Cloud point extraction–HPLC method for the determination and pharmacokinetic study of aristolochic acids in rat plasma after oral administration of Aristolochiae Fructus

Journal of Chromatography B, 953–954 (2014) 73–79

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

Cloud point extraction–HPLC method for the determination and pharmacokinetic study of aristolochic acids in rat plasma after oral administration of Aristolochiae Fructus Gang Ren a,1 , Qun Huang a,1 , Jiangang Wu b , Jinbin Yuan a,∗ , Gaihong Yang a , Zhihong Yan a,b , Shouzhuo Yao b a b

Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China State Key Laboratory of Chemo/Biosensing & Chemometrics, Chemistry & Chemical Engineering College, Hunan University, Changsha 410082, China

a r t i c l e

i n f o

Article history: Received 6 September 2013 Received in revised form 23 January 2014 Accepted 25 January 2014 Available online 8 February 2014 Keywords: Cloud point extraction HPLC Aristolochic acids Genapol X-080 Rat plasma Aristolochiae Fructus

a b s t r a c t Based on cloud-point extraction (CPE), a high performance liquid chromatography method (HPLC) was developed and validated for the determination of aristolochic acids (AAs) in rat plasma after oral administration of Aristolochiae Fructus (AF). Non-ionic surfactant Genapol X-080, an environmentally friendly solvent, was used for the micelle-mediated extraction. Various influencing factors on CPE process were investigated and optimized. AAs were extracted from rat plasma after adding 1 ml of 4.5% (v/v) surfactant in the presence of 0.2 mol/l HCl and 20 mg NaCl, and the incubation temperature and time were 50 ◦ C and 10 min, respectively. Base-line separation was obtained for the AAs in rat plasma with the optimized chromatography conditions. The detection limits (LOD) reached downward 10 ng/ml. The intra-day and inter-day precisions were less than 7.8%, the accuracies were within ±5.5%, and the average recovery factors were in the range of 94.5–105.4%. In comparison with liquid–liquid extraction, the CPE method has a considerable LOD and higher recoveries. The proposed CPE–HPLC method was specific, sensitive and reliable, and could be an effective tool for the determination of AAs in biological matrixes. With the method the pharmacokinetics of AAs were investigated successfully after oral administration of AF by rats. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Aristolochic acids (AAs) are structurally related to nitrophenanthrene carboxylic acids that occur in Aristolochiaceae plants including Aristolochia and Asarum genera, and have been proved to be nephrotoxic [1–3], carcinogenic [4,5] and mutagenic [5,6]. Therefore, most of AAs-generating herbs have been banned in many countries. However, Aristolochiae Fructus (AF, Madouling in Chinese) has been used widely for thousands of years in China and other countries with good curative effect. AF and honey-toasted AF are still listed in Chinese Pharmacopoeia [7]. Few toxicity cases have been found in the clinical use of AF, let alone honey-toasted AF, although relatively high contents of AAs are found in them [8–11]. Toxic effect of a drug is closely related with the pharmacokinetic

Abbreviations: AAs, aristolochic acids; AF, Aristolochiae Fructus; CPE, cloud-point extraction; LLE, liquid–liquid extraction. ∗ Corresponding author at: Tel.: +86 791 87118658; fax: +86 791 87118658. E-mail addresses: [email protected] (J. Yuan), [email protected] (S. Yao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jchromb.2014.01.055 1570-0232/© 2014 Elsevier B.V. All rights reserved.

characteristics of toxic compounds in it. To elucidate the fact of “low toxic” and “relatively high content of AAs”, it is necessary to investigate the pharmacokinetic characteristics of AF and honey-toasted AF. Although the nephrotoxicity [1–3] and mutagenic mechanism [6] of AAs are well studied, pharmacokinetic characteristics of AAs and related herbs are still limited [12]. Therefore, it is quite necessary to develop a specific and sensitive method to determine AAs in various complicated biosamples. A number of chromatography methods have been reported for the determination of AAs in medicinal plants, including highperformance liquid chromatography coupled with UV detection (HPLC–UV) [8,10,13], mass spectrometry (HPLC–MS) [9,14,15], and fluorescence detection (HPLC–FLD) [16–19]. In which HPLC–FLD [16,18,19] showed the best sensitivity, but pre-column derivatization procedures were needed and not suitable for some biosamples. Due to matrix effect and ion suppressions, ordinary HPLC–MS method only achieved a little higher sensitivity than HPLC–UV. UPLC–MS/MS (Ultra performance liquid chromatography–tandem mass spectrometry) showed great advantages in providing better efficiency, sensitivity, and speed over ordinary HPLC–MS [20], but it is expensive and not readily available. The best economical method

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G. Ren et al. / J. Chromatogr. B 953–954 (2014) 73–79

is HPLC–UV or HPLC–DAD (diode array detection). In order to obtain a suitable sensitivity for the complex samples or trace analysis, liquid–liquid extraction (LLE) [12,17] and solid-phase extraction (SPE) [21,22] are widely used for sample clean-up and analyte preconcentration. In the current LLE methods [12,17] for the determination of AAs in biosamples, large amounts of organic solvents (no less than 2 ml for each sample) or samples were needed. Furthermore, the LLE method is somewhat tedious and unfriendly to both the environment and operators. Therefore, there is an increasing need to develop simple and environmentally friendly methods. Cloud point extraction (CPE) offers a convenient alternative to the conventional extraction systems [23]. CPE was first introduced by Watanabe and co-workers [24]. This phenomenon happens when aqueous solutions of surfactant undergo phase separation upon certain conditions such as temperature manipulation and addition of salts or acids [24]. Two distinct phases are formed: the small volume of surfactant-rich phase and the large volume of aqueous phase. The hydrophobic analytes of the sample are extracted into the surfactant-rich phase. Compared with classical LLC, CPE has been proven to be safety, low cost, ability to concentrate solutes, easy disposal of surfactant, and low toxicity [25]. Now CPE has been extended to the extraction/preconcentration of metal ions [26,27], organic compounds [28–30], drugs [23,25,31–36] and other bioactive compounds [37–39]. In this study, non-ionic surfactant Genapol X-080 was used as CPE solvent for the extraction of AAs in rat plasma. To optimize the CPE parameters, concentration of the surfactant solution, pH of the extraction solution, equilibration time and temperature, etc, were investigated. To evaluate the feasibility of CPE in pharmacokinetics of AAs and the relative herbs, the developed CPE procedure coupled with HPLC was applied to a preliminary pharmacokinetic study following oral administration of AF by rats.

Fig. 1. Chemical structure of five AAs.

The quality control (QC) samples used in the method validation were prepared with the same procedures as the calibration standard. The grated AF was refluxed with ethanol twice (1 h each) and condensed to fluid extract with concentration equivalent to 2.0 g raw herb each ml. All the solutions were stored in a refrigerator at −20 ◦ C and brought to room temperature before use, and then the CPE procedure was applied to the samples.

2. Experimental

2.3. Cloud point extraction

2.1. Materials and reagents

An aliquot (200 ␮l) of rat plasma was pipetted into a 2.0 ml tapered centrifuge tube. Twenty microliters methanol, 20 ␮l HCl (2.0 mol/l), 20 mg NaCl and 1.0 ml 4.5% (v/v) Genapol X-080 were added to this. The tube was vortex-mixed adequately for 10 s and then incubated in a thermostatted water bath (HHS–11–2, Jiangsu, China) for 10 min at 50 ◦ C. The phase separation was accelerated by centrifugation for 5 min at 3100 × g. The water phase was then removed and the surfactant-rich phase stuck to the tube wall was obtained. The surfactant-rich phase was then diluted with methanol (150 ␮l). Co-extractants such as hydrophobic proteins were removed from the surfactant-rich phase by precipitation via centrifugation at 15,800 × g for 10 min (Sigma 3–18K, Sartorious, Germany), then 20 ␮l of the supernatant was directly analyzed by HPLC.

A. Fructus was purchased from Haixing Chinese Herbal Pieces Ltd (Bozhou, Anhui), and was authenticated as the fruits of Aristolochia contorta Bunge by Professor Wuliang Yang (Jiangxi University of Traditional Chinese Medicine, JXUTCM). Voucher specimens are preserved in the Herbarium of Pharmacognosy, School of Pharmaceutical Sciences, JXUTCM, Jiangxi, China. The crude crystals and the mixed crystals of five main AAs (Fig. 1) were isolated from the AF according to the literature [16,40]. Then, preparative chromatography was used for further separation and purification. The obtained crystals were characterized based on the chromatographic retention time, UV spectrum and electrospray MS characteristics [8,9]. The purities of all the standards were not less than 98% according to HPLC–DAD analysis [8,10]. The reagent grade non-ionic surfactant Genapol X-080 (Lot No. 48750–250ML–F) was obtained from Sigma-Alderich (St. Louis, Mo, USA) and was used without further purification. HPLC-grade methanol was from Tedia (Fairfield, OH, USA). Purified water (Wahaha Group, Hangzhou, China) was used throughout the experiments. All other chemical reagents were of analytical grade.

2.2. Sample preparation The stock solutions of the mixed standards were prepared in methanol and a series of standard solutions were obtained by successive dilution with methanol. The relative calibration standards in plasma were prepared by spiking 20 ␮l the corresponding standard solutions into 200 ␮l blank rat plasma.

2.4. Liquid–liquid extraction Twenty microliters standard solution and 20 ␮l HCl (2.0 mol/l) were added into 200 ␮l blank plasma, and vortexed with 2.0 ml acetic ether for 10 s, then the supernatant was transferred into centrifuge tube. The extraction was repeated with 1.0 ml acetic ether. The combined organic layer was evaporated to dryness under a gentle stream of nitrogen at RT. The residue was resolved in 200 ␮l methanol, and centrifuged at 15,800 × g for 10 min. The supernatant was used for chromatographic analysis. 2.5. Chromatographic analysis Chromatography was performed using an Agilent 1200 HPLC system (CA, USA), equipped with a quaternary pump, a degasser,

G. Ren et al. / J. Chromatogr. B 953–954 (2014) 73–79

a column compartment, and a UV detector. Separations were performed on an Ultimate XB-C18, 5 ␮m, 4.6 mm × 200 mm column. Methanol (A) and 0.2% (v/v) glacial acetic acid were used as the mobile phase, and a gradient elution was employed. The gradation was as follows: 0–20 min: 30–45% B; 20–40 min: 45–65% B; 40–45 min: 65–75% B; 45–50 min: 75–100% B. The other chromatographic conditions were as follows: column temperature, 30 ◦ C; flow rate: 1.0 ml/min; injection volume, 20 ␮l; detection wavelength, 254 nm.

2.6. Method validation This method was validated as per the current ICH guideline [41]. A series of the mixed standard solutions (six concentration levels) were prepared in triplicate according to the section 2.2, and the CPE procedure was applied to all samples under the optimal conditions. Calibration curve (y = ax + b) was established by plotting the peak area (y) against the concentrations (x) of the calibration solution with least square linear regression analysis. Limit of detection (LOD) and limit of quantification (LOQ) were determined when peak height was three times and ten times the background noise, respectively. The intra- and inter-day precisions and accuracies were calculated by an analysis of variance based on replicate analysis of QC samples, and the work was accompanied by a standard calibration curve on each analytical run. The precision and accuracy were required to be within ±20% (RSD%) for the lower limit of quantification (LLOQ) and within ±15% (RSD%) for other concentrations. Precisions were evaluated under the optimal conditions six times within the same day for intra-day variance and six different days for inter-day variance. The accuracy was determined by calculating the percentage deviation of the observed concentrations from the spiked concentrations and expressed as relative error (RE%). The recovery tests were studied by spiking the known content of

75

the mixed standard into the blank plasma and expressed as (mean measured concentration)/(spiked concentration) × 100%. The quality control (QC) samples were assayed under several different conditions to evaluate the stability of AAs in rat plasma. The freeze–thaw stability of the analyte was determined over three freeze–thaw cycles. In each freeze–thaw cycle, the samples were frozen and stored at −20 ◦ C for 24 h, then thawed at room temperature. To evaluate long-term stability of AAs, the plasma samples were stored at −20 ◦ C for 14 days. For the short-term stability, fresh plasma samples were kept at room temperature for 12 h before sample preparation. The stability of the prepared plasma samples was tested after keeping the samples at room temperature for 3 days. Stability was evaluated by comparing the mean concentration of the stored samples with that of those prepared freshly. The stability data were acceptable when the bias were within ±15% of the actual value. 2.7. Preliminary pharmacokinetics of AF Male SD rats (Certificate No. SCXK (Hunan) 2011-001) were purchased from Hunan Silaike Jingda Experimental Animal Ltd (Changsha, China). The University Animal Care and Welfare Committee approved all animal protocols. The rats were kept in an environmental-controlled breeding room for 1 week of acclimation period. After oral administration of A. Fructus (30 g/kg), blood samples (0.5 ml) were collected from the ocular vein at 0, 5, 10, 20, 30, 45 min and 1, 1.5, 2, 3, 5, 8, 12 h. The samples were then immediately centrifuged at 3100 × g for 10 min. The plasma obtained was stored frozen at −20 ◦ C till analysis. All statistical analyses were performed using the SPSS 11.0 software (Chicago, USA). P values <0.05 were considered statistically significant. Pharmacokinetic analysis was performed using the proprietary DAS 2.1 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China).

VWD1 A, Wavelength=254 nm (CPE070808.D) mAU 15

A

10 5 0 0

5 10 VWD1 A, Wavelength=254 nm (CPE070809.D)

15

20

25

30

35

mAU 15

2

1

B

40

3

45

min

5

4

10 5 0 0

5 10 VWD1 A, Wavelength=254 nm (CPE070806.D)

15

20

25

30

mAU 15

35

2 3

1

C

40

45

min

5

4

10 5 0 0

5 10 VWD1 A, Wavelength=254 nm (CPE070903.D)

15

20

25

30

35

40

45

min

mAU 15

D

10

5

3

1

2

5

4

0 0

5

10

15

20

25

30

35

40

45

min

Fig. 2. Typical chromatograms of AAs. (A), Blank plasma; (B), mixed standards; (C), mixed standards spiked in blank plasma; (D), plasma sample 0.5 h after oral administration of AF. Peak identification: (1), AA C; (2), 7-OH AA I; (3), AA D; (4), AA II; (5), AA I.

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G. Ren et al. / J. Chromatogr. B 953–954 (2014) 73–79 320

3. Results and discussion

AA C 7-OH AA I AA D AA II AA I

300

3.1. HPLC analysis

280 260

Peak area

Based on our previous work [8,9,16], chromatographic conditions were further improved to adapt the separation of AAs in complicated matrix. It can be seen from Fig. 2, no interference peaks in the chromatograms from endogenic metabolites of rat blood were observed, and good separation of AAs were obtained under the optimal conditions. The results indicate the proposed method has good specificity.

240 220 200 180 160

3.2. Optimization of the CPE conditions 3.2.1. Selection of the surfactant In our previous experiments, Triton X-100 was selected for the preconcentration of AAs in the Aristolochiaceae plants. But Triton X100 has strong UV absorption above 210 nm and will not suitable for the low content of AAs in biosamples. Genapol X-080, a polyoxyethylene glycol mono ether-type surfactant, does not absorb at 254 nm and will not interfere with the determination of AAs in complex matrix. So Genapol X-080 was selected for this work. 3.2.2. Effect of Genapol X-080 concentration The surfactant concentration was determined by both recovery and dilution effects in CPE. Initially, increasing the surfactant concentration will improve the recovery of the target compound. However, the increased recovery becomes insignificant beyond a certain point, and then the increase in surfactant concentration results in an increase in phase volume ratio (volume of surfactantrich phase/volume of aqueous solution). As the phase volume ratio increases, the response signal is reduced due to dilution effects. As shown in Fig. 3, 4.5% (v/v) Genapol X-080 was the suitable concentration for the following CPE procedure. 3.2.3. Effect of sample pH Solution acidity plays an important role in the CPE process, especially when ionizable compounds are extracted. In CPE the ionic form of a neutral molecule normally does not interact and bind with the micelle aggregate as strongly as its unionized form does. AAs, as weak acidic compounds, may obtain higher recoveries in acidic conditions. In this work 20 ␮l HCl (0, 0.5, 1.0, 2.0, 3.0 mol/l) was added into the sample solution, respectively. As shown in Fig. 4, no significant differences were found between the concentration ranges of

140 120

0.0

.5

1.0

1.5

2.0

2.5

3.0

3.5

Concentration of HCl (mol/l) Fig. 4. Effect of HCl concentration on the extraction efficiency (n = 3). Conditions: 4.5% Genapol X-080, 20 mg NaCl, 50 ◦ C, and 10 min. For other conditions, see text.

0.5–3.0 mol/l, except for 0 mol/l (no HCl was added). Based on the above results, HCl (20 ␮l, 2.0 mol/l) was used to control the pH of the sample for subsequent studies. 3.2.4. Effect of incubation conditions It can be seen from Fig. 5, the peak area increased rapidly with the temperature rising from 45 to 48 ◦ C, and reached the maximum values between 48 and 55 ◦ C, then a little decrease were observed. So 50 ◦ C was chosen as the working temperature. The equilibrium time ranging from 3.0 to 20 min was carefully investigated in a thermostated bath (50 ◦ C). It can be seen from the Fig. 6, the peak areas of AAs increase from 3.0 to 8 min, then keep almost constant. So 10 min is the optimal incubation time. It has been reported that addition of electrolytes can improve the separation of the surfactant-rich phase from the aqueous phase. In this work the effects of ionic strength were investigated by adding NaCl, and three kinds of phenomena were observed. First, when the addition amount of NaCl was within 10–30 mg, the surfactant-rich phase stuck to the tube wall and the phase separation was improved. Second, when the addition amounts were 40–60 mg, the surfactant-rich phase was not steady and the phase 320

300

280 260

Peak area

250

Peak area

AA C 7-OH AA I AA D AA II AA I

300

AA C 7-OH AA I AA D AA II AA I

200

240 220 200 180

150

160

1

2

3

4

5

6

7

8

9

Concentration of genapol X-080 (V/V) Fig. 3. Effect of the surfactant concentration on the extraction efficiency of AAs (n = 3). Conditions: 0.2 mol/l HCl, 20 mg NaCl, 50 ◦ C, and 10 min. For other conditions, see text.

140 40

50

60

70

80

o

Incubation temperature ( C) Fig. 5. Effect of equilibration temperature on the extraction efficiency (n = 3). Conditions: 4.5% Genapol X-080, 0.2 mol/l HCl, 20 mg NaCl, and 10 min. For other conditions, see text.

G. Ren et al. / J. Chromatogr. B 953–954 (2014) 73–79

Table 1 Linear ranges, correlation coefficients, quantification and detection limits of AAs in rat plasma.

320 AA C 7-OH AA I AA D AA II AA I

300 280

Peak area

260

77

240 220

Compound

Linear range (␮g/ml)

r2

LOQ (ng/ml)

LOD (ng/ml)

AA C 7-OH AA I AA D AA II AA I

0.082–32.8 0.064–25.5 0.061–24.5 0.042–16.6 0.065–25.8

0.9993 0.9990 0.9984 0.9995 0.9986

82 64 61 42 34

22 19 18 13 10

200

Therefore, 20 mg NaCl was selected to aid the separation of the surfactant-rich phase from the aqueous phase.

180 160

3.3. Method validation

140 120

2

4

6

8

10

12

14

16

18

20

22

Incubation time (min) Fig. 6. Effect of equilibration time on the extraction efficiency (n = 3). Conditions: 4.5% Genapol X-080, 0.2 mol/l HCl, 20 mg NaCl, and 50 ◦ C. For other conditions, see text.

separation deteriorated. Third, when the amount of NaCl was higher than 70 mg, the surfactant-rich phase was suspended on the top of the aqueous solution accompanying with the deposition of plasma protein, and the extraction efficiency became lower.

The linearity parameters of five AAs in rat plasma are summarized in Table 1. Good linearity data were obtained with correlation coefficients >0.998. LOD reached downward 10 ng/ml, and for AAs in complicated matrix such as plasma the value was satisfactory. The precisions and accuracy results are shown in Table 2. The analytical precisions were less than 7.8%, the relative errors were within ±5.5%, and the recoveries were within 94.5–105.4%. These data indicate that the CPE–HPLC method is reproducible, accurate, and reliable. The stability data under different experiment conditions are summarized in Table 3. The detection variabilities expressed as RSDs (%) were less than 10.7%, and which suggested that rat plasma

Table 2 Precision, accuracy and recovery of AAs in rat plasma (n = 6). Compound

Precision (RSD%)

Concentration (␮g/ml)

0.164 1.64 3.28 0.128 1.28 2.55 0.123 1.23 2.45 0.083 0.83 1.66 0.129 1.29 2.58

AA C

7-OH AA I

AA D

AA II

AA I

Intra-day

Inter-day

3.6 3.2 2.8 4.3 3.8 3.5 4.9 4.0 3.3 6.1 5.6 5.2 3.7 2.5 2.2

5.6 4.5 4.9 6.2 5.4 5.9 6.5 5.0 5.3 7.8 5.8 6.5 5.9 4.9 4.8

Accuracy (R.E.%)

Recovery (%)

3.7 −1.3 −2.8 −5.4 −3.8 −2.1 4.1 2.6 1.7 5.5 −2.1 −2.3 2.3 −1.9 −2.4

103.7 98.8 97.3 94.5 96.1 97.6 104.1 102.4 101.6 105.4 97.9 97.6 102.3 98.4 97.7

Table 3 Stability of AAs in rat plasma expressed as mean ± SD and RSDs (n = 6). Compound

Ca

Freeze–thaw stability b

AA C

7-OH AA I

AA D

AA II

AA I

a b

0.328 1.64 3.28 0.255 1.28 2.55 0.245 1.23 2.45 0.166 0.83 1.66 0.258 1.29 2.58

Short-term stability

Long-term stability b

Post-paraparative stability

C

RSD%

C

RSD%

C

RSD%

Cb

RSD%

0.332 ± 0.025 1.62 ± 0.112 3.18 ± 0.20 0.240 ± 0.019 1.15 ± 0.08 2.39 ± 0.18 0.250 ± 0.023 1.19 ± 0.09 2.42 ± 0.15 0.170 ± 0.013 0.800 ± 0.054 1.51 ± 0.09 0.260 ± 0.018 1.18 ± 0.061 2.47 ± 0.13

7.5 6.9 6.3 8.1 6.9 7.5 9.3 7.6 6.2 7.9 6.7 6.0 6.9 5.2 5.3

0.334 ± 0.021 1.63 ± 0.09 3.20 ± 0.18 0.241 ± 0.020 1.24 ± 0.09 2.49 ± 0.18 0.261 ± 0.021 1.25 ± 0.09 2.50 ± 0.19 0.172 ± 0.013 0.820 ± 0.042 1.63 ± 0.08 0.272 ± 0.015 1.25 ± 0.05 2.54 ± 0.12

6.3 5.5 5.6 8.3 7.3 7.2 8.1 7.2 7.9 7.6 5.1 4.9 5.7 4.0 4.7

0.325 ± 0.032 1.59 ± 0.15 3.19 ± 0.23 0.238 ± 0.025 1.16 ± 0.10 2.41 ± 0.18 0.239 ± 0.022 1.12 ± 0.11 2.40 ± 0.21 0.168 ± 0.015 0.790 ± 0.068 1.57 ± 0.12 0.261 ± 0.021 1.20 ± 0.07 2.43 ± 0.13

9.8 9.4 7.2 10.7 8.6 7.4 9.3 10.0 8.6 9.0 8.6 7.4 8.3 5.7 5.3

0.341 ± 0.020 1.61 ± 0.10 3.20 ± 0.16 0.245 ± 0.016 1.24 ± 0.06 2.50 ± 0.12 0.262 ± 0.019 1.26 ± 0.09 2.52 ± 0.15 0.182 ± 0.012 0.821 ± 0.053 1.62 ± 0.09 0.273 ± 0.014 1.28 ± 0.05 2.53 ± 0.12

6.1 6.2 5.0 6.8 5.2 4.9 7.4 7.2 5.9 6.7 6.5 5.4 5.1 3.8 4.9

C—Concentration added (␮g/ml). C—Concentration added (␮g/ml).

b

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Table 4 Comparison of the extraction recoveriesa of AAs in rat plasma (n = 3).

2500

Concentration (␮g/ml)

Recovery (%) in CPE (mean ± SD)

Recovery (%) in LLE (mean ± SD)

AA C

0.164 1.64 3.28 0.128 1.28 2.55 0.123 1.23 2.45 0.083 0.83 1.66 0.129 1.29 2.58

81.6 ± 1.7 80.5 ± 1.4 80.7 ± 1.5 82.5 ± 1.4 82.2 ± 1.6 81.8 ± 0.8 83.4 ± 1.6 81.7 ± 1.2 80.2 ± 1.3 86.2 ± 1.8 85.4 ± 1.6 84.9 ± 1.0 87.2 ± 1.7 87.1 ± 1.1 86.5 ± 1.0

68.1 ± 1.3 67.8 ± 1.3 66.9 ± 1.4 69.6 ± 1.7 69.3 ± 1.8 70.4 ± 1.2 69.9 ± 1.3 68.5 ± 1.0 69.1 ± 1.0 72.7 ± 1.4 72.4 ± 1.5 70.5 ± 1.9 70.8 ± 1.2 69.9 ± 1.1 68.5 ± 1.1

7-OH AA I

AA D

AA II

AA I

AA I AA II AA D 7-OH AA I AA C

2000

C (ng/ml)

Compound

1500

1000

500

0 0

a

Take AA I for example, extraction recovery is the peak area ratio of AA I in methanol and that in the extract from the blank plasma spiked corresponding concentration of AA I.

samples containing AAs can be handled under normal laboratory conditions without significant loss of the target compounds. 3.4. Comparison with LLE Acetic ether [12,17] has been found suitable for the extraction of AA I and/or AA II in biosamples with LLE. But relatively large amounts of samples and solvents are required in LLE with acetic ether. For pharmacokinetics application, the extraction efficiency or the method sensitivity was the primary concern. The extraction efficiency was evaluated by extraction recovery. As shown in Table 4, the higher recoveries for the five AAs were obtained with CPE when equal plasma was used. Besides the higher extraction efficiency, the CPE method uses less organic solvent, has a shorter analysis time and is easier to perform than LLE does. 3.5. Application in pharmacokinetics The total content of five AAs is 2.66 mg/g in the AF used in this work. In which, the content of AA I (0.842 mg/g raw herb) is the highest, and equals to 25.3 mg/g AA I. In pharmacokinetic study of AA I, the literature dose is about 10–15 mg/kg [20,42,43]. But taking the other AAs into consideration, the dose of AF of 30 g/kg was selected according to the preliminary experiment. After oral administration of A. Fructus, rat plasma samples were prepared and analyzed as the above descriptions. The typical chromatograms were shown in Fig. 2. Several main AAs in A. Fructus can be detected in rat blood with the proposed CPE–HPLC method. The mean plasma concentration–time profiles of AAs after oral administration of AF extract were shown in Fig. 7. The mean pharmacokinetic parameters of AA I, AA II, AA D and 7-OH AA I were summarized in Table 5. The parameters of AA C were not reported due to the insufficient data. After oral administration, AAs in blood occurred rapidly, and reached the maximum concentrations at about 30 min, then were Table 5 Pharmacokinetic parameters of AA I, AA II, AA D and 7-OH AA I after oral administration of AF. Parameters

AA I

AA II

AA D

7-OH AA I

t1/2z (h) AUC(0−∞) (ng/ml h) Tmax (min) Cmax (ng/ml) MRT(0−∞) (h) CLz /F (ml/h/kg)

1.96 1476.4 20 2056.6 1.62 20.3

0.83 271.6 30 205.8 1.26 110.4

0.75 1108.7 30 1108.6 1.23 27.1

0.74 1146.2 30 1275.2 1.20 26.2

2

4

6

8

Time (h) Fig. 7. Mean plasma concentration-time profile of five AAs in rat after oral administration of AF extract at a dose of 30 g/kg (n = 5).

eliminated rapidly. The current pharmacokinetic studies focus on AA I and/or AA II with HPLC–UV [12,42,43] or HPLC–MS [20]. In this work, the pharmacokinetic parameters of AA D and 7-OH AA I were reported at the first time. The results indicated that the present method has good separation performance and sensitivity, and could be an effective tool for further pharmacokinetics study of Aristolochiaceae plants. 4. Conclusion A simple, specific and sensitive CPE–HPLC method was developed and validated for the determination of AAs in rat plasma. Compare with LLE technique, the proposed method uses less organic solvent and is more environmentally friendly, and provides an effective replacement for the current LLE method using in biosamples. The proposed method was successfully applied to the pharmacokinetic study of AAs after oral administration of AF. The method is expected to use as a reliable tool for the determination of AAs in other biological matrixes (such as urine and tissue homogenates) and further pharmacokinetics of the other Aristolochiaceae plants. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (no. 81060326 and 81260605), Jiangxi Provincial Natural Science Foundation (no. 2010GZY0163) and Jiangxi Provincial Education Department (no. GJJ09277). References [1] A. Lemy, K.M. Wissing, S. Rorive, A. Zlotta, T. Roumeguere, M.M. Muniz, C. Decaestecker, I. Salmon, D. Abramowicz, J.L. Vanherweghem, J. Nortier, Am. J. Kidney Dis. 51 (2008) 471. [2] B. Jelakovic, S. Karanovic, I. Vukovic-Lela, F. Miller, K.L. Edwards, J. Nikolic, K. Tomic, N. Slade, B. Brdar, R.J. Turesky, Z. Stipancic, D. Dittrich, A.P. Grollman, K.G. Dickman, Kidney Int. 81 (2012) 559. [3] U. Mengs, C.D. Stotzem, Arch. Toxicol. 67 (1993) 307. [4] C.H. Chen, K.G. Dickman, M. Moriya, J. Zavadil, V.S. Sidorenko, K.L. Edwards, D.V. Gnatenko, L. Wu, R.J. Turesky, X.R. Wu, Y.S. Pu, A.P. Grollman, PNAS 109 (2012) 8241. [5] N. Feldmeyer, H.H. Schmeiser, K.R. Muehlbauer, D. Belharazem, Y. Knyazev, T. Nedelko, M. Hollstein, Mutat. Res. 608 (2006) 163. [6] M. Stiborova, E. Frei, V.M. Arlt, H.H. Schmeiser, Mutat. Res. 658 (2008) 55. [7] CPC, Chinese Pharmacopoeia, 2010 ed., China Medical Science Press, Beijing, 2010. [8] J.B. Yuan, L.H. Nie, D.Y. Zeng, X.Z. Luo, F. Tang, L. Ding, Q. Liu, M.L. Guo, S.Z. Yao, Talanta 73 (2007) 644.

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