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MS and its application to a pharmacokinetic study after oral administration of Cerebralcare Granule
Journal of Chromatography B, 1017 (2016) 28–35
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of seven alkaloids in rat plasma by UFLC-MS/MS and its application to a pharmacokinetic study after oral administration of Cerebralcare Granule Li Xiaowen a , Tong Ling b , Li Yunfei b , Sun Guoxiang a,∗ , Yang Dailin c , Sun Herry b a
School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China Tasly R&D Institute, Tianjin Tasly Group Co., Ltd., Tianjin 300402, China c China Pharmaceutical University, Nanjing 210009, China b
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 20 January 2016 Accepted 30 January 2016 Available online 26 February 2016 Keywords: Cerebralcare Granule Alkaloids UFLC-MS/MS Pharmacokinetics
a b s t r a c t An ultra fast liquid chromatography-tandem mass sepectrometry (UFLC-MS/MS) method was developed for simultaneous determination of seven active alkaloid components (tetrahydropalmatine, corydaline, ␣-allocryptopine, tetrahydroberberine, tetrahydrocoptisine, tetrahydrocolumbamine and dehydrocorydaline) in rat plasma after oral administration of Cerebralcare Granule. Plasma samples were pretreated by protein precipitation with acetronitrile containing the internal standard diazepam. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (100 × 2.1 mm, 2.6 m) with gradient elution using mobile phase consisting of acetonitrile −0.1% formic acid in water at a flow rate of 0.3 mL/min. The detection was performed on an electrospray ionization triple quadrupole tandem mass spectrometer using multiple reaction monitoring (MRM) with positive ionization mode. The established method was fully validated and proved to be sensitive and specific with lower limits of quantification (LLOQs) all less than 0.0265 ng/mL in rat plasma. Good linearities of seven alkaloids were obtained in respective concentration ranges (r > 0.9923). The intra- and inter-day precisions were below of 15% for all the seven alkaloids in terms of relative standard deviation (RSD), and the accuracies were ranged from −2.7% to 8.3% in terms of relative error (RE). Extraction recovery, matrix effect and stability were within the required limits in rat plasma. The validated method was successfully applied to investigate the pharmacokinetics of the seven alkaloids in rat plasma after oral administration of Cerebralcare Granule (CG). © 2016 Elsevier B.V. All rights reserved.
1. Introduction Traditional Chinese medicine (TCM) and their formulations have been used in Asian countries for thousands of years and played an indispensable role in prevention and treatment of diseases, especially for complicated and chronic condition [1,2]. Most of TCM formulations are taken orally, and some components persist at detectable levels after digestion and absorption. Therefore, investigation of the pharmacokinetic properties of those components in vivo can gain more in-depth insights into the active components and the mechanisms for TCM formulation, especially for the drugs working in the brain system [3,4]. Cerebralcare Granule (CG) is a widely used botanical drug for the treatment of cerebrovascular diseases including stroke,
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (S. Guoxiang). http://dx.doi.org/10.1016/j.jchromb.2016.01.062 1570-0232/© 2016 Elsevier B.V. All rights reserved.
headache and dizziness (CFDA Approved No.1002004736603642). Pharmacological studies indicated that the CG improves brain microcirculation, increases cerebral blood flow and eases vascular spasm, especially in the treatment of chronic cerebral insufficiency effects [5–8]. CG is developed from the TCM “Siwutang” and composed of eleven herbs (i.e., Radix angelica sinensis, Rhizoma Chuanxiong, Radix paeoniae alba, Rhizoma corydalis yanhusuo, etc.). Our previous work has shown that CG contains many components such as alkaloids, triterpenoids, phenolic acids and anthraquinones [9,10]. R. corydalis, a key constituent herb in CG, shows therapeutic effects of promoting blood circulation and alleviating pains [11–13]. Alkaloids in R. corydalis had been identified as the active components. Among them, tetrahydropalmatine (THP) has been used as a chemical marker for the quality control of R. corydalis in Chinese pharmacopoeia [14]. Meanwhile, alkaloids, such as tetrahydropalmatine (THP), corydaline (CDL), ␣-allocryptopine (␣-ACP), tetrahydroberberine (THB), tetrahydrocoptisine (THC), tetrahydrocolumbamine (THCB) and dehydrocorydaline (DDCL),
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Fig. 1. The product ion fragmentation modes and mass spectra of [M+H]+ of tetrahydropalmatine (A), corydaline (B), ␣-allocryptopine (C), tetrahydroberberine (D), tetrahydrocoptisine (E), tetrahydrocolumbamine (F), dehydrocorydaline (G) and diazepam, IS (H).
possess various biological activities such as antimicrobial, antiinflammatory, anti-diabetic and antioxidant ect [15–20]. Our previous work showed that all the seven alkaloids existed in the CG were absorbed into the blood and could be detected after oral administration. Therefore, it is of great value to develop an efficient and reliable method for simultaneous determination of all the seven compounds in biological matrix and to study their pharmacokinetics properties in order to advance the therapeutic application of CG. Various analytical methods based on liquid chromatography technology have been described to determine only one or two of the seven alkaloids mentioned above in biological matrices. Such as,
Ma et al., developed a LC-MS method to determine THP, protopine and palatine in rat plasma [21]. Zhang et al., developed a UFLCMS/MS method to quantify THP and scutellarin in rat plasma [22]. However, none of these analytical methods is able to simultaneously determination all the seven compounds in biological samples. In this study, a sensitive and reliable UFLC-MS/MS method using electrospray ionization (ESI) source was developed and validated to simultaneously determine the concentration of THP, CDL, ␣-ACP, THB, THC, THCB and DDCL in rat plasma for the first time. And the method was successfully applied to a pharmacokinetic study in rats following oral administration of CG.
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Fig. 2. Typical chromatograms of blank plasma (A); blank plasma sample spiked with seven analytes at LLOQ and IS (B); 2.0 h plasma sample after oral administration of Cerebralcare Granule(C); (1): tetrahydropalmatine, (2): corydaline, (3): ␣-allocryptopine, (4): tetrahydroberberine, (5): tetrahydrocoptisine, (6): tetrahydrocolumbamine, (7): dehydrocorydaline and (8): IS.
L. Xiaowen et al. / J. Chromatogr. B 1017 (2016) 28–35 Table 1 Optimized MRM parameters, collision energy (CE), declustering potential (DP), entrance potential (EP) and cell exit potential (CXP) of seven analytes and IS. Analyte
Precursor ion (m/z)
Product ion (m/z)
CE (eV)
DP (V)
EP (V)
CXP (V)
THP CDL ␣- ACP THB THC THCB DDCL IS
356.3 370.1 370.1 340.1 324.1 342.2 366.2 285.1
192.2 192.2 290.1 176.1 176.1 178.1 350.2 257.1
36 41 37 40 50 40 41 30
92 116 32 24 105 39 24 50
10 10 10 10 10 10 10 10
35 12 15 18 8 22 12 13
2. Experiment 2.1. Materials and reagents Cerebralcare Granule (batch NO. 140601) was supplied by Tianjin Tasly Pharmaceutical Co. Ltd. (Tianjin, China). The reference standards of THP, CDL, ␣-ACP, THB, THC, THCB, DDCL and diazepam (internal standards, IS, structure as Fig. 1) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile of HPLC grade were purchased from Merck (Darmstadt, Germany). The purity of all standards were above 98.0% and suitable for LC-MS/MS analysis. Formic acid of HPLC grade was obtained from Tedia Company Inc. (Beijing, China). Deionized water was prepared from a Milli-Q water purification system (Millipore, Billerica, USA). Other reagents were of analytical grade. 2.2. Apparatus and UFLC-MS/MS condition All sample analyses were performed on an ESI triple quadrupole mass spectrometer, model Triple Quad 5500 (AB SCIEX, Foster City, CA), which was controlled by Analyst® 1.6.1 software. The mass spectrometer was directly coupled with a UFLC system from Shimadzu Corporation (Shimadzu, Kyoto, Japan), which consisted of a SIL-30AC auto-sampler, a CTO-20A column heater, a CBM-20A Lite controller, a DGU-20A5 degasser, and two LC-30AD pumps. Chromatographic separation was achieved on an Phenomenex Kinetex C18 column (100 × 2.1 mm, 2.6 m). The mobile phase was a mixture of 0.1% formic acid ultrapure water (A) and acetonitrile solution (B) using a gradient elution system at a flow rate of 0.3 mL/min. It was run at 5–50% B over 0–1 min, 50–90% B over 1–3.5 min, and the composition was maintained at 90% B for 0.5 min and then returned to initial condition. All analytes were eluted rapidly within 5 min. The injection volume was 5 L and the column temperature was set at 45 ◦ C. Mass spectrometric detection was operated in positive ionization with multiple reaction monitoring (MRM) mode. The optimized ionspray voltage and source temperature were maintained at 5500 V and 550 ◦ C. High-purity nitrogen was used as nebulizing (50 psi), auxiliary (50 psi) and curtain gas (20 psi). The optimized quantitative parameters are listed in Table 1. 2.3. Preparation of standard and quality control samples Stock solutions at 1.0 mg/mL for each standard were prepared in methanol. A series of working solutions of these analytes were obtained by diluting mixed standard solution with methanol at appropriate concentrations. A quantity of diazepam was dissolved in methanol to produce the IS solution with a concentration of 50.0 ng/mL. All solutions were stored at −20 ◦ C and brought to room temperature (25 ◦ C) before use.
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Table 2 The regression equations, linear range and LLOQs of the seven analytes. Analyte
Range (ng/mL)
Calibration curves
Correlation coefficient (r)
LLOQ (ng/mL)
THP CDL ␣- ACP THB THC THCB DDCL
0.163–150 0.125–100 0.0263–25.0 0.0263–25.0 0.125–100 0.125–100 0.0625–50.0
y = 13.52x + 6.61 y = 8.79x + 4.37 y = 2.34x + 0.48 y = 22.35x + 0.08 y = 3.02x + 1.85 y = 0.43x + 0.14 y = 25.16x + 12.23
0.9964 0.9982 0.9966 0.9987 0.9923 0.9933 0.9992
0.163 0.125 0.0263 0.0263 0.125 0.125 0.0625
Calibration standards were prepared by spiking appropriate amount of the standard solutions in blank plasma (100 L) to yield final concentrations of 0.163–150 ng/mL for THP, 0.125–100 ng/mL for CDL, 0.0263–25.0 ng/mL for ␣-ACP, 0.0263–25.0 ng/mL for THB, 0.125–100 ng/mL for THC, 0.125–100 ng/mL for THCB and 0.0625–50.0 ng/mL for DDCL. Low, medium and high concentration QCs were prepared in the same way as the calibration standards (1.63, 32.5, 120 ng/mL for THP; 1.25, 25.0, 80.0 ng/mL for CDL, THC and THCB; 0.263, 6.25, 20.0 ng/mL for ␣-ACP and THB; 0.625, 12.5, 40.0 ng/mL for DDCL). QCs at concentrations of 50.0 ng/mL for IS were also prepared.
2.4. Sample preparations All samples were thawed at room temperature before analysis. To a 100 L aliquot of plasma samples, 20 L IS (diazepam, 50.0 ng/mL) and 10 L 1.0 mol/L hydrochloric acid were spiked in a 2 mL Eppendorf tube, and then vortexed for 1 min. The mixture was extracted with 2 mL ethyl acetate by vertex-mixing for 5 min and centrifugated at 12,000 rpm for 5 min. The supernatant was transferred to another vial and evaporated to dryness in vacuo at 40 ◦ C. The residue was reconstituted in 100 L mobile phase, vortex-mixed for 1 min and centrifuged at 12,000 rpm for 3 min. Finally, 5 L of the supernatant was injected into the UFLC-MS/MS system for analysis.
2.5. Method validation This method was fully validated in accordance with US-FDA Bioanalytical Method Validation Guidance and European Medicines Agency Guideline on Bioanalytical Method Validation [23,24]. by determining selectivity, linearity, precision and accuracy, recovery, matrix effect and stability. Selectivity was assessed by comparing chromatograms of six different batches of blank rat plasma with the corresponding spiked rat plasma. Linearity was assessed by weighted (1/x2 ) least-squares analysis of seven different calibration curves. Intra- and inter-day precision (the relative standard deviation, RSD) and accuracy (the relative error, RE) were determined by analysis of low, medium, and high QC samples (n = 6) on 3 different days. The stability were assessed of low, medium and high QC samples (n = 6) in three complete freeze/thaw cycles (−40–25 ◦ C), long-term sample storage (−40 ◦ C for 30 days), and bench-top (25 ◦ C for 4 h). The ready-toinjection stability of extracted samples in the autosampler rack at 4 ◦ C for 24 h was also evaluated. The matrix effect was investigated by comparing the peak areas of analytes in the post-extraction spiked blank plasma at low, medium, and high concentrations with those of the corresponding standard solutions. The extraction recovery was determined by comparing the mean peak areas of six extracted samples at low, medium, and high QC concentrations with the mean peak areas of spike-after-extraction samples.
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Table 3 precision, accuracy, recovery and matrix effect for seven analytes in rat plasma (n = 6). Analyte
Spiked (ng/mL)
THP
1.63 32.5 120
Inter-day RSD (%) 5.4 2.3 4.5
Intra-day RSD (%) 1.8 4.9 2.6
Accuracy RE (%) 0.44 7.3 6.0
Recovery (%) 89.5 ± 2.5 96.4 ± 2.8 85.8 ± 4.9
Matrix effect (%) 101.8 ± 6.5 114.3 ± 2.3 85.1 ± 0.5
CDL
1.25 25.0 80.0
5.9 9.2 5.3
3.0 5.2 3.8
0.73 2.4 7.8
89.5 ± 7.9 95.3 ± 1.9 101.3 ± 4.7
102.1 ± 5.7 105.2 ± 3.5 94.8 ± 5.5
␣ACP
0.263 6.25 20.0
5.8 4.8 6.0
4.0 9.8 5.2
−0.23 3.6 8.3
73.3 ± 3.3 67.4 ± 6.4 84.2 ± 5.4
93.8 ± 5.8 92.7 ± 3.5 86.6 ± 3.2
THB
0.263 6.25 20.0
9.7 4.5 11
7.7 1.0 1.5
0.19 3.1 −1.2
87.2 ± 4.6 79.5 ± 3.3 89.9 ± 2.3
109.5 ± 8.0 106.7 ± 2.1 97.2 ± 1.9
THC
1.25 25.0 80.0
3.1 8.7 5.6
5.3 6.9 4.2
0.12 6.1 4.4
85.4 ± 2.6 89.9 ± 3.3 87.3 ± 2.2
86.3 ± 1.4 107.7 ± 2.0 99.1 ± 3.1
THCB
1.25 25.0 80.0
2.9 4.1 4.9
8.9 4.4 3.1
−0.51 5.2 3.7
68.8 ± 4.4 78.6 ± 0.4 82.9 ± 5.2
94.4 ± 6.5 99.3 ± 6.6 101.0 ± 0.6
DDCL
0.625 12.5 40.0
12 2.6 6.9
9.3 7.7 4.1
0.80 3.0 −2.7
83.1 ± 3.3 113.1 ± 3.5 109.2 ± 1.3
93.7 ± 8.4 113.1 ± 8.7 109.2 ± 3.2
Table 4 The stability of seven analytes in rat plasma under different storage condition. (n = 6). 25 ◦ C for 4 h
Frozen for 30 days
Three-freeze-thraw cysles
4 ◦ C for 24 h
RSD (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
−7.1 3.1 2.8
10 4.5 3.1
0.51 −0.10 2.4
10.1 9.9 0.90
14 0.14 5.6
1.3 0.24 5.7
3.1 1.2 1.0
−1.5 0.26 7.4
9.1 0.92 8.2
0.31 6.0 6.9
5.6 2.2 9.1
−3.0 −9.2 1.6
5.2 7.6 3.6
1.5 2.9 5.3
9.9 9.1 2.4
−2.3 −3.4 0.8
8.5 8.1 2.3
−5.3 −0.55 0.77
3.8 9.4 6.7
−3.3 4.0 1.7
2.4 3.5 1.5
−2.4 4.6 −0.54
Analyte
Concentration (ng/mL)
RSD (%)
RE (%)
THP
1.63 32.5 120
6.6 3.0 2.2
−1.9 3.6 5.4
7.1 3.7 2.8
−1.9 5.2 2.4
7.6 4.4 1.8
CDL
1.25 25.0 80.0
4.9 8.0 3.3
−1.3 3.5 1.1
5.1 11 5.7
1.2 5.5 1.0
6.7 5.2 2.0
2.6 −3.1 6.0
␣ACP
0.265 6.25 20.0
2.0 6.3 3.3
−6.4 10 6.3
2.3 1.2 4.0
−3.9 1.5 5.7
7.3 0.72 5.1
THB
0.27 6.25 20.0
1.9 1.0 4.9
3.5 2.2 11
2.0 1.3 4.1
3.5 1.0 12
10.2 0.87 6.4
THC
1.25 25.0 80.0
8.5 5.6 1.9
2.9 0.70 −6.9
1.9 11 0.63
0.90 −6.7 4.3
10.2 7.1 1.2
THCB
1.25 25.0 80.0
2.4 1.4 3.7
4.4 −3.3 2.4
5.2 7.9 8.0
−4.0 −5.2 5.4
DDCL
0.625 12.5 40.0
3.6 7.7 8.7
−2.8 2.6 −1.9
2.8 3.4 3.7
−3.5 3.7 −1.9
RE (%)
Table 5 Pharmacokinetic parameters of analytes after oral administration of CG (8.0 g/kg) to rats (n = 6). Parameters
T1/2 (h)
THP CDL ␣-ACP THB THC THCB DDCL
6.4 6.8 11.5 14.1 12.2 8.8 5.9
± ± ± ± ± ± ±
Cmax (ng/mL) 3.2 3.8 6.6 2.9 6.4 3.9 2.7
90.0 59.0 24.9 20.1 73.9 63.5 34.5
± ± ± ± ± ± ±
31.6 36.4 13.1 12.2 33.4 28.5 19.2
Tmax (h) 0.9 0.8 0.8 0.5 0.5 0.5 0.8
± ± ± ± ± ± ±
0.3 0.2 0.3 0.2 0.3 0.4 0.1
AUC0−t (ng h/mL) 836.7 195.8 209.5 171.7 995.7 810.7 323.5
2.6. Pharmacokinetic study Male Sprague-Dawley rats (200 ± 20 g) were housed under a humidity controlled room, maintained at 22–25 ◦ C, with a 12 h
± ± ± ± ± ± ±
291.3 106.0 83.2 76.2 377.4 256.6 125.6
AUC0−∞ (ngh/mL) 936.8 229.8 228.9 196.1 1002.7 884.2 361.7
± ± ± ± ± ± ±
319.9 124.3 95.4 85.3 402.6 273.2 134.4
MRT0−24 (h)
MRT0−∞ (h)
10.52 ± 2.15 9.43 ± 1.73 8.31 ± 2.87 8.66 ± 1.28 7.25 ± 1.45 8.27 ± 2.78 9.22 ± 1.43
11.81 10.59 9.08 9.18 8.75 9.94 10.49
± ± ± ± ± ± ±
2.69 3.08 1.44 2.50 2.09 3.53 1.64
light–dark cycle. Prior to experiment treatment, all animals were fasted overnight with free access to water. All procedures involving animals were in accordance with the Regulations of Experimental
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Fig. 3. Mean plasma concentration-time profiles of tetrahydropalmatine (A), corydaline (B), ␣-allocryptopine (C), tetrahydroberberine (D), tetrahydrocoptisine (E), tetrahydrocolumbamine (F), dehydrocorydaline (G) after oral administration of CG at 8.0 g/kg to rats (n = 6).
Animal Administration issued by the State Committee of Science and Technology of People’s Republic of China. Six rats were intragastric administered with 8.0 g/kg CG (equivalent to 0.356 mg/kg of THP, 0.874 mg/kg of CDL, 0.516 mg/kg of ␣-ACP, 0.131 mg/kg of THB, 0.266 mg/kg of THC, 0.638 mg/kg of THCB and 1.74 mg/kg of DDCL) suspended in 0.5% CM C-Na (w/v). Serial blood samples (300 L) were obtained at 0 (pre-dose), 0.08, 0.25, 0.5, 0.75, 1, 1.5,
2, 3, 5, 8, 12, 24 and 30 h by puncture of the retro-orbital sinus after oral administration. All samples were placed into heparinized tubes. After centrifugation at 4500 rpm for 10 min, plasma was collected and frozen at −40 ◦ C until analysis. The pharmacokinetic parameters were calculated by DAS 2.0 pharmacokinetic program (Chinese Pharmacological Society) using non-compartmental analysis.
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3. Results and discussion 3.1. Mass spectrometry According to the direct full-scan ESI mass spectra, the ion intensities of all the seven alkaloids and IS were observed higher in the positive mode than the negative mode. The MS/MS product ion spectras of the analytes and IS are shown in Fig. 1. The presence of a bit of formic acid buffer in the mobile phase improved MS detection with good response in the positive ionization mode. Considering the factors above mentioned the optimal mobile phase was consisting of acetonitrile and 0.1% formic acid-water eluted in a simple gradient elution program with column temperature keeping at 45 ◦ C. Under the selected conditions, all the analytes were eluted rapidly within 5.0 min on a C18 column without interference from other components in rat plasma. 3.2. Optimization of extraction conditions To achieve satisfactory recoveries for each analyte, liquid–liquid extraction (LLE), solid phase extraction (SPE) and protein precipitation (PPT) were investigated respectively. Different extraction solvents (ethyl acetate, acetone, diethyl ether, and n-butyl alcohol) and their combination were tested, but no satisfactory recovery was obtained for all the alkaloids. SPE with Waters Oasis HLB cartridges (Milford, MA, USA) was costly and time-costing, not suitable for multi-sample analysis. Finally, protein precipitation (PPT) using acetonitrile was tried and found to give good and consistent recovery for all the seven alkaloids and IS. 3.3. Method validation 3.3.1. Selectivity Typical chromatograms obtained from a blank, a spiked plasma sample with the analytes and IS, and a plasma sample after an oral dose are presented in Fig. 2. The retention time for THP, CDL, ␣-ACP, THB, THC, THCB, DDCL and IS were 2.67, 2.70, 2.67, 2.68, 2.65, 2.71, 2.67, and 3.24 min, respectively. No interference of endogenous peaks exhibited in drug-free plasma indicated the high selectivity of LC–MS/MS. 3.3.2. Linearity and lower limits of quantification Regression equation, correlation coefficients, linear ranges and LLOQ of the seven analytes are shown in Table 2. All the seven components exhibited good linearity within selected concentration ranges with correlation coefficients (r) between 0.9923 and 0.9992. Due to the different structures of these alkaloids, they have different mass ionization capacity and fragmentation modes, which lead to the difference in LLOQ. The LLOQ of THP, CDL, ␣-ACP, THB, THC, THCB and DDCL were 0.163, 0.125, 0.0263, 0.0263, 0.125, 0.125, 0.0625 ng/mL, respectively. The limits were sufficient for pharmacokinetic studies. 3.3.3. Precision, accuracy and stability The precision and accuracy data for the seven analytes from the QC samples are presented in Table 3. They were evaluated by RSD and RE values, respectively. The intra- and inter- day precisions were below of 15% for the seven analytes in terms of RSD, and the accuracies were ranged from −2.7% to 8.3% in terms of RE. All the assay values were within the acceptable criteria, which indicated the overall reproducibility of the method. 3.3.4. Matrix effect and extraction recovery As shown in Table 3, the extraction recoveries of all the seven analytes were in the range 67.4–113.1%, with RSD less than 8.5%.
The mean extraction recovery of the IS was 87.2 ± 3.6%. The average matrix effect values for the seven analytes and IS were within 85.1–114.3%. No significant matrix effect for all the analytes was observed, which indicated that no co-eluting substance could influence the ionization of the analytes.
3.3.5. Stablility Six replicates of QC samples at three concentrations were used for stability evaluation of the seven analytes stored under several conditions. The results of short-term stability, long-term stability, freeze-thaw stability and auto-sampler stability are summarized in Table 4. It shows that the deviations (RE) between measured values and nominal values were all within ±13.7%, demonstrating good stability under these storage conditions.
3.3.6. Pharmacokinetic study The validated UFLC–MS/MS method was successfully applied to a pharmacokinetic study of THP, CDL, ␣-ACP, THB, THC, THCB and DDCL in rat after a single oral administration of CG at dose of 8.0 g/kg. Mean plasma concentration-time profiles of the seven analytes are illustrates in Fig. 3, and the main pharmacokinetic parameters assessed by non compartmental analysis are listed in Table 5. Following oral administration of CG, double peaks were observed in both individual and mean plasma concentration profiles of THP, CDL, ␣-ACP, THB, THC, THCB and DDCL. The first peaks of the seven alkaloids emerged at approximately 0.5–0.9 h, respectively, which suggested that both of them could be absorbed in the stomach or upper intestinal lumen and mucosa. While the second peaks emerged at about 4–8 h and those peaks concentration were lower than the first peaks. The result indicated that all of the seven alkaloids had rapid absorption and slow elimination. Meanwhile, Ma et al. [21] and Zhang et al. [22] have reported two peaks in the plasma concentration curve of THP, ␣-ACP and THB, and attributed the phenomenon to distribution, re-absorption and enterhepatic circulation. However, by comparing with the previous literature, the T1/2 and MRT0−∞ of THP, THB and THC were a litter shorter, and the ␣-ACP was longer than reported results [18]. The results might be presumably due to the different physiochemical properties of each compound or the pharmacokinetic interaction of the prescribed chemical components. Overall, the information described in the present study might be helpful for further studies on the PK evaluation of CG, and beneficial for application of this formula in clinical therapy.
4. Conclusion In the present study, a reliable and sensitive UFLC-MS/MS method was first time developed for the simultaneous quantification of 7 alkaloids in rat plasma samples following oral administration of Cerebralcare Granule. This method offers great simplicity and efficiency for high sample throughput in bioanalysis owing to a short analysis time of 5.0 min per sample and a relatively simple sample preparation procedure with one-step protein precipitation. The pharmacokinetic characteristics of 7 alkaloids had rapid absorption and slow elimination, and they all exhibited double peaks after oral administration of CG. These works could provide more in-depth insights into these active alkaloids working in vivo and would be helpful for further revealing the pharmacology and mechanisms of CG. However, since species difference usually existed in the metabolism between the rat and human, further study in human would be needed to carry out for better understanding of this formulation.
L. Xiaowen et al. / J. Chromatogr. B 1017 (2016) 28–35
Conflicts of interest The authors declare no conflict of interest. Acknowledgment This work is supported by National Key Special Project of Science and Technology for Innovation Drug of China (Grant No. 2013ZX09402202). References [1] WHO/EDM/TRM, WHO traditional medicine strategy 2002–2005, Geneva, 2002. [2] W.Y. Jiang, Therapeutic wisdom in traditional Chinese medicine: a perspective from modern science, Trends Pharmacol. Sci. 26 (2005) 558–563. [3] G. Cao, H. Cai, Y. Zhang, X.D. Cong, C.R. Zhang, B.C. Cai, Identification of metabolites of crude and processed Fructus Corni in rats by microdialysis sampling coupled with electrospray ionization linear quadrupole ion trap mass spectrometry, J. Pharm. Biomed. Anal. 56 (2011) 118–125. [4] T. Lu, J.L. Yang, X.M. Cao, P. Chen, F.F. Du, Y. Sun, Plasma and urinary tanshinol from Salvia miltiorrhiza (Danshen) can be used as pharmacokinetic markers for cardiotonic pills, a cardiovascular herbal medicine, Drug Metab. Dispos. 36 (2008) 1578–1586. [5] X.S. Xu, Z.Z. Ma, F. Wang, B.H. Hu, C.S. Wang, Y.Y. Liu, X.R. Zhao, L.H. An, X. Chang, F.L. Liao, J.Y. Fan, H. Niimi, J.Y. Han, The antioxidant Cerebralcare Granule attenuates cerebral microcirculatory disturbance during ischemia-reperfusion injury, Shock 32 (2009) 201–209. [6] F. Wang, Q. Hu, H.C. Chen, X.S. Xu, C.M. Zhou, Y.F. Zhao, B.H. Hu, X. Chang, J.Y. Han, The protective effect of Cerebralcare Granule on brain edema cerebral microcirculatory disturbance, and neuron injury in a focal cerebral ischemia rat model, Microcirculation 19 (2012) 260–272. [7] K. Sun, Q. Hu, C.M. Zhou, X.S. Xu, F. Wang, B.H. Hu, X.Y. Zhao, X. Chang, C.H. Chen, P. Huang, L.H. An, Y.Y. Liu, J.Y. Fan, C.S. Wang, L. Yang, J.Y. Han, Cerebralcare Granule® , a Chinese herb compound preparation, improves cerebral microcirculatory disorder and hippocampal CA1 neuron injury in gerbils after ischemia–reperfusion, J. Ethnopharmacol. 130 (2010) 398–406. [8] X.W. Li, L. Tong, D.X. Li, W.Y. Sun, G.X. Li, A novel strategy to identify analytical markers of Cerebralcare Granule for quality assessing by ultra-high performance chromatography and chemometric analysis, Anal. Methods 15 (2015) 6307–6317. [9] X.Y. Wang, X.H. Ma, W. Li, Y. Chu, J.H. Guo, S.M. Li, J.M. Wang, H.C. Zhang, S.P. Zhou, Y.H. Zhu, Simultaneous determination of five phenolic components and paeoniflorin in rat plasma by liquid chromatography–tandem mass spectrometry and pharmacokinetic study after oral administration of Cerebralcare Granule® , J. Pharm. Biomed. Anal. 86 (2013) 82–91. [10] X.Y. Wang, X.H. Ma, W. Li, Y. Chu, J.H. Guo, S.P. Zhou, Y.H. Zhu, Simultaneous quantitative determination of six active components in traditional chinese medicinal preparation Cerebralcare Granule® by RP-HPLC coupled with diode array detection for quality control, J. Chromatogr. Sci. 52 (2014) 814–817.
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of seven alkaloids in rat plasma by UFLC-MS/MS and its application to a pharmacokinetic study after oral administration of Cerebralcare Granule Li Xiaowen a , Tong Ling b , Li Yunfei b , Sun Guoxiang a,∗ , Yang Dailin c , Sun Herry b a
School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China Tasly R&D Institute, Tianjin Tasly Group Co., Ltd., Tianjin 300402, China c China Pharmaceutical University, Nanjing 210009, China b
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 20 January 2016 Accepted 30 January 2016 Available online 26 February 2016 Keywords: Cerebralcare Granule Alkaloids UFLC-MS/MS Pharmacokinetics
a b s t r a c t An ultra fast liquid chromatography-tandem mass sepectrometry (UFLC-MS/MS) method was developed for simultaneous determination of seven active alkaloid components (tetrahydropalmatine, corydaline, ␣-allocryptopine, tetrahydroberberine, tetrahydrocoptisine, tetrahydrocolumbamine and dehydrocorydaline) in rat plasma after oral administration of Cerebralcare Granule. Plasma samples were pretreated by protein precipitation with acetronitrile containing the internal standard diazepam. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (100 × 2.1 mm, 2.6 m) with gradient elution using mobile phase consisting of acetonitrile −0.1% formic acid in water at a flow rate of 0.3 mL/min. The detection was performed on an electrospray ionization triple quadrupole tandem mass spectrometer using multiple reaction monitoring (MRM) with positive ionization mode. The established method was fully validated and proved to be sensitive and specific with lower limits of quantification (LLOQs) all less than 0.0265 ng/mL in rat plasma. Good linearities of seven alkaloids were obtained in respective concentration ranges (r > 0.9923). The intra- and inter-day precisions were below of 15% for all the seven alkaloids in terms of relative standard deviation (RSD), and the accuracies were ranged from −2.7% to 8.3% in terms of relative error (RE). Extraction recovery, matrix effect and stability were within the required limits in rat plasma. The validated method was successfully applied to investigate the pharmacokinetics of the seven alkaloids in rat plasma after oral administration of Cerebralcare Granule (CG). © 2016 Elsevier B.V. All rights reserved.
1. Introduction Traditional Chinese medicine (TCM) and their formulations have been used in Asian countries for thousands of years and played an indispensable role in prevention and treatment of diseases, especially for complicated and chronic condition [1,2]. Most of TCM formulations are taken orally, and some components persist at detectable levels after digestion and absorption. Therefore, investigation of the pharmacokinetic properties of those components in vivo can gain more in-depth insights into the active components and the mechanisms for TCM formulation, especially for the drugs working in the brain system [3,4]. Cerebralcare Granule (CG) is a widely used botanical drug for the treatment of cerebrovascular diseases including stroke,
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (S. Guoxiang). http://dx.doi.org/10.1016/j.jchromb.2016.01.062 1570-0232/© 2016 Elsevier B.V. All rights reserved.
headache and dizziness (CFDA Approved No.1002004736603642). Pharmacological studies indicated that the CG improves brain microcirculation, increases cerebral blood flow and eases vascular spasm, especially in the treatment of chronic cerebral insufficiency effects [5–8]. CG is developed from the TCM “Siwutang” and composed of eleven herbs (i.e., Radix angelica sinensis, Rhizoma Chuanxiong, Radix paeoniae alba, Rhizoma corydalis yanhusuo, etc.). Our previous work has shown that CG contains many components such as alkaloids, triterpenoids, phenolic acids and anthraquinones [9,10]. R. corydalis, a key constituent herb in CG, shows therapeutic effects of promoting blood circulation and alleviating pains [11–13]. Alkaloids in R. corydalis had been identified as the active components. Among them, tetrahydropalmatine (THP) has been used as a chemical marker for the quality control of R. corydalis in Chinese pharmacopoeia [14]. Meanwhile, alkaloids, such as tetrahydropalmatine (THP), corydaline (CDL), ␣-allocryptopine (␣-ACP), tetrahydroberberine (THB), tetrahydrocoptisine (THC), tetrahydrocolumbamine (THCB) and dehydrocorydaline (DDCL),
L. Xiaowen et al. / J. Chromatogr. B 1017 (2016) 28–35
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Fig. 1. The product ion fragmentation modes and mass spectra of [M+H]+ of tetrahydropalmatine (A), corydaline (B), ␣-allocryptopine (C), tetrahydroberberine (D), tetrahydrocoptisine (E), tetrahydrocolumbamine (F), dehydrocorydaline (G) and diazepam, IS (H).
possess various biological activities such as antimicrobial, antiinflammatory, anti-diabetic and antioxidant ect [15–20]. Our previous work showed that all the seven alkaloids existed in the CG were absorbed into the blood and could be detected after oral administration. Therefore, it is of great value to develop an efficient and reliable method for simultaneous determination of all the seven compounds in biological matrix and to study their pharmacokinetics properties in order to advance the therapeutic application of CG. Various analytical methods based on liquid chromatography technology have been described to determine only one or two of the seven alkaloids mentioned above in biological matrices. Such as,
Ma et al., developed a LC-MS method to determine THP, protopine and palatine in rat plasma [21]. Zhang et al., developed a UFLCMS/MS method to quantify THP and scutellarin in rat plasma [22]. However, none of these analytical methods is able to simultaneously determination all the seven compounds in biological samples. In this study, a sensitive and reliable UFLC-MS/MS method using electrospray ionization (ESI) source was developed and validated to simultaneously determine the concentration of THP, CDL, ␣-ACP, THB, THC, THCB and DDCL in rat plasma for the first time. And the method was successfully applied to a pharmacokinetic study in rats following oral administration of CG.
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L. Xiaowen et al. / J. Chromatogr. B 1017 (2016) 28–35
Fig. 2. Typical chromatograms of blank plasma (A); blank plasma sample spiked with seven analytes at LLOQ and IS (B); 2.0 h plasma sample after oral administration of Cerebralcare Granule(C); (1): tetrahydropalmatine, (2): corydaline, (3): ␣-allocryptopine, (4): tetrahydroberberine, (5): tetrahydrocoptisine, (6): tetrahydrocolumbamine, (7): dehydrocorydaline and (8): IS.
L. Xiaowen et al. / J. Chromatogr. B 1017 (2016) 28–35 Table 1 Optimized MRM parameters, collision energy (CE), declustering potential (DP), entrance potential (EP) and cell exit potential (CXP) of seven analytes and IS. Analyte
Precursor ion (m/z)
Product ion (m/z)
CE (eV)
DP (V)
EP (V)
CXP (V)
THP CDL ␣- ACP THB THC THCB DDCL IS
356.3 370.1 370.1 340.1 324.1 342.2 366.2 285.1
192.2 192.2 290.1 176.1 176.1 178.1 350.2 257.1
36 41 37 40 50 40 41 30
92 116 32 24 105 39 24 50
10 10 10 10 10 10 10 10
35 12 15 18 8 22 12 13
2. Experiment 2.1. Materials and reagents Cerebralcare Granule (batch NO. 140601) was supplied by Tianjin Tasly Pharmaceutical Co. Ltd. (Tianjin, China). The reference standards of THP, CDL, ␣-ACP, THB, THC, THCB, DDCL and diazepam (internal standards, IS, structure as Fig. 1) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile of HPLC grade were purchased from Merck (Darmstadt, Germany). The purity of all standards were above 98.0% and suitable for LC-MS/MS analysis. Formic acid of HPLC grade was obtained from Tedia Company Inc. (Beijing, China). Deionized water was prepared from a Milli-Q water purification system (Millipore, Billerica, USA). Other reagents were of analytical grade. 2.2. Apparatus and UFLC-MS/MS condition All sample analyses were performed on an ESI triple quadrupole mass spectrometer, model Triple Quad 5500 (AB SCIEX, Foster City, CA), which was controlled by Analyst® 1.6.1 software. The mass spectrometer was directly coupled with a UFLC system from Shimadzu Corporation (Shimadzu, Kyoto, Japan), which consisted of a SIL-30AC auto-sampler, a CTO-20A column heater, a CBM-20A Lite controller, a DGU-20A5 degasser, and two LC-30AD pumps. Chromatographic separation was achieved on an Phenomenex Kinetex C18 column (100 × 2.1 mm, 2.6 m). The mobile phase was a mixture of 0.1% formic acid ultrapure water (A) and acetonitrile solution (B) using a gradient elution system at a flow rate of 0.3 mL/min. It was run at 5–50% B over 0–1 min, 50–90% B over 1–3.5 min, and the composition was maintained at 90% B for 0.5 min and then returned to initial condition. All analytes were eluted rapidly within 5 min. The injection volume was 5 L and the column temperature was set at 45 ◦ C. Mass spectrometric detection was operated in positive ionization with multiple reaction monitoring (MRM) mode. The optimized ionspray voltage and source temperature were maintained at 5500 V and 550 ◦ C. High-purity nitrogen was used as nebulizing (50 psi), auxiliary (50 psi) and curtain gas (20 psi). The optimized quantitative parameters are listed in Table 1. 2.3. Preparation of standard and quality control samples Stock solutions at 1.0 mg/mL for each standard were prepared in methanol. A series of working solutions of these analytes were obtained by diluting mixed standard solution with methanol at appropriate concentrations. A quantity of diazepam was dissolved in methanol to produce the IS solution with a concentration of 50.0 ng/mL. All solutions were stored at −20 ◦ C and brought to room temperature (25 ◦ C) before use.
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Table 2 The regression equations, linear range and LLOQs of the seven analytes. Analyte
Range (ng/mL)
Calibration curves
Correlation coefficient (r)
LLOQ (ng/mL)
THP CDL ␣- ACP THB THC THCB DDCL
0.163–150 0.125–100 0.0263–25.0 0.0263–25.0 0.125–100 0.125–100 0.0625–50.0
y = 13.52x + 6.61 y = 8.79x + 4.37 y = 2.34x + 0.48 y = 22.35x + 0.08 y = 3.02x + 1.85 y = 0.43x + 0.14 y = 25.16x + 12.23
0.9964 0.9982 0.9966 0.9987 0.9923 0.9933 0.9992
0.163 0.125 0.0263 0.0263 0.125 0.125 0.0625
Calibration standards were prepared by spiking appropriate amount of the standard solutions in blank plasma (100 L) to yield final concentrations of 0.163–150 ng/mL for THP, 0.125–100 ng/mL for CDL, 0.0263–25.0 ng/mL for ␣-ACP, 0.0263–25.0 ng/mL for THB, 0.125–100 ng/mL for THC, 0.125–100 ng/mL for THCB and 0.0625–50.0 ng/mL for DDCL. Low, medium and high concentration QCs were prepared in the same way as the calibration standards (1.63, 32.5, 120 ng/mL for THP; 1.25, 25.0, 80.0 ng/mL for CDL, THC and THCB; 0.263, 6.25, 20.0 ng/mL for ␣-ACP and THB; 0.625, 12.5, 40.0 ng/mL for DDCL). QCs at concentrations of 50.0 ng/mL for IS were also prepared.
2.4. Sample preparations All samples were thawed at room temperature before analysis. To a 100 L aliquot of plasma samples, 20 L IS (diazepam, 50.0 ng/mL) and 10 L 1.0 mol/L hydrochloric acid were spiked in a 2 mL Eppendorf tube, and then vortexed for 1 min. The mixture was extracted with 2 mL ethyl acetate by vertex-mixing for 5 min and centrifugated at 12,000 rpm for 5 min. The supernatant was transferred to another vial and evaporated to dryness in vacuo at 40 ◦ C. The residue was reconstituted in 100 L mobile phase, vortex-mixed for 1 min and centrifuged at 12,000 rpm for 3 min. Finally, 5 L of the supernatant was injected into the UFLC-MS/MS system for analysis.
2.5. Method validation This method was fully validated in accordance with US-FDA Bioanalytical Method Validation Guidance and European Medicines Agency Guideline on Bioanalytical Method Validation [23,24]. by determining selectivity, linearity, precision and accuracy, recovery, matrix effect and stability. Selectivity was assessed by comparing chromatograms of six different batches of blank rat plasma with the corresponding spiked rat plasma. Linearity was assessed by weighted (1/x2 ) least-squares analysis of seven different calibration curves. Intra- and inter-day precision (the relative standard deviation, RSD) and accuracy (the relative error, RE) were determined by analysis of low, medium, and high QC samples (n = 6) on 3 different days. The stability were assessed of low, medium and high QC samples (n = 6) in three complete freeze/thaw cycles (−40–25 ◦ C), long-term sample storage (−40 ◦ C for 30 days), and bench-top (25 ◦ C for 4 h). The ready-toinjection stability of extracted samples in the autosampler rack at 4 ◦ C for 24 h was also evaluated. The matrix effect was investigated by comparing the peak areas of analytes in the post-extraction spiked blank plasma at low, medium, and high concentrations with those of the corresponding standard solutions. The extraction recovery was determined by comparing the mean peak areas of six extracted samples at low, medium, and high QC concentrations with the mean peak areas of spike-after-extraction samples.
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Table 3 precision, accuracy, recovery and matrix effect for seven analytes in rat plasma (n = 6). Analyte
Spiked (ng/mL)
THP
1.63 32.5 120
Inter-day RSD (%) 5.4 2.3 4.5
Intra-day RSD (%) 1.8 4.9 2.6
Accuracy RE (%) 0.44 7.3 6.0
Recovery (%) 89.5 ± 2.5 96.4 ± 2.8 85.8 ± 4.9
Matrix effect (%) 101.8 ± 6.5 114.3 ± 2.3 85.1 ± 0.5
CDL
1.25 25.0 80.0
5.9 9.2 5.3
3.0 5.2 3.8
0.73 2.4 7.8
89.5 ± 7.9 95.3 ± 1.9 101.3 ± 4.7
102.1 ± 5.7 105.2 ± 3.5 94.8 ± 5.5
␣ACP
0.263 6.25 20.0
5.8 4.8 6.0
4.0 9.8 5.2
−0.23 3.6 8.3
73.3 ± 3.3 67.4 ± 6.4 84.2 ± 5.4
93.8 ± 5.8 92.7 ± 3.5 86.6 ± 3.2
THB
0.263 6.25 20.0
9.7 4.5 11
7.7 1.0 1.5
0.19 3.1 −1.2
87.2 ± 4.6 79.5 ± 3.3 89.9 ± 2.3
109.5 ± 8.0 106.7 ± 2.1 97.2 ± 1.9
THC
1.25 25.0 80.0
3.1 8.7 5.6
5.3 6.9 4.2
0.12 6.1 4.4
85.4 ± 2.6 89.9 ± 3.3 87.3 ± 2.2
86.3 ± 1.4 107.7 ± 2.0 99.1 ± 3.1
THCB
1.25 25.0 80.0
2.9 4.1 4.9
8.9 4.4 3.1
−0.51 5.2 3.7
68.8 ± 4.4 78.6 ± 0.4 82.9 ± 5.2
94.4 ± 6.5 99.3 ± 6.6 101.0 ± 0.6
DDCL
0.625 12.5 40.0
12 2.6 6.9
9.3 7.7 4.1
0.80 3.0 −2.7
83.1 ± 3.3 113.1 ± 3.5 109.2 ± 1.3
93.7 ± 8.4 113.1 ± 8.7 109.2 ± 3.2
Table 4 The stability of seven analytes in rat plasma under different storage condition. (n = 6). 25 ◦ C for 4 h
Frozen for 30 days
Three-freeze-thraw cysles
4 ◦ C for 24 h
RSD (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
−7.1 3.1 2.8
10 4.5 3.1
0.51 −0.10 2.4
10.1 9.9 0.90
14 0.14 5.6
1.3 0.24 5.7
3.1 1.2 1.0
−1.5 0.26 7.4
9.1 0.92 8.2
0.31 6.0 6.9
5.6 2.2 9.1
−3.0 −9.2 1.6
5.2 7.6 3.6
1.5 2.9 5.3
9.9 9.1 2.4
−2.3 −3.4 0.8
8.5 8.1 2.3
−5.3 −0.55 0.77
3.8 9.4 6.7
−3.3 4.0 1.7
2.4 3.5 1.5
−2.4 4.6 −0.54
Analyte
Concentration (ng/mL)
RSD (%)
RE (%)
THP
1.63 32.5 120
6.6 3.0 2.2
−1.9 3.6 5.4
7.1 3.7 2.8
−1.9 5.2 2.4
7.6 4.4 1.8
CDL
1.25 25.0 80.0
4.9 8.0 3.3
−1.3 3.5 1.1
5.1 11 5.7
1.2 5.5 1.0
6.7 5.2 2.0
2.6 −3.1 6.0
␣ACP
0.265 6.25 20.0
2.0 6.3 3.3
−6.4 10 6.3
2.3 1.2 4.0
−3.9 1.5 5.7
7.3 0.72 5.1
THB
0.27 6.25 20.0
1.9 1.0 4.9
3.5 2.2 11
2.0 1.3 4.1
3.5 1.0 12
10.2 0.87 6.4
THC
1.25 25.0 80.0
8.5 5.6 1.9
2.9 0.70 −6.9
1.9 11 0.63
0.90 −6.7 4.3
10.2 7.1 1.2
THCB
1.25 25.0 80.0
2.4 1.4 3.7
4.4 −3.3 2.4
5.2 7.9 8.0
−4.0 −5.2 5.4
DDCL
0.625 12.5 40.0
3.6 7.7 8.7
−2.8 2.6 −1.9
2.8 3.4 3.7
−3.5 3.7 −1.9
RE (%)
Table 5 Pharmacokinetic parameters of analytes after oral administration of CG (8.0 g/kg) to rats (n = 6). Parameters
T1/2 (h)
THP CDL ␣-ACP THB THC THCB DDCL
6.4 6.8 11.5 14.1 12.2 8.8 5.9
± ± ± ± ± ± ±
Cmax (ng/mL) 3.2 3.8 6.6 2.9 6.4 3.9 2.7
90.0 59.0 24.9 20.1 73.9 63.5 34.5
± ± ± ± ± ± ±
31.6 36.4 13.1 12.2 33.4 28.5 19.2
Tmax (h) 0.9 0.8 0.8 0.5 0.5 0.5 0.8
± ± ± ± ± ± ±
0.3 0.2 0.3 0.2 0.3 0.4 0.1
AUC0−t (ng h/mL) 836.7 195.8 209.5 171.7 995.7 810.7 323.5
2.6. Pharmacokinetic study Male Sprague-Dawley rats (200 ± 20 g) were housed under a humidity controlled room, maintained at 22–25 ◦ C, with a 12 h
± ± ± ± ± ± ±
291.3 106.0 83.2 76.2 377.4 256.6 125.6
AUC0−∞ (ngh/mL) 936.8 229.8 228.9 196.1 1002.7 884.2 361.7
± ± ± ± ± ± ±
319.9 124.3 95.4 85.3 402.6 273.2 134.4
MRT0−24 (h)
MRT0−∞ (h)
10.52 ± 2.15 9.43 ± 1.73 8.31 ± 2.87 8.66 ± 1.28 7.25 ± 1.45 8.27 ± 2.78 9.22 ± 1.43
11.81 10.59 9.08 9.18 8.75 9.94 10.49
± ± ± ± ± ± ±
2.69 3.08 1.44 2.50 2.09 3.53 1.64
light–dark cycle. Prior to experiment treatment, all animals were fasted overnight with free access to water. All procedures involving animals were in accordance with the Regulations of Experimental
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Fig. 3. Mean plasma concentration-time profiles of tetrahydropalmatine (A), corydaline (B), ␣-allocryptopine (C), tetrahydroberberine (D), tetrahydrocoptisine (E), tetrahydrocolumbamine (F), dehydrocorydaline (G) after oral administration of CG at 8.0 g/kg to rats (n = 6).
Animal Administration issued by the State Committee of Science and Technology of People’s Republic of China. Six rats were intragastric administered with 8.0 g/kg CG (equivalent to 0.356 mg/kg of THP, 0.874 mg/kg of CDL, 0.516 mg/kg of ␣-ACP, 0.131 mg/kg of THB, 0.266 mg/kg of THC, 0.638 mg/kg of THCB and 1.74 mg/kg of DDCL) suspended in 0.5% CM C-Na (w/v). Serial blood samples (300 L) were obtained at 0 (pre-dose), 0.08, 0.25, 0.5, 0.75, 1, 1.5,
2, 3, 5, 8, 12, 24 and 30 h by puncture of the retro-orbital sinus after oral administration. All samples were placed into heparinized tubes. After centrifugation at 4500 rpm for 10 min, plasma was collected and frozen at −40 ◦ C until analysis. The pharmacokinetic parameters were calculated by DAS 2.0 pharmacokinetic program (Chinese Pharmacological Society) using non-compartmental analysis.
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3. Results and discussion 3.1. Mass spectrometry According to the direct full-scan ESI mass spectra, the ion intensities of all the seven alkaloids and IS were observed higher in the positive mode than the negative mode. The MS/MS product ion spectras of the analytes and IS are shown in Fig. 1. The presence of a bit of formic acid buffer in the mobile phase improved MS detection with good response in the positive ionization mode. Considering the factors above mentioned the optimal mobile phase was consisting of acetonitrile and 0.1% formic acid-water eluted in a simple gradient elution program with column temperature keeping at 45 ◦ C. Under the selected conditions, all the analytes were eluted rapidly within 5.0 min on a C18 column without interference from other components in rat plasma. 3.2. Optimization of extraction conditions To achieve satisfactory recoveries for each analyte, liquid–liquid extraction (LLE), solid phase extraction (SPE) and protein precipitation (PPT) were investigated respectively. Different extraction solvents (ethyl acetate, acetone, diethyl ether, and n-butyl alcohol) and their combination were tested, but no satisfactory recovery was obtained for all the alkaloids. SPE with Waters Oasis HLB cartridges (Milford, MA, USA) was costly and time-costing, not suitable for multi-sample analysis. Finally, protein precipitation (PPT) using acetonitrile was tried and found to give good and consistent recovery for all the seven alkaloids and IS. 3.3. Method validation 3.3.1. Selectivity Typical chromatograms obtained from a blank, a spiked plasma sample with the analytes and IS, and a plasma sample after an oral dose are presented in Fig. 2. The retention time for THP, CDL, ␣-ACP, THB, THC, THCB, DDCL and IS were 2.67, 2.70, 2.67, 2.68, 2.65, 2.71, 2.67, and 3.24 min, respectively. No interference of endogenous peaks exhibited in drug-free plasma indicated the high selectivity of LC–MS/MS. 3.3.2. Linearity and lower limits of quantification Regression equation, correlation coefficients, linear ranges and LLOQ of the seven analytes are shown in Table 2. All the seven components exhibited good linearity within selected concentration ranges with correlation coefficients (r) between 0.9923 and 0.9992. Due to the different structures of these alkaloids, they have different mass ionization capacity and fragmentation modes, which lead to the difference in LLOQ. The LLOQ of THP, CDL, ␣-ACP, THB, THC, THCB and DDCL were 0.163, 0.125, 0.0263, 0.0263, 0.125, 0.125, 0.0625 ng/mL, respectively. The limits were sufficient for pharmacokinetic studies. 3.3.3. Precision, accuracy and stability The precision and accuracy data for the seven analytes from the QC samples are presented in Table 3. They were evaluated by RSD and RE values, respectively. The intra- and inter- day precisions were below of 15% for the seven analytes in terms of RSD, and the accuracies were ranged from −2.7% to 8.3% in terms of RE. All the assay values were within the acceptable criteria, which indicated the overall reproducibility of the method. 3.3.4. Matrix effect and extraction recovery As shown in Table 3, the extraction recoveries of all the seven analytes were in the range 67.4–113.1%, with RSD less than 8.5%.
The mean extraction recovery of the IS was 87.2 ± 3.6%. The average matrix effect values for the seven analytes and IS were within 85.1–114.3%. No significant matrix effect for all the analytes was observed, which indicated that no co-eluting substance could influence the ionization of the analytes.
3.3.5. Stablility Six replicates of QC samples at three concentrations were used for stability evaluation of the seven analytes stored under several conditions. The results of short-term stability, long-term stability, freeze-thaw stability and auto-sampler stability are summarized in Table 4. It shows that the deviations (RE) between measured values and nominal values were all within ±13.7%, demonstrating good stability under these storage conditions.
3.3.6. Pharmacokinetic study The validated UFLC–MS/MS method was successfully applied to a pharmacokinetic study of THP, CDL, ␣-ACP, THB, THC, THCB and DDCL in rat after a single oral administration of CG at dose of 8.0 g/kg. Mean plasma concentration-time profiles of the seven analytes are illustrates in Fig. 3, and the main pharmacokinetic parameters assessed by non compartmental analysis are listed in Table 5. Following oral administration of CG, double peaks were observed in both individual and mean plasma concentration profiles of THP, CDL, ␣-ACP, THB, THC, THCB and DDCL. The first peaks of the seven alkaloids emerged at approximately 0.5–0.9 h, respectively, which suggested that both of them could be absorbed in the stomach or upper intestinal lumen and mucosa. While the second peaks emerged at about 4–8 h and those peaks concentration were lower than the first peaks. The result indicated that all of the seven alkaloids had rapid absorption and slow elimination. Meanwhile, Ma et al. [21] and Zhang et al. [22] have reported two peaks in the plasma concentration curve of THP, ␣-ACP and THB, and attributed the phenomenon to distribution, re-absorption and enterhepatic circulation. However, by comparing with the previous literature, the T1/2 and MRT0−∞ of THP, THB and THC were a litter shorter, and the ␣-ACP was longer than reported results [18]. The results might be presumably due to the different physiochemical properties of each compound or the pharmacokinetic interaction of the prescribed chemical components. Overall, the information described in the present study might be helpful for further studies on the PK evaluation of CG, and beneficial for application of this formula in clinical therapy.
4. Conclusion In the present study, a reliable and sensitive UFLC-MS/MS method was first time developed for the simultaneous quantification of 7 alkaloids in rat plasma samples following oral administration of Cerebralcare Granule. This method offers great simplicity and efficiency for high sample throughput in bioanalysis owing to a short analysis time of 5.0 min per sample and a relatively simple sample preparation procedure with one-step protein precipitation. The pharmacokinetic characteristics of 7 alkaloids had rapid absorption and slow elimination, and they all exhibited double peaks after oral administration of CG. These works could provide more in-depth insights into these active alkaloids working in vivo and would be helpful for further revealing the pharmacology and mechanisms of CG. However, since species difference usually existed in the metabolism between the rat and human, further study in human would be needed to carry out for better understanding of this formulation.
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