Journal of Chromatography B, 973 (2014) 104–109
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Rapid and sensitive analysis of parishin and its metabolites in rat plasma using ultra high performance liquid chromatography-fluorescence detection Chunlan Tang, Li Wang, Mengchun Cheng, Xiaozhe Zhang, Xiaoyan Liu, Hongbin Xiao ∗ Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, China
a r t i c l e
i n f o
Article history: Received 20 January 2014 Accepted 13 August 2014 Available online 20 August 2014 Keywords: Parishin UHPLC-FLD Metabolites Pharmacokinetics
a b s t r a c t A simple, rapid and sensitive ultra high performance liquid chromatography with fluorescence detection (UHPLC-FLD) method was developed and validated for quantification of parishin and its metabolites in rats. Plasma samples were prepared by protein precipitation and then analyzed using UHPLC-FLD system. Repeated optimization showed that parishin and its metabolites, including gastrodin, p-hydroxybenzyl alcohol, parishin B and parishin C, could be sensitively detected based on the autofluorescence when excitation and emission wavelengths were set at 225 nm and 295 nm, respectively. The limit of detections (LODs) of GAS, HBA, PB, PC and PA reached 0.6, 0.8, 1, 1 and 1 ng/mL, respectively. The linearity for all targets was within the range 2.5–5000 ng/mL and the correlation coefficient (r2 ) was larger than 0.999. Importantly, our method was almost free from matrix effects and the recoveries were higher than 80%. Additionally, our method also had high precision and accuracy for all analytes, presenting RSDs and REs within ±6% and ±14%, respectively. Finally, the validated UHPLC-FLD method was successfully applied for studying the pharmacokinetics of parishin following intragastrically administration in rats. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Gastrodia elata blume (GEB), the tuber of the orchid, is a traditional Chinese herb widely used against convulsant [1], vertigo, paralysis, epilepsy, tetanus [2,3], asthma and immune dysfunctions [4,5]. Phytochemical studies have identified a number of bioactive components in Gastrodia elata, such as parishin, parishin B, parishin C and gastrodin [6–8]. These parishins are considered to be responsible to neuroprotective effect of Gastrodia elata [9,10]. In addition, parishin can be degenerated in vivo, leading to the production of gastrodin, p-hydroxybenzyl alcohol, parishin B and parishin C as its dominant metabolites [11]. Despite these interesting activities of parishin, there is little pharmacokinetic information available for parishin. Our present study thus aimed to develop a novel analytical method that allowed studying the pharmacokinetics of parishin and dominant metabolites. Various methods, for example HPLC [12], CE [13] or LC-ESIMS [14], have been reported for the determination of gastrodin and p-hydroxybenzyl alcohol in biological samples. Most of these
∗ Corresponding author. Tel.: +86 411 8437 9756; fax: +86 411 8437 9667. E-mail addresses: [email protected], [email protected] (H. Xiao). http://dx.doi.org/10.1016/j.jchromb.2014.08.020 1570-0232/© 2014 Elsevier B.V. All rights reserved.
methods were either lack the sensitivity for bioanalytical work or required extensive sample preparation with potentially insufficient recovery. HPLC-UV method was established for pharmacokinetic study of gastrodin in human plasma after an oral administration of gastrodin capsule [15], while the reported limit of quantification was 50 ng/mL, which was insufficient for monitoring the pharmacokinetics of other target analytes at low plasma concentration. To improve sensitivity, LC-ESI-MS with post-column addition of ammonium hydroxide was employed to determine gastrodin and p-hydroxybenzyl alcohol in rat blood, brain and bile [16]. However, in addition to post-column setup required, this method offered relatively poor recovery for the two analytes, implying extra cleanup procedures were possibly necessary to remove interfering substances that could cause ionization suppression during LC–MS analysis. We reasoned that FLD probably provided high sensitivity because all the analytes have natural fluorescent properties. We thus sought to develop a rapid and sensitive method for simultaneous determination of parishin and its four metabolites by combined use of UHPLC and FLD. The results showed that the limit of detection of analytes could reach as low as 1 ng/mL in a total run time of only 10 min. The method was proved quite robust and suitable for the use in pharmacokinetic study of parishin.
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2. Experimental
2.5. Analytical method validation
2.1. Chemicals and reagents
The validation of this method was carried out with regard to specificity, linearity, precision, accuracy, stability, recovery and matrix effect.
Parishin (PA > 98%), parishin B (PB > 95%), parishin C (PC > 95%), gastrodin (GAS > 98%) and p-hydroxybenzyl alcohol (HBA > 98%) was isolated and purified from the dried roots of Gastrodia elata and identified in our lab [6]. The structures of the standards were described in Fig. 1. Thiamphenicol as internal standard (IS) was purchased from Meilun Biotech Co., Ltd. Ethylenediaminetetraacetic acid disodium salt (Na2 EDTA) was purchased from Fluka. Acetonitrile, methanol and formic acid of chromatographic grade were obtained by Burdick & Jackson (USA). Ultrapure water was obtained by the Milli-Q water purification system (Millipore, Bedford, MA).
2.2. Instrumentation Analysis was carried out on an UHPLC-FLD system, which was equipped with a 1260 binary pump, an autosampler, degasser and fluorescence detector (Agilent, Germany). Separation of parishin and the metabolites was performed on a reverse phase Agilent ZORBAX SB-C18 UHPLC column (3.0 × 100 mm, 1.8 m) with column oven 60 ◦ C. The elution gradient was carried out with a binary solvent system consisting of 0.5% formic acid aqueous solution (A) and methanol (B) at a flow rate of 0.35 mL/min. Optimized separation of all targets was obtained according to a linear gradient that increased from 8% to 40% B in 5 min and held for additional 5 min. The temperature of the autosampler was set at 4 ◦ C, and a 20 L aliquot of each sample was injected for analysis. The excitation and emission wavelength of fluorescence analysis were set at 225 nm and 295 nm, respectively.
2.3. Preparation of standard solutions PA, PB, PC, GAS, HBA and IS powders were separately dissolved in 8% methanol–water at a final concentration of 1 mg/mL. Working standard solutions were prepared by serial dilution of primary stock solutions with 8% methanol-water to obtain concentrations of 3.2, 6, 10, 20, 100, 200, 1000, 2000, 10,000 and 20,000 ng/mL, respectively. The internal standard (IS) working solution of 6 g/mL was prepared by diluting 60 L of the stock solution to 10 mL with 8% methanol–water. All solutions were kept at 4 ◦ C in refrigerator and were brought to room temperature prior to use. Calibration standard samples of PA, PB, PC, GAS, HBA at the concentration ranges of 0.8–5000 ng/mL were prepared by diluting serial working standards and IS with blank plasma. The final concentration of IS was 1.5 g/mL. Quality control (QC) samples were prepared with blank plasma at 40 ng/mL, 100 ng/mL and 1 g/mL of low, medium and high concentration levels, which were used in developed analytical methods.
2.4. Sample preparation The plasma samples were thawed to 4 ◦ C before processing. An aliquot of 100 L plasma sample was transferred to an eppendorf tube, 12.5 L IS and 300 L of ice-cold methanol was added to precipitate the plasma proteins and vortex-mixed for 5 min. The samples were centrifuged at 15,000 rpm for 5 min. The supernatant was transferred and evaporated to dryness under gentle stream of nitrogen at 50 ◦ C. The residue was reconstituted in 50 L of initial mobile phase and 20 L aliquot was injected for analysis.
2.5.1. Specificity The specificity was evaluated by comparing the chromatograms of blank plasma, standard mixture containing IS, blank plasma spiked with IS and standard mixture, and rat plasma sample obtained 1 h after intragastric administration of PA. 2.5.2. Linearity, LLOQ and LOD Ten-level calibration series of analytes with plasma concentrations 0.8, 1.5, 2.5, 5, 25, 50, 250, 500, 2500, 5000 ng/mL were prepared. Calibration curve was conducted by linear regression of the peak area ratio (y) of targets to internal standard, versus spiked concentration (x) in ng/mL, respectively. The limit of detection (LOD) was estimated as the concentration yielding a signal to noise of 3. The lower limit of quantification (LLOQ) were defined as the lowest concentration standard in the calibration curve with the signal-to-noise ratio over 10 and was analyzed with accuracy within ±20% and a precision ≤20%. 2.5.3. Precision and accuracy The precision and accuracy were determined by analyzing QC samples for three concentration levels. Each level was analyzed five times on the same day for intra-day precision and accuracy, and on five consecutive days for inter-day precision and accuracy. Precision was expressed as relative standard deviation (RSD) at each concentration level and accuracy was calculated by relative error (RE). For accuracy, the criterion for the acceptability of data should not exceed 15% for each concentration level. Similarly the value for precision also should not deviate by ±15% from the nominal concentration. 2.5.4. Recovery and Matrix effect The recovery was determined by comparing peak area of rat plasma samples spiked with three different concentrations of standard mixture before and after extraction. Each time, the concentrations of added standards reached a QC level. The matrix effect was evaluated by comparing the peak area of the analytes that were spiked into the post-precipitation matrix with those of standard solutions in the absence of matrix for three QC levels. Five replicates for each QC level were performed. 2.5.5. Stability The stability was performed by triplicate assay at three QC concentration levels under various storage or handing conditions, including stability of plasma samples at −80 ◦ C for one month (longterm), stability of plasma samples at room temperature for 6 h (short-term), stability after three freeze–thaw cycles and stability of extracted samples storage in the working temperature (4 ◦ C in autosampler) for 24 h throughout the duration of a typical sequence of chromatographic analyses. It was evaluated by comparing the measured concentration with those of the respective freshly prepared QC samples. 2.6. Pharmacokinetic study of parishin The experiments were performed on 7-week-old female Sprague–Dawley rats weighing approximately 200 g, which were obtained from the Laboratory Animal Center in Dalian Medical University (Dalian, China). All experimental protocols on animals were carried out according to the guidelines of the Committee on the Care and Use of Laboratory Animals of China. The rats were fasted for 24 h
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Fig. 1. Molecular structures of parishin and its metabolites.
prior to the study and allowed water ad libitum. Serial blood samples were collected into Eppendorf tube containing Na2 EDTA as an anticoagulant at pre-defined time points as 0, 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h and 8 h after an intragastric administration of PA at a dose of 375 mg/kg. Blood samples were promptly centrifuged at 3000 rpm for 10 min and plasma fraction was separated and stored at −80 ◦ C until analyzed. In the case that the concentration of an analyte was beyond the upper limit of quantitation, plasma samples were diluted with blank plasma.
40% methanol in 5 min and held for additional 5 min (Fig. 2). In addition, the pretreatment process of plasma in our method was extremely simplified, in which only immediate protein precipitation was used. And the comparison of acetonitrile and methanol was conducted to examine the extraction efficiency. The results showed that methanol allowed higher recovery compared with acetonitrile.
3. Results and discussion
Regarding the IS selection, drugs such as helicid and thiamphenicol were tested according to structural similarity and fluorescence capacity. Finally, thiamphenicol was selected as IS, because of being a fluorescent compound, an appropriate retention time and a good resolution from PA and its metabolites under the chromatographic condition.
3.1. Optimization of fluorescence method Most organic molecules with rigid planar structure have strong fluorescence. From the structures of the five targets (Fig. 1), we predicted their fluorescence properties and further verified their natural fluorescence by fluorescence spectroscopy analysis. Afterwards, the excitation and emission wavelength were optimized by online scanning the excitation wavelengths (multi-wavelength excitation mode) and emission wavelengths (multi-wavelength emission mode) using a step width of 5 nm, response time of 4 s and PMT-gain of 10. The results were presented in an isoabsorbance plot, which was one technique to present the peak intensity, wavelength and time as a contour map. The contour map presented the spectral information in a series of isoabsorptive, concentric lines in the wavelength and time plane, which allowed for all spectral information to be visibly inspected. From the isoabsorbance plots (see details in supplementary data), we found that the maximum excitation and emission wavelengths were 225 nm and 310 nm, respectively. However, an obvious interfering peak was co-eluted with PB at retention time of 8.2 min, and this issue was unsolvable by optimization of chromatographic conditions (see supplementary data). As a compromise, the excitation and emission wavelength were further set to 225 nm and 295 nm, respectively. Using these parameters, the sensitivity of analytes remained high, while the interfering peak disappeared. 3.2. Optimization of chromatographic conditions The chromatographic conditions were optimized through several trials to achieve good resolution and symmetric sharp peaks for both analytes and IS, as well as a short run time. In order to reduce the total run time, a column packed with sub 2-m particles was selected. During the optimization of mobile phase, methanol–water and acetonitrile–water were investigated. A better separation was obtained using methanol–water compared with acetonitrile–water. And 0.5% formic acid as the modifier was adopted to avoid tailing. Optimized separation of all targets was obtained according to a linear gradient that increased from 8% to
3.3. Selection of the internal standard
4. Validation of the method 4.1. Specificity Specificity was assessed by comparing the chromatograms of blank plasma, standard mixture containing IS, blank plasma spiked with standard mixture and IS, and rat plasma sample obtained 1 h after intragastric administration of PA. Representative chromatograms were presented in Fig. 1. It can be seen that GAS, HBA, PB, PC, PA and IS were sufficiently separated and no interfering peak was observed at the retention times of these compounds. 5. Linearity, LLOQ and LOD The calibration model was evaluated from a ten-concentration level plasma series with five replicates at each level. After processing the calibration data, the regression curve presented high linearity with a coefficient (r2 ) larger than 0.999 within the concentration range from 2.5 to 5000 ng/mL, and demonstrated acceptable precisions (RSD%, 0.15–14.76) and accuracies (RE%, −15.00 to 14.55) at these eight concentration levels (see supplementary data). The linear regression equation of calibration curves for GAS, HBA, PB, PC, PA was y = 0.0079x−0.0125, y = 0.0075x−0.0255, y = 0.0018x−0.0017, y = 0.0021x−0.0020 and y = 0.0023x + 0.0047, respectively. In the equation, y indicated the peak area ratio between an analytical compound and IS, while x indicated the concentration of this analyte. In addition, the LODs of GAS, HBA, PB, PC and PA were 0.6 ng/mL, 0.8 ng/mL, 1 ng/mL, 1 ng/mL and 1 ng/mL, respectively. The LLOQs of all targets were 2.5 ng/mL, which were considerable improvement over that of commonly used HPLCUV (limit of quantification for GAS: 50 ng/mL) [15] and HPLC–MS (limit of quantification for HBA: 830 ng/mL) [14]. Therefore, it was
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Fig. 2. Representative chromatograms of blank rat plasma (A), mixed standard solution with PA, PB, PC, GAS, HBA at 2.5 ng/mL and IS (B), rat plasma with PA, PB, PC, GAS, HBA at 2.5 ng/mL and IS (C), and rat plasma sample at 1 h after intragastric administration of PA at 375 mg/kg (D).
sufficient for pharmacokinetic study of PA following an intragastric administration of PA to rats.
the present method was reliable and reproducible for the quantitative analysis of PA and its metabolites in plasma samples.
6. Precision and accuracy
6.1.1. Recovery and matrix effect
The precision and accuracy results at low, medium and high concentrations of PA and its metabolites in rat plasma are demonstrated in Table 1. The results showed that precisions and accuracies at three concentration levels (5 replicates) were within ±6% and ±14%, respectively. The high precision and accuracy indicated that
Table 1 summarizes the matrix effect and recovery of measured compounds in plasma. The values of matrix effects for all targets were close to 1 and recoveries were over 80%, indicating that the matrix effect could be ignored and the method could be kept free from endogenous substances, without the additional
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Table 1 Intra- and inter-day precision and accuracy, matrix effect and recovery of GAS, HBA, PB, PC and PA in rat plasma. Compound
Concentration (ng/mL)
Precision and accuracy
Matrix effect
Intraday (n = 5)
Recovery
Interday (n = 5)
Mean ± SD
RSD (%)
RE (%)
Mean ± SD
RSD (%)
RE (%)
Mean ± SD (%)
RSD (%)
Mean ± SD (%)
RSD (%)
40 100 1000
44.70 ± 0.61 107.00 ± 1.61 1027.37 ± 2.87
1.37 1.51 0.28
11.7 7.00 2.74
42.47 ± 1.08 105.39 ± 3.38 981.74 ± 3.85
2.54 3.21 0.39
6.18 5.34 −1.82
95.73 ± 1.31 102.39 ± 1.07 99.19 ± 1.08
1.37 1.04 1.09
95.14 ± 4.90 100.28 ± 0.61 98.36 ± 1.76
5.15 0.61 1.79
HBA
40 100 1000
40.90 ± 0.56 98.91 ± 1.16 942.80 ± 4.38
1.37 1.17 0.46
2.24 −1.09 −5.72
39.08 ± 0.79 97.97 ± 2.79 907.65 ± 3.09
2.00 2.84 0.34
−1.89 −2.02 −9.24
100.88 ± 1.03 103.33 ± 1.86 99.06 ± 1.74
1.02 1.80 1.76
99.45 ± 2.14 101.59 ± 1.69 98.49 ± 2.40
2.15 1.67 2.43
PB
40 100 1000
38.47 ± 1.72 101.41 ± 4.41 1091.80 ± 8.47
4.48 4.34 0.78
−3.81 1.41 9.18
37.79 ± 1.49 105.69 ± 3.96 1016.93 ± 24.65
3.96 3.75 2.42
−5.52 5.69 1.61
93.16 ± 2.70 104.56 ± 1.40 99.79 ± 0.97
2.89 1.34 0.97
90.25 ± 8.10 102.20 ± 0.27 98.70 ± 0.95
8.97 0.26 0.96
PC
40 100 1000
45.24 ± 1.85 102.05 ± 3.03 942.11 ± 7.57
4.10 2.97 0.80
13.10 2.05 5.79
45.72 ± 1.77 103.1752 ± 5.62 900.79 ± 15.11
3.87 5.44 1.68
14.3 3.17 −9.92
92.70 ± 2.78 104.97 ± 1.41 100.12 ± 0.99
3.00 1.34 0.99
88.47 ± 6.55 104.66 ± 2.42 98.78 ± 1.37
7.40 2.31 1.38
PA
40 100 1000
39.81 ± 0.37 98.27 ± 3.09 950.58 ± 6.43
0.93 3.15 0.68
−0.47 −1.73 −4.94
2.30 2.11 3.39
−1.25 −3.58 0.95
103.46 ± 0.77 102.36 ± 1.37 99.10 ± 2.01
0.75 1.34 2.03
104.62 ± 7.01 100.08 ± 0.26 100.66 ± 1.56
6.70 0.26 1.55
39.50 ± 0.91 96.42 ± 2.04 966.09 ± 5.76
Table 2 Stability of GAS, HBA, PB, PC and PA in rat plasma. Compound
Concentration (ng/mL)
Mean ± SD
RSD (%)
RE (%)
Mean ± SD
RSD (%)
RE (%)
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
RE (%)
GAS
40 100 1000
41.74 ± 3.67 100.94 ± 0.69 980.42 ± 4.20
8.78 0.68 0.43
4.36 0.94 −1.96
44.11 ± 1.02 101.67 ± 0.44 978.18 ± 4.97
2.32 0.44 0.51
10.27 1.67 −2.18
44.57 ± 0.61 100.54 ± 0.06 968.99 ± 1.92
1.37 0.06 0.20
11.43 0.53 −3.10
44.42 ± 0.36 106.48 ± 1.89 1027.10 ± 3.29
0.82 1.77 0.32
11.05 6.48 2.71
HBA
40 100 1000
42.68 ± 2.64 100.44 ± 0.99 974.76 ± 2.74
6.18 0.99 0.28
6.70 0.44 −2.52
44.34 ± 0.76 100.73 ± 0.53 971.43 ± 4.11
1.71 0.52 0.42
10.87 0.73 −2.86
44.80 ± 0.36 99.48 ± 0.29 963.38 ± 3.50
0.80 0.30 0.36
12.01 −3.66 −3.66
40.85 ± 0.47 98.41 ± 1.12 944.90 ± 3.60
1.15 1.14 0.38
2.12 −1.58 −5.51
PB
40 100 1000
41.62 ± 3.10 97.88 ± 0.97 907.03 ± 3.98
7.45 0.99 0.44
4.04 −2.12 −9.30
40.91 ± 0.84 96.99 ± 0.43 905.94 ± 2.74
2.05 0.44 0.30
2.28 −3.00 −9.41
40.96 ± 0.65 96.56 ± 1.44 899.75 ± 2.19
1.59 1.49 0.24
2.39 −3.44 −10.03
39.16 ± 1.84 98.26 ± 1.24 1088.17 ± 9.13
4.71 1.26 0.84
−2.10 −1.73 8.82
PC
40 100 1000
43.58 ± 2.57 102.95 ± 1.28 937.48 ± 3.03
5.89 1.24 0.32
8.96 2.95 −6.25
46.77 ± 1.68 100.98 ± 0.85 936.17 ± 6.01
3.59 0.84 0.64
16.94 0.98 −6.38
46.21 ± 1.08 101.00 ± 1.08 929.58 ± 1.11
2.33 1.07 0.12
15.53 1.00 −7.04
45.86 ± 2.19 101.08 ± 3.84 939.11 ± 8.27
4.78 3.80 0.88
14.65 1.08 −6.09
PA
40 100 1000
42.21 ± 2.29 97.19 ± 5.50 914.31 ± 8.04
5.43 5.66 0.88
5.53 −2.81 −8.57
43.41 ± 0.90 91.00 ± 1.17 918.53 ± 5.99
2.08 1.29 0.65
8.52 −9.00 −8.14
43.59 ± 0.69 89.18 ± 0.86 910.14 ± 1.92
1.59 0.96 0.21
8.99 −10.82 −8.99
38.66 ± 2.45 98.60 ± 1.46 946.92 ± 2.20
6.34 1.48 0.23
−3.34 −1.40 −5.30
Short-term
Freeze–thaw
Long-term
24 h in autosampler RE (%)
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GAS
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concentration of 6.3 g/mL at 45 min. HBA, PB and PC reached the peak concentrations of 28, 955 and 592 ng/mL at 60, 60 and 30 min, respectively. GAS, HBA, PB and PC were eliminated rapidly in vivo and the concentrations of four metabolites fell to 343.2, 6.9, 40.7 and 25.0 ng/mL 8 h after the administration of parishin. 8. Conclusion
Fig. 3. Mean plasma concentration–time profile of GAS, HBA, PB, PC and PA after intragastric administration of PA at 375 mg/kg. PA, PB, PC, GAS corresponded with primary axis and HBA with secondary axis (n = 3).
cleanup procedures such as SPE and LLE before injection that was often necessary in LC–MS analysis owing to the existence of significant interference or ion suppression from endogenous substance in biological matrix. 7. Stability The stability results of short-term, three freeze–thaw, long-term and storage in the working temperature at three QC levels were summarized in Table 2. Briefly, three freeze–thaw cycles, long-term storage at −80 ◦ C and ambient temperature storage of the QC samples up to 6 h prior to sample preparation appeared to have no effect on the quantitation of all analytes except PC at the concentration of 40 ng/mL for three freeze–thaw cycles and long-term storage at −80 ◦ C. Thus, we supposed that three freeze–thaw cycles and longterm storage might influence quantitative analysis of PC at the low concentration in rat plasma. So the samples of PC are best not to be repeatedly frozen–thawed and long-term stored. In addition, extracted analytes were allowed to remain in the working temperature (4 ◦ C in autosampler) for 24 h throughout the duration of a typical sequence of chromatographic analyses. 7.1. Pharmacokinetic study of parishin The successful application of our developed method to pharmacokinetic study of parishin in rat plasma we performed. The plasma concentration–time profiles of GAS, HBA, PB, PC and PA after an intragastric administration of PA using a dose of 375 mg/kg are shown in Fig. 3. The concentration of PA and metabolites was readily measurable in plasma sample. The results showed that parishin was completely metabolized to GAS, HBA, PB and PC 5 min after the administration of parishin. GAS reached a peak
This study presented a rapid, sensitive and robust UHPLC-FLD method for simultaneous analysis of parishin and its metabolites in rat plasma for the first time. The analytical parameters, including limit of detection, recovery and linear range, precision, accuracy, matrix effect and stability, matched the requirements for monitoring all target compounds. The application of this method allowed successfully determining the pharmacokinetics of parishin, which showed the rapid degeneration of parishin in vivo and fast increases of metabolites in plasma of rats. Acknowledgments This work is financially supported by the National Natural Science Foundation of China (Grant No. 81001629). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2014.08.020. References [1] C.L. Hsieh, S.Y. Chiang, K.S. Cheng, Y.H. Lin, N.Y. Tang, C.J. Lee, C.Z. Pon, C.T. Hsieh, Am. J. Chinese Med. 29 (2001) 331–341. [2] Y. Zhang, NHBA isolated from Gastrodia elata exerts sedative and hypnotic effects in sodium pentobarbital-treated mice (vol 102, pg 450, 2012), Pharmacology Biochemistry and Behavior, 102 (2012) 593-593. [3] C.L. Huang, J.M. Yang, K.C. Wang, Y.C. Lee, Y.L. Lin, Y.C. Yang, N.K. Huang, J. Ethnopharmacol. 138 (2011) 162–168. [4] Y.W. Jang, J.Y. Lee, C.J. Kim, Int. Immunopharmacol. 10 (2010) 147–154. [5] S.M. Hwang, Y.J. Lee, D.G. Kang, H.S. Lee, Am. J. Chinese Med. 37 (2009) 395–406. [6] L. Wang, H.B. Xiao, X.M. Liang, L.X. Wei, J. Sep. Sci. 30 (2007) 1488–1495. [7] S.Y. Huang, G.Q. Li, J.G. Shi, S.Y. Mo, S.J. Wang, Y.C. Yang, J. Asian Nat. Prod. Res. 6 (2004) 49–61. [8] H.B. Li, F. Chen, J. Chromatogr. A 1052 (2004) 229–232. [9] C.M. Shu, C.G. Chen, D.P. Zhang, H.P. Guo, H. Zhou, J. Zong, Z.Y. Bian, X. Dong, J. Dai, Y. Zhang, Q.Z. Tang, Mol. Cell. Biochem. 359 (2012) 9–16. [10] X.H. Zeng, Y. Zhang, S.M. Zhang, X.X. Zheng, Biol. Pharm. Bull. 30 (2007) 801–804. [11] L. Wang, Y.J. Lei, H.B. Xiao, 37th International Symposium on High Performance Liquid Phase Separations and Related Techniques, 2011. [12] X.D. Yang, J. Zhu, R. Yang, J.P. Liu, L. Li, H.B. Zhang, Nat. Prod. Res. 21 (2007) 180–186. [13] Y.K. Zhao, Q.E. Cao, Y.Q. Xiang, Z.D. Hu, J. Chromatogr. A 849 (1999) 277–283. [14] W. Zhang, Y.X. Sheng, J.L. Zhang, Phytomedicine 15 (2008) 844–850. [15] X.H. Ju, Y. Shi, N. Liu, D.M. Guo, X. Cui, J. Chromatogr. B 878 (2010) 1982–1986. [16] L.C. Lin, Y.F. Chen, W.C. Lee, Y.T. Wu, T.H. Tsai, J. Pharmaceut. Biomed. 48 (2008) 909–917.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Rapid and sensitive analysis of parishin and its metabolites in rat plasma using ultra high performance liquid chromatography-fluorescence detection Chunlan Tang, Li Wang, Mengchun Cheng, Xiaozhe Zhang, Xiaoyan Liu, Hongbin Xiao ∗ Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, China
a r t i c l e
i n f o
Article history: Received 20 January 2014 Accepted 13 August 2014 Available online 20 August 2014 Keywords: Parishin UHPLC-FLD Metabolites Pharmacokinetics
a b s t r a c t A simple, rapid and sensitive ultra high performance liquid chromatography with fluorescence detection (UHPLC-FLD) method was developed and validated for quantification of parishin and its metabolites in rats. Plasma samples were prepared by protein precipitation and then analyzed using UHPLC-FLD system. Repeated optimization showed that parishin and its metabolites, including gastrodin, p-hydroxybenzyl alcohol, parishin B and parishin C, could be sensitively detected based on the autofluorescence when excitation and emission wavelengths were set at 225 nm and 295 nm, respectively. The limit of detections (LODs) of GAS, HBA, PB, PC and PA reached 0.6, 0.8, 1, 1 and 1 ng/mL, respectively. The linearity for all targets was within the range 2.5–5000 ng/mL and the correlation coefficient (r2 ) was larger than 0.999. Importantly, our method was almost free from matrix effects and the recoveries were higher than 80%. Additionally, our method also had high precision and accuracy for all analytes, presenting RSDs and REs within ±6% and ±14%, respectively. Finally, the validated UHPLC-FLD method was successfully applied for studying the pharmacokinetics of parishin following intragastrically administration in rats. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Gastrodia elata blume (GEB), the tuber of the orchid, is a traditional Chinese herb widely used against convulsant [1], vertigo, paralysis, epilepsy, tetanus [2,3], asthma and immune dysfunctions [4,5]. Phytochemical studies have identified a number of bioactive components in Gastrodia elata, such as parishin, parishin B, parishin C and gastrodin [6–8]. These parishins are considered to be responsible to neuroprotective effect of Gastrodia elata [9,10]. In addition, parishin can be degenerated in vivo, leading to the production of gastrodin, p-hydroxybenzyl alcohol, parishin B and parishin C as its dominant metabolites [11]. Despite these interesting activities of parishin, there is little pharmacokinetic information available for parishin. Our present study thus aimed to develop a novel analytical method that allowed studying the pharmacokinetics of parishin and dominant metabolites. Various methods, for example HPLC [12], CE [13] or LC-ESIMS [14], have been reported for the determination of gastrodin and p-hydroxybenzyl alcohol in biological samples. Most of these
∗ Corresponding author. Tel.: +86 411 8437 9756; fax: +86 411 8437 9667. E-mail addresses: [email protected], [email protected] (H. Xiao). http://dx.doi.org/10.1016/j.jchromb.2014.08.020 1570-0232/© 2014 Elsevier B.V. All rights reserved.
methods were either lack the sensitivity for bioanalytical work or required extensive sample preparation with potentially insufficient recovery. HPLC-UV method was established for pharmacokinetic study of gastrodin in human plasma after an oral administration of gastrodin capsule [15], while the reported limit of quantification was 50 ng/mL, which was insufficient for monitoring the pharmacokinetics of other target analytes at low plasma concentration. To improve sensitivity, LC-ESI-MS with post-column addition of ammonium hydroxide was employed to determine gastrodin and p-hydroxybenzyl alcohol in rat blood, brain and bile [16]. However, in addition to post-column setup required, this method offered relatively poor recovery for the two analytes, implying extra cleanup procedures were possibly necessary to remove interfering substances that could cause ionization suppression during LC–MS analysis. We reasoned that FLD probably provided high sensitivity because all the analytes have natural fluorescent properties. We thus sought to develop a rapid and sensitive method for simultaneous determination of parishin and its four metabolites by combined use of UHPLC and FLD. The results showed that the limit of detection of analytes could reach as low as 1 ng/mL in a total run time of only 10 min. The method was proved quite robust and suitable for the use in pharmacokinetic study of parishin.
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2. Experimental
2.5. Analytical method validation
2.1. Chemicals and reagents
The validation of this method was carried out with regard to specificity, linearity, precision, accuracy, stability, recovery and matrix effect.
Parishin (PA > 98%), parishin B (PB > 95%), parishin C (PC > 95%), gastrodin (GAS > 98%) and p-hydroxybenzyl alcohol (HBA > 98%) was isolated and purified from the dried roots of Gastrodia elata and identified in our lab [6]. The structures of the standards were described in Fig. 1. Thiamphenicol as internal standard (IS) was purchased from Meilun Biotech Co., Ltd. Ethylenediaminetetraacetic acid disodium salt (Na2 EDTA) was purchased from Fluka. Acetonitrile, methanol and formic acid of chromatographic grade were obtained by Burdick & Jackson (USA). Ultrapure water was obtained by the Milli-Q water purification system (Millipore, Bedford, MA).
2.2. Instrumentation Analysis was carried out on an UHPLC-FLD system, which was equipped with a 1260 binary pump, an autosampler, degasser and fluorescence detector (Agilent, Germany). Separation of parishin and the metabolites was performed on a reverse phase Agilent ZORBAX SB-C18 UHPLC column (3.0 × 100 mm, 1.8 m) with column oven 60 ◦ C. The elution gradient was carried out with a binary solvent system consisting of 0.5% formic acid aqueous solution (A) and methanol (B) at a flow rate of 0.35 mL/min. Optimized separation of all targets was obtained according to a linear gradient that increased from 8% to 40% B in 5 min and held for additional 5 min. The temperature of the autosampler was set at 4 ◦ C, and a 20 L aliquot of each sample was injected for analysis. The excitation and emission wavelength of fluorescence analysis were set at 225 nm and 295 nm, respectively.
2.3. Preparation of standard solutions PA, PB, PC, GAS, HBA and IS powders were separately dissolved in 8% methanol–water at a final concentration of 1 mg/mL. Working standard solutions were prepared by serial dilution of primary stock solutions with 8% methanol-water to obtain concentrations of 3.2, 6, 10, 20, 100, 200, 1000, 2000, 10,000 and 20,000 ng/mL, respectively. The internal standard (IS) working solution of 6 g/mL was prepared by diluting 60 L of the stock solution to 10 mL with 8% methanol–water. All solutions were kept at 4 ◦ C in refrigerator and were brought to room temperature prior to use. Calibration standard samples of PA, PB, PC, GAS, HBA at the concentration ranges of 0.8–5000 ng/mL were prepared by diluting serial working standards and IS with blank plasma. The final concentration of IS was 1.5 g/mL. Quality control (QC) samples were prepared with blank plasma at 40 ng/mL, 100 ng/mL and 1 g/mL of low, medium and high concentration levels, which were used in developed analytical methods.
2.4. Sample preparation The plasma samples were thawed to 4 ◦ C before processing. An aliquot of 100 L plasma sample was transferred to an eppendorf tube, 12.5 L IS and 300 L of ice-cold methanol was added to precipitate the plasma proteins and vortex-mixed for 5 min. The samples were centrifuged at 15,000 rpm for 5 min. The supernatant was transferred and evaporated to dryness under gentle stream of nitrogen at 50 ◦ C. The residue was reconstituted in 50 L of initial mobile phase and 20 L aliquot was injected for analysis.
2.5.1. Specificity The specificity was evaluated by comparing the chromatograms of blank plasma, standard mixture containing IS, blank plasma spiked with IS and standard mixture, and rat plasma sample obtained 1 h after intragastric administration of PA. 2.5.2. Linearity, LLOQ and LOD Ten-level calibration series of analytes with plasma concentrations 0.8, 1.5, 2.5, 5, 25, 50, 250, 500, 2500, 5000 ng/mL were prepared. Calibration curve was conducted by linear regression of the peak area ratio (y) of targets to internal standard, versus spiked concentration (x) in ng/mL, respectively. The limit of detection (LOD) was estimated as the concentration yielding a signal to noise of 3. The lower limit of quantification (LLOQ) were defined as the lowest concentration standard in the calibration curve with the signal-to-noise ratio over 10 and was analyzed with accuracy within ±20% and a precision ≤20%. 2.5.3. Precision and accuracy The precision and accuracy were determined by analyzing QC samples for three concentration levels. Each level was analyzed five times on the same day for intra-day precision and accuracy, and on five consecutive days for inter-day precision and accuracy. Precision was expressed as relative standard deviation (RSD) at each concentration level and accuracy was calculated by relative error (RE). For accuracy, the criterion for the acceptability of data should not exceed 15% for each concentration level. Similarly the value for precision also should not deviate by ±15% from the nominal concentration. 2.5.4. Recovery and Matrix effect The recovery was determined by comparing peak area of rat plasma samples spiked with three different concentrations of standard mixture before and after extraction. Each time, the concentrations of added standards reached a QC level. The matrix effect was evaluated by comparing the peak area of the analytes that were spiked into the post-precipitation matrix with those of standard solutions in the absence of matrix for three QC levels. Five replicates for each QC level were performed. 2.5.5. Stability The stability was performed by triplicate assay at three QC concentration levels under various storage or handing conditions, including stability of plasma samples at −80 ◦ C for one month (longterm), stability of plasma samples at room temperature for 6 h (short-term), stability after three freeze–thaw cycles and stability of extracted samples storage in the working temperature (4 ◦ C in autosampler) for 24 h throughout the duration of a typical sequence of chromatographic analyses. It was evaluated by comparing the measured concentration with those of the respective freshly prepared QC samples. 2.6. Pharmacokinetic study of parishin The experiments were performed on 7-week-old female Sprague–Dawley rats weighing approximately 200 g, which were obtained from the Laboratory Animal Center in Dalian Medical University (Dalian, China). All experimental protocols on animals were carried out according to the guidelines of the Committee on the Care and Use of Laboratory Animals of China. The rats were fasted for 24 h
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Fig. 1. Molecular structures of parishin and its metabolites.
prior to the study and allowed water ad libitum. Serial blood samples were collected into Eppendorf tube containing Na2 EDTA as an anticoagulant at pre-defined time points as 0, 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h and 8 h after an intragastric administration of PA at a dose of 375 mg/kg. Blood samples were promptly centrifuged at 3000 rpm for 10 min and plasma fraction was separated and stored at −80 ◦ C until analyzed. In the case that the concentration of an analyte was beyond the upper limit of quantitation, plasma samples were diluted with blank plasma.
40% methanol in 5 min and held for additional 5 min (Fig. 2). In addition, the pretreatment process of plasma in our method was extremely simplified, in which only immediate protein precipitation was used. And the comparison of acetonitrile and methanol was conducted to examine the extraction efficiency. The results showed that methanol allowed higher recovery compared with acetonitrile.
3. Results and discussion
Regarding the IS selection, drugs such as helicid and thiamphenicol were tested according to structural similarity and fluorescence capacity. Finally, thiamphenicol was selected as IS, because of being a fluorescent compound, an appropriate retention time and a good resolution from PA and its metabolites under the chromatographic condition.
3.1. Optimization of fluorescence method Most organic molecules with rigid planar structure have strong fluorescence. From the structures of the five targets (Fig. 1), we predicted their fluorescence properties and further verified their natural fluorescence by fluorescence spectroscopy analysis. Afterwards, the excitation and emission wavelength were optimized by online scanning the excitation wavelengths (multi-wavelength excitation mode) and emission wavelengths (multi-wavelength emission mode) using a step width of 5 nm, response time of 4 s and PMT-gain of 10. The results were presented in an isoabsorbance plot, which was one technique to present the peak intensity, wavelength and time as a contour map. The contour map presented the spectral information in a series of isoabsorptive, concentric lines in the wavelength and time plane, which allowed for all spectral information to be visibly inspected. From the isoabsorbance plots (see details in supplementary data), we found that the maximum excitation and emission wavelengths were 225 nm and 310 nm, respectively. However, an obvious interfering peak was co-eluted with PB at retention time of 8.2 min, and this issue was unsolvable by optimization of chromatographic conditions (see supplementary data). As a compromise, the excitation and emission wavelength were further set to 225 nm and 295 nm, respectively. Using these parameters, the sensitivity of analytes remained high, while the interfering peak disappeared. 3.2. Optimization of chromatographic conditions The chromatographic conditions were optimized through several trials to achieve good resolution and symmetric sharp peaks for both analytes and IS, as well as a short run time. In order to reduce the total run time, a column packed with sub 2-m particles was selected. During the optimization of mobile phase, methanol–water and acetonitrile–water were investigated. A better separation was obtained using methanol–water compared with acetonitrile–water. And 0.5% formic acid as the modifier was adopted to avoid tailing. Optimized separation of all targets was obtained according to a linear gradient that increased from 8% to
3.3. Selection of the internal standard
4. Validation of the method 4.1. Specificity Specificity was assessed by comparing the chromatograms of blank plasma, standard mixture containing IS, blank plasma spiked with standard mixture and IS, and rat plasma sample obtained 1 h after intragastric administration of PA. Representative chromatograms were presented in Fig. 1. It can be seen that GAS, HBA, PB, PC, PA and IS were sufficiently separated and no interfering peak was observed at the retention times of these compounds. 5. Linearity, LLOQ and LOD The calibration model was evaluated from a ten-concentration level plasma series with five replicates at each level. After processing the calibration data, the regression curve presented high linearity with a coefficient (r2 ) larger than 0.999 within the concentration range from 2.5 to 5000 ng/mL, and demonstrated acceptable precisions (RSD%, 0.15–14.76) and accuracies (RE%, −15.00 to 14.55) at these eight concentration levels (see supplementary data). The linear regression equation of calibration curves for GAS, HBA, PB, PC, PA was y = 0.0079x−0.0125, y = 0.0075x−0.0255, y = 0.0018x−0.0017, y = 0.0021x−0.0020 and y = 0.0023x + 0.0047, respectively. In the equation, y indicated the peak area ratio between an analytical compound and IS, while x indicated the concentration of this analyte. In addition, the LODs of GAS, HBA, PB, PC and PA were 0.6 ng/mL, 0.8 ng/mL, 1 ng/mL, 1 ng/mL and 1 ng/mL, respectively. The LLOQs of all targets were 2.5 ng/mL, which were considerable improvement over that of commonly used HPLCUV (limit of quantification for GAS: 50 ng/mL) [15] and HPLC–MS (limit of quantification for HBA: 830 ng/mL) [14]. Therefore, it was
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Fig. 2. Representative chromatograms of blank rat plasma (A), mixed standard solution with PA, PB, PC, GAS, HBA at 2.5 ng/mL and IS (B), rat plasma with PA, PB, PC, GAS, HBA at 2.5 ng/mL and IS (C), and rat plasma sample at 1 h after intragastric administration of PA at 375 mg/kg (D).
sufficient for pharmacokinetic study of PA following an intragastric administration of PA to rats.
the present method was reliable and reproducible for the quantitative analysis of PA and its metabolites in plasma samples.
6. Precision and accuracy
6.1.1. Recovery and matrix effect
The precision and accuracy results at low, medium and high concentrations of PA and its metabolites in rat plasma are demonstrated in Table 1. The results showed that precisions and accuracies at three concentration levels (5 replicates) were within ±6% and ±14%, respectively. The high precision and accuracy indicated that
Table 1 summarizes the matrix effect and recovery of measured compounds in plasma. The values of matrix effects for all targets were close to 1 and recoveries were over 80%, indicating that the matrix effect could be ignored and the method could be kept free from endogenous substances, without the additional
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Table 1 Intra- and inter-day precision and accuracy, matrix effect and recovery of GAS, HBA, PB, PC and PA in rat plasma. Compound
Concentration (ng/mL)
Precision and accuracy
Matrix effect
Intraday (n = 5)
Recovery
Interday (n = 5)
Mean ± SD
RSD (%)
RE (%)
Mean ± SD
RSD (%)
RE (%)
Mean ± SD (%)
RSD (%)
Mean ± SD (%)
RSD (%)
40 100 1000
44.70 ± 0.61 107.00 ± 1.61 1027.37 ± 2.87
1.37 1.51 0.28
11.7 7.00 2.74
42.47 ± 1.08 105.39 ± 3.38 981.74 ± 3.85
2.54 3.21 0.39
6.18 5.34 −1.82
95.73 ± 1.31 102.39 ± 1.07 99.19 ± 1.08
1.37 1.04 1.09
95.14 ± 4.90 100.28 ± 0.61 98.36 ± 1.76
5.15 0.61 1.79
HBA
40 100 1000
40.90 ± 0.56 98.91 ± 1.16 942.80 ± 4.38
1.37 1.17 0.46
2.24 −1.09 −5.72
39.08 ± 0.79 97.97 ± 2.79 907.65 ± 3.09
2.00 2.84 0.34
−1.89 −2.02 −9.24
100.88 ± 1.03 103.33 ± 1.86 99.06 ± 1.74
1.02 1.80 1.76
99.45 ± 2.14 101.59 ± 1.69 98.49 ± 2.40
2.15 1.67 2.43
PB
40 100 1000
38.47 ± 1.72 101.41 ± 4.41 1091.80 ± 8.47
4.48 4.34 0.78
−3.81 1.41 9.18
37.79 ± 1.49 105.69 ± 3.96 1016.93 ± 24.65
3.96 3.75 2.42
−5.52 5.69 1.61
93.16 ± 2.70 104.56 ± 1.40 99.79 ± 0.97
2.89 1.34 0.97
90.25 ± 8.10 102.20 ± 0.27 98.70 ± 0.95
8.97 0.26 0.96
PC
40 100 1000
45.24 ± 1.85 102.05 ± 3.03 942.11 ± 7.57
4.10 2.97 0.80
13.10 2.05 5.79
45.72 ± 1.77 103.1752 ± 5.62 900.79 ± 15.11
3.87 5.44 1.68
14.3 3.17 −9.92
92.70 ± 2.78 104.97 ± 1.41 100.12 ± 0.99
3.00 1.34 0.99
88.47 ± 6.55 104.66 ± 2.42 98.78 ± 1.37
7.40 2.31 1.38
PA
40 100 1000
39.81 ± 0.37 98.27 ± 3.09 950.58 ± 6.43
0.93 3.15 0.68
−0.47 −1.73 −4.94
2.30 2.11 3.39
−1.25 −3.58 0.95
103.46 ± 0.77 102.36 ± 1.37 99.10 ± 2.01
0.75 1.34 2.03
104.62 ± 7.01 100.08 ± 0.26 100.66 ± 1.56
6.70 0.26 1.55
39.50 ± 0.91 96.42 ± 2.04 966.09 ± 5.76
Table 2 Stability of GAS, HBA, PB, PC and PA in rat plasma. Compound
Concentration (ng/mL)
Mean ± SD
RSD (%)
RE (%)
Mean ± SD
RSD (%)
RE (%)
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
RE (%)
GAS
40 100 1000
41.74 ± 3.67 100.94 ± 0.69 980.42 ± 4.20
8.78 0.68 0.43
4.36 0.94 −1.96
44.11 ± 1.02 101.67 ± 0.44 978.18 ± 4.97
2.32 0.44 0.51
10.27 1.67 −2.18
44.57 ± 0.61 100.54 ± 0.06 968.99 ± 1.92
1.37 0.06 0.20
11.43 0.53 −3.10
44.42 ± 0.36 106.48 ± 1.89 1027.10 ± 3.29
0.82 1.77 0.32
11.05 6.48 2.71
HBA
40 100 1000
42.68 ± 2.64 100.44 ± 0.99 974.76 ± 2.74
6.18 0.99 0.28
6.70 0.44 −2.52
44.34 ± 0.76 100.73 ± 0.53 971.43 ± 4.11
1.71 0.52 0.42
10.87 0.73 −2.86
44.80 ± 0.36 99.48 ± 0.29 963.38 ± 3.50
0.80 0.30 0.36
12.01 −3.66 −3.66
40.85 ± 0.47 98.41 ± 1.12 944.90 ± 3.60
1.15 1.14 0.38
2.12 −1.58 −5.51
PB
40 100 1000
41.62 ± 3.10 97.88 ± 0.97 907.03 ± 3.98
7.45 0.99 0.44
4.04 −2.12 −9.30
40.91 ± 0.84 96.99 ± 0.43 905.94 ± 2.74
2.05 0.44 0.30
2.28 −3.00 −9.41
40.96 ± 0.65 96.56 ± 1.44 899.75 ± 2.19
1.59 1.49 0.24
2.39 −3.44 −10.03
39.16 ± 1.84 98.26 ± 1.24 1088.17 ± 9.13
4.71 1.26 0.84
−2.10 −1.73 8.82
PC
40 100 1000
43.58 ± 2.57 102.95 ± 1.28 937.48 ± 3.03
5.89 1.24 0.32
8.96 2.95 −6.25
46.77 ± 1.68 100.98 ± 0.85 936.17 ± 6.01
3.59 0.84 0.64
16.94 0.98 −6.38
46.21 ± 1.08 101.00 ± 1.08 929.58 ± 1.11
2.33 1.07 0.12
15.53 1.00 −7.04
45.86 ± 2.19 101.08 ± 3.84 939.11 ± 8.27
4.78 3.80 0.88
14.65 1.08 −6.09
PA
40 100 1000
42.21 ± 2.29 97.19 ± 5.50 914.31 ± 8.04
5.43 5.66 0.88
5.53 −2.81 −8.57
43.41 ± 0.90 91.00 ± 1.17 918.53 ± 5.99
2.08 1.29 0.65
8.52 −9.00 −8.14
43.59 ± 0.69 89.18 ± 0.86 910.14 ± 1.92
1.59 0.96 0.21
8.99 −10.82 −8.99
38.66 ± 2.45 98.60 ± 1.46 946.92 ± 2.20
6.34 1.48 0.23
−3.34 −1.40 −5.30
Short-term
Freeze–thaw
Long-term
24 h in autosampler RE (%)
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GAS
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concentration of 6.3 g/mL at 45 min. HBA, PB and PC reached the peak concentrations of 28, 955 and 592 ng/mL at 60, 60 and 30 min, respectively. GAS, HBA, PB and PC were eliminated rapidly in vivo and the concentrations of four metabolites fell to 343.2, 6.9, 40.7 and 25.0 ng/mL 8 h after the administration of parishin. 8. Conclusion
Fig. 3. Mean plasma concentration–time profile of GAS, HBA, PB, PC and PA after intragastric administration of PA at 375 mg/kg. PA, PB, PC, GAS corresponded with primary axis and HBA with secondary axis (n = 3).
cleanup procedures such as SPE and LLE before injection that was often necessary in LC–MS analysis owing to the existence of significant interference or ion suppression from endogenous substance in biological matrix. 7. Stability The stability results of short-term, three freeze–thaw, long-term and storage in the working temperature at three QC levels were summarized in Table 2. Briefly, three freeze–thaw cycles, long-term storage at −80 ◦ C and ambient temperature storage of the QC samples up to 6 h prior to sample preparation appeared to have no effect on the quantitation of all analytes except PC at the concentration of 40 ng/mL for three freeze–thaw cycles and long-term storage at −80 ◦ C. Thus, we supposed that three freeze–thaw cycles and longterm storage might influence quantitative analysis of PC at the low concentration in rat plasma. So the samples of PC are best not to be repeatedly frozen–thawed and long-term stored. In addition, extracted analytes were allowed to remain in the working temperature (4 ◦ C in autosampler) for 24 h throughout the duration of a typical sequence of chromatographic analyses. 7.1. Pharmacokinetic study of parishin The successful application of our developed method to pharmacokinetic study of parishin in rat plasma we performed. The plasma concentration–time profiles of GAS, HBA, PB, PC and PA after an intragastric administration of PA using a dose of 375 mg/kg are shown in Fig. 3. The concentration of PA and metabolites was readily measurable in plasma sample. The results showed that parishin was completely metabolized to GAS, HBA, PB and PC 5 min after the administration of parishin. GAS reached a peak
This study presented a rapid, sensitive and robust UHPLC-FLD method for simultaneous analysis of parishin and its metabolites in rat plasma for the first time. The analytical parameters, including limit of detection, recovery and linear range, precision, accuracy, matrix effect and stability, matched the requirements for monitoring all target compounds. The application of this method allowed successfully determining the pharmacokinetics of parishin, which showed the rapid degeneration of parishin in vivo and fast increases of metabolites in plasma of rats. Acknowledgments This work is financially supported by the National Natural Science Foundation of China (Grant No. 81001629). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2014.08.020. References [1] C.L. Hsieh, S.Y. Chiang, K.S. Cheng, Y.H. Lin, N.Y. Tang, C.J. Lee, C.Z. Pon, C.T. Hsieh, Am. J. Chinese Med. 29 (2001) 331–341. [2] Y. Zhang, NHBA isolated from Gastrodia elata exerts sedative and hypnotic effects in sodium pentobarbital-treated mice (vol 102, pg 450, 2012), Pharmacology Biochemistry and Behavior, 102 (2012) 593-593. [3] C.L. Huang, J.M. Yang, K.C. Wang, Y.C. Lee, Y.L. Lin, Y.C. Yang, N.K. Huang, J. Ethnopharmacol. 138 (2011) 162–168. [4] Y.W. Jang, J.Y. Lee, C.J. Kim, Int. Immunopharmacol. 10 (2010) 147–154. [5] S.M. Hwang, Y.J. Lee, D.G. Kang, H.S. Lee, Am. J. Chinese Med. 37 (2009) 395–406. [6] L. Wang, H.B. Xiao, X.M. Liang, L.X. Wei, J. Sep. Sci. 30 (2007) 1488–1495. [7] S.Y. Huang, G.Q. Li, J.G. Shi, S.Y. Mo, S.J. Wang, Y.C. Yang, J. Asian Nat. Prod. Res. 6 (2004) 49–61. [8] H.B. Li, F. Chen, J. Chromatogr. A 1052 (2004) 229–232. [9] C.M. Shu, C.G. Chen, D.P. Zhang, H.P. Guo, H. Zhou, J. Zong, Z.Y. Bian, X. Dong, J. Dai, Y. Zhang, Q.Z. Tang, Mol. Cell. Biochem. 359 (2012) 9–16. [10] X.H. Zeng, Y. Zhang, S.M. Zhang, X.X. Zheng, Biol. Pharm. Bull. 30 (2007) 801–804. [11] L. Wang, Y.J. Lei, H.B. Xiao, 37th International Symposium on High Performance Liquid Phase Separations and Related Techniques, 2011. [12] X.D. Yang, J. Zhu, R. Yang, J.P. Liu, L. Li, H.B. Zhang, Nat. Prod. Res. 21 (2007) 180–186. [13] Y.K. Zhao, Q.E. Cao, Y.Q. Xiang, Z.D. Hu, J. Chromatogr. A 849 (1999) 277–283. [14] W. Zhang, Y.X. Sheng, J.L. Zhang, Phytomedicine 15 (2008) 844–850. [15] X.H. Ju, Y. Shi, N. Liu, D.M. Guo, X. Cui, J. Chromatogr. B 878 (2010) 1982–1986. [16] L.C. Lin, Y.F. Chen, W.C. Lee, Y.T. Wu, T.H. Tsai, J. Pharmaceut. Biomed. 48 (2008) 909–917.
