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Simultaneous quantification of two canthinone alkaloids of Picrasma quassioides in rat plasma by liquid chromatography–tandem mass spectrometry and its application to a rat pharmacokinetic study
Accepted Manuscript Title: Simultaneous quantification of two canthinone alkaloids of Picrasma quassioides in rat plasma by liquid chromatography-tandem mass spectrometry and its application to a rat pharmacokinetic study Author: Yuanyuan Shi Chunyan Hong Jian Xu Xiaoling Yang Ning Xie Feng Feng Wenyuan Liu PII: DOI: Reference:
S1570-0232(15)00093-8 http://dx.doi.org/doi:10.1016/j.jchromb.2015.02.008 CHROMB 19319
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
24-10-2014 16-1-2015 8-2-2015
Please cite this article as: Y. Shi, C. Hong, J. Xu, X. Yang, N. Xie, F. Feng, W. Liu, Simultaneous quantification of two canthinone alkaloids of Picrasma quassioides in rat plasma by liquid chromatography-tandem mass spectrometry and its application to a rat pharmacokinetic study, Journal of Chromatography B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.02.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Simultaneous quantification of two canthinone alkaloids of Picrasma quassioides in rat plasma by liquid chromatography-tandem mass spectrometry and its
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application to a rat pharmacokinetic study
Yuanyuan Shi a, Chunyan Hong a, Jian Xu b, Xiaoling Yang c, Ning Xie c, Feng Feng b*, Wenyuan
a
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Liu a, d*
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009,
b
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China
Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing,
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210009, China
Jiangxi Qingfeng Pharmaceutical Corporation, Ganzhou, China
d
Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical
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c
University), Ministry of Education
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Professor Wenyuan Liu
d
* Corresponding author
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Department of Pharmaceutical Analysis, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China
Email: [email protected]
Professor Feng Feng
Department of Natural Medicinal Chemistry, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China
Email: [email protected]
ABBREVIATIONS CID, collision-induced dissociation; FDA, Food and Drug Administration; IS, internal standard; LLOQ, lower limit of quantification; MRM, multiple reaction monitoring; QC, quality control; RE, relative error; RSD, relative standard deviation; SRM, selected reaction monitoring; ULOQ, upper limit of quantification;
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Canthinone alkaloids are characteristic components of P. quassioides.
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A LC-MS/MS method was developed for quantification of two
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alkaloids in rat plasma.
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Plasma samples were prepared for analysis using a simple liquid–
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liquid extraction.
ng/mL for two analytes.
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Method was validated over the range of 1.25−900 ng/mL and 0.5−800
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faster than another.
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Both canthinones were rapidly absorbed and one was eliminated
ABSTRACT
Picrasma quassioides (D. Don) Benn. is used in traditional Chinese medicine for
the treatment of inflammation. Characteristic components of the medicinal extract are canthinone alkaloids. In this study, a sensitive and rapid liquid chromatography with tandem mass spectrometry method has been developed for simultaneous quantification of two major canthinone alkaloids, 5-hydroxy-4-methoxycanthin-6-one and 4,5-dimethoxycanthin-6-one, in rat plasma after oral administration of P.
quassioides extract (200 mg/kg). The chromatographic separation was performed on a
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C18 column using acetonitrile-aqueous 0.1% formic acid (90:10, v/v) as the mobile phase. Plasma samples were prepared for analysis using a simple liquid–liquid extraction with ethyl acetate. Analytes were detected using tandem mass spectrometry
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in positive multiple reaction monitoring mode. Method validation revealed excellent linearity over the range 1.25−900 ng/mL for 5-hydroxy-4-methoxycanthin-6-one and
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0.5−800 ng/mL for 4,5-dimethoxycanthin-6-one with satisfactory intra- and inter-day precision, accuracy and recovery. Samples were stable under the conditions tested.
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The pharmacokinetic profiles of the analytes in rats showed that both canthinones
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than 5-hydroxy-4-methoxycanthin-6-one.
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were rapidly absorbed and that 4,5-dimethoxycanthin-6-one was eliminated faster
Keywords
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Picrasma quassioides (D. Don) Benn.; Canthinone alkaloids; HPLC−MS/MS;
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Pharmacokinetics; Method validation
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1. Introduction Picrasma quassioides (D. Don) Benn., used in traditional Chinese medicine, is mainly distributed in Southern China, Korea and Japan [1, 2]. It has been reported that
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P. quassioides has activity in an animal model of ulcerative colitis [3], inhibits tobacco mosaic virus [4] and has anti-inflammatory and antioxidant properties [5]. It
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is commonly employed as a gastrointestinal drug and vermifuge and has been used in
the treatment of sore throat, diarrhea, eczema, gastroenteritis and snake bite.
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Moreover, recent research demonstrates that P. quassioides induces caspase-
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dependent apoptosis in Hep-2 cells and decreases their viability, suggesting that it may have potential for the treatment of human cervical cancer [6]. P. quassioides has
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also been used as an important ingredient in a number of Chinese herbal preparations including Kumu injection, Kumu mixture, and Xiaoyan Lidan tablets (used to reduce
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inflammation and normalize gallbladder function) [2]. A variety of chemical
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components such as alkaloids, quassinoids and triterpenoids have been identified in P. quassioides. Alkaloids are responsible for the majority of the therapeutic effects, such
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as antipyretic, anti-inflammatory, antibacterial and antitumor activities, and have been used as marker compounds for quality control of P. quassioides medicines [2, 6].
Some canthinone alkaloids have been isolated and identified from this herb in our lab, such as 5-hydroxy-4-methoxycanthin-6-one, 4, 5-dimethoxycanthin-6-one, 3methylcanthin-5, 6-dione-β-carboline-1-carboxylic acid and 1-ethanoyl-β-carboline.
Among them, 5-hydroxy-4-methoxycanthin-6-one and 4, 5-dimethoxycanthin-6-one are the most abundant ones. Recent studies have revealed that both the compounds have a bacteriostatic effect on pneumococci [7, 8] and an inhibitory effect on porcine neutrophils [9]. Despite the pharmacological activities of these canthinone alkaloids, very little is known about their pharmacokinetics or metabolism. Therefore the two
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compounds,
5-hydroxy-4-methoxycanthin-6-one and 4, 5-dimethoxycanthin-6-one
were chosen for quantization in this study. According to the literature, several methods have been developed for the
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identification and quantification of canthinone alkaloids in P. quassioides. Most of these methods use high performance liquid chromatography (HPLC) with ultraviolet
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(UV) [10–12], mass spectrometry (MS) or diode array detection-mass spectrometry
[2]. However, none of these methods has been used for pharmacokinetic studies. Thus,
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the aim of the present study was to develop a rapid, sensitive and specific analytical
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method for simultaneous determination of the two major alkaloids in biological samples. We have used our newly developed method to evaluate the pharmacokinetics
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of these two compounds in rats. 2. Material and methods
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2.1. Reagents and chemicals
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P. quassioides was provided by Anguo City Teradyne Chinese Medicinal Herbs Co. Ltd. (Hebei, China) and authenticated by Professor Feng Feng (Department of
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Natural Medicinal Chemistry, China Pharmaceutical University). Reference standards of 5-hydroxy-4-methoxycanthin-6-one (purity > 98.0%), 4,5-dimethoxycanthin-6-one (purity > 98.0%) and wogonin (purity > 99.5%, internal standard, IS) (Fig. 1) were separated in our laboratory and identified using high resolution MS, UV, IR and NMR spectroscopy. HPLC grade acetonitrile was purchased from Shanghai Xingke Biochemistry Co. Limited (Shanghai, China). Ethyl acetate and other chemicals and solvents were of analytical grade. Deionized water was prepared with a Milli-Q water system (Millipore, USA). 2.2. LC−MS/MS equipment A Shimadzu LC-2010 series HPLC system (Shimadzu, Kyoto, Japan), equipped
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with quaternary pump, vacuum degasser, auto-sampler and column heater-cooler was connected by an electrospray ionization (ESI) interface to a Thermo Finnigan TSQ AM tandem mass spectrometer (Thermo Finnigan, San Jose, CA, USA). Data were
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acquired using Xcalibur 1.1 software (Thermo Finnigan, San Jose, CA, USA). 2.3. Chromatographic conditions
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A HYPERSIL ODS-2 C18 column (100 × 4.6 mm i.d., 5 μm particle size) with a
guard column (Hanbon, China) was used. The mobile phase consisted of solvent A
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(acetonitrile) and solution B (0.1% formic acid in water) (90:10, v/v) delivered at a
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flow rate of 1 mL/min with a split ratio of 1:3. The injection volume was 20 μL and the run time was 5 min with the effluent directed into the mass detector between 2.0
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and 5.0 min using a switching valve. The column was thoroughly washed with water/acetontrile mixtures and finally stored in acetonitrile after daily analysis
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cycle.(~100 samples).
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2.4. Mass spectrometric conditions
The MS was set in positive multiple reaction monitoring (MRM) mode and the
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tuning parameters were optimized via a syringe pump. The precursor ion, corresponding product ion and collision-induced dissociation (CID) for each compound were optimized. MS transitions were m/z 266.90→251.80 for 5-hydroxy-4methoxycanthin-6-one, m/z 281.01→236.89 for 4, 5-dimethoxycanthin-6-one and m/z 284.91→269.94 for IS. Other MS conditions were also optimized and set as follows:
spray voltage, 3.5 kV; sheath gas pressure, 40 arbitrary units; auxiliary gas pressure, 5 arbitrary units; capillary temperature, 350 °C; collision gas (argon) pressure, 1.4 mTorr; source CID voltage, 12 V. 2.5. Extraction of P. quassioides P. quassioides was crushed and extracted by refluxing with 70% methanol for 2 h.
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The mixture was filtered and the filtrate evaporated under reduced pressure. The extract was dried at 45 °C for 4 h and stored at 4 °C for use. The quantities of the two target compounds in the extract were determined by an HPLC method developed in
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our laboratory. The extract was accurately weighed and dissolved in 0.5% sodium
to rats. 2.6. Standard solution and quality control (QC) samples standards
of
5-hydroxy-4-methoxycanthin-6-one,
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Reference
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carboxymethylcellulose (CMC-Na) at a concentration of 40 mg/mL for administration
4,5-
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dimethoxycanthin-6-one and IS were separately weighed and dissolved in methanol to prepare stock solutions with concentrations of 200 μg/mL, 1 mg/mL and 1 mg/mL,
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respectively. A series of standard solutions were obtained by further dilution of the stock solution with methanol. Calibration samples were prepared by adding the series
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standard solutions (5 μL) and IS stock solutions (5 μL) to blank rat plasma (100 μL)
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to obtain concentrations of 1.25, 5, 50, 125, 250, 500 and 900 ng/mL for 5-hydroxy-4methoxycanthin-6-one, 0.5, 5, 50, 125, 250, 500 and 800 ng/mL for 4,5-
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dimethoxycanthin-6-one and 50 ng/mL for IS. QC samples were prepared independently at concentrations of 1.8, 18 and 800 ng/mL for 5-hydroxy-4methoxycanthin-6-one and 0.9, 18 and 600 ng/mL for 4, 5-dimethoxycanthin-6-one using the same method as for the calibration samples. All stock solutions and working solutions were stored at 4 °C until use. 2.7. Sample preparation
All samples were thawed at room temperature before analysis. IS solution (5 μL; 2 μg/mL) and 5 μL of methanol were added to 100 μL of the plasma sample in a 2 mL Eppendorf tube. The mixture was vortexed for 15 s and then extracted with ethyl acetate (1 mL) by vortex-mixing for 10 min and centrifugation at 17,000 g for 5 min.
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The supernatant liquor (800 μL) was evaporated to dryness at 37.5 °C under a stream of nitrogen. The residue was reconstituted in 50 μL of methanol by vortex-mixing for 2 min, then centrifuged at 17000 g for 10 min. Finally, the supernatant liquor (20 μL)
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was injected into the LC−MS/MS system for analysis. 2.8. Method validation
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The method was fully validated according to the Food and Drug Administration
(LLOQ), accuracy, precision, recovery and stability.
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2.8.1. Selectivity
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(FDA) guidelines [13] for its selectivity, linearity, the lower limit of quantification
The selectivity was evaluated by comparing chromatograms of blank rat plasma
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(from six individual rats) with an LLOQ sample and a plasma sample collected 1 h after administration of P. quassioides extract. The areas of any interference peaks
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2.8.2. Linearity and LLOQ
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should be ≤ 20% of each analyte at the LLOQ.
A seven-point linear calibration curve was prepared over a range of 1.25–900
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ng/mL for 5-hydroxy-4-methoxycanthin-6-one and 0.5–800 ng/mL for 4,5dimethoxycanthin-6-one in duplicate on three different days to demonstrate linearity. A least-squares linear regression method (1/x2 weighting) was applied to determine
the slope, intercept and coefficient of determination (r2) of the linear regression
equation. The concentrations of content-unknown samples were determined by interpolation from the calibration curve. The calibration curve was established using the Bioavailability Program Package software (BAPP, Version 2.2, Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University).The LLOQ was evaluated by analyzing six plasma samples spiked with the analyte at the lowest concentration in the calibration curve with accuracy (relative error, RE) and precision
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(relative standard deviation, RSD) ≤ 20%. 2.8.3. Precision and accuracy Six QC samples at three concentration levels (low, middle and high QC level)
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were pretreated and measured in the same day (intra-day accuracy and precision) and on three consecutive days (inter-day accuracy and precision). Concentrations were
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calculated using calibration curves obtained daily. Accuracy (RE) was calculated using the formula RE% = [(measured value − theoretical value)]/theoretical value ×
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100. The inter- and intra-day precision was expressed as the RSD. The precision and
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accuracy were judged to be acceptable when the RE was within ± 15%. 2.8.4. Extraction recovery and matrix effect
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The extraction recovery of each analyte was calculated by comparing the peak areas of analytes from an extracted sample with those of a post-extraction spiked
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sample at low, medium and high (n = 6) concentrations. The matrix effect was
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determined by comparing the peak areas obtained from a post-extraction spiked sample with those of the pure samples prepared in mobile phase containing equivalent
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amounts of the analyte at low, medium and high (n = 6) concentrations. 2.8.5. Carryover
Two processed, blank matrix samples were injected immediately after the upper
limit of quantification (ULOQ) standard to evaluate carryover in the LC−MS/MS method. The response in the first blank matrix of 5-hydroxy-4-methoxycanthin-6-one and 4,5-dimethoxycanthin-6-one should be ≤ 20% of the response of an LLOQ sample. 2.8.6. Stability Stability samples were prepared by spiking blank plasma with each analyte to obtain the same concentrations as the QC levels (low, medium and high). Short-term
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stability was investigated after QC samples had been left at room temperature for 6 h. Long-term stability was assessed after storing QC samples at −20 °C for 20 days. QC samples were also analyzed following three cycles of freezing (−20 °C) and thawing
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(ambient temperature). Furthermore, the processed sample stability of the analytes was investigated at three QC concentration levels (n = 6) after 24 h storage in the
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auto-sampler at 4 °C. Samples were considered to be stable if the deviation from the nominal concentration was within ± 15.0%.
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2.9. Pharmacokinetic study
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Five healthy male Sprague-Dawley rats (200−250 g) (Certificate No. SCXK2008-0016) were obtained from Shanghai Super-B&K Laboratory Animal Co.
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Ltd. (Shanghai, China) and provided with free access to food and water. Animals were housed under controlled conditions (temperature: 21 ± 2 °C; relative humidity: 50 ±
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10%) with a natural light-dark cycle. They were acclimated in the laboratory for at
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least 1 week prior to the experiment. After dosing, the rats were fasted for the first 4 h but had free access to water. The pharmacokinetic study was approved by the Animal
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Ethics Committee of the China Pharmaceutical University (Nanjing, China) and conformed to the Guide for Care and Use of Laboratory Animals published by the US National Institute of Health [14]. Approximately 0.2 mL blood samples were collected by orbital sinus bleeding in heparinized 2 mL Eppendorf tubes at 0, 0.08, 0.25, 0.5, 0.75, 1, 2, 3, 6, 12 and 24 h after intragastric dosing (200 mg/kg extract) based on previous rat pharmacology studies [15]. Plasma was obtained by centrifugation at 1000 g for 10 min and stored at −20 °C until analysis. Pharmacokinetic parameters were determined using the Drug and Statistics (DAS) software (version 2.1, Mathematical Pharmacology Professional Committee of China, Shanghai, China) with a non-compartmental model.
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3. Results and discussion 3.1. Optimization of mass spectrometry conditions In this study, an ESI interface was chosen and set at positive mode. A number of
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different compounds were examined and wogonin was selected as the internal standard because of its similar retention time, polarity and MS behavior to those of the
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target compounds. The precursor and product ions of the two canthinones and IS in selected reaction monitoring (SRM) mode were selected from their characteristic
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mass spectra by syringe pump infusion using each standard solution. Their MS spectra
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were recorded and are shown in Fig. 1. The [M+H]+ ions for 5-hydroxy-4methoxycanthin-6-one, 4, 5-dimethoxycanthin-6-one and IS were at m/z 266.90,
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281.01 and 284.94, respectively. These [M+H]+ ions were used as the precursors to select product ions formed by CID and the major product ions were at m/z 251.80,
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236.89 and 269.91, respectively. The SRM transitions of m/z 266.90→251.80 for 5-
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hydroxy-4-methoxycanthin-6-one, m/z 281.01→236.89 for 4,5-dimethoxycanthin-6one and m/z 284.94→269.91 for IS were selected to optimize the CID and other MS
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parameters.
3.2. Optimization of chromatographic conditions Mixtures of acetonitrile with water and different buffers, such as formic acid,
ammonium formate and acetic acid, were investigated to identify the optimal mobile phase that could produce the best sensitivity, efficiency and peak shape. It was found that the addition of 0.1% formic acid improved the response of the analytes and performed better than other buffers. A HYPERSIL ODS-2 C18 analytical column using acetonitrile-water (0.1% formic acid) (90:10, v/v) was found to achieve suitable resolution and a shorter run time than using either acetonitrile–water (0.1% formic acid) (80:20, v/v) or acetonitrile–water (0.1% formic acid) (70:30, v/v). Under the
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selected conditions, the representative chromatograms of a plasma blank, a plasma blank spiked with the analytes and IS, a carryover blank, an LLOQ sample and a rat plasma sample are shown in Fig. 2.
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3.3. Sample preparation Liquid–liquid extraction for sample preparation produced a cleaner background
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and higher recovery compared to a protein precipitation method. Different extraction
solvents such as ethyl acetate, diethyl ether and n-butanol were investigated. Good
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sensitivity, acceptable recovery and a clear supernatant were obtained when using
3.4. Method validation
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3.4.1. Selectivity, linearity and LLOQ
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ethyl acetate as the extraction solvent.
Under optimized LC−MS/MS conditions, the run time for each injection was
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only 5 min. The selected reaction monitoring MS/MS mode was highly selective and
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no significant interference by endogenous entities was observed (Fig. 2). Calibration curves for each analyte in rat plasma were constructed using concentrations of
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1.25−900 ng/mL for 5-hydroxy-4-methoxycanthin-6-one and 0.5–800 ng/mL for 4, 5dimethoxycanthin-6-one. Typical standard curves for the two compounds are shown in Table 1. The coefficient of determination (r2) was found to be >0.99. The LLOQs of
the two compounds were 1.25 ng/mL (n= 6, mean ± SD was 1.30 ± 0.14, RE = 4.34%) and 0.5 ng/mL (n= 6, mean ± SD was 0.54 ± 0.05, RE = 7.46%), respectively, in this study.
3.4.2. Precision and accuracy Table 2 summarizes the intra- and inter-day precision and accuracy for each analyte. The intra- and inter-day precision was less than 11.07% for each QC level of the analytes and accuracy was between −9.29% and 2.21%, indicating that the method
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has satisfactory accuracy, precision and reproducibility. 3.4.3. Extraction recovery, matrix effect and carryover The extraction recoveries and matrix effects for the analytes are shown in Table 3.
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At all three QC concentration levels the extraction recoveries were between 91.18% and 104.24%. The observed matrix effect ranged from 94.23% to 107.38%,
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demonstrating that there was not a significant matrix effect. A chromatogram of a carryover blank sample is presented in Fig. 2, showing that no carryover was observed
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with the chosen settings.
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3.4.4. Stability
The stability of the analytes in rat plasma during 6 h at room temperature, for up
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to 20 days at −20 °C, during three freeze–thaw cycles and over 24 h in an autosampler is summarized in Table 4. The concentrations of the analytes in rat plasma
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under different storage conditions were 100 ± 15% of the QC levels, demonstrating
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that 5-hydroxy-4-methoxycanthin-6-one and 4, 5-dimethoxycanthin-6-one have good stability under these conditions.
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3.5. Pharmacokinetic study
The pharmacokinetic data for 5-hydroxy-4-methoxycanthin-6-one and 4, 5-
dimethoxycanthin-6-one are shown in Table 5. In this study, the behaviors of the two analytes were similar (Fig. 3). The plasma concentration of 4,5-dimethoxycanthin-6one was below the LLOQ after 6 h. Both compounds were rapidly absorbed and 4,5dimethoxycanthin-6-one was eliminated faster than 5-hydroxy-4-methoxycanthin-6one in rat. The Tmax, Cmax, AUC0–t, AUC0–∞ and T1/2,z (terminal elimination half-life) in rats were 1.10 ± 0.55 h, 341.04 ± 196.56 μg/L, 2235.06 ± 627.66 μg h/L, 2388.75 ± 644.28 μg h/L and 6.21 ± 2.38 h for 5-hydroxy-4-methoxycanthin-6-one and 0.65 ± 0.34 h, 21.35 ± 7.85 μg/L, 39.92 ± 18.50 μg h/L, 40.12 ± 18.50 μg h/L and 3.30 ± 1.55
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h for 4,5-dimethoxycanthin-6-one.
4. Conclusion In this study, an LC−MS/MS method for the quantification of 5-hydroxy-4-
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methoxycanthin-6-one and 4,5-dimethoxycanthin-6-one in rat plasma has been developed and fully validated. The established method is simple, rapid, sensitive and
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reliable. Plasma samples were subjected to liquid–liquid extraction with ethyl acetate
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for analysis. The method was applied to a pharmacokinetic study of the two compounds after oral administration of P. quassioides to rats. Pharmacokinetic
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profiles of the analytes showed that both were absorbed quickly and that 4, 5dimethoxycanthin-6-one was eliminated faster than 5-hydroxy-4-methoxycanthin-6-
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one in the rat.
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Acknowledgments
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This work financially was supported by the National Natural Science Foundation of China (Grant No.81373956 and Grant No. 81274064) and the Priority Academic
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Program Development of Jiangsu Higher Education Institutions.
References
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[7]H.Y. Fan, D. Qi, M. Yang, H. Fang, K. Liu, F. Zhao. Phytomedicine, 20(2013)319– 323.
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[8]M. Chen, Y.H. Fan, S.J. Dai, K. Liu. Yantai, Shandong, China, 27 August-29 August 2006.
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[9]J.F. Liu, M. Shao, J.Y. Li, C.C. Yu, K. Liu, L.J. Wu. Morden Chin Med,
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[11]J.J. Yu, W.N. Zhao, X.X. Zhang, Q. Su, T. Sun, L. Chen, J. He, J.W. Sun. Northwest Pharm J,27 (2012)402-403 .
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[12]W.D. Huang, Y.X. Song, W.Q. Lv. Chin Hosp Pharm, 30(2010)1066-1067.
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[13]Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, Center for Drug and
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Medicine,
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2013http://www.fda.gov/cder/guidance/index.htm [14] US NIH. Guide for the care and use of laboratory animals. http://grants.nih.gov /grants/ olaw/ Guide-for-the-care-and-use-of-laboratoryanimals.pdf
[15] W.N. Zhao, Q. Su, J. He, L.N. Zhao, R.M. Xie, W.J. Sun. Pharmacol Clin Chin Mater Med. 28 (2012) 108-111.
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Figure legends Figure 1. Product ion mass spectra, chemical structures, monitored transitions of the two
analytes
and
IS
(A)
5-hydroxy-4-methoxycanthin-6-one,
(B)
4,5-
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dimethoxycanthin-6-one and (C) wogonin (IS). Figure 2. Representative SRM chromatograms (A) Chromatograms of blank rat
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plasma, (B) Plasma sample at 1 h after oral dose of 200 mg/kg P. quassioides extract, (C) Carryover blank sample, (D) Calibration standard (250 ng/mL 5-hydroxy-4-
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methoxycanthin-6-one and 4,5-dimethoxycanthin-6-one), (E) Blank plasma spiked
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with the analytes at LLOQ and IS.
Figure 3. Mean (±SD; n = 5) plasma concentration-time profile of 5-hydroxy-4and
4,5-dimethoxycanthin-6-one
in
rats
after
oral
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methoxycanthin-6-one
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te
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administration of P. quassioides extract (200 mg/kg).
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te
d
M
an
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cr
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Figure1
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Ac
ce
pt
ed
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Figure2
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pt
ed
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Figure3
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Table1
Table 1. The regression equations and LLOQ of the two analyses (n = 6).
Component
Linear range Regression equation (ng/mL)
5-hydroxy-4-metho 1.25-900 xycanthin-6-one
Nominal Mean ± concentration SD of LLOQ (LLOQ) (ng/ml)
Mean ± SD (curve)
r
Y=42.32x-0.548
0.993
Y=52.75x-0.876
0.996
Y=41.82x-0.602
0.997
RE Intra-day (%) RSD (%)
Y=(40.07± 4.82)x+(0.17±0.95)
1.25
1.30±0.14 4.34
10.94
Y=(13.37± 3.77)x-(0.02±0.13)
0.5
0.54±0.05 7.46
10.20
0.5-800
Y=11.58x-0.170
0.994
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ed
M
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Y=17.70x+0.055 0.994
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Y=10.82x+0.043 0.994 4,5-dimethoxycant hin-6-one
Page 20 of 24
Tables
Table 2. Accuracy and precision for the two analytes (n = 6). Intra-day (n=6) RSD (%)
R.E (%)
Mean ± SD (ng/mL)
RSD (%)
R.E (%)
1.8
1.71±0.04
2.44
-4.99
1.81±0.17
9.26
0.66
18.0
18.33±1.37
7.48
1.84
17.40±0.82
4.73
-3.32
800.0
769.06±64.99
8.45
-3.87
759.15±29.00
3.82
-5.11
0.9
0.83±0.02
2.66
-7.72
0.92±0.10
11.07
2.21
18.0
17.31±1.13
6.50
-3.82
16.33±0.97
5.91
-9.29
600.0
598.84±40.84
6.82
-0.19
612.10±19.95
3.26
2.02
Ac
ce pt
ed
M
an
us
4,5-dimetho xycanthin-6one
Mean ± SD (ng/mL)
ip t
5-hydroxy-4 -methoxycan thin-6-one
Concentration (ng/mL)
cr
Component
Inter-day (n=3)
Page 21 of 24
Tables
Table 3. The extraction recovery and matrix effect of the two analytes (n = 6).
Extraction recovery
4,5-dimethoxycanthin-6-one
100.57±7.98 91.18±5.74 94.48±2.10 104.24±6.49 98.53±4.84 102.22±9.01 101.29±3.65
Mean ± SD (%)
RSD (%)
7.93 6.29 2.22 6.23 4.91 8.81 3.61
94.23±7.04 94.84±3.75 101.93±5.36 107.38±8.98 101.41±3.45 95.99±3.28 106.17±6.52
7.47 3.95 5.26 8.36 3.4 3.42 6.14
Ac
ce pt
ed
M
an
us
Wogonin(IS)
1.8 18.0 800.0 0.9 18.0 600.0 50.0
RSD (%)
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5-hydroxy-4-methoxycanthin -6-one
Concentration Mean ± SD （ng/mL） (%)
cr
Component
Matrix effect
Page 22 of 24
cr
ip t
Tables
us
Table 4. Stability of the two analytes in rat plasma (n = 6). Short-term stability
(ng/mL)
-6-one
(at -4°C for 24 h)
Mean ± SD(ng/mL) RSD(%) RE(%) Mean ± SD(ng/mL) RSD(%) RE(%) Mean ± SD(ng/mL) RSD(%) RE(%) Mean ± SD(ng/mL) RSD(%) RE(%)
1.8
1.90±0.09
4.70
5.65
1.99±0.05
2.66
10.59
1.99±0.18
9.28
10.49
1.72±0.18
10.52
-4.43
18.0
19.60±1.22
6.22
8.91
18.31±0.96
5.22
1.72
19.20±1.87
9.72
6.67
17.12±1.94
11.35
-4.9
800.0
813.28±31.93
3.93
1.66
780.21±21.80
2.79
-2.47
798.83±13.78
1.72
-0.15
781.91±40.82
5.22
-2.26
0.9
0.96±0.04
4.40
6.22
0.80±0.02
3.04
-10.91
0.94±0.07
7.17
4.14
0.90±0.06
7.01
-0.17
18.0
18.22±0.32
1.74
1.23
17.59±0.49
2.77
-2.25
17.44±0.43
2.47
-3.10
16.29±0.71
4.36
-9.5
600.0
593.92±7.94
1.34
-1.01
608.07±13.28
2.18
1.35
617.57±17.09
2.77
2.93
635.07±56.71
8.93
5.85
M
4,5-dimethoxycanthin
Post-preparative stability
ep te
canthin-6-one
Long-term stability (at -20°C for 20 days)
Ac c
5-hydroxy-4-methoxy
an
Concentration
d
Component
Three freeze–thaw cycles
(at room temperature for 6 h)
Page 23 of 24
Tables
Table 5. PK parameters of the two analytes in rats after oral administration of P. quassioides extract (n = 5). 4,5-dimethoxycanthin-6-one
2235.06 ±627.66 2388.75±644.28 17132.66± 7250.99 22489.83± 10550.53 7.74 ±2.43 9.53±3.75 6.21 ±2.38 1.10 ±0.55 88.89± 24.76 825.23±513.61 341.04±196.56
39.92 ±18.50 40.12±18.50 125.50 ±59.17 131.45±59.92 3.17 ±0.49 3.32 ±0.61 3.30 ±1.55 0.65 ±0.34 5791.41±2233.98 29743.49± 21408.59 21.35±7.85
ip t
AUC0-t / μg×h×L AUC0-∞ /μg×h×L-1 AUMC0-t AUMC0-∞ MRT0-t / h MRT0-∞ / h t1/2z / h Tmax / h CLz/F / L×h-1×kg-1 Vz/F / L×kg-1 Cmax /μg×L-1
5-hydroxy-4-methoxycanthin-6-one
cr
-1
us
PK parameters
Ac
ce pt
ed
M
an
AUC: Area under the plasma concentration-time curve; AUMC: Area under the first moment curve; MRT: Mean residenc time; T1/2: Elimination half-life; Tmax: Time of maximumconcentration; Cmax: Maximum plasma concentration;
Page 24 of 24
S1570-0232(15)00093-8 http://dx.doi.org/doi:10.1016/j.jchromb.2015.02.008 CHROMB 19319
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
24-10-2014 16-1-2015 8-2-2015
Please cite this article as: Y. Shi, C. Hong, J. Xu, X. Yang, N. Xie, F. Feng, W. Liu, Simultaneous quantification of two canthinone alkaloids of Picrasma quassioides in rat plasma by liquid chromatography-tandem mass spectrometry and its application to a rat pharmacokinetic study, Journal of Chromatography B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.02.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Simultaneous quantification of two canthinone alkaloids of Picrasma quassioides in rat plasma by liquid chromatography-tandem mass spectrometry and its
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application to a rat pharmacokinetic study
Yuanyuan Shi a, Chunyan Hong a, Jian Xu b, Xiaoling Yang c, Ning Xie c, Feng Feng b*, Wenyuan
a
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Liu a, d*
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009,
b
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China
Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing,
an
210009, China
Jiangxi Qingfeng Pharmaceutical Corporation, Ganzhou, China
d
Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical
M
c
University), Ministry of Education
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Professor Wenyuan Liu
d
* Corresponding author
Ac ce p
Department of Pharmaceutical Analysis, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China
Email: [email protected]
Professor Feng Feng
Department of Natural Medicinal Chemistry, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China
Email: [email protected]
ABBREVIATIONS CID, collision-induced dissociation; FDA, Food and Drug Administration; IS, internal standard; LLOQ, lower limit of quantification; MRM, multiple reaction monitoring; QC, quality control; RE, relative error; RSD, relative standard deviation; SRM, selected reaction monitoring; ULOQ, upper limit of quantification;
Page 1 of 24
Canthinone alkaloids are characteristic components of P. quassioides.
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A LC-MS/MS method was developed for quantification of two
cr
alkaloids in rat plasma.
us
Plasma samples were prepared for analysis using a simple liquid–
an
liquid extraction.
ng/mL for two analytes.
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Method was validated over the range of 1.25−900 ng/mL and 0.5−800
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te
faster than another.
d
Both canthinones were rapidly absorbed and one was eliminated
ABSTRACT
Picrasma quassioides (D. Don) Benn. is used in traditional Chinese medicine for
the treatment of inflammation. Characteristic components of the medicinal extract are canthinone alkaloids. In this study, a sensitive and rapid liquid chromatography with tandem mass spectrometry method has been developed for simultaneous quantification of two major canthinone alkaloids, 5-hydroxy-4-methoxycanthin-6-one and 4,5-dimethoxycanthin-6-one, in rat plasma after oral administration of P.
quassioides extract (200 mg/kg). The chromatographic separation was performed on a
Page 2 of 24
C18 column using acetonitrile-aqueous 0.1% formic acid (90:10, v/v) as the mobile phase. Plasma samples were prepared for analysis using a simple liquid–liquid extraction with ethyl acetate. Analytes were detected using tandem mass spectrometry
ip t
in positive multiple reaction monitoring mode. Method validation revealed excellent linearity over the range 1.25−900 ng/mL for 5-hydroxy-4-methoxycanthin-6-one and
cr
0.5−800 ng/mL for 4,5-dimethoxycanthin-6-one with satisfactory intra- and inter-day precision, accuracy and recovery. Samples were stable under the conditions tested.
us
The pharmacokinetic profiles of the analytes in rats showed that both canthinones
M
than 5-hydroxy-4-methoxycanthin-6-one.
an
were rapidly absorbed and that 4,5-dimethoxycanthin-6-one was eliminated faster
Keywords
d
Picrasma quassioides (D. Don) Benn.; Canthinone alkaloids; HPLC−MS/MS;
Ac ce p
te
Pharmacokinetics; Method validation
Page 3 of 24
1. Introduction Picrasma quassioides (D. Don) Benn., used in traditional Chinese medicine, is mainly distributed in Southern China, Korea and Japan [1, 2]. It has been reported that
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P. quassioides has activity in an animal model of ulcerative colitis [3], inhibits tobacco mosaic virus [4] and has anti-inflammatory and antioxidant properties [5]. It
cr
is commonly employed as a gastrointestinal drug and vermifuge and has been used in
the treatment of sore throat, diarrhea, eczema, gastroenteritis and snake bite.
us
Moreover, recent research demonstrates that P. quassioides induces caspase-
an
dependent apoptosis in Hep-2 cells and decreases their viability, suggesting that it may have potential for the treatment of human cervical cancer [6]. P. quassioides has
M
also been used as an important ingredient in a number of Chinese herbal preparations including Kumu injection, Kumu mixture, and Xiaoyan Lidan tablets (used to reduce
d
inflammation and normalize gallbladder function) [2]. A variety of chemical
te
components such as alkaloids, quassinoids and triterpenoids have been identified in P. quassioides. Alkaloids are responsible for the majority of the therapeutic effects, such
Ac ce p
as antipyretic, anti-inflammatory, antibacterial and antitumor activities, and have been used as marker compounds for quality control of P. quassioides medicines [2, 6].
Some canthinone alkaloids have been isolated and identified from this herb in our lab, such as 5-hydroxy-4-methoxycanthin-6-one, 4, 5-dimethoxycanthin-6-one, 3methylcanthin-5, 6-dione-β-carboline-1-carboxylic acid and 1-ethanoyl-β-carboline.
Among them, 5-hydroxy-4-methoxycanthin-6-one and 4, 5-dimethoxycanthin-6-one are the most abundant ones. Recent studies have revealed that both the compounds have a bacteriostatic effect on pneumococci [7, 8] and an inhibitory effect on porcine neutrophils [9]. Despite the pharmacological activities of these canthinone alkaloids, very little is known about their pharmacokinetics or metabolism. Therefore the two
Page 4 of 24
compounds,
5-hydroxy-4-methoxycanthin-6-one and 4, 5-dimethoxycanthin-6-one
were chosen for quantization in this study. According to the literature, several methods have been developed for the
ip t
identification and quantification of canthinone alkaloids in P. quassioides. Most of these methods use high performance liquid chromatography (HPLC) with ultraviolet
cr
(UV) [10–12], mass spectrometry (MS) or diode array detection-mass spectrometry
[2]. However, none of these methods has been used for pharmacokinetic studies. Thus,
us
the aim of the present study was to develop a rapid, sensitive and specific analytical
an
method for simultaneous determination of the two major alkaloids in biological samples. We have used our newly developed method to evaluate the pharmacokinetics
M
of these two compounds in rats. 2. Material and methods
d
2.1. Reagents and chemicals
te
P. quassioides was provided by Anguo City Teradyne Chinese Medicinal Herbs Co. Ltd. (Hebei, China) and authenticated by Professor Feng Feng (Department of
Ac ce p
Natural Medicinal Chemistry, China Pharmaceutical University). Reference standards of 5-hydroxy-4-methoxycanthin-6-one (purity > 98.0%), 4,5-dimethoxycanthin-6-one (purity > 98.0%) and wogonin (purity > 99.5%, internal standard, IS) (Fig. 1) were separated in our laboratory and identified using high resolution MS, UV, IR and NMR spectroscopy. HPLC grade acetonitrile was purchased from Shanghai Xingke Biochemistry Co. Limited (Shanghai, China). Ethyl acetate and other chemicals and solvents were of analytical grade. Deionized water was prepared with a Milli-Q water system (Millipore, USA). 2.2. LC−MS/MS equipment A Shimadzu LC-2010 series HPLC system (Shimadzu, Kyoto, Japan), equipped
Page 5 of 24
with quaternary pump, vacuum degasser, auto-sampler and column heater-cooler was connected by an electrospray ionization (ESI) interface to a Thermo Finnigan TSQ AM tandem mass spectrometer (Thermo Finnigan, San Jose, CA, USA). Data were
ip t
acquired using Xcalibur 1.1 software (Thermo Finnigan, San Jose, CA, USA). 2.3. Chromatographic conditions
cr
A HYPERSIL ODS-2 C18 column (100 × 4.6 mm i.d., 5 μm particle size) with a
guard column (Hanbon, China) was used. The mobile phase consisted of solvent A
us
(acetonitrile) and solution B (0.1% formic acid in water) (90:10, v/v) delivered at a
an
flow rate of 1 mL/min with a split ratio of 1:3. The injection volume was 20 μL and the run time was 5 min with the effluent directed into the mass detector between 2.0
M
and 5.0 min using a switching valve. The column was thoroughly washed with water/acetontrile mixtures and finally stored in acetonitrile after daily analysis
d
cycle.(~100 samples).
te
2.4. Mass spectrometric conditions
The MS was set in positive multiple reaction monitoring (MRM) mode and the
Ac ce p
tuning parameters were optimized via a syringe pump. The precursor ion, corresponding product ion and collision-induced dissociation (CID) for each compound were optimized. MS transitions were m/z 266.90→251.80 for 5-hydroxy-4methoxycanthin-6-one, m/z 281.01→236.89 for 4, 5-dimethoxycanthin-6-one and m/z 284.91→269.94 for IS. Other MS conditions were also optimized and set as follows:
spray voltage, 3.5 kV; sheath gas pressure, 40 arbitrary units; auxiliary gas pressure, 5 arbitrary units; capillary temperature, 350 °C; collision gas (argon) pressure, 1.4 mTorr; source CID voltage, 12 V. 2.5. Extraction of P. quassioides P. quassioides was crushed and extracted by refluxing with 70% methanol for 2 h.
Page 6 of 24
The mixture was filtered and the filtrate evaporated under reduced pressure. The extract was dried at 45 °C for 4 h and stored at 4 °C for use. The quantities of the two target compounds in the extract were determined by an HPLC method developed in
ip t
our laboratory. The extract was accurately weighed and dissolved in 0.5% sodium
to rats. 2.6. Standard solution and quality control (QC) samples standards
of
5-hydroxy-4-methoxycanthin-6-one,
us
Reference
cr
carboxymethylcellulose (CMC-Na) at a concentration of 40 mg/mL for administration
4,5-
an
dimethoxycanthin-6-one and IS were separately weighed and dissolved in methanol to prepare stock solutions with concentrations of 200 μg/mL, 1 mg/mL and 1 mg/mL,
M
respectively. A series of standard solutions were obtained by further dilution of the stock solution with methanol. Calibration samples were prepared by adding the series
d
standard solutions (5 μL) and IS stock solutions (5 μL) to blank rat plasma (100 μL)
te
to obtain concentrations of 1.25, 5, 50, 125, 250, 500 and 900 ng/mL for 5-hydroxy-4methoxycanthin-6-one, 0.5, 5, 50, 125, 250, 500 and 800 ng/mL for 4,5-
Ac ce p
dimethoxycanthin-6-one and 50 ng/mL for IS. QC samples were prepared independently at concentrations of 1.8, 18 and 800 ng/mL for 5-hydroxy-4methoxycanthin-6-one and 0.9, 18 and 600 ng/mL for 4, 5-dimethoxycanthin-6-one using the same method as for the calibration samples. All stock solutions and working solutions were stored at 4 °C until use. 2.7. Sample preparation
All samples were thawed at room temperature before analysis. IS solution (5 μL; 2 μg/mL) and 5 μL of methanol were added to 100 μL of the plasma sample in a 2 mL Eppendorf tube. The mixture was vortexed for 15 s and then extracted with ethyl acetate (1 mL) by vortex-mixing for 10 min and centrifugation at 17,000 g for 5 min.
Page 7 of 24
The supernatant liquor (800 μL) was evaporated to dryness at 37.5 °C under a stream of nitrogen. The residue was reconstituted in 50 μL of methanol by vortex-mixing for 2 min, then centrifuged at 17000 g for 10 min. Finally, the supernatant liquor (20 μL)
ip t
was injected into the LC−MS/MS system for analysis. 2.8. Method validation
cr
The method was fully validated according to the Food and Drug Administration
(LLOQ), accuracy, precision, recovery and stability.
an
2.8.1. Selectivity
us
(FDA) guidelines [13] for its selectivity, linearity, the lower limit of quantification
The selectivity was evaluated by comparing chromatograms of blank rat plasma
M
(from six individual rats) with an LLOQ sample and a plasma sample collected 1 h after administration of P. quassioides extract. The areas of any interference peaks
te
2.8.2. Linearity and LLOQ
d
should be ≤ 20% of each analyte at the LLOQ.
A seven-point linear calibration curve was prepared over a range of 1.25–900
Ac ce p
ng/mL for 5-hydroxy-4-methoxycanthin-6-one and 0.5–800 ng/mL for 4,5dimethoxycanthin-6-one in duplicate on three different days to demonstrate linearity. A least-squares linear regression method (1/x2 weighting) was applied to determine
the slope, intercept and coefficient of determination (r2) of the linear regression
equation. The concentrations of content-unknown samples were determined by interpolation from the calibration curve. The calibration curve was established using the Bioavailability Program Package software (BAPP, Version 2.2, Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University).The LLOQ was evaluated by analyzing six plasma samples spiked with the analyte at the lowest concentration in the calibration curve with accuracy (relative error, RE) and precision
Page 8 of 24
(relative standard deviation, RSD) ≤ 20%. 2.8.3. Precision and accuracy Six QC samples at three concentration levels (low, middle and high QC level)
ip t
were pretreated and measured in the same day (intra-day accuracy and precision) and on three consecutive days (inter-day accuracy and precision). Concentrations were
cr
calculated using calibration curves obtained daily. Accuracy (RE) was calculated using the formula RE% = [(measured value − theoretical value)]/theoretical value ×
us
100. The inter- and intra-day precision was expressed as the RSD. The precision and
an
accuracy were judged to be acceptable when the RE was within ± 15%. 2.8.4. Extraction recovery and matrix effect
M
The extraction recovery of each analyte was calculated by comparing the peak areas of analytes from an extracted sample with those of a post-extraction spiked
d
sample at low, medium and high (n = 6) concentrations. The matrix effect was
te
determined by comparing the peak areas obtained from a post-extraction spiked sample with those of the pure samples prepared in mobile phase containing equivalent
Ac ce p
amounts of the analyte at low, medium and high (n = 6) concentrations. 2.8.5. Carryover
Two processed, blank matrix samples were injected immediately after the upper
limit of quantification (ULOQ) standard to evaluate carryover in the LC−MS/MS method. The response in the first blank matrix of 5-hydroxy-4-methoxycanthin-6-one and 4,5-dimethoxycanthin-6-one should be ≤ 20% of the response of an LLOQ sample. 2.8.6. Stability Stability samples were prepared by spiking blank plasma with each analyte to obtain the same concentrations as the QC levels (low, medium and high). Short-term
Page 9 of 24
stability was investigated after QC samples had been left at room temperature for 6 h. Long-term stability was assessed after storing QC samples at −20 °C for 20 days. QC samples were also analyzed following three cycles of freezing (−20 °C) and thawing
ip t
(ambient temperature). Furthermore, the processed sample stability of the analytes was investigated at three QC concentration levels (n = 6) after 24 h storage in the
cr
auto-sampler at 4 °C. Samples were considered to be stable if the deviation from the nominal concentration was within ± 15.0%.
us
2.9. Pharmacokinetic study
an
Five healthy male Sprague-Dawley rats (200−250 g) (Certificate No. SCXK2008-0016) were obtained from Shanghai Super-B&K Laboratory Animal Co.
M
Ltd. (Shanghai, China) and provided with free access to food and water. Animals were housed under controlled conditions (temperature: 21 ± 2 °C; relative humidity: 50 ±
d
10%) with a natural light-dark cycle. They were acclimated in the laboratory for at
te
least 1 week prior to the experiment. After dosing, the rats were fasted for the first 4 h but had free access to water. The pharmacokinetic study was approved by the Animal
Ac ce p
Ethics Committee of the China Pharmaceutical University (Nanjing, China) and conformed to the Guide for Care and Use of Laboratory Animals published by the US National Institute of Health [14]. Approximately 0.2 mL blood samples were collected by orbital sinus bleeding in heparinized 2 mL Eppendorf tubes at 0, 0.08, 0.25, 0.5, 0.75, 1, 2, 3, 6, 12 and 24 h after intragastric dosing (200 mg/kg extract) based on previous rat pharmacology studies [15]. Plasma was obtained by centrifugation at 1000 g for 10 min and stored at −20 °C until analysis. Pharmacokinetic parameters were determined using the Drug and Statistics (DAS) software (version 2.1, Mathematical Pharmacology Professional Committee of China, Shanghai, China) with a non-compartmental model.
Page 10 of 24
3. Results and discussion 3.1. Optimization of mass spectrometry conditions In this study, an ESI interface was chosen and set at positive mode. A number of
ip t
different compounds were examined and wogonin was selected as the internal standard because of its similar retention time, polarity and MS behavior to those of the
cr
target compounds. The precursor and product ions of the two canthinones and IS in selected reaction monitoring (SRM) mode were selected from their characteristic
us
mass spectra by syringe pump infusion using each standard solution. Their MS spectra
an
were recorded and are shown in Fig. 1. The [M+H]+ ions for 5-hydroxy-4methoxycanthin-6-one, 4, 5-dimethoxycanthin-6-one and IS were at m/z 266.90,
M
281.01 and 284.94, respectively. These [M+H]+ ions were used as the precursors to select product ions formed by CID and the major product ions were at m/z 251.80,
d
236.89 and 269.91, respectively. The SRM transitions of m/z 266.90→251.80 for 5-
te
hydroxy-4-methoxycanthin-6-one, m/z 281.01→236.89 for 4,5-dimethoxycanthin-6one and m/z 284.94→269.91 for IS were selected to optimize the CID and other MS
Ac ce p
parameters.
3.2. Optimization of chromatographic conditions Mixtures of acetonitrile with water and different buffers, such as formic acid,
ammonium formate and acetic acid, were investigated to identify the optimal mobile phase that could produce the best sensitivity, efficiency and peak shape. It was found that the addition of 0.1% formic acid improved the response of the analytes and performed better than other buffers. A HYPERSIL ODS-2 C18 analytical column using acetonitrile-water (0.1% formic acid) (90:10, v/v) was found to achieve suitable resolution and a shorter run time than using either acetonitrile–water (0.1% formic acid) (80:20, v/v) or acetonitrile–water (0.1% formic acid) (70:30, v/v). Under the
Page 11 of 24
selected conditions, the representative chromatograms of a plasma blank, a plasma blank spiked with the analytes and IS, a carryover blank, an LLOQ sample and a rat plasma sample are shown in Fig. 2.
ip t
3.3. Sample preparation Liquid–liquid extraction for sample preparation produced a cleaner background
cr
and higher recovery compared to a protein precipitation method. Different extraction
solvents such as ethyl acetate, diethyl ether and n-butanol were investigated. Good
us
sensitivity, acceptable recovery and a clear supernatant were obtained when using
3.4. Method validation
M
3.4.1. Selectivity, linearity and LLOQ
an
ethyl acetate as the extraction solvent.
Under optimized LC−MS/MS conditions, the run time for each injection was
d
only 5 min. The selected reaction monitoring MS/MS mode was highly selective and
te
no significant interference by endogenous entities was observed (Fig. 2). Calibration curves for each analyte in rat plasma were constructed using concentrations of
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1.25−900 ng/mL for 5-hydroxy-4-methoxycanthin-6-one and 0.5–800 ng/mL for 4, 5dimethoxycanthin-6-one. Typical standard curves for the two compounds are shown in Table 1. The coefficient of determination (r2) was found to be >0.99. The LLOQs of
the two compounds were 1.25 ng/mL (n= 6, mean ± SD was 1.30 ± 0.14, RE = 4.34%) and 0.5 ng/mL (n= 6, mean ± SD was 0.54 ± 0.05, RE = 7.46%), respectively, in this study.
3.4.2. Precision and accuracy Table 2 summarizes the intra- and inter-day precision and accuracy for each analyte. The intra- and inter-day precision was less than 11.07% for each QC level of the analytes and accuracy was between −9.29% and 2.21%, indicating that the method
Page 12 of 24
has satisfactory accuracy, precision and reproducibility. 3.4.3. Extraction recovery, matrix effect and carryover The extraction recoveries and matrix effects for the analytes are shown in Table 3.
ip t
At all three QC concentration levels the extraction recoveries were between 91.18% and 104.24%. The observed matrix effect ranged from 94.23% to 107.38%,
cr
demonstrating that there was not a significant matrix effect. A chromatogram of a carryover blank sample is presented in Fig. 2, showing that no carryover was observed
us
with the chosen settings.
an
3.4.4. Stability
The stability of the analytes in rat plasma during 6 h at room temperature, for up
M
to 20 days at −20 °C, during three freeze–thaw cycles and over 24 h in an autosampler is summarized in Table 4. The concentrations of the analytes in rat plasma
d
under different storage conditions were 100 ± 15% of the QC levels, demonstrating
te
that 5-hydroxy-4-methoxycanthin-6-one and 4, 5-dimethoxycanthin-6-one have good stability under these conditions.
Ac ce p
3.5. Pharmacokinetic study
The pharmacokinetic data for 5-hydroxy-4-methoxycanthin-6-one and 4, 5-
dimethoxycanthin-6-one are shown in Table 5. In this study, the behaviors of the two analytes were similar (Fig. 3). The plasma concentration of 4,5-dimethoxycanthin-6one was below the LLOQ after 6 h. Both compounds were rapidly absorbed and 4,5dimethoxycanthin-6-one was eliminated faster than 5-hydroxy-4-methoxycanthin-6one in rat. The Tmax, Cmax, AUC0–t, AUC0–∞ and T1/2,z (terminal elimination half-life) in rats were 1.10 ± 0.55 h, 341.04 ± 196.56 μg/L, 2235.06 ± 627.66 μg h/L, 2388.75 ± 644.28 μg h/L and 6.21 ± 2.38 h for 5-hydroxy-4-methoxycanthin-6-one and 0.65 ± 0.34 h, 21.35 ± 7.85 μg/L, 39.92 ± 18.50 μg h/L, 40.12 ± 18.50 μg h/L and 3.30 ± 1.55
Page 13 of 24
h for 4,5-dimethoxycanthin-6-one.
4. Conclusion In this study, an LC−MS/MS method for the quantification of 5-hydroxy-4-
ip t
methoxycanthin-6-one and 4,5-dimethoxycanthin-6-one in rat plasma has been developed and fully validated. The established method is simple, rapid, sensitive and
cr
reliable. Plasma samples were subjected to liquid–liquid extraction with ethyl acetate
us
for analysis. The method was applied to a pharmacokinetic study of the two compounds after oral administration of P. quassioides to rats. Pharmacokinetic
an
profiles of the analytes showed that both were absorbed quickly and that 4, 5dimethoxycanthin-6-one was eliminated faster than 5-hydroxy-4-methoxycanthin-6-
M
one in the rat.
d
Acknowledgments
te
This work financially was supported by the National Natural Science Foundation of China (Grant No.81373956 and Grant No. 81274064) and the Priority Academic
Ac ce p
Program Development of Jiangsu Higher Education Institutions.
References
[1]China Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of China, Peoples Medicinal Publishing House, Beijing, 2010, p. 186. [2]H. Liao, Z. Lai, J. Su, Y. Yi, Y. Li, X. Lai, Z. Su, Z. Lin. J. Sep. Sci. 35(2012) 2193-2202 . [3]J. Liu, M. Shao, D. Zhai, K. Liu, L. Wu. Planta Med. 75(2009)142-145 . [4]J. Chen, X. Yan, J. Dong, P. Sang, X. Fang, Y. Di, Z. Zhang, X. Hao. Benn. J Agr Food Chem. 57 (2009)6590-6595.
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[5]Y. Yin, M. Wang. Nat Prod Sci.17(2011)5-9 . [6]H.E. Lee, E.S. Choi, J.A. Shin, L.H. Kim, N.P. Cho, S.D. Cho. Cell Biochem Funct, 32 (2014)229-235.
ip t
[7]H.Y. Fan, D. Qi, M. Yang, H. Fang, K. Liu, F. Zhao. Phytomedicine, 20(2013)319– 323.
cr
[8]M. Chen, Y.H. Fan, S.J. Dai, K. Liu. Yantai, Shandong, China, 27 August-29 August 2006.
us
[9]J.F. Liu, M. Shao, J.Y. Li, C.C. Yu, K. Liu, L.J. Wu. Morden Chin Med,
an
11(2009)30-33 .
[10]W.Q. Lv, W.D. Huang, Y.X. Song. Chin Tradit Herbal Drugs,40 (2009)1321-1323.
M
[11]J.J. Yu, W.N. Zhao, X.X. Zhang, Q. Su, T. Sun, L. Chen, J. He, J.W. Sun. Northwest Pharm J,27 (2012)402-403 .
d
[12]W.D. Huang, Y.X. Song, W.Q. Lv. Chin Hosp Pharm, 30(2010)1066-1067.
te
[13]Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, Center for Drug and
Research
Ac ce p
Evaluation
Center
for
Veterinary
Medicine,
September
2013http://www.fda.gov/cder/guidance/index.htm [14] US NIH. Guide for the care and use of laboratory animals. http://grants.nih.gov /grants/ olaw/ Guide-for-the-care-and-use-of-laboratoryanimals.pdf
[15] W.N. Zhao, Q. Su, J. He, L.N. Zhao, R.M. Xie, W.J. Sun. Pharmacol Clin Chin Mater Med. 28 (2012) 108-111.
Page 15 of 24
Figure legends Figure 1. Product ion mass spectra, chemical structures, monitored transitions of the two
analytes
and
IS
(A)
5-hydroxy-4-methoxycanthin-6-one,
(B)
4,5-
ip t
dimethoxycanthin-6-one and (C) wogonin (IS). Figure 2. Representative SRM chromatograms (A) Chromatograms of blank rat
cr
plasma, (B) Plasma sample at 1 h after oral dose of 200 mg/kg P. quassioides extract, (C) Carryover blank sample, (D) Calibration standard (250 ng/mL 5-hydroxy-4-
us
methoxycanthin-6-one and 4,5-dimethoxycanthin-6-one), (E) Blank plasma spiked
an
with the analytes at LLOQ and IS.
Figure 3. Mean (±SD; n = 5) plasma concentration-time profile of 5-hydroxy-4and
4,5-dimethoxycanthin-6-one
in
rats
after
oral
M
methoxycanthin-6-one
Ac ce p
te
d
administration of P. quassioides extract (200 mg/kg).
Page 16 of 24
Ac ce p
te
d
M
an
us
cr
ip t
Figure1
Page 17 of 24
Ac
ce
pt
ed
M
an
us
cr
i
Figure2
Page 18 of 24
Ac
ce
pt
ed
M
an
us
cr
i
Figure3
Page 19 of 24
Table1
Table 1. The regression equations and LLOQ of the two analyses (n = 6).
Component
Linear range Regression equation (ng/mL)
5-hydroxy-4-metho 1.25-900 xycanthin-6-one
Nominal Mean ± concentration SD of LLOQ (LLOQ) (ng/ml)
Mean ± SD (curve)
r
Y=42.32x-0.548
0.993
Y=52.75x-0.876
0.996
Y=41.82x-0.602
0.997
RE Intra-day (%) RSD (%)
Y=(40.07± 4.82)x+(0.17±0.95)
1.25
1.30±0.14 4.34
10.94
Y=(13.37± 3.77)x-(0.02±0.13)
0.5
0.54±0.05 7.46
10.20
0.5-800
Y=11.58x-0.170
0.994
Ac
ce pt
ed
M
an
us
cr
Y=17.70x+0.055 0.994
ip t
Y=10.82x+0.043 0.994 4,5-dimethoxycant hin-6-one
Page 20 of 24
Tables
Table 2. Accuracy and precision for the two analytes (n = 6). Intra-day (n=6) RSD (%)
R.E (%)
Mean ± SD (ng/mL)
RSD (%)
R.E (%)
1.8
1.71±0.04
2.44
-4.99
1.81±0.17
9.26
0.66
18.0
18.33±1.37
7.48
1.84
17.40±0.82
4.73
-3.32
800.0
769.06±64.99
8.45
-3.87
759.15±29.00
3.82
-5.11
0.9
0.83±0.02
2.66
-7.72
0.92±0.10
11.07
2.21
18.0
17.31±1.13
6.50
-3.82
16.33±0.97
5.91
-9.29
600.0
598.84±40.84
6.82
-0.19
612.10±19.95
3.26
2.02
Ac
ce pt
ed
M
an
us
4,5-dimetho xycanthin-6one
Mean ± SD (ng/mL)
ip t
5-hydroxy-4 -methoxycan thin-6-one
Concentration (ng/mL)
cr
Component
Inter-day (n=3)
Page 21 of 24
Tables
Table 3. The extraction recovery and matrix effect of the two analytes (n = 6).
Extraction recovery
4,5-dimethoxycanthin-6-one
100.57±7.98 91.18±5.74 94.48±2.10 104.24±6.49 98.53±4.84 102.22±9.01 101.29±3.65
Mean ± SD (%)
RSD (%)
7.93 6.29 2.22 6.23 4.91 8.81 3.61
94.23±7.04 94.84±3.75 101.93±5.36 107.38±8.98 101.41±3.45 95.99±3.28 106.17±6.52
7.47 3.95 5.26 8.36 3.4 3.42 6.14
Ac
ce pt
ed
M
an
us
Wogonin(IS)
1.8 18.0 800.0 0.9 18.0 600.0 50.0
RSD (%)
ip t
5-hydroxy-4-methoxycanthin -6-one
Concentration Mean ± SD （ng/mL） (%)
cr
Component
Matrix effect
Page 22 of 24
cr
ip t
Tables
us
Table 4. Stability of the two analytes in rat plasma (n = 6). Short-term stability
(ng/mL)
-6-one
(at -4°C for 24 h)
Mean ± SD(ng/mL) RSD(%) RE(%) Mean ± SD(ng/mL) RSD(%) RE(%) Mean ± SD(ng/mL) RSD(%) RE(%) Mean ± SD(ng/mL) RSD(%) RE(%)
1.8
1.90±0.09
4.70
5.65
1.99±0.05
2.66
10.59
1.99±0.18
9.28
10.49
1.72±0.18
10.52
-4.43
18.0
19.60±1.22
6.22
8.91
18.31±0.96
5.22
1.72
19.20±1.87
9.72
6.67
17.12±1.94
11.35
-4.9
800.0
813.28±31.93
3.93
1.66
780.21±21.80
2.79
-2.47
798.83±13.78
1.72
-0.15
781.91±40.82
5.22
-2.26
0.9
0.96±0.04
4.40
6.22
0.80±0.02
3.04
-10.91
0.94±0.07
7.17
4.14
0.90±0.06
7.01
-0.17
18.0
18.22±0.32
1.74
1.23
17.59±0.49
2.77
-2.25
17.44±0.43
2.47
-3.10
16.29±0.71
4.36
-9.5
600.0
593.92±7.94
1.34
-1.01
608.07±13.28
2.18
1.35
617.57±17.09
2.77
2.93
635.07±56.71
8.93
5.85
M
4,5-dimethoxycanthin
Post-preparative stability
ep te
canthin-6-one
Long-term stability (at -20°C for 20 days)
Ac c
5-hydroxy-4-methoxy
an
Concentration
d
Component
Three freeze–thaw cycles
(at room temperature for 6 h)
Page 23 of 24
Tables
Table 5. PK parameters of the two analytes in rats after oral administration of P. quassioides extract (n = 5). 4,5-dimethoxycanthin-6-one
2235.06 ±627.66 2388.75±644.28 17132.66± 7250.99 22489.83± 10550.53 7.74 ±2.43 9.53±3.75 6.21 ±2.38 1.10 ±0.55 88.89± 24.76 825.23±513.61 341.04±196.56
39.92 ±18.50 40.12±18.50 125.50 ±59.17 131.45±59.92 3.17 ±0.49 3.32 ±0.61 3.30 ±1.55 0.65 ±0.34 5791.41±2233.98 29743.49± 21408.59 21.35±7.85
ip t
AUC0-t / μg×h×L AUC0-∞ /μg×h×L-1 AUMC0-t AUMC0-∞ MRT0-t / h MRT0-∞ / h t1/2z / h Tmax / h CLz/F / L×h-1×kg-1 Vz/F / L×kg-1 Cmax /μg×L-1
5-hydroxy-4-methoxycanthin-6-one
cr
-1
us
PK parameters
Ac
ce pt
ed
M
an
AUC: Area under the plasma concentration-time curve; AUMC: Area under the first moment curve; MRT: Mean residenc time; T1/2: Elimination half-life; Tmax: Time of maximumconcentration; Cmax: Maximum plasma concentration;
Page 24 of 24