Research article Received: 7 November 2013,
Revised: 24 April 2014,
Accepted: 17 May 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3273
Development of a sensitive LC-MS/MS method for simultaneous quantification of eleven constituents in rat serum and its application to a pharmacokinetic study of a Chinese medicine Shengmai injection Shuyu Zhana,b, Qing Shaoa, Xiaohui Fana* and Zheng Lic* ABSTRACT: A sensitive LC-MS/MS method was developed and validated for simultaneous quantification of 11 constituents, ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B, in rat serum using digoxin as the internal standard (IS). The serum samples were pretreated and extracted with a two-step liquid–liquid extraction. Chromatographic separation was achieved on a C18 analytical column with a proper gradient elution using 0.02% acetic acid aqueous solution and 0.02% acetic acid–acetonitrile as mobile phase at a flow rate of 0.5 mL/min. MS detection was performed using multiple reaction monitoring via an electrospray ionization source. Good linearity was observed in the validated concentration range for every analyte (r2 ≥0.9929), and the lower limits of quantitation of the analytes were in the range of 0.044–1.190 ng/mL in rat serum. Intra- and inter-day precisions were 75.8%.The validated method was successfully applied to a pharmacokinetic study of all analytes in rats after single intravenous administration of Shengmai injection. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: LC-MS/MS; Shengmai injection; ginsenoside; ophiopogonin; lignan; pharmacokinetics
Introduction Shengmai injection, originating from a traditional Chinese medicine formula composed of Panax ginseng, Ophiopogon japonicas and Schisandra chinensis, is widely used in China for the treatment of cardiac emergency, such as coronary atherosclerotic cardiopathy, viral myocarditis and myocardial infarction (Zheng et al., 2011; Chen et al., 2012; Liu et al., 2012). Chemical and pharmacological studies have demonstrated that ginsenosides, ophiopogonins and lignan isolated from ginseng, Ophiopogon and Schisandra, respectively, are the major constituents responsible for the pharmacological activities. Ginsenosides have been widely reported to exhibit anti-inflammatory (Chai et al., 2008; Wang et al., 2012) and anti-oxidative (Xie et al., 2006; Zhu et al., 2009; Li et al., 2012) effects, to increase vessel dilatation through the activation of nitric oxide (NO) (Scott et al., 2001; Ma et al., 2006), to suppress vascular neointimal hyperplasia (Gao et al., 2011; Zhang et al., 2012) and to promote angiogenesis (Huang et al., 2005; Cheung et al., 2011). Ophiopogonins were also reported to have anti-inflammatory (Huang et al., 2008) and anti-oxidative (Qian et al., 2010) effects and antithrombotic activities (Kou et al., 2006). Lignans of Schisandra, such as Schisandrin and Schizandrin B, have also been found to possess antiinflammatory and anti-oxidative effects (Chiu et al., 2008; Guo et al., 2008; Checker et al., 2012). All these biological effects of ginsenosides, ophiopogonins and lignan in Shengmai injection could be responsible for its protective effects in the cardiovascular system.
Biomed. Chromatogr. (2014)
At present, Shengmai injection has been applied widely in clinical treatment. However, its pharmacokinetics properties have not been well studied. Previous studies on pharmacokinetic have been reported to simultaneously determine ginsenosides Rg1, ginsenosides Re, ginsenosides Rd, ginsenosides Rb1, ginsenosides Rf or ophiopogonin D in plasma after intravenous injection of Shenmai injection, a formula composed of red ginseng and Ophiopogon (Yu et al., 2007; Xia et al., 2008a, 2008b). The main active components derived from each herb can be used as markers for pharmacokinetic study of multiple-herb products. To the best of our knowledge, there is no report yet on the
* Correspondence to: Xiaohui Fan, Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China. Email: [email protected] Zheng Li, State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China. Email: [email protected] a
Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b
Department of Pharmaceutics, Medical College of Jiaxing University, Jiaxing 314001, China
c
State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China Abbreviations used: LLE, liquid–liquid extraction; MRM, multiple reaction monitoring.
Copyright © 2014 John Wiley & Sons, Ltd.
S. Zhan et al. pharmacokinetics of ginsenosides, ophiopogonins and lignans of Schisandra together in rat serum after intravenous administration of Shengmai injection. In this paper, an LC-MS/MS method was developed and validated for simultaneous quantification of 11 active constituents (ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rg2, ginsenoside Rb1, ginsenoside Rd, ginsenoside Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B) in rat serum. The method exhibited high specificity and sensitivity for determining the trace constituents of Shengmai injection in rat serum. The validated method was then successfully applied to a pharmacokinetic study of all analytes in rats after a single intravenous administration of Shengmai injection. The pharmacokinetic profile of Shengmai injection in rats was obtained according to a pharmacokinetic study of the constituents mentioned above. The results provided supporting data for evaluating the efficacy and clinical application of Shengmai injection.
was set at 0.5 mL/min. The sample injection volume was 20 μL. Ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D and digoxin were determined in negative ionization mode, and schisandrin, schisandrol B and schizandrin B were determined in positive ionization mode. The analytes were quantified by multiple-reaction monitoring (MRM) mode. Mass spectrometry was operated with an optimized ion spray voltage at 4200 V for negative ionization mode and 4200 V for positive ionization mode, turbo spray temperature 650°C, and collision gas at 12 psi. The curtain gas, nebulizer gas (gas 1) and auxiliary gas (gas 2) were at 10, 60 and 70 psi, respectively. The precursorto-product ion pairs, the optimized declustering potential and collision energy for each analyte are shown in Fig. 1. Ginsenoside Rg1 799.4/637.5 Da DP: -138 V; CE: -37 V
Ginsenoside Re 945.6/637.4 Da DP: -132 V; CE: -56 V
Materials and methods Chemicals, reagents and materials
Ginsenoside Rf 799.3/475.2 Da DP: -133 V; CE: -56 V
Ginsenoside Rg2 783.8/475.3 Da DP: -155 V; CE: -56 V Ginsenoside Rb1 1108.0/178.8 Da DP: -145 V; CE: -69 V
Intensity, cps
Ginsenoside Rg1 (Rg1), ginsenoside Re (Re), ginsenoside Rf (Rf), ginsenoside Rg2 (Rg2), ginsenoside Rb1 (Rb1), ginsenoside Rd (Rd) and ginsenoside Rc (Rc) were supplied by Jilin University (Changchun, China). Ophiopogonin D (oph D), schisandrin, schisandrol B and schizandrin B were purchased from Shanghai Winherb Medical S&T Development Co. Ltd (Shanghai, China). Shengmai injection was supplied by SZYY Group Pharmaceutical Limited (Jiangyan, Jiangsu, China). Digoxin (internal standard, IS) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile, methanol (Merck, Germany) and acetic acid (Roe Scientific Inc.) were of HPLC grade. Water was produced using a Milli-Q water system (Millipore, Bedford, MA, USA). All the other reagents were obtained from commercial sources and were of analytical grade.
Ginsenoside Rd 945.5/621.6 Da DP: -170 V; CE: -58 V
Animals
Ginsenoside Rc 1077.8/191.0 Da DP: -155 V; CE: -76 V
Male Sprague–Dawley rats, weighing 260–300 g, were bought from Slaccas Experiment Animal Limited of Shanghai (Shanghai, China). The studies were approved by The Animal Ethic Review Committees of Zhejiang University. The rats were maintained in an air-conditioned animal quarter at a temperature of 22 ± 2°C and a relative humidity of 50 ± 10%. The animals were allowed free access to food and water before the experiment, and then fasted with free access to water for 12 h prior to each experiment.
Ophiopogonin D 853.4/721.4 Da DP: -152 V; CE: -42 V Schisandrin 432.8/384.0 Da DP: 78 V; CE: 28 V
Chromatographic and mass spectrometry conditions The LC-MS/MS system was composed of an Agilent 1200 liquid chromatography system, equipped with a binary pump, a vacuum degasser unit, an autosampler and a triple-quadrupole tandem mass spectrometer (API 4000, Applied Biosystems, Foster City, USA). The chromatographic separation was achieved on a Phenomenex Kinetex C18 column (50 × 4.6 mm, 2.6 μm), and the column temperature was set at 40°C. The mobile phase consisted of 0.02% acetic acid aqueous solution (A) and 0.02% acetic acid–acetonitrile (B) using a gradient elution of 25% B at 0–5 min, 25–40% B at 5–10 min, 40–60% B at 10–20 min, 60–70% B at 20–27 min and 70–75% B at 27–32 min, and the re-equilibration time of gradient elution was 7 min. The flow rate
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Schisandrol B 399.7/369.1 Da DP: 100 V; CE: 27 V
Schizandrin B 402.3/301.2 Da DP: 60 V; CE: 33 V
m/z, Da Figure 1. The product ion scan spectra, the precursor-to-product ion pairs, declustering potential and collision energy of ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B.
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. (2014)
Simultaneous quantification of eleven herbal constituents in rat serum
Figure 2. Representative multiple reaction monitoring chromatograms of ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D (oph D), schisandrin, schisandrol B, schizandrin B and IS (digoxin). (A) blank serum; (B) blank serum spiked with the 11 analytes and IS; (C) 0.08 h serum sample after intravenous administration of Shengmai injection.
Biomed. Chromatogr. (2014)
Copyright © 2014 John Wiley & Sons, Ltd.
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S. Zhan et al. Before extraction, the water-saturated n-butyl alcohol was mixed with IS stock solution (the ultimate concentration was 35.5 ng/mL) to form extract solution. The two-step extraction comprised:
Preparation of calibration standards and quality control samples The stock solutions of ginsenoside Rg1 (1.19 mg/mL), Re (0.99 mg/mL), Rf (1.09 mg/mL), Rg2 (1.65 mg/mL), Rb1 (5.60 mg/mL), Rd (1.45 mg/mL), Rc (1.52 mg/mL), ophiopogonin D (1.31 mg/mL), schisandrin (4.58 mg/mL), schisandrol B (1.30 mg/mL) and schizandrin B (1.08 mg/mL) were prepared in methanol. Then, the appropriate amounts of the 11 stock solutions were mixed and diluted with methanol to a final mixed standard solution. A series of working solutions of these analytes were obtained by diluting mixed standard solution with methanol at appropriate concentrations. The digoxin (IS) stock solution (0.44 mg/mL) was also prepared in the methanol. All solutions were stored at 4°C. To prepare calibration curves, each working solution (300 μL) was evaporated to dryness in the Thermo Scientific SpeedVac concentrator. The residue was then spiked with a 300 μL drug-free serum sample and mixed to form calibration standards in concentrations ranging from 1.190 to 1487.5 ng/mL for ginsenoside Rg1, from 0.165 to 619.1 ng/mL for Re, from 0.109 to 408.8 ng/mL for Rf, from 0.083 to 309.4 ng/mL for Rg2, from 0.280 to 1050.0 ng/mL for Rb1, from 0.145 to 543.8 ng/mL for Rd, from 0.152 to 570.0 ng/mL for Rc, from 0.044 to 54.4 ng/mL for ophiopogonin D, from 0.229 to 572.3 ng/mL for schisandrin, from 0.260 to 325.0 ng/mL for schisandrol B and from 0.144 to 349.9 ng/mL for schizandrin B in rat serum. To validate this method, quality control (QC) serum samples containing all 11 analytes at 2.98, 449.82 and 899.64 ng/mL for ginsenoside Rg1, at 0.40, 199.98 and 399.96 ng/mL for Re, at 0.30, 149.98 and 299.97 ng/mL for Rf, at 0.20, 149.82 and 299.64 ng/mL for Rg2, at 2.50, 499.52 and 999.04 ng/mL for Rb1, at 0.40, 249.98 and 499.96 ng/mL for Rd, at 0.40, 249.89 and 499.78 ng/mL for Rc, at 0.08, 2.50 and 5.00 ng/mL for ophiopogonin D, at 0.43, 194.12 and 388.25 ng/mL for schisandrin, at 0.60, 110.24 and 219.96 ng/mL for schisandrol B, and at 1.00, 149.90 and 299.81 ng/mL for schizandrin B were prepared in the same manner. The calibration standard solutions and QC sample were prepared for every analysis batch.
(1) To each 300 μL aliquot of serum sample, 1.5 mL extract solution was added. Then, the mixture was shaken on a vortex-mixer at 1500 rpm for 20 min. After centrifugation at 12,000 rpm for 10 min, 1mL of the supernatant was transferred to a clean tube. (2) The preciptitate in the tube was re-mixed with 1mL extract solution, and then the mixture was shaken on a vortexmixer at 1500 rpm for 10 min. After centrifugation at 12,000 rpm for 10 min, 0.8 mL of the supernatant was combined with the 1mL supernatant from step one. The supernatant was evaporated to dryness in the Thermo Scientific SpeedVac concentrator. The obtained residue was dissolved in 60 μL of 80% methanol and centrifuged at 12,000 rpm for another 10 min. Finally, an aliquot of 20 μL of the solution was injected into the LC-MS/MS system for analysis.
Method validation Specificity. The specificity of the method was evaluated by comparing the MRM chromatograms of blank rat serum from six different rats. Each blank serum was tested for endogenous interference using previously described LC-MS/MS method.
Sample preparation
Linearity, low limit of quantification and low limit of detection. The calibration curves were prepared by assaying standard serum samples as described above, and each level sample was prepared and assayed in duplicate on three separate days. The calibration curves were plotted by the peak area ratio (analyte/IS, y) vs the analyte concentration (x) using a 1/x2 weighted linear least-squares regression model. The lower limit of quantification (LLOQ) was defined as the lowest concentration on the calibration curve that produced a signal-to-noise (S/N) ratio of at least 10 with an acceptable accuracy (recovery within 80–120%) and precision (RSD, below 20%). The lower limit of detection (LLOD) was defined as the lowest concentration that produced a peak distinguishable from background noise (S/N ratio of 3:1) for the analytes.
In order to improve extraction efficiency, a two-step liquid–liquid extraction (LLE) with water-saturated n-butyl alcohol was applied.
Precision and accuracy. The precision and accuracy were evaluated by analyzing six replicates of QC samples at low, medium
Table 1. The linearity and lower limits of quantitation and detection (LLOQ and LLOD) of the assay for the 11 analytes Analyte
Ginsenoside Rg1 Ginsenoside Re Ginsenoside Rf Ginsenoside Rg2 Ginsenoside Rb1 Ginsenoside Rd Ginsenoside Rc Ophiopogonin D Schisandrin Schisandrol B Schizandrin B
Linear range (ng/mL)
1.190–1487.5 0.165–619.1 0.109–408.8 0.083–309.4 0.280–1050.0 0.145–543.8 0.152–570.0 0.044–54.4 0.229–572.3 0.260–325.0 0.144–349.9
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Regression equation
y= y= y= y= y= y= y= y= y= y= y=
0.0293x + 4.5274 0.0577x + 2.8364 0.1101x + 4.2256 0.1133x + 1.1734 0.0476x + 4.5820 0.0565x + 2.2027 0.0687x + 3.2216 0.1286x + 0.1807 0.1280x + 0.0027 0.0764x + 0.6209 0.0986x +0.1028
r2
0.9960 0.9950 0.9929 0.9986 0.9984 0.9940 0.9974 0.9997 0.9956 0.9994 0.9959
LLOQ Concentration (ng/mL)
Accuracy (recovery, %)
Precision (RSD%, n = 5)
1.190 0.165 0.109 0.083 0.280 0.145 0.152 0.044 0.229 0.260 0.144
108.0 114.2 108.4 100.9 97.9 110.6 105.9 94.1 90.5 108.5 101.4
4.9 7.3 3.1 1.4 9.6 9.3 8.1 8.4 9.6 13.1 12.3
Copyright © 2014 John Wiley & Sons, Ltd.
LLOD (ng/mL)
0.595 0.099 0.036 0.028 0.093 0.048 0.051 0.022 0.152 0.130 0.072
Biomed. Chromatogr. (2014)
Simultaneous quantification of eleven herbal constituents in rat serum Table 2. Precision and accuracy for the assay of the 11 analytes in rat serum (n = 6) Analyte
Concentration spiked (ng/mL)
Ginsenoside Rg1
Ginsenoside Re
Ginsenoside Rf
Ginsenoside Rg2
Ginsenoside Rb1
Ginsenoside Rd
Ginsenoside Rc
Ophiopogonin D
Schisandrin
Schisandrol B
Schizandrin B
2.98 449.82 899.64 0.40 199.98 399.96 0.30 149.98 299.97 0.20 149.82 299.64 2.50 499.52 999.04 0.40 249.98 499.96 0.40 249.89 499.78 0.08 2.50 5.00 0.43 194.12 388.25 0.60 110.24 219.96 1.00 149.90 299.81
Intra-day Concentration measured (ng/mL) 2.74 507.40 859.77 0.41 213.95 367.02 0.32 158.89 309.96 0.20 159.13 320.90 2.65 521.15 1027.07 0.45 275.10 529.50 0.42 272.37 519.46 0.08 2.46 5.26 0.44 202.14 363.79 0.50 123.42 225.98 0.85 136.91 293.70
± 0.17 ± 21.20 ± 41.55 ± 0.03 ± 13.07 ± 21.65 ± 0.03 ± 10.81 ± 21.63 ± 0.02 ± 7.93 ± 15.46 ± 0.09 ± 21.24 ± 72.13 ± 0.03 ± 11.64 ± 14.29 ± 0.04 ± 16.02 ± 34.09 ± 0.01 ± 0.15 ± 0.36 ± 0.01 ± 16.71 ± 14.32 ± 0.03 ± 4.58 ± 5.81 ± 0.05 ± 11.92 ± 27.65
Inter-day
Precision (%)
Accuracy (%)
6.1 4.2 4.8 7.9 6.1 5.9 8.2 6.8 7.0 7.6 5.0 4.8 3.4 4.1 7.0 6.7 4.2 2.7 10.2 5.9 6.6 13.2 6.2 6.8 1.9 8.3 3.9 7.3 3.7 2.6 5.5 8.7 9.4
92.2 112.8 95.6 103.5 107.0 91.8 105.8 105.9 103.3 103.3 106.2 107.1 105.9 104.3 102.8 112.5 110.0 105.9 104.2 109.0 103.9 96.9 98.4 105.3 102.2 104.1 93.7 85.9 112.0 102.7 85.1 91.3 98.0
Concentration measured (ng/mL) 3.03 ± 475.50 ± 911.39 ± 0.39 ± 208.70 ± 414.35 ± 0.31 ± 156.95 ± 310.87 ± 0.20 ± 155.94 ± 317.08 ± 2.59 ± 523.03 ± 1032.85 ± 0.43 ± 260.31 ± 510.11 ± 0.41 ± 256.66 ± 501.67 ± 0.08 ± 2.51 ± 5.23 ± 0.48 ± 201.03 ± 377.54 ± 0.59 ± 117.49 ± 217.81 ± 0.97 ± 151.25 ± 285.23 ±
Precision (%)
Accuracy (%)
9.7 3.3 4.5 12.3 7.3 6.6 11.1 3.2 5.2 6.6 3.0 3.7 8.9 5.2 2.8 14.1 4.5 4.7 11.9 4.7 2.6 8.9 5.7 6.6 9.1 6.2 7.4 9.9 8.6 4.4 9.7 8.9 7.7
101.7 105.7 101.3 99.4 104.4 103.6 102.9 104.6 103.6 99.1 104.1 105.8 103.9 104.7 103.4 106.6 104.1 102.0 103.1 102.7 100.4 98.4 100.3 104.7 110.6 103.6 97.2 98.7 106.6 99.0 97.1 100.9 95.1
0.29 15.83 41.19 0.05 15.15 27.20 0.03 5.02 16.31 0.01 4.63 11.77 0.23 27.34 29.07 0.06 11.72 23.88 0.05 11.98 12.97 0.01 0.14 0.35 0.04 12.50 27.83 0.06 10.09 9.61 0.09 13.49 21.90
Table 3. The extraction recovery and matrix effect of 11 analytes at low, medium and high conentration levels in rat serum (mean ± SD, n = 6) Analyte
Extraction recovery (%) Low
Ginsenoside Rg1 Ginsenoside Re Ginsenoside Rf Ginsenoside Rg2 Ginsenoside Rb1 Ginsenoside Rd Ginsenoside Rc Ophiopogonin D Schisandrin Schisandrol B Schizandrin B
Biomed. Chromatogr. (2014)
87.2 87.3 81.5 83.0 81.8 85.6 83.4 84.8 79.8 83.3 85.0
± ± ± ± ± ± ± ± ± ± ±
11.5 6.4 7.2 5.7 10.2 9.0 10.9 7.6 3.6 4.8 10.8
Medium 87.6 86.5 89.3 90.4 84.1 83.7 86.0 79.5 84.3 82.8 88.1
± ± ± ± ± ± ± ± ± ± ±
2.7 3.5 1.4 1.9 4.3 1.5 4.0 7.2 4.4 4.1 9.6
Matrix effect (%) High 92.5 89.9 90.1 87.6 89.7 85.7 88.9 75.8 87.0 92.7 85.1
± ± ± ± ± ± ± ± ± ± ±
5.8 6.5 9.7 7.6 6.8 6.4 9.9 9.9 8.7 9.3 3.3
Low 85.6 ± 86.3 ± 87.2 ± 85.7 ± 101.8 ± 83.8 ± 90.6 ± 45.8 ± 89.3 ± 86.9 ± 85.6 ±
Copyright © 2014 John Wiley & Sons, Ltd.
9.7 9.3 5.2 5.7 11.5 4.6 13.1 2.7 6.3 6.2 9.1
Medium 88.0 ± 88.2 ± 85.6 ± 87.6 ± 97.0 ± 82.0 ± 91.1 ± 48.0 ± 99.3 ± 101.0 ± 102.0 ±
8.2 9.1 6.8 8.5 8.6 6.5 5.3 8.3 8.2 7.7 12.1
High 86.0 84.2 82.5 79.1 89.4 81.4 88.8 41.3 89.9 85.6 96.4
± 9.4 ± 10.4 ± 9.0 ± 7.1 ± 8.2 ± 6.9 ± 10.4 ± 5.4 ± 10.3 ± 7.7 ± 8.7
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S. Zhan et al. and high concentration levels as described above. The intra-day precision and accuracy of the assays were determined in the same day by analyzing six replicates at each concentration level. The inter-day precision and accuracy were evaluated on six consecutive days at each concentration level. The precision values expressed as RSD were required to be 300 μL of serum was obtained and immediately stored at 20°C until analysis.
Results and discussion LC-MS/MS method
Concentration (ng/mL)
In order to obtain optimum MS conditions, the standard solutions of the analytes and IS were infused into the mass spectrometer separately, and the optimized mass parameters such as declustering potential and collision energy were obtained. The mass transitions of precursor to product ion at m/z 799.4/637.5 for ginsenoside Rg1, 945.6/637.4 for Re, 799.3/475.2 for Rf, 783.8/ 475.3 for Rg2, 1108.0/178.8 for Rb1, 945.5/621.6 for Rd, 1077.8/ 191.0 for Rc, 853.4/721.4 for ophiopogonin D, 432.8/384.0 for schisandrin, 399.7/369.1 for schisandrol B, 402.3/301.2 for schizandrin B and 779.2/650.0 for IS were chosen for the quantification studies. The product ion mass spectrum of the analytes iss shown in Fig. 1. To evaluate the sensitivity of the method, the 10000.0
Rg1 Re Rf Rg2
1000.0
1000.0
100.0
MS ion source parameters, such as ion spray voltage, turbo spray temperature, collision gas, curtain gas, nebulizer gas (gas 1) and auxiliary gas (gas 2), were further investigated to obtain the optimal results for quantification. The chromatographic conditions, especially the additive and the gradient of mobile phase, could affect the separation and the peak shape of ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B. It was found that the optimal separations and peak responses for the analytes and IS were obtained by adding 0.02% acetic acid in mobile phase with an appropriate gradient elution. Sample preparation In this study, the one-step LLE with 1.5 mL extract solution was first tested to extract the analytes and IS, and the results demonstrated that some analytes, such as ophiopogonin D and schizandrin B, exhibited relatively low extraction recovery (60.6 and 38.3%, respectively). Therefore, the method of two-step LLE was used to improve extraction efficiency in order to guarantee the extraction recovery of all analytes >75%. Method validation Specificity. Under the above LC-MS/MS conditions, the representative chromatograms of a blank serum sample, a blank serum sample spiked with 11 analytes and IS, and a serum sample from a rat at 0.08 h after intravenous administration of Shengmai injection are shown in Fig. 2. The retention times of ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B, schizandrin B and IS were 10.54, 10.60, 13.38, 14.26, 13.70, 15.25, 14.08, 20.88, 18.29, 19.83, 29.59 and 13.14 min, respectively. No significant interferences from endogenous substances in the blank serum sample were observed at the retention times of analytes and IS. Good specificity and selectivity were thus observed for the assay method. Linearity, LLOQ and LLOD. The linear ranges, regression equations, correlation coefficients, LLOQ and LLOD of the assay are shown in Table 1. Good linearity was obtained in the validated concentration range (r 2 ≥0.9929). The accuracy (recovery, %) and precision (RSD, %) of LLOQs in the method were 90.5–114.2 and
Revised: 24 April 2014,
Accepted: 17 May 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3273
Development of a sensitive LC-MS/MS method for simultaneous quantification of eleven constituents in rat serum and its application to a pharmacokinetic study of a Chinese medicine Shengmai injection Shuyu Zhana,b, Qing Shaoa, Xiaohui Fana* and Zheng Lic* ABSTRACT: A sensitive LC-MS/MS method was developed and validated for simultaneous quantification of 11 constituents, ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B, in rat serum using digoxin as the internal standard (IS). The serum samples were pretreated and extracted with a two-step liquid–liquid extraction. Chromatographic separation was achieved on a C18 analytical column with a proper gradient elution using 0.02% acetic acid aqueous solution and 0.02% acetic acid–acetonitrile as mobile phase at a flow rate of 0.5 mL/min. MS detection was performed using multiple reaction monitoring via an electrospray ionization source. Good linearity was observed in the validated concentration range for every analyte (r2 ≥0.9929), and the lower limits of quantitation of the analytes were in the range of 0.044–1.190 ng/mL in rat serum. Intra- and inter-day precisions were 75.8%.The validated method was successfully applied to a pharmacokinetic study of all analytes in rats after single intravenous administration of Shengmai injection. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: LC-MS/MS; Shengmai injection; ginsenoside; ophiopogonin; lignan; pharmacokinetics
Introduction Shengmai injection, originating from a traditional Chinese medicine formula composed of Panax ginseng, Ophiopogon japonicas and Schisandra chinensis, is widely used in China for the treatment of cardiac emergency, such as coronary atherosclerotic cardiopathy, viral myocarditis and myocardial infarction (Zheng et al., 2011; Chen et al., 2012; Liu et al., 2012). Chemical and pharmacological studies have demonstrated that ginsenosides, ophiopogonins and lignan isolated from ginseng, Ophiopogon and Schisandra, respectively, are the major constituents responsible for the pharmacological activities. Ginsenosides have been widely reported to exhibit anti-inflammatory (Chai et al., 2008; Wang et al., 2012) and anti-oxidative (Xie et al., 2006; Zhu et al., 2009; Li et al., 2012) effects, to increase vessel dilatation through the activation of nitric oxide (NO) (Scott et al., 2001; Ma et al., 2006), to suppress vascular neointimal hyperplasia (Gao et al., 2011; Zhang et al., 2012) and to promote angiogenesis (Huang et al., 2005; Cheung et al., 2011). Ophiopogonins were also reported to have anti-inflammatory (Huang et al., 2008) and anti-oxidative (Qian et al., 2010) effects and antithrombotic activities (Kou et al., 2006). Lignans of Schisandra, such as Schisandrin and Schizandrin B, have also been found to possess antiinflammatory and anti-oxidative effects (Chiu et al., 2008; Guo et al., 2008; Checker et al., 2012). All these biological effects of ginsenosides, ophiopogonins and lignan in Shengmai injection could be responsible for its protective effects in the cardiovascular system.
Biomed. Chromatogr. (2014)
At present, Shengmai injection has been applied widely in clinical treatment. However, its pharmacokinetics properties have not been well studied. Previous studies on pharmacokinetic have been reported to simultaneously determine ginsenosides Rg1, ginsenosides Re, ginsenosides Rd, ginsenosides Rb1, ginsenosides Rf or ophiopogonin D in plasma after intravenous injection of Shenmai injection, a formula composed of red ginseng and Ophiopogon (Yu et al., 2007; Xia et al., 2008a, 2008b). The main active components derived from each herb can be used as markers for pharmacokinetic study of multiple-herb products. To the best of our knowledge, there is no report yet on the
* Correspondence to: Xiaohui Fan, Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China. Email: [email protected] Zheng Li, State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China. Email: [email protected] a
Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b
Department of Pharmaceutics, Medical College of Jiaxing University, Jiaxing 314001, China
c
State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China Abbreviations used: LLE, liquid–liquid extraction; MRM, multiple reaction monitoring.
Copyright © 2014 John Wiley & Sons, Ltd.
S. Zhan et al. pharmacokinetics of ginsenosides, ophiopogonins and lignans of Schisandra together in rat serum after intravenous administration of Shengmai injection. In this paper, an LC-MS/MS method was developed and validated for simultaneous quantification of 11 active constituents (ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rg2, ginsenoside Rb1, ginsenoside Rd, ginsenoside Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B) in rat serum. The method exhibited high specificity and sensitivity for determining the trace constituents of Shengmai injection in rat serum. The validated method was then successfully applied to a pharmacokinetic study of all analytes in rats after a single intravenous administration of Shengmai injection. The pharmacokinetic profile of Shengmai injection in rats was obtained according to a pharmacokinetic study of the constituents mentioned above. The results provided supporting data for evaluating the efficacy and clinical application of Shengmai injection.
was set at 0.5 mL/min. The sample injection volume was 20 μL. Ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D and digoxin were determined in negative ionization mode, and schisandrin, schisandrol B and schizandrin B were determined in positive ionization mode. The analytes were quantified by multiple-reaction monitoring (MRM) mode. Mass spectrometry was operated with an optimized ion spray voltage at 4200 V for negative ionization mode and 4200 V for positive ionization mode, turbo spray temperature 650°C, and collision gas at 12 psi. The curtain gas, nebulizer gas (gas 1) and auxiliary gas (gas 2) were at 10, 60 and 70 psi, respectively. The precursorto-product ion pairs, the optimized declustering potential and collision energy for each analyte are shown in Fig. 1. Ginsenoside Rg1 799.4/637.5 Da DP: -138 V; CE: -37 V
Ginsenoside Re 945.6/637.4 Da DP: -132 V; CE: -56 V
Materials and methods Chemicals, reagents and materials
Ginsenoside Rf 799.3/475.2 Da DP: -133 V; CE: -56 V
Ginsenoside Rg2 783.8/475.3 Da DP: -155 V; CE: -56 V Ginsenoside Rb1 1108.0/178.8 Da DP: -145 V; CE: -69 V
Intensity, cps
Ginsenoside Rg1 (Rg1), ginsenoside Re (Re), ginsenoside Rf (Rf), ginsenoside Rg2 (Rg2), ginsenoside Rb1 (Rb1), ginsenoside Rd (Rd) and ginsenoside Rc (Rc) were supplied by Jilin University (Changchun, China). Ophiopogonin D (oph D), schisandrin, schisandrol B and schizandrin B were purchased from Shanghai Winherb Medical S&T Development Co. Ltd (Shanghai, China). Shengmai injection was supplied by SZYY Group Pharmaceutical Limited (Jiangyan, Jiangsu, China). Digoxin (internal standard, IS) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile, methanol (Merck, Germany) and acetic acid (Roe Scientific Inc.) were of HPLC grade. Water was produced using a Milli-Q water system (Millipore, Bedford, MA, USA). All the other reagents were obtained from commercial sources and were of analytical grade.
Ginsenoside Rd 945.5/621.6 Da DP: -170 V; CE: -58 V
Animals
Ginsenoside Rc 1077.8/191.0 Da DP: -155 V; CE: -76 V
Male Sprague–Dawley rats, weighing 260–300 g, were bought from Slaccas Experiment Animal Limited of Shanghai (Shanghai, China). The studies were approved by The Animal Ethic Review Committees of Zhejiang University. The rats were maintained in an air-conditioned animal quarter at a temperature of 22 ± 2°C and a relative humidity of 50 ± 10%. The animals were allowed free access to food and water before the experiment, and then fasted with free access to water for 12 h prior to each experiment.
Ophiopogonin D 853.4/721.4 Da DP: -152 V; CE: -42 V Schisandrin 432.8/384.0 Da DP: 78 V; CE: 28 V
Chromatographic and mass spectrometry conditions The LC-MS/MS system was composed of an Agilent 1200 liquid chromatography system, equipped with a binary pump, a vacuum degasser unit, an autosampler and a triple-quadrupole tandem mass spectrometer (API 4000, Applied Biosystems, Foster City, USA). The chromatographic separation was achieved on a Phenomenex Kinetex C18 column (50 × 4.6 mm, 2.6 μm), and the column temperature was set at 40°C. The mobile phase consisted of 0.02% acetic acid aqueous solution (A) and 0.02% acetic acid–acetonitrile (B) using a gradient elution of 25% B at 0–5 min, 25–40% B at 5–10 min, 40–60% B at 10–20 min, 60–70% B at 20–27 min and 70–75% B at 27–32 min, and the re-equilibration time of gradient elution was 7 min. The flow rate
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Schisandrol B 399.7/369.1 Da DP: 100 V; CE: 27 V
Schizandrin B 402.3/301.2 Da DP: 60 V; CE: 33 V
m/z, Da Figure 1. The product ion scan spectra, the precursor-to-product ion pairs, declustering potential and collision energy of ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B.
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. (2014)
Simultaneous quantification of eleven herbal constituents in rat serum
Figure 2. Representative multiple reaction monitoring chromatograms of ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D (oph D), schisandrin, schisandrol B, schizandrin B and IS (digoxin). (A) blank serum; (B) blank serum spiked with the 11 analytes and IS; (C) 0.08 h serum sample after intravenous administration of Shengmai injection.
Biomed. Chromatogr. (2014)
Copyright © 2014 John Wiley & Sons, Ltd.
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S. Zhan et al. Before extraction, the water-saturated n-butyl alcohol was mixed with IS stock solution (the ultimate concentration was 35.5 ng/mL) to form extract solution. The two-step extraction comprised:
Preparation of calibration standards and quality control samples The stock solutions of ginsenoside Rg1 (1.19 mg/mL), Re (0.99 mg/mL), Rf (1.09 mg/mL), Rg2 (1.65 mg/mL), Rb1 (5.60 mg/mL), Rd (1.45 mg/mL), Rc (1.52 mg/mL), ophiopogonin D (1.31 mg/mL), schisandrin (4.58 mg/mL), schisandrol B (1.30 mg/mL) and schizandrin B (1.08 mg/mL) were prepared in methanol. Then, the appropriate amounts of the 11 stock solutions were mixed and diluted with methanol to a final mixed standard solution. A series of working solutions of these analytes were obtained by diluting mixed standard solution with methanol at appropriate concentrations. The digoxin (IS) stock solution (0.44 mg/mL) was also prepared in the methanol. All solutions were stored at 4°C. To prepare calibration curves, each working solution (300 μL) was evaporated to dryness in the Thermo Scientific SpeedVac concentrator. The residue was then spiked with a 300 μL drug-free serum sample and mixed to form calibration standards in concentrations ranging from 1.190 to 1487.5 ng/mL for ginsenoside Rg1, from 0.165 to 619.1 ng/mL for Re, from 0.109 to 408.8 ng/mL for Rf, from 0.083 to 309.4 ng/mL for Rg2, from 0.280 to 1050.0 ng/mL for Rb1, from 0.145 to 543.8 ng/mL for Rd, from 0.152 to 570.0 ng/mL for Rc, from 0.044 to 54.4 ng/mL for ophiopogonin D, from 0.229 to 572.3 ng/mL for schisandrin, from 0.260 to 325.0 ng/mL for schisandrol B and from 0.144 to 349.9 ng/mL for schizandrin B in rat serum. To validate this method, quality control (QC) serum samples containing all 11 analytes at 2.98, 449.82 and 899.64 ng/mL for ginsenoside Rg1, at 0.40, 199.98 and 399.96 ng/mL for Re, at 0.30, 149.98 and 299.97 ng/mL for Rf, at 0.20, 149.82 and 299.64 ng/mL for Rg2, at 2.50, 499.52 and 999.04 ng/mL for Rb1, at 0.40, 249.98 and 499.96 ng/mL for Rd, at 0.40, 249.89 and 499.78 ng/mL for Rc, at 0.08, 2.50 and 5.00 ng/mL for ophiopogonin D, at 0.43, 194.12 and 388.25 ng/mL for schisandrin, at 0.60, 110.24 and 219.96 ng/mL for schisandrol B, and at 1.00, 149.90 and 299.81 ng/mL for schizandrin B were prepared in the same manner. The calibration standard solutions and QC sample were prepared for every analysis batch.
(1) To each 300 μL aliquot of serum sample, 1.5 mL extract solution was added. Then, the mixture was shaken on a vortex-mixer at 1500 rpm for 20 min. After centrifugation at 12,000 rpm for 10 min, 1mL of the supernatant was transferred to a clean tube. (2) The preciptitate in the tube was re-mixed with 1mL extract solution, and then the mixture was shaken on a vortexmixer at 1500 rpm for 10 min. After centrifugation at 12,000 rpm for 10 min, 0.8 mL of the supernatant was combined with the 1mL supernatant from step one. The supernatant was evaporated to dryness in the Thermo Scientific SpeedVac concentrator. The obtained residue was dissolved in 60 μL of 80% methanol and centrifuged at 12,000 rpm for another 10 min. Finally, an aliquot of 20 μL of the solution was injected into the LC-MS/MS system for analysis.
Method validation Specificity. The specificity of the method was evaluated by comparing the MRM chromatograms of blank rat serum from six different rats. Each blank serum was tested for endogenous interference using previously described LC-MS/MS method.
Sample preparation
Linearity, low limit of quantification and low limit of detection. The calibration curves were prepared by assaying standard serum samples as described above, and each level sample was prepared and assayed in duplicate on three separate days. The calibration curves were plotted by the peak area ratio (analyte/IS, y) vs the analyte concentration (x) using a 1/x2 weighted linear least-squares regression model. The lower limit of quantification (LLOQ) was defined as the lowest concentration on the calibration curve that produced a signal-to-noise (S/N) ratio of at least 10 with an acceptable accuracy (recovery within 80–120%) and precision (RSD, below 20%). The lower limit of detection (LLOD) was defined as the lowest concentration that produced a peak distinguishable from background noise (S/N ratio of 3:1) for the analytes.
In order to improve extraction efficiency, a two-step liquid–liquid extraction (LLE) with water-saturated n-butyl alcohol was applied.
Precision and accuracy. The precision and accuracy were evaluated by analyzing six replicates of QC samples at low, medium
Table 1. The linearity and lower limits of quantitation and detection (LLOQ and LLOD) of the assay for the 11 analytes Analyte
Ginsenoside Rg1 Ginsenoside Re Ginsenoside Rf Ginsenoside Rg2 Ginsenoside Rb1 Ginsenoside Rd Ginsenoside Rc Ophiopogonin D Schisandrin Schisandrol B Schizandrin B
Linear range (ng/mL)
1.190–1487.5 0.165–619.1 0.109–408.8 0.083–309.4 0.280–1050.0 0.145–543.8 0.152–570.0 0.044–54.4 0.229–572.3 0.260–325.0 0.144–349.9
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Regression equation
y= y= y= y= y= y= y= y= y= y= y=
0.0293x + 4.5274 0.0577x + 2.8364 0.1101x + 4.2256 0.1133x + 1.1734 0.0476x + 4.5820 0.0565x + 2.2027 0.0687x + 3.2216 0.1286x + 0.1807 0.1280x + 0.0027 0.0764x + 0.6209 0.0986x +0.1028
r2
0.9960 0.9950 0.9929 0.9986 0.9984 0.9940 0.9974 0.9997 0.9956 0.9994 0.9959
LLOQ Concentration (ng/mL)
Accuracy (recovery, %)
Precision (RSD%, n = 5)
1.190 0.165 0.109 0.083 0.280 0.145 0.152 0.044 0.229 0.260 0.144
108.0 114.2 108.4 100.9 97.9 110.6 105.9 94.1 90.5 108.5 101.4
4.9 7.3 3.1 1.4 9.6 9.3 8.1 8.4 9.6 13.1 12.3
Copyright © 2014 John Wiley & Sons, Ltd.
LLOD (ng/mL)
0.595 0.099 0.036 0.028 0.093 0.048 0.051 0.022 0.152 0.130 0.072
Biomed. Chromatogr. (2014)
Simultaneous quantification of eleven herbal constituents in rat serum Table 2. Precision and accuracy for the assay of the 11 analytes in rat serum (n = 6) Analyte
Concentration spiked (ng/mL)
Ginsenoside Rg1
Ginsenoside Re
Ginsenoside Rf
Ginsenoside Rg2
Ginsenoside Rb1
Ginsenoside Rd
Ginsenoside Rc
Ophiopogonin D
Schisandrin
Schisandrol B
Schizandrin B
2.98 449.82 899.64 0.40 199.98 399.96 0.30 149.98 299.97 0.20 149.82 299.64 2.50 499.52 999.04 0.40 249.98 499.96 0.40 249.89 499.78 0.08 2.50 5.00 0.43 194.12 388.25 0.60 110.24 219.96 1.00 149.90 299.81
Intra-day Concentration measured (ng/mL) 2.74 507.40 859.77 0.41 213.95 367.02 0.32 158.89 309.96 0.20 159.13 320.90 2.65 521.15 1027.07 0.45 275.10 529.50 0.42 272.37 519.46 0.08 2.46 5.26 0.44 202.14 363.79 0.50 123.42 225.98 0.85 136.91 293.70
± 0.17 ± 21.20 ± 41.55 ± 0.03 ± 13.07 ± 21.65 ± 0.03 ± 10.81 ± 21.63 ± 0.02 ± 7.93 ± 15.46 ± 0.09 ± 21.24 ± 72.13 ± 0.03 ± 11.64 ± 14.29 ± 0.04 ± 16.02 ± 34.09 ± 0.01 ± 0.15 ± 0.36 ± 0.01 ± 16.71 ± 14.32 ± 0.03 ± 4.58 ± 5.81 ± 0.05 ± 11.92 ± 27.65
Inter-day
Precision (%)
Accuracy (%)
6.1 4.2 4.8 7.9 6.1 5.9 8.2 6.8 7.0 7.6 5.0 4.8 3.4 4.1 7.0 6.7 4.2 2.7 10.2 5.9 6.6 13.2 6.2 6.8 1.9 8.3 3.9 7.3 3.7 2.6 5.5 8.7 9.4
92.2 112.8 95.6 103.5 107.0 91.8 105.8 105.9 103.3 103.3 106.2 107.1 105.9 104.3 102.8 112.5 110.0 105.9 104.2 109.0 103.9 96.9 98.4 105.3 102.2 104.1 93.7 85.9 112.0 102.7 85.1 91.3 98.0
Concentration measured (ng/mL) 3.03 ± 475.50 ± 911.39 ± 0.39 ± 208.70 ± 414.35 ± 0.31 ± 156.95 ± 310.87 ± 0.20 ± 155.94 ± 317.08 ± 2.59 ± 523.03 ± 1032.85 ± 0.43 ± 260.31 ± 510.11 ± 0.41 ± 256.66 ± 501.67 ± 0.08 ± 2.51 ± 5.23 ± 0.48 ± 201.03 ± 377.54 ± 0.59 ± 117.49 ± 217.81 ± 0.97 ± 151.25 ± 285.23 ±
Precision (%)
Accuracy (%)
9.7 3.3 4.5 12.3 7.3 6.6 11.1 3.2 5.2 6.6 3.0 3.7 8.9 5.2 2.8 14.1 4.5 4.7 11.9 4.7 2.6 8.9 5.7 6.6 9.1 6.2 7.4 9.9 8.6 4.4 9.7 8.9 7.7
101.7 105.7 101.3 99.4 104.4 103.6 102.9 104.6 103.6 99.1 104.1 105.8 103.9 104.7 103.4 106.6 104.1 102.0 103.1 102.7 100.4 98.4 100.3 104.7 110.6 103.6 97.2 98.7 106.6 99.0 97.1 100.9 95.1
0.29 15.83 41.19 0.05 15.15 27.20 0.03 5.02 16.31 0.01 4.63 11.77 0.23 27.34 29.07 0.06 11.72 23.88 0.05 11.98 12.97 0.01 0.14 0.35 0.04 12.50 27.83 0.06 10.09 9.61 0.09 13.49 21.90
Table 3. The extraction recovery and matrix effect of 11 analytes at low, medium and high conentration levels in rat serum (mean ± SD, n = 6) Analyte
Extraction recovery (%) Low
Ginsenoside Rg1 Ginsenoside Re Ginsenoside Rf Ginsenoside Rg2 Ginsenoside Rb1 Ginsenoside Rd Ginsenoside Rc Ophiopogonin D Schisandrin Schisandrol B Schizandrin B
Biomed. Chromatogr. (2014)
87.2 87.3 81.5 83.0 81.8 85.6 83.4 84.8 79.8 83.3 85.0
± ± ± ± ± ± ± ± ± ± ±
11.5 6.4 7.2 5.7 10.2 9.0 10.9 7.6 3.6 4.8 10.8
Medium 87.6 86.5 89.3 90.4 84.1 83.7 86.0 79.5 84.3 82.8 88.1
± ± ± ± ± ± ± ± ± ± ±
2.7 3.5 1.4 1.9 4.3 1.5 4.0 7.2 4.4 4.1 9.6
Matrix effect (%) High 92.5 89.9 90.1 87.6 89.7 85.7 88.9 75.8 87.0 92.7 85.1
± ± ± ± ± ± ± ± ± ± ±
5.8 6.5 9.7 7.6 6.8 6.4 9.9 9.9 8.7 9.3 3.3
Low 85.6 ± 86.3 ± 87.2 ± 85.7 ± 101.8 ± 83.8 ± 90.6 ± 45.8 ± 89.3 ± 86.9 ± 85.6 ±
Copyright © 2014 John Wiley & Sons, Ltd.
9.7 9.3 5.2 5.7 11.5 4.6 13.1 2.7 6.3 6.2 9.1
Medium 88.0 ± 88.2 ± 85.6 ± 87.6 ± 97.0 ± 82.0 ± 91.1 ± 48.0 ± 99.3 ± 101.0 ± 102.0 ±
8.2 9.1 6.8 8.5 8.6 6.5 5.3 8.3 8.2 7.7 12.1
High 86.0 84.2 82.5 79.1 89.4 81.4 88.8 41.3 89.9 85.6 96.4
± 9.4 ± 10.4 ± 9.0 ± 7.1 ± 8.2 ± 6.9 ± 10.4 ± 5.4 ± 10.3 ± 7.7 ± 8.7
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S. Zhan et al. and high concentration levels as described above. The intra-day precision and accuracy of the assays were determined in the same day by analyzing six replicates at each concentration level. The inter-day precision and accuracy were evaluated on six consecutive days at each concentration level. The precision values expressed as RSD were required to be 300 μL of serum was obtained and immediately stored at 20°C until analysis.
Results and discussion LC-MS/MS method
Concentration (ng/mL)
In order to obtain optimum MS conditions, the standard solutions of the analytes and IS were infused into the mass spectrometer separately, and the optimized mass parameters such as declustering potential and collision energy were obtained. The mass transitions of precursor to product ion at m/z 799.4/637.5 for ginsenoside Rg1, 945.6/637.4 for Re, 799.3/475.2 for Rf, 783.8/ 475.3 for Rg2, 1108.0/178.8 for Rb1, 945.5/621.6 for Rd, 1077.8/ 191.0 for Rc, 853.4/721.4 for ophiopogonin D, 432.8/384.0 for schisandrin, 399.7/369.1 for schisandrol B, 402.3/301.2 for schizandrin B and 779.2/650.0 for IS were chosen for the quantification studies. The product ion mass spectrum of the analytes iss shown in Fig. 1. To evaluate the sensitivity of the method, the 10000.0
Rg1 Re Rf Rg2
1000.0
1000.0
100.0
MS ion source parameters, such as ion spray voltage, turbo spray temperature, collision gas, curtain gas, nebulizer gas (gas 1) and auxiliary gas (gas 2), were further investigated to obtain the optimal results for quantification. The chromatographic conditions, especially the additive and the gradient of mobile phase, could affect the separation and the peak shape of ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B. It was found that the optimal separations and peak responses for the analytes and IS were obtained by adding 0.02% acetic acid in mobile phase with an appropriate gradient elution. Sample preparation In this study, the one-step LLE with 1.5 mL extract solution was first tested to extract the analytes and IS, and the results demonstrated that some analytes, such as ophiopogonin D and schizandrin B, exhibited relatively low extraction recovery (60.6 and 38.3%, respectively). Therefore, the method of two-step LLE was used to improve extraction efficiency in order to guarantee the extraction recovery of all analytes >75%. Method validation Specificity. Under the above LC-MS/MS conditions, the representative chromatograms of a blank serum sample, a blank serum sample spiked with 11 analytes and IS, and a serum sample from a rat at 0.08 h after intravenous administration of Shengmai injection are shown in Fig. 2. The retention times of ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B, schizandrin B and IS were 10.54, 10.60, 13.38, 14.26, 13.70, 15.25, 14.08, 20.88, 18.29, 19.83, 29.59 and 13.14 min, respectively. No significant interferences from endogenous substances in the blank serum sample were observed at the retention times of analytes and IS. Good specificity and selectivity were thus observed for the assay method. Linearity, LLOQ and LLOD. The linear ranges, regression equations, correlation coefficients, LLOQ and LLOD of the assay are shown in Table 1. Good linearity was obtained in the validated concentration range (r 2 ≥0.9929). The accuracy (recovery, %) and precision (RSD, %) of LLOQs in the method were 90.5–114.2 and
