Journal of Chromatography B, 945–946 (2014) 185–192

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Simultaneous determination of imperatorin and its metabolite xanthotoxol in rat plasma by using HPLC–ESI-MS coupled with hollow fiber liquid phase microextraction Juan Zhang a,1 , Min Zhang b,1 , Shan Fu a , Tao Li a , Shuang Wang a , Minmin Zhao a , Weijing Ding a , Chunying Wang a , Qiao Wang a,∗ a b

Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang, 050017, PR China Quality Control Office, Hebei Provincial Chest Hospital, Shijiazhuang, 050041, PR China

a r t i c l e

i n f o

Article history: Received 12 October 2013 Accepted 25 November 2013 Available online 3 December 2013 Keywords: Imperatorin Xanthotoxol Hollow fiber liquid phase microextraction Pharmacokinetics HPLC–ESI-MS

a b s t r a c t The objective of the present study was to develop a new method for the simultaneous quantitation of imperatorin and its metabolite xanthotoxol in rat plasma. The samples were prepared with hollow fiber liquid phase microextraction (HF-LPME). The optimized extraction procedure was acquired by assessing extraction solvent, length of the fiber, agitation rate, extraction temperature and time. A comparison of sample pretreatment ways between HF-LPME and deproteinization with methanol was performed, which demonstrated less ion suppression and better sensitivity of HF-LPME. Analytes were separated on a C18 column with a gradient elution consisted of methanol and water containing 1 mmol/L ammonium acetate. The detection was accomplished by electrospray ionization (ESI) source operating in the positive ionization mode. Selected-multiple-reaction monitoring (SMRM) scanning was employed, which guaranteed a higher sensitivity compared with MRM mode. Calibration curves were linear over investigated ranges with correlation coefficients greater than 0.9979. Precision varied from 0.26% to 14%, and the accuracy varied within ±5.5%. The developed method was successfully applied to the pharmacokinetic research of imperatorin and its metabolite xanthotoxol after oral administration of imperatorin to rats. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Imperatorin, an active natural furocoumarin, originates from many medicinal plants such as Glehnia littoralis Fr. Schmidt ex Miq and Peucedanum Praeruptorum Dunn. It has been reported that imperatorin possesses many pharmacologic actions including anticonvulsion [1], anti-inflammatory [2], vasodilation [3], inhibiting the activity of cytochrome P450 enzyme [4] and anti-hypertension [5]. Evaluation of the pharmacokinetic properties of imperatorin is quite necessary due to the high importance in determining whether imperatorin can be pursued as a chemical entity. To date, some studies on imperatorin pharmacokinetics in rat plasma [5–9] and rat liver [9] after oral administration of imperatorin have been reported. There have also been a few reports on imperatorin metabolites. In 2012, two metabolites of imperatorin, imperatorin

∗ Corresponding author at: Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang 050017, PR China. Tel.: +86 311 86265625; fax: +86 311 86266419. E-mail address: [email protected] (Q. Wang). 1 These authors contributed equally to this work 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.11.050

hydroxylate and imperatorin epoxide, were found and quantitated in dog plasma after intravenous administration of imperatorin [10]. Recently, 51 metabolites of imperatorin have been identified in rat urine in our lab [11], in which we found that xanthotoxol displayed obvious and stronger response among these metabolites. Moreover, during our preliminary experiment, xanthotoxol was also mainly detected in rat plasma after oral administration of imperatorin. These suggested that xanthotoxol was one of the main metabolites of imperatorin and it would be informative to employ xanthotoxol as one of marker compounds for pharmacokinetic study of imperatorin. Until now, xanthotoxol has not yet been determined either alone or along with imperatorin in rat plasma. Some methods, including HPLC-UV [6,7,9], GC–MS [5] and LC–MS [8,10], have been developed to quantitate imperatorin and metabolites in plasma after oral administration of imperatorin. Among these methods, LC–MS method was more attractive than other methods due to its high sensitivity and specificity. Nevertheless, this method was still faced with the challenge of matrix effect [12–14]. Various sample preparation methods, such as liquid–liquid extraction (LLE) [15–17] and solid-phase extraction (SPE) [18–20] have been established to reduce the matrix effect. However, they also suffer from some drawbacks, such as high labor intensity, high cost and large mounts of toxic solvent. In the year of

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1999, HF-LPME was introduced [21] and afterwards, this method was widely applied to the analysis of biological samples [22–34] because it not only can avoid above shortcomings but also has the advantage of excellent sample cleanup ability. In the present study, a HPLC–ESI-MS coupled with HF-LPME method was first developed to the determination of imperatorin and xanthotoxol in rat plasma. The extraction procedure was optimized by assessing extraction solvent, length of the fiber, agitation rate, extraction temperature and time. A comparison of sample pretreatment ways between HF-LPME and deproteinization with methanol was performed, which demonstrated less ion suppression and better sensitivity of HF-LPME. The method was then applied to the pharmacokinetic study of imperatorin and its metabolite xanthotoxol after oral administration of imperatorin. 2. Experimental 2.1. Reagents and materials Imperatorin (purity ≥ 98%) was purchased from Shanghai Sunny Biotech Co., Ltd. Xanthotoxol (purity ≥ 98%) was purchased from Shanghai Tauto Biotech (Shanghai, China) Scopoletin (purity ≥ 98%) was obtained from National Institutes for Food and Drug Control, Beijing, China. n-Propanol, n-butanol n-pentanol and acetone were bought from Tianjin Yongda Chemical Reagent Co., Ltd. Hexylalcohol, n-heptanol and n-octyl alcohol were bought from Tianjin Forever Chemical Co., Ltd. Acetic ether was bought from Tianjin Hedong District Red Rock Reagent Factory. HPLC grade methanol was provided by J.T. Baker. Ammonium acetate and formic acid were from Dikma Technologies Incorporation. Ultrapurified water was purchased from Hangzhou Wahaha Group Co. Ltd. Polyvinylidene difluoride hollow fiber (pore: 0.2 ␮m; inner diameter: 0.7 mm) was provided by Tianjin Motmo Membrane Technology Co. Ltd.

Analyst software (versions 1.5.2) was used for data acquisition and processing. 2.3. Preparation of standard solution The appropriate amount of imperatorin and xanthotoxol was exactly weighed and dissolved respectively in methanol to prepare stock standard solution. Then, the two stock solutions were mixed and diluted with methanol to prepare a final mixed standard solution containing 4890 ng/mL of imperatorin and 706.6 ng/mL of xanthotoxol, respectively. A series of working solutions of the two analytes were freshly prepared by diluting mixed standard solution with methanol at the appropriate ratios to yield concentration from 38.20 to 4890 ng/mL for imperatorin and from 5.520 to 706.6 ng/mL for xanthotoxol, respectively. The internal standard solution was prepared by dissolving scopoletin with methanol to 617.3 ng/mL. For the validation of the method, three concentration levels of standard solution containing imperatorin (76.40, 305.6 and 2445 ng/mL) and xanthotoxol (11.04, 44.16 and 353.3 ng/mL) were used for preparing QC plasma samples. 2.4. Animals Six male Sprague–Dawley rats (weighed 250 ± 20 g, Experimental Animal Research Center, Hebei Medical University, China) were used in this study. The rats were housed under controlled environmental conditions (temperature: 25 ± 2 ◦ C; humidity: 55 ± 5%; 12-h dark/12-hlight cycle) for at least 5 days before the experiments. The animals were fed with food and water ad libitum and were fasted overnight prior to the test. All animal experiments were carried out according to the Guidelines for the Care and Use of Laboratory Animals, and approved by the Animal Ethics Committee of Hebei Medical University. 2.5. Drug administration and blood sampling

2.2. Instrumentation and conditions 2.2.1. Liquid chromatography Agilent 1200 liquid chromatography system (Agilent, USA) equipped with a quaternary solvent delivery system, an autosampler and a column compartment was used. The analytes were separated on an Agilent Zorbax SB-C18 column (150 mm × 4.6 mm, 5 ␮m). The column temperature was maintained at 25 ◦ C. The mobile phase consisted of methanol (A) and water containing 1 mmol/L ammonium acetate (B). The gradient condition was as follows: 50% A linear gradient to 95% A over 5 min, held at 95% A for the next 3.5 min, and returned to 50% A over 0.1 min. The flow rate was set at 0.8 mL/min and the injection volume was 10 ␮L. 2.2.2. Mass spectrometer The mass spectrometer was a QTRAPTM 3200 with Turbo V sources and Turbo Ionspray interface from Applied Biosystems (Applied Biosystems, Foster City, CA, USA). The turboionspray interface operated in positive ionization mode was used. Typical source conditions were set as follows: ion spray needle voltage 5500 V; turbo spray temperature 650 ◦ C; nebulizer gas 60 psi; heater gas 65 psi; curtain gas 25 psi. Interface heater was on. Nitrogen was used in all cases. Analytes were quantified under selected-multiple-reaction monitoring (SMRM) mode using the following precursor-to-product ion pair and parameters: Imperatorin, m/z 271.1 → 203.2 with DP 25 V and CE 15 eV, and the selected time was 7.32 min; Xanthotoxol, m/z 203.2 → 147.1 with DP 20 V and CE 32 eV, and the selected time was 4.38 min; Scopoletin (IS) m/z 193.1 → 133.1 with DP 49 V and CE 29 eV, and the selected time was 3.48 min; the detection windows of all the analytes were 60 s. All of the dwell time was set at 50 ms. Applied Biosystems/MDS Sciex

Imperatorin was suspended in 0.3% CMC-Na and orally administrated to rats at a dose of 80 mg/kg. Blood samples were collected at 0.083, 0.33, 0.67, 1, 1.5, 2, 4, 6, 9, 12 and 24 h after dosing, 0.3 mL of venous blood samples were collected at the rat’s medial angle of eye with heparinized capillary and then put in 1.5 mL heparinized tube. The blood samples were then centrifuged at 4000 rpm for 5 min at room temperature, and the plasma was separated and stored at −80 ◦ C for later analysis. 2.6. Pretreatment procedure of the samples 2.6.1. Real samples 20 ␮L of internal standard solution was placed in a sample vial with a screw top and evaporated to dryness at room temperature under a gentle stream of nitrogen. 100 ␮L of thawed plasma sample was added to the vial and then diluted with 2% NaCl solution to a total volume of 1.5 mL. After the mixture was vortexed for 2 min (served as aqueous B), a magnetic stirrer bar was put into the vial. Before using, the hollow fiber was ultrasonically cleaned in acetone-water (50:50) for 10 min in order to remove any contaminants and then dried in air. Immediately afterwards, the hollow fiber was cut manually into 5 cm length pieces, and then the pieces were immersed in n-heptanol (organic solvent, acceptor phase) for around 1 min in order to impregnate its pores with organic solvent. Then the fiber paper was employed to blot the organic solvent on the ektexine gently. 20 ␮L of n-heptanol was injected into the hollow fiber using a microsyringe and a knot was tied at the length of 5 cm. Then the part of fiber under the knot was then immersed in the diluted plasma sample, afterwards, the sample vial was stirred at room temperature with a magnetic stirrer to facilitate for 50 min.

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At the end of the extraction time, the fiber was removed from the sample and its closed end was cut and the extract was blown off by a microsyringe, followed by being eluted using 200 ␮L of ethyl acetate. Both the n-heptanol and ethyl acetate solutions were placed in a 1.5 mL centrifuge tube. The mixture was evaporated to dryness at room temperature under a gentle stream of nitrogen. The residue was dissolved in 20 ␮L of methanol-water (50:50), and the mixture was vortexed for 2 min. After centrifugation at 14000 rpm/min for 10 min, 10 ␮L aliquot of the supermatant (served as aqueous A) was injected into the LC–MS/MS system for analysis. 2.6.2. QC samples 20 ␮L internal standard solution and 50 ␮L standard solution (three concentration levels) were placed in a sample vial with a screw top and the mixture was evaporated to dryness at room temperature under a gentle stream of nitrogen. 100 ␮L of thawed blank plasma was added to the vial, and then three concentration QC samples for imperatorin (76.40, 305.6 and 2445 ng/mL) and xanthotoxol (11.04, 44.16 and 353.3 ng/mL) were obtained. The QC samples were pretreated as described in Section 2.6.1. 2.7. Method validation 2.7.1. Specificity Samples of rat blank plasma, blank plasma spiked with analytes and IS, and plasma after gavage of imperatorin and xanthotoxol were tested. 2.7.2. Calibration curve and limit of quantitation The calibration plasma samples were prepared as described in Section 2.6.2. The ratios of peak area of the analytes to that of IS were plotted against nominal concentrations of the analytes and standard curves were in the form of y = ax + b (1/x2 weighed). The values of limit of quantitation (LOQ) were defined as the amount that could be detected with a signal-to-noise ratio of 10. 2.7.3. Accuracy and precision Intra- and inter-day precision and accuracy of the analytical method were estimated by analyzing six replicates of QC samples at low, medium and high concentration levels pretreated as described in Section 2.6.2 on three different days. Each concentration was calculated using a calibration curve constructed on the same day. The accuracy was evaluated in terms of relative error (RE) and the precision for intra-day and inter-day was evaluated by relative standard deviation (RSD). 2.7.4. Stability The stability of each analyte in rat plasma was investigated by subjecting QC samples to various conditions. Long-term stability was evaluated by freezing QC samples at −80 ◦ C for 10 days. The autosampler stability was assessed by placing processed QC samples in the autosampler at 22 ◦ C for 24 h. For freeze-thaw stability, QC plasma samples were subjected to three cycles from −80 ◦ C to room temperature. 2.7.5. Matrix effect Matrix effect experiments were investigated by continuous post column infusion (10 ␮L/min) of standard solutions of three analytes, respectively. During the infusion, blank plasma samples were injected to LC–MS/MS analysis under the chromatographic conditions as described in Section 2.2, and the influence of blank plasma samples on MS/MS responses of the MRM transitions for imperatorin, xanthotoxol and scopoletin was monitored.

Fig. 1. The product ion scan spectra, chemical structures, monitored transitions, declustering potential (DP) and collision energy (CE) of imperatorin, xanthotoxol and scopoletin (IS).

2.7.6. Carry-over Carry-over was assessed by injecting blank samples immediately after the ULOQ (upper limit of quantification, 2445 ng/mL for imperatorin and 353.3 ng/mL for xanthotoxol) sample of the standard curve, the acceptance criterion of the carry-over is the peak area of each analyte in the blank sample account for less than 20% of the sample of lowest concentration on the calibration curve [35]. 3. Results and discussion 3.1. Mass spectrometry The first step in developing the HPLC–MS method was to select precursor ions and product ions of the analytes and IS for SMRM mode analysis from the characteristic mass spectra by syringe pump infusion. The positive electrospray interface was used for high sensitivity, and reproducibility and fragmentation were obtained. In the full scan mass spectra of imperatorin, xanthotoxol and IS, precursor ions [M+H]+ of m/z 271.1, m/z 203.2 and m/z 193.1 were stable and exhibited higher abundance, respectively. Under the product ion scan mode, the most intensive ions were [M+H-C5 H8 ]+ (m/z 203.2) for imperatorin, [M+H-2CO]+ (m/z 147.1) for xanthotoxol and [M+H-CO-OCH3 ]+ (m/z 133.1) for IS. Fig. 1 shows the product ions scan spectra of the analytes and IS. In the present study, SMRM mode was used. It could not only provide much higher sensitivity for all the targeting analytes, but also dramatically broaden their dynamic ranges [36]. In this mode, the selection of retention time and detection window of great significance, for this desirable strategy and should promise a repeatable peak for all the target analytes. In our experiment, the retention time for each analyte was ensured first and all the analytes could be eluted in 60 s, so the detection window was set at 60 s. 3.2. Chromatography The mobile phase plays an important role in retention behavior and peak shape. The electrolyte-free mobile phase water/methanol and electrolyte mobile phase (different electrolytes including acetic acid, formic acid and ammonium acetate in water phase) were tested. It was found that better sensitivity and peak shape could be obtained by adding 1 mmol ammonium acetate to water phase.

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For xanthotoxol is a metabolite of imperatorin, the polarity of xanthotoxol is stronger than imperatorin, which made the retention time of imperatorin much longer than that of xanthotoxol. Thus, we chose a gradient elution to obtain a suitable retention of the two analytes in a chromatogram.

the peak areas of two analytes kept decreasing as the temperature increased from 25 ◦ C (room temperature) to 40 ◦ C. When the temperature exceeded 40 ◦ C, the peak areas began to increase and reach its maximum at 45 ◦ C, which was similar to that of 25 ◦ C. Therefore, the extraction temperature was held at 25 ◦ C in further experiments.

3.3. Optimization of the HF-LPME procedure In order to develop a HF-LPME method for the determination of imperatorin and xanthotoxol in plasma samples, some conditions influencing extraction efficiency, such as extraction solvent, length of the fiber, agitation rate, extraction temperature and time were investigated and optimized. 3.3.1. Selection of the organic extraction solvent It was important to choose a suitable organic extraction solvent in LPME method, and some universalistic requirements should be met. First of all, those selected solvents should ensure high enrichment for those target analytes, and then they should be of low volatility to prevent evaporation, and low polarity to ensure compatibility with the hollow fiber and immiscible with water. For the reasons mentioned above, acetone, chloroform, n-propanol, nbutanol, n-pentanol, hexylalcohol, n-heptanol and n-octyl alcohol were selected to investigate the extraction efficiency. As a result, acetone and chloroform were eliminated because of their strong volatility, and n-propanol was not chosen for the reason that it could be soluble with water. The results are shown in Fig. 2(A). It can be seen that the highest chromatographic peak areas of the two target analytes were obtained when n-heptanol was used. Thus, n-heptanol was selected for subsequent experiments. 3.3.2. Effect of length of the fiber Fiber length affects the experiments by means of different volumes of the extraction solvent. The length of the hollow fiber was separately set at 5, 8, 10, 12, 15 cm and the corresponding extraction solvent volumes were 20, 30, 40, 50, 60 ␮L, respectively. Each sample was redissolved by the corresponding volume of methyl alcohol–water (50:50). Fig. 2(B) shows that higher chromatographic peak area was obtained when lower volume of n-heptanol was used. But too small volume might result in little extraction solvent received in the end. Based on these results, a hollow fiber of 5 cm length was used for the optimization of the parameters. 3.3.3. Effect of stirring speed Agitation promises new and fresh interface between aqueous phase and organic phase continuously and improves mass transfer of target compounds through the organic solvent in the pores of the fiber, thus the extraction can be accelerated by stirring of the sample solution. But a too high stirring rate may not only cause air bubbles to attach to the fiber surface and limit the mass transfer of analytes but also enhance the diffusion/dissolution of analytes from organic liquid on membrane into sample solution, which caused the decrease in extraction efficiency. In addition, it can also lead to the loss of organic liquid impregnated in the membrane [37]. The reasons mentioned above make a fact that high stirring speed can decrease the extraction ability. All the experiments were carried out at stirring speeds ranging from 225 to 900 rpm. The results are shown in Fig. 2(C). It suggested that the peak areas of analytes reached the highest at 675 rpm. 3.3.4. Selection of temperature Owing to the influences on the rates of mass transfer partition coefficients of the analytes, temperature plays an important role in the extraction process. A series of extraction temperatures were studied. The results are shown in Fig. 2(D), and it can be seen that

3.3.5. Selection of dilution times Plasma sample must be diluted prior to experiments for the reasons that sample is limited and plasma is too sticky to be operated using hollow fiber which may result in blocking up the pores of hollow fiber. Nevertheless, dilution may influence extraction efficiency by decreasing the concentration of analytes, thus it is essential to choose a suitable dilution times. The experiments were studied by diluting 100 ␮L plasma to 1 mL, 1.25 mL, 1.5 mL, 2 mL and 2.5 mL by water. It can be seen from Fig. 2(E) that when diluted to1.5 mL, namely the times of dilution was 15, analytes presented the maximum response, so the times of dilution of 15 was used in our further experiments. 3.3.6. Salt effect Based on the properties of target analytes, it was inferred that ionic strength of aqueous solution may have effects on extraction efficiency [38]. Studies were performed to investigate the salt effect on HF-LPME by diluting 100 ␮L plasma with NaCl solution which containing 0, 1, 2, 3, 4, and 5% (w/v) NaCl. It was found that the responses of the analytes increased with the increase of NaCl concentration until 2%, and then decreased and the results are shown in Fig. 2(F). This phenomenon might result from the fact that proper concentrations of NaCl could reduce the affinity of coumarins in donor phase and increase extraction efficiency. Therefore, the excess of the ionic strength could affect diffusion of the analytes into the organic phase because of the electrostatic interaction between salt ions and analytes in solution. So, 2% NaCl aqueous solution was chosen in our experiment. 3.3.7. Selection of extraction time Extraction time affects extraction efficiency. Hence, the tendency of peak responses of analytes with extraction time was studied. Fig. 2(G) shows that the peak areas of analytes were increased obviously with increased extraction time from 20 to 50 min, and then the plot showed a little decrease. Therefore, an extraction time of 50 min was employed in further experiments. To sum up, the optimum conditions of extraction were as follows: 100 ␮L plasma was diluted to 1.5 mL by 2% (w/v) NaCl aqueous, and then the analytes were extracted by n-heptanol for 50 min with 675 rpm at room temperature. 3.4. Enrichment factor of analytes Enrichment factor (EF) was defined as the ratio of the analyte peak area after extraction to that without extraction. In the present study, EF was calculated by PA /PB , where PA and PB were the peak areas of each analyte in aqueous A and aqueous B (aqueous A and aqueous B were described in Section 2.6.), respectively. The EF of imperatorin and xanthotoxol were 20.48 and 21.51, respectively. 3.5. Method validation The selectivity of the method toward endogenous plasma matrix was evaluated in six rat plasma. The typical chromatograms of blank plasma, plasma sample spiked two analytes at the LOQ and IS, and the plasma sample from a rat after oral administration of imperatorin are shown in Fig. 3. A conclusion can be drawn that no endogenous components in rat plasma were observed to produce

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Fig. 2. Optimization of the HF-LPME procedure. (A) Selection of the organic extraction solvent (conditions: length of the fiber, 12 cm; stirring speed, 450 rpm; extraction temperature, 25 ◦ C; the times of dilution,15; NaCl, 0%; extraction time, 30 min, respectively). (B) Effect of length of the fiber (conditions: extraction solvent, heptanol; stirring speed, 450 rpm; extraction temperature, 25 ◦ C; the times of dilution, 15; NaCl, 0%; extraction time, 30 min, respectively). (C) Effect of stirring speed (conditions: length of the fiber, 5 cm; extraction temperature, 25 ◦ C; the times of dilution, 15; NaCl, 0%; extraction time, 30 min, respectively). (D) Selection of temperature (conditions: extraction solvent, heptanol; length of the fiber, 5 cm; stirring speed, 675 rpm; the times of dilution, 15; NaCl, 0%; extraction time, 30 min, respectively). (E) Selection of dilution times (conditions: extraction solvent, heptanol; length of the fiber, 5 cm; stirring speed, 675 rpm extraction temperature, 25 ◦ C; NaCl, 0%; extraction time, 30 min, respectively). (F) Salt effect (conditions: extraction solvent, heptanol; length of the fiber, 5 cm; stirring speed, 675 rpm; extraction temperature, 25 ◦ C; the times of dilution, 15; extraction time, 30 min, respectively). (G) Selection of extraction time (conditions: extraction solvent, heptanol; length of the fiber, 5 cm; stirring speed, 675 rpm; extraction temperature, 25 ◦ C; the times of dilution, 15; NaCl, 2%, respectively).

Fig. 3. Representative SMRM chromatograms of imperatorin, xanthotoxol and scopoletin (IS). Each column referred to different constituents (A: blank plasma; B: blank plasma spiked with two analytes at LOQ and IS; C: sample plasma 4 h after a single oral administration of imperatorin).

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Table 1 The intra- and inter-day accuracies and precisions of imperatorin and xanthotoxol in rat plasma at low, medium, and high concentration levels. Compounds

Spiked conc. (ng/ml)

Intra-day (n = 6)

Inter-day (n = 3)

Measured conc. (ng/ml)

Accuracy (%)

Precision (%)

Measured conc. (ng/ml)

Accuracy (%)

Precision (%)

Imperatorin

38.20 152.8 1222

40.30 156.3 1177

5.5 2.3 −3.7

3.6 2.0 3.8

38.53 153.8 1213

0.85 0.67 −0.72

14 14 1.8

Xanthotoxol

5.520 22.08 176.6

5.820 24.01 174.1

5.4 8.7 −1.4

0.46 2.3 0.26

5.580 22.66 166.7

1.2 2.7 −5.5

3.1 3.2 2.6

any interfering peaks. The retention times of imperatorin, xanthotoxol and IS were 7.32 min, 4.38 min and 3.48 min, respectively. All the peaks of the analytes and IS in plasma samples were unambiguously identified by comparison of retention time, parent and product ion with reference standards. The regression equations and correlation coefficients (r2 ) of imperatorin and xanthotoxol were y = 0.00241x + 0.00802, r2 = 0.9983, and y = 0.0027x + 0.00335, r2 = 0.9979, respectively. The calibration curves were linear in the ranges of 19.10–2445 ng/mL for imperatorin and 2.760–335.8 ng/mL for xanthotoxol, respectively. LOQ of the two compounds were 0.7000 ng/mL and 1.730 ng/mL for imperatorin and xanthotoxol, respectively. Table 1 summarizes the intra- and inter-day precisions and accuracies of imperatorin and xanthotoxol at three concentration levels (low, middle and high). The intra- and inter-day precisions (RSD) of these analytes were all less than 5.6% and 14%. The accuracies (RE) were within 15%. As seen in Table 2, the analytes in rat plasma were stable for 10 days at −80 ◦ C, 24 h after pretreatment at room temperature and three freeze (−80 ◦ C)-thaw (room temperature) cycles, and all RE values between post-storage and initial QC samples were

within ±15%, which demonstrated a good stability of imperatorin and xanthotoxol over all steps of the determination. It can be seen from Fig. 4 that ion suppression occurred from 1.5 min to 2.5 min, while the earliest retention time in this experiment was 3.48 min, which indicated that all the measurement were free of the influence of plasma matrix. When blank samples were injected immediately after the ULOQ sample of the standard curve, a slight carry-over of imperatorin was found. An aqueous solution of methanol-water (50:50) containing 1% formic acid was used to wash the needle, by which the adhesion of imperatorin on the needle ektexine might be decreased. As a result, the peak area of analytes obtained from the blank sample injected after the ULOQ sample was lower than that of 20% of the average of three LLOQ samples, while the peak area of IS was lower than that of 5% of standard curve. It was shown that the carry-over would not influence the detection.

3.6. Comparison of HF-LPME and deproteinization with methanol Deproteinization with methanol is a traditional and common method for the pretreatment of plasma samples. In this study, a

Fig. 4. Post-column infusion chromatograms of imperatorin, xanthotoxol, and scopoletin (IS) for LC–ESI-MS/MS analysis of methyl alcohol (A), blank sample pretreated by HF-LPME (B), and blank sample pretreated by methanol protein deposition (C). The mobile phases used in the experiments were 1 mmol ammonium and methanol with a flow rate of 0.8 ml/min.

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Table 2 Stability of imperatorin and xanthotoxol in rat plasma (n = 3). Compounds

Spiked conc. (ng/ml)

Long-term stability (10 days at −80 ◦ C)

Autosampler stability (24 h at room temperature)

Freeze–thaw stability (three cycles)

Measured conc.a (ng/ml)

Accuracy (%)

Measured conc.a (ng/ml)

Accuracy (%)

Measured conc.a (ng/ml)

Accuracy (%)

Imperatorin

38.20 152.8 1222

40.64 ± 1.8 163.8 ± 4.0 1230 ± 4.1

6.4 7.2 0.64

38.87 ± 3.0 140.8 ± 5.8 1163 ± 88

1.8 −7.9 −4.9

40.20 ± 2.0 141.3 ± 2.2 1206 ± 14

5.3 −7.6 −1.3

Xanthotoxol

5.520 22.08 176.6

6.078 ± 0.20 20.39 ± 2.1 187.3 ± 5.3

11 −7.7 6.1

5.880 ± 2.2 21.87 ± 0.89 184.3 ± 1.3

6.6 −0.95 4.4

6.067 ± 0.28 22.08 ± 1.9 182.4 ± 4.4

10 −4.4 3.3

a

Mean ± standard deviation.

Table 3 Pharmacokinetic parameters of imperatorin and xanthotoxol after oral gavaged of imperatorin. Analytes

T1/2 (h)

Cmax (ng/mL)

Tmax (h)

Ke (1/h)

AUC0→t (ng h/mL)

AUC0→∞ (ng h/mL)

Imperatorin Xanthotoxol

7.93 7.95

441.8 56.6

1.01 4.0004

0.079 0.1163

91777.59 25484.5

106851 29952.9

T1/2 : biological half-time; Cmax : peak concentration; Tmax : peak time; Ke : elimination rate constant; AUC: area under concentration–time curve.

comparison of sample pretreatment ways between HF-LPME and deproteinization with methanol was also performed. Six spiked samples (152.8 ng/mL for imperatorin and 22.08 ng/mL for xanthotoxol) were divided into two groups. Three of them were treated with HF-LPME described in Section 2.6.2 and the other three were operated by deproteinization with methanol. The process of deproteinization with methanol was as follows: 100 ␮L of spiked sample and 300 ␮L of methanol were placed in a 1.5 mL centrifuge tube and the mixture were vortexed for 2 min. After centrifugation at 14000 rpm for 10 min, 10 ␮L aliquot of the supernatant was injected into the LC–MS/MS system for analysis. Peak areas were used to compare the sensitivity, the average peak areas of imperatorin and xanthotoxol were 4.77 e4 and 2.39 e3 respectively for the method of deproteinization with methanol, but 4.08 e5 and 2.38 e4 respectively for the method of HF-LPME. The increase in peak area revealed that HF-LPME could concentrate the analytes in the samples which improved the sensitivity of the method. Actually, this new method guaranteed a lower LOQ 0.7000 ng/mL compared with either GC [5] or LC–MS/MS method [8] which both were 1.000 ng/mL. Fig. 4 describes matrix effect of samples pretreated by HF-LPME and deproteinization with methanol. It can be seen that obvious reversal peaks were observed in the chromatograms of plasma samples pretreated by deproteinization with methanol, which was much more remarkable than that of plasma samples pretreated by HF-LPME. This indicated that it was easy for deproteinization with methanol to produce significant ion suppression for all the analytes, while HF-LPME could obtain much clearer sample and create little matrix effect. This could contribute to improving the lifetime of mass spectrometer and shortening the retention time of analytes. 3.7. Pharmacokinetic study This validated HPLC–ESI-MS coupled with HF-LPME method was successfully applied to the simultaneous determination of imperatorin and xanthotoxol in rat plasma after oral gavage of 80 mg imperatorin per kilogram of bodyweight. The mean plasma concentration-time profiles of imperatorin and xanthotoxol are illustrated in Fig. 5 and the pharmacokinetic parameters are presented in Table 3. As seen from Table 3, T1/2 was calculated to be 7.93 h and 9.13 h for imperatorin and xanthotoxol respectively, and the T1/2 of imperatorin was similar to the result reported in 2010 [9].

Fig. 5. Mean plasma concentration–time curves of imperatorin and xanthotoxol after gavage of imperatorin.

Imperatorin was absorbed quickly after oral administration and acquired the maximum concentration at 1.01 h, which was consistent with the conclusion in previous paper [5]. The metabolite xanthotoxol occurred at 5 min with a tenuous signal and increased slowly within 2 h, and then reached a maximum concentration at 4.00 h. The values of the Ke were 0.079 and 0.078 for imperatorin and xanthotoxol respectively and the former agreed with the outcome of Guangchao Ding [8]. In addition, a double-peak phenomenon was presented in concentration-time profiles of xanthotoxol. The first peak appeared at about 4 h, while the second peak appeared at about 9 h which was much lower than the first one. 4. Conclusion For the first time, a sensitive and specific HPLC–ESI-MS method coupled with HF-LPME was developed for the simultaneous quantification of imperatorin and its metabolite xanthotoxol in rat plasma. The developed method showed significantly weakened ion suppression effects and guaranteed a lower LOQ compared with other methods. It was proved to be suitable for the pharmacokinetic

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study of imperatorin and xanthotoxol. In addition, the quantification and pharmacokinetic parameters of xanthotoxol in rat plasma were studied for the first time in present study.

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Acknowledgement

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The authors sincerely thank financial supports from the National Natural Science Foundation of China (81102412), the Ministry of Education Key Project of Science and Technology Foundation of China (211021), and the Natural Science Foundation of Hebei Province of China (C2011206158, 08B031).

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Simultaneous determination of imperatorin and its metabolite xanthotoxol in rat plasma by using HPLC-ESI-MS coupled with hollow fiber liquid phase microextraction.

The objective of the present study was to develop a new method for the simultaneous quantitation of imperatorin and its metabolite xanthotoxol in rat ...
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