flavones. It can be inferred that herb extract could have a effect to the absorption of five flavone compounds in rat plasma. According to prior phytochemistry study, many iridoid glycosides were separated and identified in V. officinalis L. such as verbascoside, verbenalin, hastatoside, aucubin, and gentiopicroside (Calvo et al., 1997; Makboul, 1986; Rimpler et al., 1979; Muller et al., 2004). The main reason of double peaks suggested a
8000
Concentration (ng/mL)
Luteolin
15 days storage at −20 ◦ C
Freeze-thaw cycles
luteolin kaempferol
6000
apigenin quercetol
4000
isorhamnetin
2000 0
0
2
4
6
8
10
12
14
Time (h) Fig. 4. Mean ± S.D. (n = 6) plasma concentration–time curves of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rats after oral administration of Verbena officinalis L. extract.
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Table 5 Mean pharmacokinetic parameters for luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma after oral administration of Verbena officinalis L. extract (n = 6). Parameters
Mean ± S.D. Luteolin
T1/2 (h) Ke (1/h) Tmax1 (h) Cmax1 (g/mL) Tmax2 (h) Cmax2 (g/mL) AUC0−t (g h/mL) AUC0−∞ (g h/mL) MRT0−∞ (h) Vz/F (L/kg) Cl/F (L/kg/h)
4.02 0.18 0.48 1.23 3.97 1.62 6.38 10.67 9.67 3.32 0.20
± ± ± ± ± ± ± ± ± ± ±
Kaempferol 0.81 0.04 2.14 0.23 1.48 0.19 0.31 0.34 3.88 2.05 0.06
7.65 0.09 0.25 0.35 4.05 0.59 3.62 5.14 12.75 7.06 0.57
± ± ± ± ± ± ± ± ± ± ±
0.71 0.01 0.13 0.02 0.46 0.09 0.25 0.85 1.43 2.93 0.11
possible enterohepatic recirculation (Wan et al., 2007; Zhang et al., 2009). However, this hypothesis needs further investigation. The information described above might be helpful for further studies on the pharmacokinetics of V. officinalis L., and beneficial for the application of this TCM in clinical therapy. 4. Conclusions This paper described a simple, sensitive and validated LC–MS/MS method for simultaneous determination of luteolin, kaempferol, apigenin, quercetol and isorhamnetin after solvent extraction/acid hydrolysis in rat plasma after oral administration of V. officinalis L. extract, and investigated on their pharmacokinetic studies as well, with a short run time of 5 min. This method was sensitive, with high accuracy and met all requirements in bioanalytical method. It was successfully applied to the pharmacokinetic studies of active components of V. officinalis L. extract, which maybe provide some references to the apprehension of the action mechanism and clinical application of V. officinalis L. This is the first report of pharmacokinetic studies of luteolin, kaempferol, apigenin, quercetol and isorhamnetin together in vivo following the oral administration of V. officinalis L. extract. Acknowledgement We thank the financial support from Department of Science and Technology of Hebei Province of China (09276423D) for this work. References Calvo, M.I., 2006. Anti-inflammatory and analgesic activity of the topical preparation of Verbena officinalis L. Journal of Ethnopharmacology 107, 380–382. Calvo, M.I., San Julian, A., Fernandez, M., 1997. Identification of the major compounds in extracts of Verbena officinalis L. (Verbenaceae) by HPLC with post-column derivatization. Chromatographia 46, 241–244. Chen, G.M., Zhang, J.Y., Liu, H.M., et al., 2006a. Studies on chemical constituents of flavonoid from Verbena officinals. Journal of Chinese Medicinal Materials 29, 677–678. Chen, G.M., Zhang, J.Y., Hong, G.F., et al., 2006b. Determination of flavonoids content for Verbena officinalis. Chinese Journal of Modern Applied Pharmacy 3, 798–799.
Apigenin 3.30 0.22 0.97 3.07 4.33 6.69 25.88 28.77 5.95 0.54 0.12
± ± ± ± ± ± ± ± ± ± ±
0.83 0.05 1.08 0.26 0.58 0.27 0.49 0.21 0.57 0.12 0.01
Quercetol 4.55 0.15 1.04 0.79 2.99 6.36 13.44 15.81 5.06 3.44 0.21
± ± ± ± ± ± ± ± ± ± ±
0.49 0.02 4.25 0.15 0.48 0.77 0.79 0.27 1.03 2.53 0.04
Isorhamnetin 5.56 0.13 0.25 0.71 4.02 0.82 2.71 3.25 7.09 4.28 0.52
± ± ± ± ± ± ± ± ± ± ±
1.32 0.03 0.16 0.05 0.34 0.12 0.09 0.33 1.53 0.41 0.16
Deepak, M., Handa, S.S., 2000. Anti-inflammatory activity and chemical composition of extracts of Verbena officinalis. Phytotherapy Research 14, 463– 465. Deng, J.G., Zhou, X.L., 2005. The study of chemical constituent and pharmacologic action in Verbena officinalis L. Guangxi Journal of Traditional Chinese Medica 28, 63–65. Fernandez, M., 1981. Las plantas en la medicina popular navarra 1. Navarra humeda del NO. Sociedad de EstudiosVascos-Eusko Ikaskuntza, Pamplona. Gui, C.H., Tang, R.J., 1985. The study of preventing cough active component in Verbena officinalis L. Bulletin of Chinese Material Medica 10, 35. Jin, W.J., Zhang, Z.D., 2007. Progress of Verbena officinalis L. Lishizhen Medicine and Materia Medica Research 18, 693–694. Li, L.P., Jiang, H.D., Zeng, S., et al., 2005. Simultaneous determination of luteolin and apigenin in dog plasma by RP-HPLC. Journal of Pharmaceutical and Biomedical Analysis 37, 615–620. Liu, H.M., Bao, F.Y., Yan, X.B., 2002. Studies on chemical constituents of Verbena officinalis. Chinese Traditional and Herbal Drugs 33, 492–494. Makboul, A.M., 1986. Chemical constituents of Verbena officinalis. Fitoterapia 57, 50–51. Muller, A., Ganzera, M., Stuppner, H., 2004. Analysis of the aerial parts of Verbena officinalis L. by Micellar Electrokinetic Capillary Chromatography. Chromatographia 60, 193–197. National Commission Chinese Pharmacopoeia of the People’s Republic of China, vol. 1. Chemical Industry press, Beijing, 2005, p. 35. Rimpler, H., Schaefer, B.Z., Hastatoside, 1979. a new iridoid from Verbena hastata L. and Verbena officinalis L. Zeitschrift fur Naturforschung 3, 311–318. Tian, Q., Zhao, Y.M., Luan, X.H., 2005. Study on the chemical constituents of Verbena officinalis. China Journal of Chinese Materia Medica 30, 268–269. Wan, L.L., Guo, C., Yu, Q., et al., 2007. Quantitative determination of apigenin and its metabolism in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry. Journal of Chromatography B 855, 286–289. Wichtl, M., Bisset, N.G. (Eds.), 1994. Herbal Drugs and Phytopharmaceuticals. Medpharm Scientific Publishers, Stuttgart. Xin, F., Jin, Y.S., Xu, W., et al., 2008. Study on the chemical constituents of Verbena officinalis. Modern Chinese Medicine 10, 21–23. Yuan, Z.F., Duan, K.F., Zhang, L.T., et al., 2009. Simultaneous determination of verbenalin and hastatoside in Verbena officinalis L. with RP-HPLC. Chinese Traditional and Herbal Drugs 40, 818–820. Zhang, T., Lv, Z.M., Ruan, J.L., et al., 2002. The exploration of the biosynthesis route of the iridoid glucosides in Verbane officinalis. Journal of Jianghan University (Medical Edition) 30, 16–17. Zhang, Y., Zhou, J.P., Huo, M.R., 2007. A data analysis program in pharmacokinetics base on Microsoft Excel – development and validation of PKSlover 1.0. Journal of Mathematical Medicine 20, 58–61. Zhang, Q.L., Zhang, Y.J., Zhang, Z.T., et al., 2009. Sensitive determination of kaempferol in rat plasma by high-performance liquid chromatography with chemiluminescence detection and application to a pharmacokinetic study. Journal of Chromatography B 877, 3595–3600.
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Review
LC–MS/MS determination and pharmacokinetic study of five flavone components after solvent extraction/acid hydrolysis in rat plasma after oral administration of Verbena officinalis L. extract Kunfeng Duan, Zhifang Yuan, Wei Guo, Yan Meng, Yang Cui, Dezhi Kong, Lantong Zhang ∗ , Na Wang Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang 050017, China
a r t i c l e
i n f o
Article history: Received 21 April 2010 Received in revised form 18 December 2010 Accepted 5 January 2011 Available online 8 January 2011 Keywords: Verbena officinalis L. extract HPLC–MS/MS Pharmacokinetics
a b s t r a c t Ethnopharmacological relevance: Traditional Chinese medicine (TCM) has been used in clinical practice for several thousand years. TCM has played an indispensable role in the prevention and treatment of diseases, especially the complicated and chronic ones. Pharmacokinetic study on active constituents in herbal preparations is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of TCM. Aim of the study: A selective and sensitive HPLC–MS/MS method was first developed and validated for the determination of luteolin, kaempferol, apigenin, quercetol, and isorhamnetin in rat plasma. Materials and methods: The LC system consisted of an Agilent Technologies Series 1200 system (Agilent, USA) equipped with an automatic degasser, a quaternary pump, and an autosampler. Chromatographic separations were performed on a Waters SunFireTM C18 column (150 mm × 4.6 mm, 5 m), and the column temperature was maintained at 25 ◦ C and the sample injection volume was 20 L. The current LC–MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability. Results: The validated method was successfully applied to monitoring the concentrations and pharmacokinetic studies of five flavone compounds in rat plasma after a single oral administration of Verbena officinalis L. extract with a dosage of 8.0 mL/kg. The time to reach the maximum plasma concentration (Tmax1 ) was 0.48 ± 2.14 h for luteolin, 0.25 ± 0.13 h for kaempferol, 0.97 ± 1.08 h for apigenin, 1.04 ± 4.25 h for quercetol and 0.25 ± 0.16 h for isorhamnetin, and the maximum plasma concentration (Tmax2 ) was 3.97 ± 1.48 h, 4.05 ± 0.46 h, 4.33 ± 0.58 h, 2.99 ± 0.48 h and 4.02 ± 0.34 h. The elimination half-time (t1/2 ) of luteolin, kaempferol, apigenin, quercetol and isorhamnetin was 4.02 ± 0.81, 7.65 ± 0.71, 3.30 ± 0.83, 4.55 ± 0.49 and 5.56 ± 1.32 h, respectively. Conclusions: This paper described a simple, sensitive and validated LC–MS/MS method for simultaneous determination of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma after oral administration of V. officinalis L. extract, and investigated on their pharmacokinetic studies as well, with a short run time of 5 min. © 2011 Published by Elsevier Ireland Ltd.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chemicals and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Instrumentation and analytical conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Preparation of V. officinalis L. extract in the administration solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Preparation of calibration standards and quality control (QC) samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Application to pharmacokinetic study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author. Tel.: +86 311 86266419; fax: +86 311 86266419. E-mail address: [email protected] (L. Zhang). 0378-8741/$ – see front matter © 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2011.01.002
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2.7. Method validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Mass spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Liquid chromatographic conditions and matrix effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Selection of IS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Method validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1. Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2. Linearity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.3. Precision, accuracy and extraction recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.4. Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Application in pharmacokinetic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Traditional Chinese medicine (TCM) has been used in clinical practice for several thousand years. TCM has played an indispensable role in the prevention and treatment of diseases, especially the complicated and chronic ones. Pharmacokinetic study on active constituents in herbal preparations is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of TCM. Verbena officinalis L., which is known as Mabiancao mostly grows in South China, is a famous traditional Chinese medicine (TCM) and widely used for clearing away heat and detoxicating, promoting blood circulation and removing blood stasis, inducing diuresis and excreting dampness based on the Chinese medical theory (National Commission Chinese Pharmacopoeia of the People’s Republic of China, 2005). It also can be used in folk medicine as a diuretic, expectorant and anti-rheumatic (Wichtl and Bisset, 1994). In Navarra, Spain, it is used extensively in traditional medicine mainly because of its anti-inflammatory topical applications (Fernandez, 1981; Deepak and Handa, 2000; Calvo, 2006). In 1985, it was the first time to report V. officinalis L. had antitussive effect which was associated with the components including verbenalin by Gui Chenghui in China (Gui and Tang, 1985). However, as we know, one or several categories of ingredients contribute to the pharmacodyamic material basis in most traditional Chinese medicine. Up to now, the total extract of V. officinalis L. and its ethyl acetate and n-butyl alcohol parts had significant antitussive and anti-inflammatory effects (Deng and Zhou, 2005; Jin and Zhang, 2007). Phytochemical investigation found that V. officinalis L. contained many constituents such as flavonoids, iridoid glycosides, phenylpropanoid glycoside, sterols, triterpenes and glycoconjugate (Rimpler et al., 1979; Makboul, 1986; Calvo et al., 1997; Liu et al., 2002; Muller et al., 2004). It has been reported that flavonoids and iridoid glycosides are important active constituents most contributing to the pharmacological efficacy of V. officinalis L. (Zhang et al., 2002; Xin et al., 2008). Pharmacological tests have revealed that the major active compounds of anti-inflammatory are flavonoids mainly including luteolin, kaempferol, apigenin, quercetol and isorhamnetin (Tian et al., 2005; Chen et al., 2006a,b). Their chemical structures are shown in Fig. 1. To date, there has many preliminary researches on the quantitative analysis of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in plant material, natural products and preparations using HPLC, HPLC-MS and MEKC (Zhang et al., 2002; Yuan et al., 2009). In contrast, only few papers have been reported on HPLC or HPLC–MS methods for the pharmacokinetic study of luteolin in rabbits and apigenin in rats, independently (Li et al., 2005). As luteolin, kaempferol, apigenin, quercetol and isorhamnetin are the major active components of V. officinalis L., it is essential to develop a method to determine all
204 204 204 204 204 205 205 205 205 205 206 206 208 208 208
five of them in plasma samples for the purpose of studying the mechanism of action. In the present study we developed and validated a sensitive, specific and accurate method for the simultaneous determination of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma. The method was successfully applied to evaluate the pharmacokinetics of luteolin, kaempferol, apigenin, quercetol and isorhamnetin after oral administration of V. officinalis L. extract to rats. 2. Experimental 2.1. Chemicals and reagents Five standards, luteolin; kaempferol; apigenin; quercetol and isorhamnetin (Fig. 1), were purchased from the National Institute for Control of Biological and Pharmaceutical Products (Beijing, China). SMZ (Sulfamethoxazolum) as internal standard was identified and supplied by Hebei Medical University (Hebei, China). Methanol (Tedia, Fairfield, USA), acetonitrile (Tedia, Fairfield, USA) and formic acid (Kermel, Tianjin, China) were of HPLC grade. The distilled water was prepared from demineralized water and used throughout the experiments. All other chemical solvents were of analytical grade from Beijing Chemical Factory (Beijing, China). V. officinalis L. was purchased from a traditional Chinese medicinal store in Shijiazhuang, China and authenticated by Professor Ding Zhao (Key Laboratory of Modern Chinese Medicines, School of Pharmacy, Hebei Medical University, Shijiazhuang, China). 2.2. Instrumentation and analytical conditions The LC system consisted of an Agilent Technologies Series 1200 system (Agilent, USA) equipped with an automatic degasser, a quaternary pump, and an autosampler. Chromatographic separations were performed on a Waters SunFireTM C18 column (150 mm × 4.6 mm, 5 m), and the column temperature was maintained at 25 ◦ C and the sample injection volume was 20 L. The mobile phase consisted of acetonitrile and 0.1% aqueous formic acid using gradient elution (0–2 min, 45–80% acetonitrile; 2–5 min, 80% acetonitrile; 5–5.1 min 80–45% acetonitrile) and was delivered at a flow rate of 0.8 mL/min. Determination was performed with 3200 QTRAPTM system from Applied Biosystems/MDS Sciex (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion trap mass spectrometer equipped with Turbo V sources and TurboIonspray interface. The analytes were determined in negative ionization mode by monitoring the precursor–product combination in multiple reaction monitoring (MRM) mode. The ion source temperature was held at 650 ◦ C. Target ions were monitored at m/z 285.0
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Fig. 1. Chemical structures of luteolin (A), kaempferol (B), apigenin (C), quercetol (D), isorhamnetin (E) and IS (F).
for luteolin, 285.0 for kaempferol, 269.0 for apigenin, 301.1 for quercetol, 315.1 for isorhamnetin and 252.0 for IS. The data acquisition and peak integration were performed using analyst software (Version 1.4.2). 2.3. Preparation of V. officinalis L. extract in the administration solution The dried powder of V. officinalis L. (100 g) was extracted twice by refluxing with 80% ethanol (1:8 and then 1:6, w/v) for 3 h and the extraction solutions were combined to be filtered, concentrated to 100 mL under reduced pressure and then precipitated with the same volume water. The precipitate was discarded and the ethanol was removed under the condition of waterbath. The solution was then extracted with same volume acetoacetate for 3 times. The acetoacetate was removed under reduced pressure and the residue was dissolved in water. The administration solution was obtained and stored at 4 ◦ C before use. To calculate the administered dose, the contents of five flavones in administration solution were quantitatively determined by external standardization using the same chromatography conditions as described above. The contents of luteolin, kaempferol, apigenin, quercetol and isorhamnetin were 0.206, 0.288, 0.332, 0.162 and 0.094 mg/mL in extract, respectively. 2.4. Preparation of calibration standards and quality control (QC) samples Stock solution of luteolin, kaempferol, apigenin, quercetol, isorhamnetin and IS was prepared in methanol at the concentration of 530 g/mL, 220 g/mL, 941 g/mL, 196 g/mL, 520 g/mL and 360 g/mL. A series of standard mixture working solutions with concentrations 0.005–2.50 g/mL for luteolin, 0.005–2.50 g/mL for kaempferol, 0.020–10.0 g/mL for apigenin, 0.025–12.5 g/mL for quercetol and 0.020–10.0 g/mL for isorhamnetin were obtained by diluting the mixture of the stock standard solutions with methanol. The working solution of IS (3.60 g/mL) was prepared by diluting SMZ. All the working solutions were kept at 4 ◦ C. Then assay standard samples were prepared by spiking 100 L blank rat plasma with standard mixture working solutions (20 L) and IS working solution (20 L), in which the concentrations were 0.1, 0.25, 1.0, 5.0, 20.0, and 50.0 g/mL for luteolin, 0.1, 0.25,
1.0, 5.0, 20.0, and 50.0 g/mL for kaempferol, 0.4, 1.0, 4.0, 20.0, 80.0, and 200.0 g/mL for apigenin, 0.5, 1.25, 5.0, 25.0, 100.0, and 250.0 g/mL for quercetol and 0.4, 1.0, 4.0, 20.0, 80.0, and 200.0 g/mL for isorhamnetin. Quality control (QC) samples (luteolin 0.0125, 0.250, and 2.0 g/mL, kaempferol 0.0125, 0.250, and 2.00 g/mL, apigenin 0.050, 1.00, and 8.00 g/mL, quercetol 0.0625, 1.25, and 10.0 g/mL, isorhamnetin 0.050, 1.00, and 8.00 g/mL) were independently prepared in the same manner. The working and assay standard solutions were prepared everyday. 2.5. Sample preparation Plasma samples (100 L each) were treated with 10 mmol/L ascorbic acid before storage at −70 ◦ C. For clean-up, the thawed plasma samples were vortex-mixed with 20 L of IS and 280 L of methanol–hydrochloric acid (25%) (4:1, v/v). The solution was centrifuged at 12,000 rpm for 10 min to separate the protein from the organic phase, then the upper supernatant was treated at 90 ◦ C for 30 min, and then the supernatant was collected and centrifuged at 12,000 rpm for another 10 min. At last 20 L of the resulting supernatant was applied to LC–MS/MS analysis. 2.6. Application to pharmacokinetic study Male Sprague–Dawley rats (250 ± 20 g) were supplied by Lab Animal Center of Heibei Medical University (Shijiazhuang, China) and kept in an environmentally controlled breeding room (temperature: 24 ± 2 ◦ C, humidity: 60 ± 5%, 12 h dark–light cycle) for at least 5 days before starting the experiments. They were fed with food and water ad libitum and fasted overnight prior to the test. Blood samples of about 0.3 mL were collected into heparinized centrifuge tubes from the fossa orbitalis vein at 0, 5, 15, 30, 60, 90, 120, 150, 180, 240, 360, 480, and 720 min in 6 healthy rats after single oral administration of V. officinalis L. extract (10 mL/kg). Then the samples were centrifuged at 4000 rpm for 10 min and the separated plasma samples were frozen in polypropylene tubes at −20 ◦ C until analysis. PK parameters including area under concentration–time curve (AUC), maximum plasma concentration (Cmax ), time to reach the maximum concentrations (Tmax ), the plasma half-life (T1/2 ), the area under the plasma concentration–time curve from zero to the time of the final measurable sample (AUC0−t ), the area under the
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plasma concentration–time curve from zero to infinity (AUC0−∞ ) and mean residence time (MRT) were estimated using PKSlover 1.0 (Zhang et al., 2007).
and 252.0/155.9 for IS. The product ion scan spectra were shown in Fig. 2. 3.2. Liquid chromatographic conditions and matrix effect
2.7. Method validation
3. Results and discussion
The chromatographic condition was optimized to improve peak shape, increase signal response of analytes and shorten run time. In the previous study, acetonitrile was chosen as the organic solvent because it provided a higher sensitivity and lower background noise than methanol. Comparative studies showed that the use of a mobile phase containing an extremely low concentration of formic acid dramatically improved the electrospray ionization efficiency of the analytes in the negative ion mode. We tested different buffers including formic acid (0.01%, 0.05%, and 0.1%), ammonium acetate (0.2, 1, and 2 mmol/L) and acetic acid (0.01%, 0.05%, and 0.1%) to identify the optimal mobile phase that produced the best sensitivity and efficiency. The results indicated that the intensity of the five analytes peak strength increased dramatically when 0.01% formic acid was added into mobile phase. Therefore acetonitrile–0.01% formic acid was used as the mobile phase with an acceptable run time of 5.0 min. The matrix effect (ME) was examined by comparing the mean peak areas of the analytes between two different sets of samples. In set 1, reference standards were dissolved in water (100 L), methanol–hydrochloric acid (25%) (4:1, v/v) (260 L) and five replicates were analyzed. In set 2, blank plasma samples (100 L) were extracted and then spiked with the same concentrations of analytes. Samples of both sets were prepared at three QC levels for the analytes (n = 6). The ME was defined as following: ME% = 100 × set 2/set1. Matrix effect results were shown in Table 4. The average matrix effect values were 96.8%, 101.7% and 92.4% for luteolin, 94.8%, 97.5% and 98.4% for kaempferol, 96.9%, 97.8% and 87.8% for apigenin, 107.6%, 102.9% and 96.2% for quercetol and 104.6%, 94.8% and 101.5% for isorhamnetin at the three QC concentration levels. Results, which were described as percentage in concentration of diluted samples to working standard solutions, were no less than 85% or more than 115% for all the six analytes, and showed that there was no significant difference between the peak areas of samples prepared from rat plasma and from water which indicated that no co-eluting unseen compounds significantly influenced the ionization of analytes and IS.
3.1. Mass spectra
3.3. Sample preparation
Luteolin, kaempferol, apigenin, quercetol, and isorhamnetin were at first characterized according to their mass spectra from syringe pump infusion analysis, respectively. Meanwhile, their precursor ions and product ions were ascertained for use in MRM. The electrospray interface (ESI) was employed for good sensitivity and fragmentation were obtained. In our experiment, we discovered that the negative ion mode was more sensitive for flavonol analysis. Positive mode also tested, but the sensitivity obtained was not satisfactory for all of the six analytes and IS. Thus, negative ion mode was finally employed. The optimum declustering potential were −70 V for luteolin, −67 V for kaempferol, −58 V for apigenin, −50 V for quercetol, −52 V for isorhamnetin and −30 V for IS. The molecular ions of luteolin, kaempferol, apigenin, quercetol, isorhamnetin and SMZ IS were fragmented at collision energy of −47 eV, −83 eV, −50 eV, −30 eV, −30 eV and −20 eV using nitrogen as collision gas. In the full scan mass spectra, the protonated molecular ions [M−H]− were stable and exhibited higher abundance, thus [M−H]− were chosen as the precursor ions for MS/MS fragmentation analysis of the analytes. The optimized mass transition ion-pairs (m/z) for quantitation were 285.0/133.0 for luteolin, 285.0/92.9 for kaempferol, 269.0/117.0 for apigenin, 301.1/151.0 for quercetol, 315.1/300.0 for isorhamnetin,
Commonly, the used extraction techniques mainly including liquid–liquid extraction (LLE), protein precipitation (PPT) and solid phase extraction (SPE). The method of SPE has good extraction recovery and reproducibility, however, this extraction method is complicated and costly so that it is not suitable for the processing of multiple samples in a limited amount of time for bioequivalence and pharmacokinetic studies. In the course of our method development, we found that liquid–liquid extraction technique had a low recovery when ethyl acetate, dichloromethane, n-butanol or their mixture in different combinations and ratios used as extraction solvents. Therefore, plasma samples were subjected to a simple protein precipitation procedure. Finally, methanol–25% hydrochloric acid (4:1, v/v) appeared to be optimal, especially for five analytes and IS. The determined concentration included the free flavones and that after acid hydrolysis. During the experiment, it was found that the five flavones were hardly to be detected after plasma precipitation only by organic solvents. The extract was a complex system, may be some compounds result in the induction of metabolic enzymes, the determined flavones rapidly transformed to their metabolites. Therefore, the total concentration of the five flavones was determined after solvent extraction/acid hydrolysis. So hydrolysis was added. At last a simple one-step protein
The current LC–MS/MS assay was validated for linearity, intraday and inter-day precisions, accuracy, extraction recovery and stability. The calibration plasma samples were prepared by adding 20 L of working solutions, 20 L of IS solution to 100 L of blank plasma and extracted as described in Section 2.5. Calibration curves were prepared by assaying standard plasma samples at six concentrations. Weighted (1/x) linear least-squares regression method was used to determine the slope, intercept and correlation coefficient. The limits of detection (LOD) and limits of quantification (LOQ) were determined at a signal-to-noise ratio of about 3 and 10 by analyzing the diluted standard solution. Three validation batches, each containing six replicates of QC samples at low, medium and high concentration levels (0.0125, 0.250, and 2.00 g/mL for luteolin, 0.0125, 0.250, and 2.00 g/mL for kaempferol, 0.050, 1.00, and 8.00 g/mL for apigenin, 0.0625, 1.25, and 10.0 g/mL for quercetol and 0.050, 1.00, and 8.00 g/mL for isorhamnetin), were assayed to assess the precision and accuracy of the method on three different days. The intra-day and inter-day precisions were defined as relative standard deviation (R.S.D.) and the accuracy was assessed by comparing the measured concentration with its true value. The extraction recovery was determined in sets of six replicate quality control samples by measuring the amount of each compound recovered after extraction and calculated by comparing the peak areas of the extracted samples with that of the unextracted standard solutions (the solvent was changed to water) containing an equivalent amount of the analytes. The stability of analytes in rat plasma was evaluated by analyzing QC samples at three concentrations exposed to different conditions (room temperature, 24 h; −20 ◦ C, 15 days; freeze-thaw cycles). Stability was assessed by comparing the mean concentration of the stored QC samples.
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1.0e5
132.9
4.6e6
205
92.9
B
A
285.0
285.0 100
200
117.0
4.6e6
100
300
150.9
1.7e6
C
200
300
D
269.0
100
200
300.9
300.0
1.35e6
100
300
252.0
315.1 100
200
300
300
F
155.9
2.0e7
E
200
400
100
200
300
Fig. 2. MS and product ion spectra of the compounds. Luteolin (A), kaempferol (B), apigenin (C), quercetol (D), isorhamnetin (E) and IS (F).
precipitation was executed, and high recovery and good precision and accuracy were obtained. 3.4. Selection of IS An internal standard should be used when performing pharmacokinetic study. An appropriate internal standard will control for extraction, HPLC injection and ionization variability. We investigated several compounds served as suitable IS, such as paeoniflorin, puerarin, and orientin. As these compounds are also flavone, their physicochemical features and ionization characteristics are similar to those of analytes. The result showed that SMZ was more appropriate for IS, because it provided clean chromatography and shorter retention time and no significant direct interference was observed. Moreover, SMZ is not a component of biological samples after oral administration of V. officinalis L. extract. 3.5. Method validation 3.5.1. Specificity The typical chromatograms of plasma sample from a rat after oral administration of V. officinalis L. extract are shown in Fig. 3. Four channels were used for recording and the retention times of luteolin, kaempferol, apigenin, quercetol, isorhamnetin and IS were
3.22, 3.80, 3.70, 3.30, 3.85 and 3.18 min, respectively. All the peaks of the analytes and IS were detected with excellent resolution as well as peak shapes, and no interfering peaks were observed at the retention times of the analytes. At the same time, due to the high selectivity of MRM mode, there was no endogenous interference and matrix effect on ionization. 3.5.2. Linearity The regression equations, correlation coefficients, test ranges, LODs and LOQs are shown in Table 1. The results showed that there was excellent correlation between the ratio of peak area and concentration for each compound within the test ranges. The LODs were 2.0, 8.0, 2.5, 7.5 and 9.0 ng/mL for luteolin, kaempferol, apigenin, quercetol and isorhamnetin, and the LOQs were less than 25 ng/mL, indicating that this method is sensitive for the quantitative evaluation of the three compounds. 3.5.3. Precision, accuracy and extraction recovery The assay precision and accuracy results are shown in Table 2. The intra- and inter-day precisions and accuracy were determined by the replicate analyses of QC samples (n = 6) at three level concentrations (low, middle, and high). The intra- and inter-day precisions (R.S.D.) of these analytes were all less than 9.1% and 10.8%. The results of extraction recovery are shown in Table 4. The mean
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Fig. 3. Typical MRM chromatograms of analytes (A–E) and IS (F) in rat plasma sample colleted at 30 min after oral administration of Verbena officinalis L. extract. Luteolin (A), kaempferol (B), apigenin (C), quercetol (D), isorhamnetin (E) and IS (F).
extraction recoveries of luteolin, kaempferol, apigenin, quercetol and isorhamnetin ranged from 95.8% to 106.2%, from 83.9% to 105.7%, from 102.1% to 104.5%, from 81.5% to 102.8% and from 95.6% to 108.1%, respectively. The results demonstrated that the values were all within the acceptable range and the method was accurate and precise.
3.5.4. Stability Stability data are summarized in Table 3 and indicated that the three analytes in plasma were stable for 24 h in autosampler condition after preparation, at least three freeze-thaw cycles. Moreover, the results of the stability showed that all the investigated compounds were stable for at least 4 weeks when kept frozen at −20 ◦ C (Table 4).
3.6. Application in pharmacokinetic studies The validated method was successfully applied to monitoring the concentrations and pharmacokinetic studies of five flavone compounds after solvent extraction/acid hydrolysis in rat plasma after a single oral administration of V. officinalis L. extract with a dosage of 8.0 mL/kg. The mean plasma concentration–time curves (n = 6) of luteolin, kaempferol, apigenin, quercetol and isorhamnetin are shown in Fig. 4. The pharmacokinetic parameters are listed in Table 5. These five analytes exhibited consistent tendency in plasma concentration–time profiles. The time to reach the maximum plasma concentration (Tmax1 ) was 0.48 ± 2.14 h for luteolin, 0.25 ± 0.13 h for kaempferol, 0.97 ± 1.08 h for apigenin, 1.04 ± 4.25 h for quercetol and 0.25 ± 0.16 h for isorhamnetin, and the maximum plasma
Table 1 Calibration curves, LODs and LLOQs of luteolin, kaempferol, apigenin, quercetol and isorhamnetin. Components
Regression equation
Luteolin Kaempferol Apigenin Quercetol Isorhamnetin
Y = 2.1764X − 0.0695 Y = 0.7365X − 0.1575 Y = 3.5734X − 0.0593 Y = 0.1291X + 0.0172 Y = 0.1728X + 0.0034
Test range (g/mL) 0.005–2.50 0.005–2.50 0.020–10.0 0.025–12.5 0.020–10.0
r2
LOD (ng/mL)
LOQ (ng/mL)
0.9975 0.9974 0.9931 0.9987 0.9958
2 8 2.5 7.5 9
5 5 20 25 20
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Table 2 Precision and accuracy for the determination of luteolin, kaempferol, apigenin, quercetol and isorhamnetin. Components
Spiked concentration (g/mL)
Intra-day (n = 6)
Inter-day (n = 6)
Measured concentration (g/mL, mean ± S.D.) Luteolin
Kaempferol
Apigenin
Quercetol
Isorhamnetin
0.0125 0.250 2.00 0.0125 0.250 2.00 0.050 1.00 8.00 0.0625 1.25 10.0 0.050 1.00 8.00
0.0116 0.255 1.95 0.0105 0.237 2.08.0 0.0521 1.03 8.11 0.0626 1.27 10.1 0.0520 0.972 8.09
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.8 17.0 52.3 0.5 19.8 29.3 0.6 5.7 13.4 1.8 3.4 18.6 0.3 4.9 25.7
R.S.D. (%)
RE (%)
Measured concentration (g/mL, mean ± S.D.)
8.5 3.2 4.0 2.8 3.0 1.8 2.3 6.7 1.9 3.6 7.3 4.2 1.7 2.4 9.1
−1.7 1.8 −1.4 −4.6 3.9 −1.6 −3.0 5.0 −3.1 −2.8 6.9 2.0 −1.3 1.2 −0.8
0.0116 0.264 2.08.6 0.0099 0.271 2.12 0.0522 1.04 8.13 0.0621 1.24 10.1 0.0517 0.925 8.13
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.0 21.5 34.6 0.7 22.1 13.5 0.4 5.8 18.3 1.3 2.8 13.2 0.5 9.4 18.4
R.S.D. (%)
RE (%)
10.8 4.7 3.5 7.5 3.9 2.3 4.9 6.8 2.5 3.8 7.2 2.9 3.0 8.4 5.7
−2.3 1.1 −2.6 −4.1 2.7 2.0 −3.5 4.6 1.8 −3.7 4.3 −8.3 −6.8 1.9 3.2
Table 3 Stability of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma (n = 3). Components
Accuracy (%, mean ± S.D.)
Concentration added (g/mL)
24 h at room temperature
Kaempferol
Apigenin
Quercetol
Isorhamnetin
0.0125 0.250 2.00 0.0125 0.250 2.00 0.050 1.00 8.00 0.0625 1.25 10.0 0.050 1.00 8.00
97.42 102.69 97.35 95.48 102.47 98.25 98.27 104.34 97.36 101.92 98.17 103.44 99.45 97.24 101.58
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.1 4.1 3.7 2.7 4.4 5.1 1.8 2.0 8.3 5.0 2.8 5.3 7.6 7.3 8.4
concentration (Tmax2 ) was 3.97 ± 1.48 h, 4.05 ± 0.46 h, 4.33 ± 0.58 h, 2.99 ± 0.48 h and 4.02 ± 0.34 h. The elimination half-time (T1/2 ) of luteolin, kaempferol, apigenin, quercetol and isorhamnetin was 4.02 ± 0.81, 7.65 ± 0.71, 3.30 ± 0.83, 4.55 ± 0.49 and 5.56 ± 1.32 h, respectively. Following oral administration of V. officinalis L. extract, double peaks were observed in mean plasma concentration curves
Table 4 The mean recovery and matrix effect of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma (n = 6). Components
Luteolin
Kaempferol
Apigenin
Quercetol
Isorhamnetin
Concentration (g/mL)
Recovery (%, mean ± S.D.)
0.0125 0.250 2.00 0.0125 0.250 2.00 0.050 1.00 8.00 0.0625 1.25 10.0 0.050 1.00 8.00
95.80 106.2 104.8 83.9 96.3 105.7 102.1 104.6 104.5 81.5 102.8 93.4 108.1 105.7 95.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.2 6.3 1.6 8.7 12.1 1.7 5.3 2.8 0.8 8.5 5.6 5.1 1.8 2.6 4.8
98.46 103.4 96.84 97.26 101.52 95.17 99.15 101.71 97.14 98.37 99.09 104.82 97.65 98.37 102.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.5 2.2 1.8 2.8 3.1 3.8 2.4 1.4 2.6 4.3 9.2 2.8 3.4 1.9 6.7
4.3 5.4 5.9 6.7 5.3 2.0 5.2 1.7 1.8 2.8 1.9 4.7 5.1 1.6 2.8
103.83 106.01 99.37 98.15 97.63 105.48 98.69 102.47 98.35 97.06 104.85 97.54 98.23 103.58 104.19
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.6 3.9 2.8 1.5 6.8 4.7 4.6 3.9 5.4 6.1 6.5 8.0 2.9 4.5 6.4
10000
Matrix effect (%, mean ± S.D.) 96.80 101.7 92.4 94.8 97.5 98.4 96.9 97.8 87.8 107.6 102.9 96.2 104.6 94.8 101.5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
of all five individual flavones. It can be inferred that herb extract could have a effect to the absorption of five flavone compounds in rat plasma. According to prior phytochemistry study, many iridoid glycosides were separated and identified in V. officinalis L. such as verbascoside, verbenalin, hastatoside, aucubin, and gentiopicroside (Calvo et al., 1997; Makboul, 1986; Rimpler et al., 1979; Muller et al., 2004). The main reason of double peaks suggested a
8000
Concentration (ng/mL)
Luteolin
15 days storage at −20 ◦ C
Freeze-thaw cycles
luteolin kaempferol
6000
apigenin quercetol
4000
isorhamnetin
2000 0
0
2
4
6
8
10
12
14
Time (h) Fig. 4. Mean ± S.D. (n = 6) plasma concentration–time curves of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rats after oral administration of Verbena officinalis L. extract.
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Table 5 Mean pharmacokinetic parameters for luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma after oral administration of Verbena officinalis L. extract (n = 6). Parameters
Mean ± S.D. Luteolin
T1/2 (h) Ke (1/h) Tmax1 (h) Cmax1 (g/mL) Tmax2 (h) Cmax2 (g/mL) AUC0−t (g h/mL) AUC0−∞ (g h/mL) MRT0−∞ (h) Vz/F (L/kg) Cl/F (L/kg/h)
4.02 0.18 0.48 1.23 3.97 1.62 6.38 10.67 9.67 3.32 0.20
± ± ± ± ± ± ± ± ± ± ±
Kaempferol 0.81 0.04 2.14 0.23 1.48 0.19 0.31 0.34 3.88 2.05 0.06
7.65 0.09 0.25 0.35 4.05 0.59 3.62 5.14 12.75 7.06 0.57
± ± ± ± ± ± ± ± ± ± ±
0.71 0.01 0.13 0.02 0.46 0.09 0.25 0.85 1.43 2.93 0.11
possible enterohepatic recirculation (Wan et al., 2007; Zhang et al., 2009). However, this hypothesis needs further investigation. The information described above might be helpful for further studies on the pharmacokinetics of V. officinalis L., and beneficial for the application of this TCM in clinical therapy. 4. Conclusions This paper described a simple, sensitive and validated LC–MS/MS method for simultaneous determination of luteolin, kaempferol, apigenin, quercetol and isorhamnetin after solvent extraction/acid hydrolysis in rat plasma after oral administration of V. officinalis L. extract, and investigated on their pharmacokinetic studies as well, with a short run time of 5 min. This method was sensitive, with high accuracy and met all requirements in bioanalytical method. It was successfully applied to the pharmacokinetic studies of active components of V. officinalis L. extract, which maybe provide some references to the apprehension of the action mechanism and clinical application of V. officinalis L. This is the first report of pharmacokinetic studies of luteolin, kaempferol, apigenin, quercetol and isorhamnetin together in vivo following the oral administration of V. officinalis L. extract. Acknowledgement We thank the financial support from Department of Science and Technology of Hebei Province of China (09276423D) for this work. References Calvo, M.I., 2006. Anti-inflammatory and analgesic activity of the topical preparation of Verbena officinalis L. Journal of Ethnopharmacology 107, 380–382. Calvo, M.I., San Julian, A., Fernandez, M., 1997. Identification of the major compounds in extracts of Verbena officinalis L. (Verbenaceae) by HPLC with post-column derivatization. Chromatographia 46, 241–244. Chen, G.M., Zhang, J.Y., Liu, H.M., et al., 2006a. Studies on chemical constituents of flavonoid from Verbena officinals. Journal of Chinese Medicinal Materials 29, 677–678. Chen, G.M., Zhang, J.Y., Hong, G.F., et al., 2006b. Determination of flavonoids content for Verbena officinalis. Chinese Journal of Modern Applied Pharmacy 3, 798–799.
Apigenin 3.30 0.22 0.97 3.07 4.33 6.69 25.88 28.77 5.95 0.54 0.12
± ± ± ± ± ± ± ± ± ± ±
0.83 0.05 1.08 0.26 0.58 0.27 0.49 0.21 0.57 0.12 0.01
Quercetol 4.55 0.15 1.04 0.79 2.99 6.36 13.44 15.81 5.06 3.44 0.21
± ± ± ± ± ± ± ± ± ± ±
0.49 0.02 4.25 0.15 0.48 0.77 0.79 0.27 1.03 2.53 0.04
Isorhamnetin 5.56 0.13 0.25 0.71 4.02 0.82 2.71 3.25 7.09 4.28 0.52
± ± ± ± ± ± ± ± ± ± ±
1.32 0.03 0.16 0.05 0.34 0.12 0.09 0.33 1.53 0.41 0.16
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