Original Paper Received: January 29, 2015 Accepted after revision: March 19, 2015 Published online: June 12, 2015
Pharmacology 2015;96:49–54 DOI: 10.1159/000381784
Determination of Caudatin in Rat Plasma by UPLC-MS/MS: Application to a Preclinical Pharmacokinetic Study Qiqi Zhu a Yuanyuan Hu a Yuanyuan Shan b Yiyan Wang a Xiaolong Wu a Baiping Mao a Ren-Shan Ge a
Departments of a Anaesthesiology, b Pathology, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
Key Words Caudatin · UPLC-MS/MS · Rat plasma · Pharmacokinetics
Abstract In this study, a simple, sensitive, and robust analytical method based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of caudatin in rat plasma using carbamazepine as internal standard (IS). This method was linear over the concentration range 2.5–300 ng/ml with a lower limit of quantification (LLOQ) of 2.5 ng/ml. Inter- and intra-day precision (RSD%) were all within 10% and the accuracy (RE%) was equal or lower than 5%. Recoveries of caudatin and IS were more than 80% and matrix effects were not significant. Stability studies showed that caudatin was stable under a variety of storage conditions. The method was successfully applied to a pharmacokinetic study involving oral administra© 2015 S. Karger AG, Basel tion of caudatin to rats.
Introduction
Many natural products have been used as cancer chemotherapeutic agents due to their excellent pharmacological activities and low toxicity. C-21 steroidal glyco© 2015 S. Karger AG, Basel 0031–7012/15/0962–0049$39.50/0 E-Mail [email protected] www.karger.com/pha
sides is one species of important biological active compounds widely found in the plants of the Asclepiadaceae family, which have extensive pharmacological effects such as inhibiting proliferation and invasion of tumor cells [1, 2]. As one species of C-21 steroidal glycosides, caudatin (fig. 1) is mainly isolated from the root of Cynanchum bungei Decne, a traditional Chinese medicine and health food, which has potent anti-hepatitis B virus (HBV) activity [3, 4]. Moreover, many lines of evidence have shown that caudatin induced apoptosis of many cancer cells [5–9]. Currently, several analytical methods exist for quantifying caudatin in the plants and biological samples by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) [10–12]. Quantification and pharmacokinetics studies on constituents of Traditional Chinese Medicine in plasma are required to offer suitable references in clinical application. Even a publication has reported the caudatin concentrations in biological fluids, but the method had several drawbacks, such as long analysis time, requiring large volumes of plasma samples and time-consuming gradient elution [12]. Therefore, to characterize the pharmacokinetic properties of caudatin, it is very necessary to develop an accurate and selective bioanalytical method for the determination of caudatin in plasma. As a result of recent advances in analytical techniques, ultra-performance liquid chromatography coupled with Ren-Shan Ge Department of Anaesthesiology The Second Affiliated Hospital, Wenzhou Medical University Wenzhou 325027, Zhejiang (China) E-Mail raygee0828 @ 163.com
Downloaded by: Kungliga Tekniska Hogskolan 198.143.54.65 - 1/28/2016 3:01:28 AM
O O O
HO
OH
N
H
a
Materials and Methods Chemicals and Reagents Caudatin (purity >98%) was purchased from Chengdu Mansite Pharmaceutical Co., Ltd. (Chengdu, China). Carbamazepine (IS, purity >98%) was purchased from Sigma-Aldrich (St. Louis, Mo., USA). Acetonitrile, methanol, and formic acid were of HPLC grade and were purchased from Merck Company (Darmstadt, Germany). Ultrapure water, prepared by a Milli-Q Reagent water system (Millipore, Mass., USA), was used throughout the study. UPLC-MS/MS Conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, Mass., USA) with an Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm particle size) and inline 0.2 μm stainless steel frit filter (Waters Corp., Milford, Mass., USA). A gradient program was employed with the mobile phase combining solvent A (0.1% formic acid in water) and solvent B (acetonitrile) as follows: 30–30% B (0–0.3 min), 30–90% B (0.3–0.5 min), 90–90% B (0.5–1.8 min),
50
Pharmacology 2015;96:49–54 DOI: 10.1159/000381784
b
HO
tandem mass spectrometry (UPLC-MS/MS) has emerged as an efficient analytical tool with improved sensitivity, selectivity, and specificity. The usefulness of this technique was demonstrated for a wide range of applications in bioanalytical, environmental, and pharmaceutical research [13–15]. Thus, in the present work, a highly selective and rapid UPLC-MS/MS method was developed and fully validated as per the USFDA guidelines for measurement of caudatin in rat plasma using carbamazepine as internal standard (IS). The method offered a small turnaround time for analysis, high sensitivity for the analyte, and utilization of only 100 μl of rat plasma for sample processing with a simple one-step protein precipitation by acetonitrile. The method was free from endogenous matrix interference and was successfully applied to a pharmacokinetic study in rats.
NH2
90–30% B (1.8–2.0 min). A subsequent re-equilibration time (1 min) was performed before the next injection. The flow rate was 0.40 ml/min and the injection volume was 6 μl. The column and sample temperature were maintained at 40 and 4 ° C, respectively. An AB Sciex QTRAP 5500 triple quadruple mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Ont., Canada) was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) modes under unit mass resolution (0.7 amu) in the mass analyzers. The dwell time was set to 200 ms for each MRM transition. The MRM transitions were m/z 345.4→255.4 and m/z 237.1→194.2 for caudatin and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 60 psig; ion spray voltage, 5.0 kV; and temperature, 500 ° C. Data acquiring and processing were performed using analyst software (version 1.5, AB Sciex).
Standard Solutions, Calibration Standards and Quality Control (QC) Sample Standard stock solutions of caudatin and carbamazepine (IS) were prepared in methanol at 1 mg/ml. Then, the stock solutions were diluted with methanol to obtain fresh standard working solution. Calibration standards were prepared by adding corresponding working solutions in drug-free plasma. The final concentrations of caudatin in plasma were 2.5, 5, 10, 25, 50, 100, 200, and 300 ng/ml, respectively. IS was diluted with acetonitrile to 100 ng/ml. Low-, mid-, and high-level quality control (QC) samples containing 5, 80, and 240 ng/ml of caudatin, were prepared in a manner similar to that used for the preparation of calibration samples. All stock solutions, working solutions, calibration standards, and QC samples were stored at –20 ° C and were brought to room temperature before analysis.
Sample Preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5 ml centrifuge tube, an aliquot of 200 μl of the IS working solution (100 ng/ml in acetonitrile) was added to 100 μl of plasma sample. The mixture was vortexed for 30 s followed by centrifugation at 13,000 g for 10 min at room temperature. A 6 μl aliquot of each supernatant was injected into the UPLC-MS/MS system for the analysis.
Zhu/Hu/Shan/Wang/Wu/Mao/Ge
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Fig. 1. The chemical structures of caudatin and IS in the present study: caudatin (a); carbamazepine (IS) (b).
O
OH
Method Validation The method was validated for specificity, linearity, accuracy, precision, recovery, and stability according to the literature for validation of bioanalytical methods (US Food and Drug Administration, 2001). Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC plasma samples. Specificity The specificity of the method was evaluated by analyzing six different sources of rat blank plasma samples and plasma samples with caudatin and IS to investigate endogenous interferences at the peak region of caudatin and IS. The chromatograms of blank plasma samples were compared with those obtained from blank plasma samples spiked with caudatin and IS and plasma samples after oral administration of caudatin. Linearity and LLOQ Calibration curves (y = a + bx) were generated by plotting the peak area ratio (y) of the analyte to IS versus the nominal concentration (x) of the analyte with weighted (1/x2) least square linear regression. The LLOQ was defined as the lowest concentration on the calibration curve where a signal-to-noise (S/N) was at least 10. The acceptance criteria for accuracy and precision of calibration curve data were 80–120% of the nominal concentrations and relative standard deviation (RSD) of ±20% of the nominal concentration at the LLOQ, respectively. Accuracy and Precision Intra- and inter-day accuracy and precision of the method were estimated by analyzing six replicates of QC samples at concentrations of 5, 80, and 240 ng/ml on the same day and on three consecutive days. Accuracy was expressed as relative error (RE%), which should be within the limits of ±15% at all concentrations. The precision was expressed as the relative standard deviation (RSD%), which should not exceed 15% at all concentrations. Recovery and Matrix Effect Recovery was evaluated by comparing the peak area responses of caudatin and IS in six replicates of QC samples at three different concentration levels with those of the analytes added to post-extracted blank plasma at equivalent concentrations. Matrix effects were evaluated by comparing the peak area responses of the analytes in processed blank plasma extract with those of the same analytes present in pure water at equivalent concentrations. Stability The stability of caudatin in rat plasma was investigated using quintuplicates of QC samples (5, 80, and 240 ng/ml). For bench top stability study, QC samples were kept at room temperature for 12 h prior to extraction. Stability in post-preparative samples was evaluated after leaving in the autosampler at 4 ° C for 24 h. The freeze-thaw stability of caudatin was assessed by analyzing plasma samples after three freeze-thaw cycles (–20 to 25 ° C). Long-term stability was tested by analyzing QC samples stored at –20 ° C for 30 days. Dilution stability was assessed using a spiked sample with the concentration that was higher than the upper limit of quantification samples. The result was obtained by comparing the backcalculated value of the sample after being diluted 5 times with the nominal value.
Determination of Caudatin in Rat Plasma by UPLC-MS/MS
Results and Discussion
Method Development and Optimization During the method development, a number of UPLC columns were evaluated including the Acquity BEH C18, Ultimate XB-C8, Ultimate XB-C18, Thermo Accucore XL C18, Hanbon Dubhe C18, and Shiseido Proteonavi C4 columns. Surprisingly, only the Acquity BEH C18 column, which was originally developed for the analysis of peptides and proteins, gave suitable peaks despite using different mobile phases both isocratically and by gradient elution. Mobile phases containing either acetonitrile-water or methanol-water behaved similarly, but the addition of formic acid enhanced the intensity of peaks and improved peak shape. Finally, gradient elution using acetonitrile and 0.1% (v/v) formic acid gave satisfactory peak shapes for caudatin and IS with retention times of 1.66 and 1.41 min, respectively. Protein precipitation is a relatively simple and highthroughput method capable of sample preparation. Initially, methanol and acetonitrile were tested using ratios of 3:1 for methanol:plasma and 2:1 for acetonitrile:plasma. And, the presence of acetonitrile had better recovery than that of methanol. Thus, acetonitrile was used for protein precipitation. Finally, concentration of the sample under a high-speed centrifugation improved the detection limit with no obvious matrix effects for analyte and IS.
Assay Validation Specificity Figure 2 shows the representative chromatograms of blank plasma, a plasma sample spiked with caudatin and Pharmacology 2015;96:49–54 DOI: 10.1159/000381784
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Application to a Pharmacokinetic Study Male Sprague-Dawley rats (180–220 g) were obtained from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) used to study the pharmacokinetics of caudatin. All 6 rats were housed at the Wenzhou Medical University Laboratory Animal Research Center. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical College and were in accordance with the Guide for the Care and Use of Laboratory Animals. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.3 ml) were collected from the tail vein into heparinized 1.5 ml polythene tubes at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, and 6 h after oral administration of caudatin (12.0 mg/kg). The samples were immediately centrifuged at 4,000 g for 8 min. The plasma obtained (100 μl) was stored at –20 ° C until analysis. Plasma caudatin concentration versus time data for each rat was analyzed by DAS (Drug and Statistics) software (version 2.0, Wenzhou Medical University, China).
100
0
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1.00
1.50
100
0
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a
1.00
1.50
Time
0
2.00
0
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1.00
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1.41
%
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% 0
0
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IS
100
% 0
0
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100 %
%
% 0
Caudatin
100
0
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b
1.00
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0
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c
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1.00 Time
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Fig. 2. Representative chromatograms of caudatin and IS in rat plasma samples. a A blank plasma sample; b blank plasma sample spiked with caudatin and carbamazepine, the internal standard (IS); c a rat plasma sample taken 60 min after oral administration of 12.0 mg/kg caudatin in rats.
Table 1. Precision and accuracy of method for the determination of caudatin in rat plasma (n = 6)
Caudatin
Concentration added, ng/ml
Intra-day precision mean ± SD
RSD, %
RE, %
mean ± SD
RSD, %
RE, %
5 80 240
5.2±0.5 80.7±7.3 241.2±12.1
9.0 9.1 5.0
4.3 0.8 0.5
4.8±0.4 76.2±6.9 230.8±18.1
9.1 9.0 7.8
–4.3 –4.8 –3.8
IS as well as a sample plasma, which was obtained at 60 min after oral administration of caudatin (12.0 mg/kg). Caudatin and IS were eluted at about 1.66 and 1.41 min, respectively. The assay was free of interference from endogenous peaks in plasma at the retention times of caudatin and IS. Linearity and LLOQ The assay was linear over the concentration range of 2.5–300 ng/ml with a typical calibration curve equation of y = 0.0020x + 0.0052 and correlation coefficient of r2 = 0.996. The LLOQ of caudatin in rat plasma was found to be 2.5 ng/ml, which was sufficient for the pharmacokinetics study. The precision and accuracy at LLOQ were 9.8 and 109.1%, respectively. Accuracy and Precision The intra- and inter-assay variations were found to be within accepted limits. The intra- and inter-day precisions of caudatin at three QC concentrations were within 9.1% (table 1). Assay accuracy was found to be within 52
Pharmacology 2015;96:49–54 DOI: 10.1159/000381784
Inter-day precision
±4.3%. The results indicate that the present method is reliable and reproducible for the quantitative determination of caudatin. Recovery and Matrix Effect The results of recovery and matrix effect studies are shown in table 2. The recovery of caudatin was in the range of 81.2–86.2% with matrix effect within the range of 98.7– 102.0% at the three QC concentration levels. The recovery of IS was 83.5% with a matrix effect of 95.8%. The results indicate the reasonable recoveries with no obvious suppression or enhancement of ionization of either caudatin or IS. Stability The autosampler, room temperature, freeze-thaw, and long-term (30-day) stability results indicate that the analyte was stable under the storage conditions described above since the bias in concentration was within ±15% of nominal values, and the established method was suitable for the pharmacokinetic study. This result is within the FDA acceptance criteria. Zhu/Hu/Shan/Wang/Wu/Mao/Ge
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Analytes
300
Caudatin
Concentration (ng/ml)
250 200 150 100 50 0 0
Fig. 3. Mean plasma concentration time
1
2
profile after oral administration of 12.0 mg/kg caudatin in 6 rats.
4
5
6
Analytes Concentration Recovery, % added, ng/ml
Matrix effect, %
Caudatin
5 80 240
86.2±6.3 81.2±3.9 81.5±7.2
7.3 4.8 8.8
98.8±5.0 5.1 98.7±6.2 6.3 102.0±5.4 5.3
IS
100
83.5±5.2
6.2
95.8±4.1 4.3
mean ± SD RSD, % mean ± SD RSD, %
Table 3. The main pharmacokinetic parameters after oral administration of 12.0 mg/kg caudatin in 6 rats
Parameters
Caudatin
t1/2, h MRT, h CL, l/h/kg Cmax, ng/ml AUC0→t, ng/ml·h AUC0→∞, ng/ml·h
1.16±0.19 1.64±0.09 22.1±3.9 256.5±20.6 841.5±160.9 845.8±178.7
MRT = Mean retention time; AUC = area under curve; CL = clearance.
Application of the Method in a Pharmacokinetic Study The method was applied to a pharmacokinetic study in rats. The mean plasma concentration-time curve after oral administration of 12.0 mg/kg caudatin was shown in
figure 3. The main pharmacokinetic parameters from non-compartment model analysis are summarized in table 3. After oral administration of caudatin, a mean maximum plasma concentration (Cmax) was found to be 256.5 ± 20.6 ng/ml. The plasma concentration of caudatin decreased rapidly and was eliminated from plasma with a terminal half-life of 1.16 ± 0.19 h. The initial rapid decline in the plasma concentration indicates that the compound might have left the plasma and been distributed into the other tissues, but further studies will be conducted to confirm these findings.
Conclusions
A UPLC-MS/MS method for the determination of caudatin in rat plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of caudatin level in rat plasma using the UPLC-MS/MS method. The method offered sample preparation with a simple one-step precipitation of plasma protein by acetonitrile and shorter run time of 2.0 min. The method meets the requirement of high sample throughput in bioanalysis and was successfully applied to the pharmacokinetic study of caudatin in rats.
Conflicts of interest The authors report no conflicts of interest.
Pharmacology 2015;96:49–54 DOI: 10.1159/000381784
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Table 2. Recovery and matrix effect of caudatin and IS (n = 6)
Determination of Caudatin in Rat Plasma by UPLC-MS/MS
3 Time (h)
References
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Pharmacology 2015;96:49–54 DOI: 10.1159/000381784
through modulating GSK3β/β-catenin pathway. J Cell Biochem 2012;113:3403–3410. Wang YQ, Zhang SJ, Lu H, Yang B, Ye LF, Zhang RS: A C 21-steroidal glycoside isolated from the roots of cynanchum auriculatum induces cell cycle arrest and apoptosis in human gastric cancer SGC-7901 cells. Evid Based Complement Alternat Med 2013;2013: 180839. Luo Y, Sun Z, Li Y, Liu L, Cai X, Li Z: Caudatin inhibits human hepatoma cell growth and metastasis through modulation of the Wnt/βcatenin pathway. Oncol Rep 2013; 30: 2923– 2928. Li X, Zhang X, Liu X, Tan Z, Yang C, Ding X, Hu X, Zhou J, Xiang S, Zhou C, Zhang J: Caudatin induces cell apoptosis in gastric cancer cells through modulation of Wnt/β-catenin signaling. Oncol Rep 2013;30:677–684. Qi LW, Gu XJ, Li P, Liang Y, Hao H, Wang G: Structural characterization of pregnane glycosides from cynanchum auriculatum by liquid chromatography on a hybrid ion trap time-of-flight mass spectrometer. Rapid Commun Mass Spectrom 2009; 23: 2151– 2160. Xu W, Luo H, Zhang Y, Shan L, Li H, Yang M, Liu R, Zhang W: Simultaneous determination of five main active bufadienolides of chan su
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in rat plasma by liquid chromatography tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2007; 859: 157– 163. Peng Y, Li Y, Wang D, Liu X, Zhang J, Qian S, Duan J: Determination of caudatin-2,6-dideoxy-3-O-methy-beta-d-cymaropyranoside in rat plasma using liquid chromatography-tandem mass spectrometry. Biomed Chromatogr 2008;22:575–580. Vijaya Bhaskar Reddy A, Venugopal N, Madhavi G, Gangadhara Reddy K, Madhavi V: A selective and sensitive UPLC-MS/MS approach for trace level quantification of four potential genotoxic impurities in zolmitriptan drug substance. J Pharm Biomed Anal 2013;84:84–89. Xiong W, Tao X, Pang S, Yang X, Tang G, Bian Z: Separation and quantitation of three acidic herbicide residues in tobacco and soil by dispersive solid-phase extraction and UPLC-MS/ MS. J Chromatogr Sci 2014;52:1326–1331. Agnesod D, De Nicolò A, Simiele M, Mohamed Abdi A, Boglione L, Di Perri G, D’Avolio A: Development and validation of a useful UPLCMS/MS method for quantification of total and phosphorylated-ribavirin in peripheral blood mononuclear cells of HCV+ patients. J Pharm Biomed Anal 2014;90:119–126.
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1 Zhang R, Liu Y, Wang Y, Ye Y, Li X: Cytotoxic and apoptosis-inducing properties of auriculoside A in tumor cells. Chem Biodivers 2007;4:887–892. 2 Peng YR, Ding YF, Wei YJ, Shu B, Li YB, Liu XD: Caudatin-2,6-dideoxy-3-O-methy-βD-cymaropyranoside 1 induced apoptosis through caspase 3-dependent pathway in human hepatoma cell line SMMC7721. Phytother Res 2011;25:631–637. 3 Wang LJ, Geng CA, Ma YB, Luo J, Huang XY, Chen H, Zhou NJ, Zhang XM, Chen JJ: Design, synthesis, and molecular hybrids of caudatin and cinnamic acids as novel anti-hepatitis B virus agents. Eur J Med Chem 2012;54: 352–365. 4 Wang LJ, Geng CA, Ma YB, Huang XY, Luo J, Chen H, Guo RH, Zhang XM, Chen JJ: Synthesis, structure-activity relationships and biological evaluation of caudatin derivatives as novel anti-hepatitis B virus agents. Bioorg Med Chem 2012;20:2877–2888. 5 Fei HR, Chen HL, Xiao T, Chen G, Wang FZ: Caudatin induces cell cycle arrest and caspase-dependent apoptosis in HepG2 cell. Mol Biol Rep 2012;39:131–138. 6 Fei HR, Cui LY, Zhang ZR, Zhao Y, Wang FZ: Caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis
Pharmacology 2015;96:49–54 DOI: 10.1159/000381784
Determination of Caudatin in Rat Plasma by UPLC-MS/MS: Application to a Preclinical Pharmacokinetic Study Qiqi Zhu a Yuanyuan Hu a Yuanyuan Shan b Yiyan Wang a Xiaolong Wu a Baiping Mao a Ren-Shan Ge a
Departments of a Anaesthesiology, b Pathology, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
Key Words Caudatin · UPLC-MS/MS · Rat plasma · Pharmacokinetics
Abstract In this study, a simple, sensitive, and robust analytical method based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of caudatin in rat plasma using carbamazepine as internal standard (IS). This method was linear over the concentration range 2.5–300 ng/ml with a lower limit of quantification (LLOQ) of 2.5 ng/ml. Inter- and intra-day precision (RSD%) were all within 10% and the accuracy (RE%) was equal or lower than 5%. Recoveries of caudatin and IS were more than 80% and matrix effects were not significant. Stability studies showed that caudatin was stable under a variety of storage conditions. The method was successfully applied to a pharmacokinetic study involving oral administra© 2015 S. Karger AG, Basel tion of caudatin to rats.
Introduction
Many natural products have been used as cancer chemotherapeutic agents due to their excellent pharmacological activities and low toxicity. C-21 steroidal glyco© 2015 S. Karger AG, Basel 0031–7012/15/0962–0049$39.50/0 E-Mail [email protected] www.karger.com/pha
sides is one species of important biological active compounds widely found in the plants of the Asclepiadaceae family, which have extensive pharmacological effects such as inhibiting proliferation and invasion of tumor cells [1, 2]. As one species of C-21 steroidal glycosides, caudatin (fig. 1) is mainly isolated from the root of Cynanchum bungei Decne, a traditional Chinese medicine and health food, which has potent anti-hepatitis B virus (HBV) activity [3, 4]. Moreover, many lines of evidence have shown that caudatin induced apoptosis of many cancer cells [5–9]. Currently, several analytical methods exist for quantifying caudatin in the plants and biological samples by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) [10–12]. Quantification and pharmacokinetics studies on constituents of Traditional Chinese Medicine in plasma are required to offer suitable references in clinical application. Even a publication has reported the caudatin concentrations in biological fluids, but the method had several drawbacks, such as long analysis time, requiring large volumes of plasma samples and time-consuming gradient elution [12]. Therefore, to characterize the pharmacokinetic properties of caudatin, it is very necessary to develop an accurate and selective bioanalytical method for the determination of caudatin in plasma. As a result of recent advances in analytical techniques, ultra-performance liquid chromatography coupled with Ren-Shan Ge Department of Anaesthesiology The Second Affiliated Hospital, Wenzhou Medical University Wenzhou 325027, Zhejiang (China) E-Mail raygee0828 @ 163.com
Downloaded by: Kungliga Tekniska Hogskolan 198.143.54.65 - 1/28/2016 3:01:28 AM
O O O
HO
OH
N
H
a
Materials and Methods Chemicals and Reagents Caudatin (purity >98%) was purchased from Chengdu Mansite Pharmaceutical Co., Ltd. (Chengdu, China). Carbamazepine (IS, purity >98%) was purchased from Sigma-Aldrich (St. Louis, Mo., USA). Acetonitrile, methanol, and formic acid were of HPLC grade and were purchased from Merck Company (Darmstadt, Germany). Ultrapure water, prepared by a Milli-Q Reagent water system (Millipore, Mass., USA), was used throughout the study. UPLC-MS/MS Conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, Mass., USA) with an Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm particle size) and inline 0.2 μm stainless steel frit filter (Waters Corp., Milford, Mass., USA). A gradient program was employed with the mobile phase combining solvent A (0.1% formic acid in water) and solvent B (acetonitrile) as follows: 30–30% B (0–0.3 min), 30–90% B (0.3–0.5 min), 90–90% B (0.5–1.8 min),
50
Pharmacology 2015;96:49–54 DOI: 10.1159/000381784
b
HO
tandem mass spectrometry (UPLC-MS/MS) has emerged as an efficient analytical tool with improved sensitivity, selectivity, and specificity. The usefulness of this technique was demonstrated for a wide range of applications in bioanalytical, environmental, and pharmaceutical research [13–15]. Thus, in the present work, a highly selective and rapid UPLC-MS/MS method was developed and fully validated as per the USFDA guidelines for measurement of caudatin in rat plasma using carbamazepine as internal standard (IS). The method offered a small turnaround time for analysis, high sensitivity for the analyte, and utilization of only 100 μl of rat plasma for sample processing with a simple one-step protein precipitation by acetonitrile. The method was free from endogenous matrix interference and was successfully applied to a pharmacokinetic study in rats.
NH2
90–30% B (1.8–2.0 min). A subsequent re-equilibration time (1 min) was performed before the next injection. The flow rate was 0.40 ml/min and the injection volume was 6 μl. The column and sample temperature were maintained at 40 and 4 ° C, respectively. An AB Sciex QTRAP 5500 triple quadruple mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Ont., Canada) was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) modes under unit mass resolution (0.7 amu) in the mass analyzers. The dwell time was set to 200 ms for each MRM transition. The MRM transitions were m/z 345.4→255.4 and m/z 237.1→194.2 for caudatin and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 60 psig; ion spray voltage, 5.0 kV; and temperature, 500 ° C. Data acquiring and processing were performed using analyst software (version 1.5, AB Sciex).
Standard Solutions, Calibration Standards and Quality Control (QC) Sample Standard stock solutions of caudatin and carbamazepine (IS) were prepared in methanol at 1 mg/ml. Then, the stock solutions were diluted with methanol to obtain fresh standard working solution. Calibration standards were prepared by adding corresponding working solutions in drug-free plasma. The final concentrations of caudatin in plasma were 2.5, 5, 10, 25, 50, 100, 200, and 300 ng/ml, respectively. IS was diluted with acetonitrile to 100 ng/ml. Low-, mid-, and high-level quality control (QC) samples containing 5, 80, and 240 ng/ml of caudatin, were prepared in a manner similar to that used for the preparation of calibration samples. All stock solutions, working solutions, calibration standards, and QC samples were stored at –20 ° C and were brought to room temperature before analysis.
Sample Preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5 ml centrifuge tube, an aliquot of 200 μl of the IS working solution (100 ng/ml in acetonitrile) was added to 100 μl of plasma sample. The mixture was vortexed for 30 s followed by centrifugation at 13,000 g for 10 min at room temperature. A 6 μl aliquot of each supernatant was injected into the UPLC-MS/MS system for the analysis.
Zhu/Hu/Shan/Wang/Wu/Mao/Ge
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Fig. 1. The chemical structures of caudatin and IS in the present study: caudatin (a); carbamazepine (IS) (b).
O
OH
Method Validation The method was validated for specificity, linearity, accuracy, precision, recovery, and stability according to the literature for validation of bioanalytical methods (US Food and Drug Administration, 2001). Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC plasma samples. Specificity The specificity of the method was evaluated by analyzing six different sources of rat blank plasma samples and plasma samples with caudatin and IS to investigate endogenous interferences at the peak region of caudatin and IS. The chromatograms of blank plasma samples were compared with those obtained from blank plasma samples spiked with caudatin and IS and plasma samples after oral administration of caudatin. Linearity and LLOQ Calibration curves (y = a + bx) were generated by plotting the peak area ratio (y) of the analyte to IS versus the nominal concentration (x) of the analyte with weighted (1/x2) least square linear regression. The LLOQ was defined as the lowest concentration on the calibration curve where a signal-to-noise (S/N) was at least 10. The acceptance criteria for accuracy and precision of calibration curve data were 80–120% of the nominal concentrations and relative standard deviation (RSD) of ±20% of the nominal concentration at the LLOQ, respectively. Accuracy and Precision Intra- and inter-day accuracy and precision of the method were estimated by analyzing six replicates of QC samples at concentrations of 5, 80, and 240 ng/ml on the same day and on three consecutive days. Accuracy was expressed as relative error (RE%), which should be within the limits of ±15% at all concentrations. The precision was expressed as the relative standard deviation (RSD%), which should not exceed 15% at all concentrations. Recovery and Matrix Effect Recovery was evaluated by comparing the peak area responses of caudatin and IS in six replicates of QC samples at three different concentration levels with those of the analytes added to post-extracted blank plasma at equivalent concentrations. Matrix effects were evaluated by comparing the peak area responses of the analytes in processed blank plasma extract with those of the same analytes present in pure water at equivalent concentrations. Stability The stability of caudatin in rat plasma was investigated using quintuplicates of QC samples (5, 80, and 240 ng/ml). For bench top stability study, QC samples were kept at room temperature for 12 h prior to extraction. Stability in post-preparative samples was evaluated after leaving in the autosampler at 4 ° C for 24 h. The freeze-thaw stability of caudatin was assessed by analyzing plasma samples after three freeze-thaw cycles (–20 to 25 ° C). Long-term stability was tested by analyzing QC samples stored at –20 ° C for 30 days. Dilution stability was assessed using a spiked sample with the concentration that was higher than the upper limit of quantification samples. The result was obtained by comparing the backcalculated value of the sample after being diluted 5 times with the nominal value.
Determination of Caudatin in Rat Plasma by UPLC-MS/MS
Results and Discussion
Method Development and Optimization During the method development, a number of UPLC columns were evaluated including the Acquity BEH C18, Ultimate XB-C8, Ultimate XB-C18, Thermo Accucore XL C18, Hanbon Dubhe C18, and Shiseido Proteonavi C4 columns. Surprisingly, only the Acquity BEH C18 column, which was originally developed for the analysis of peptides and proteins, gave suitable peaks despite using different mobile phases both isocratically and by gradient elution. Mobile phases containing either acetonitrile-water or methanol-water behaved similarly, but the addition of formic acid enhanced the intensity of peaks and improved peak shape. Finally, gradient elution using acetonitrile and 0.1% (v/v) formic acid gave satisfactory peak shapes for caudatin and IS with retention times of 1.66 and 1.41 min, respectively. Protein precipitation is a relatively simple and highthroughput method capable of sample preparation. Initially, methanol and acetonitrile were tested using ratios of 3:1 for methanol:plasma and 2:1 for acetonitrile:plasma. And, the presence of acetonitrile had better recovery than that of methanol. Thus, acetonitrile was used for protein precipitation. Finally, concentration of the sample under a high-speed centrifugation improved the detection limit with no obvious matrix effects for analyte and IS.
Assay Validation Specificity Figure 2 shows the representative chromatograms of blank plasma, a plasma sample spiked with caudatin and Pharmacology 2015;96:49–54 DOI: 10.1159/000381784
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Application to a Pharmacokinetic Study Male Sprague-Dawley rats (180–220 g) were obtained from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) used to study the pharmacokinetics of caudatin. All 6 rats were housed at the Wenzhou Medical University Laboratory Animal Research Center. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical College and were in accordance with the Guide for the Care and Use of Laboratory Animals. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.3 ml) were collected from the tail vein into heparinized 1.5 ml polythene tubes at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, and 6 h after oral administration of caudatin (12.0 mg/kg). The samples were immediately centrifuged at 4,000 g for 8 min. The plasma obtained (100 μl) was stored at –20 ° C until analysis. Plasma caudatin concentration versus time data for each rat was analyzed by DAS (Drug and Statistics) software (version 2.0, Wenzhou Medical University, China).
100
0
0.50
1.00
1.50
100
0
0.50
1.00
1.50
0.50
a
1.00
1.50
Time
0
2.00
0
0.50
1.00
1.50
2.00
1.41
%
100
% 0
0
2.00
IS
100
% 0
0
2.00
1.66
100 %
%
% 0
Caudatin
100
0
0.50
b
1.00
1.50
0
2.00
c
Time
0
0.50
1.00 Time
1.50
2.00
Fig. 2. Representative chromatograms of caudatin and IS in rat plasma samples. a A blank plasma sample; b blank plasma sample spiked with caudatin and carbamazepine, the internal standard (IS); c a rat plasma sample taken 60 min after oral administration of 12.0 mg/kg caudatin in rats.
Table 1. Precision and accuracy of method for the determination of caudatin in rat plasma (n = 6)
Caudatin
Concentration added, ng/ml
Intra-day precision mean ± SD
RSD, %
RE, %
mean ± SD
RSD, %
RE, %
5 80 240
5.2±0.5 80.7±7.3 241.2±12.1
9.0 9.1 5.0
4.3 0.8 0.5
4.8±0.4 76.2±6.9 230.8±18.1
9.1 9.0 7.8
–4.3 –4.8 –3.8
IS as well as a sample plasma, which was obtained at 60 min after oral administration of caudatin (12.0 mg/kg). Caudatin and IS were eluted at about 1.66 and 1.41 min, respectively. The assay was free of interference from endogenous peaks in plasma at the retention times of caudatin and IS. Linearity and LLOQ The assay was linear over the concentration range of 2.5–300 ng/ml with a typical calibration curve equation of y = 0.0020x + 0.0052 and correlation coefficient of r2 = 0.996. The LLOQ of caudatin in rat plasma was found to be 2.5 ng/ml, which was sufficient for the pharmacokinetics study. The precision and accuracy at LLOQ were 9.8 and 109.1%, respectively. Accuracy and Precision The intra- and inter-assay variations were found to be within accepted limits. The intra- and inter-day precisions of caudatin at three QC concentrations were within 9.1% (table 1). Assay accuracy was found to be within 52
Pharmacology 2015;96:49–54 DOI: 10.1159/000381784
Inter-day precision
±4.3%. The results indicate that the present method is reliable and reproducible for the quantitative determination of caudatin. Recovery and Matrix Effect The results of recovery and matrix effect studies are shown in table 2. The recovery of caudatin was in the range of 81.2–86.2% with matrix effect within the range of 98.7– 102.0% at the three QC concentration levels. The recovery of IS was 83.5% with a matrix effect of 95.8%. The results indicate the reasonable recoveries with no obvious suppression or enhancement of ionization of either caudatin or IS. Stability The autosampler, room temperature, freeze-thaw, and long-term (30-day) stability results indicate that the analyte was stable under the storage conditions described above since the bias in concentration was within ±15% of nominal values, and the established method was suitable for the pharmacokinetic study. This result is within the FDA acceptance criteria. Zhu/Hu/Shan/Wang/Wu/Mao/Ge
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Analytes
300
Caudatin
Concentration (ng/ml)
250 200 150 100 50 0 0
Fig. 3. Mean plasma concentration time
1
2
profile after oral administration of 12.0 mg/kg caudatin in 6 rats.
4
5
6
Analytes Concentration Recovery, % added, ng/ml
Matrix effect, %
Caudatin
5 80 240
86.2±6.3 81.2±3.9 81.5±7.2
7.3 4.8 8.8
98.8±5.0 5.1 98.7±6.2 6.3 102.0±5.4 5.3
IS
100
83.5±5.2
6.2
95.8±4.1 4.3
mean ± SD RSD, % mean ± SD RSD, %
Table 3. The main pharmacokinetic parameters after oral administration of 12.0 mg/kg caudatin in 6 rats
Parameters
Caudatin
t1/2, h MRT, h CL, l/h/kg Cmax, ng/ml AUC0→t, ng/ml·h AUC0→∞, ng/ml·h
1.16±0.19 1.64±0.09 22.1±3.9 256.5±20.6 841.5±160.9 845.8±178.7
MRT = Mean retention time; AUC = area under curve; CL = clearance.
Application of the Method in a Pharmacokinetic Study The method was applied to a pharmacokinetic study in rats. The mean plasma concentration-time curve after oral administration of 12.0 mg/kg caudatin was shown in
figure 3. The main pharmacokinetic parameters from non-compartment model analysis are summarized in table 3. After oral administration of caudatin, a mean maximum plasma concentration (Cmax) was found to be 256.5 ± 20.6 ng/ml. The plasma concentration of caudatin decreased rapidly and was eliminated from plasma with a terminal half-life of 1.16 ± 0.19 h. The initial rapid decline in the plasma concentration indicates that the compound might have left the plasma and been distributed into the other tissues, but further studies will be conducted to confirm these findings.
Conclusions
A UPLC-MS/MS method for the determination of caudatin in rat plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of caudatin level in rat plasma using the UPLC-MS/MS method. The method offered sample preparation with a simple one-step precipitation of plasma protein by acetonitrile and shorter run time of 2.0 min. The method meets the requirement of high sample throughput in bioanalysis and was successfully applied to the pharmacokinetic study of caudatin in rats.
Conflicts of interest The authors report no conflicts of interest.
Pharmacology 2015;96:49–54 DOI: 10.1159/000381784
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Table 2. Recovery and matrix effect of caudatin and IS (n = 6)
Determination of Caudatin in Rat Plasma by UPLC-MS/MS
3 Time (h)
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