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Determination of matrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies
Talanta 59 (2003) 965 /971 www.elsevier.com/locate/talanta
Determination of matrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies Xinan Wu a,b, Fumiyoshi Yamashita b, Mitsuru Hashida b, Xingguo Chen a,*, Zhide Hu a b
a Department of Chemistry, Lanzhou University, Lanzhou 730000, People’s Republic of China Department of Drug Delivery research, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
Received 23 August 2002; received in revised form 20 November 2002; accepted 9 December 2002
Abstract A simple high-performance liquid chromatography (HPLC) method is described for the determination of matrine in rat plasma. The plasma was deproteinized with acetonitrile that contained an internal standard (phenacetin) and was separated from the aqueous layer by adding sodium chloride. Matrine was extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC (column: YMC-pack ODS-A, 5 mm, 150 /4.6 mm, I.D.; eluent: acetonitrile /0.02 mol ammonium acetate buffer /triethylamine (35:65:0.035, v/v/v) and ultraviolet detection (220 nm). The limit of quantitation for matrine was 200 ng ml 1 in plasma, and the recovery was greater than 89%. The assay was linear from 0.5 to 50.0 mg ml 1. Variation over the range of the standard curve was less than 6%. The method was used to determine the concentration /time profiles of matrine in the plasma following oral administration of matrine aqueous solution or bolus injection from which the fractions of matrine reaching the systemic circulation were estimated by a deconvolution method for the first time. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Matrine; HPLC /UV; Rat plasma
1. Introduction The Chinese have used herbs for a wide variety of medical treatments for several thousand years.
* Corresponding author. Fax: /86-931-891-2582. E-mail address: [email protected] (X. Chen).
Ku-dou-zi is the dried roots of Sophora alopecuroides L. and a commonly used Chinese herbal drug. It possesses antipyretic, anti-inflammatory, analgesic effects and is used to treat acute or chronic gastroenteritis [1]. One of the major components of S. alopecuroides is matrine [2] (structure shown in Fig. 1), which has been shown to have protective effect on the lipopolysacchride-
0039-9140/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0039-9140(03)00009-2
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2. Experiment 2.1. Chemicals and reagents Fig. 1. Structure of matrine.
reduced liver injury [3], and on restraint and water immersion stress ulcer in mice [4]. Matrine has been also shown to possess an anti-inflammatory property [5], a significant effect on the inhibition of proliferation cells and inducing differentiation in K-562 cells [6], and to have anti-arrhythmic effect [7]. The discovery of matrine’s various pharmacological effects has been well described, as well as their body fates [8,9]. However, these pharmacokinetics studies have been focused only on the processes after intravenous injection. In spite that Ku-dou-zi is usually administered orally, there is a lack of information concerning the extent to which matrine is absorbed following oral administration. Because most Chinese medicines are administered orally in the form of a crude extract in clinical use, the absorption rate of the active components in gastrointestinal tract would be used as a suitable reference in clinical application. Several methods for the determination of matrine in Plasma have been described in the literature. This includes gas chromatography [10] high-performance liquid chromatography (HPLC) [9]. However, these methods either lack sensitivity or are too complex in the samples pretreatment. In addition, they are not applicable to the quantitation of matrine in rat plasma because only a small amount of blood can been sampled at given interval time in pharmacokinetic study. To obtain the available pharmacokinetic parameters of matrine, a simple, sensitive high performance liquid chromatographic method was developed for the determination of matrine in rat plasma after oral administration or bolus injection of matrine aqueous solution from which the fractions of matrine reaching the systemic circulation were estimated by a deconvolution method.
Matrine was of analytical grade for using as analysis and of drug grade as animal experiment, purchased from the Chinese Medicine Control Institute (Beijing, China) and the Yanchi Pharmaceutical Factory (Ningxia, China), respectively. Acetonitrile and water were of HPLC grade, obtained from Kanto Chemical Co. Inc. (Tokyo, Japan). Ammonium acetate and phenacetin (special grade) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Triethylamine (special grade) was purchased from Sigma / Aldrich Co. (Tokyo, Japan). 2.2. HPLC system The HPLC analysis was carried out using a Shimadzu LC-6A HPLC system (Kyoto, Japan), equipped with SPD-A UV /VIS detector and SIL6B autoinjector. A YMC-pack ODS-A column, 4.6 /150 mm, 5 mm (YMC Co, Ltd, Kyoto, Japan) was used. The mobile phase was acetonitrile /0.02 mol l 1 ammonium acetate buffer / triethylamine (35:65:0.035, v/v/v), filtered through a 0.45-mm Millipore filter and degassed prior to use. The flow rate was 0.9 ml min 1. Detection was performed at a wavelength of 220 nm at room temperature. The sample injection volume was 20 ml. 2.3. Animals and blood sampling Male Wistar-strain rats (180 /210 g) were obtained from SLC Inc. (Shizuoka, Japan), and fasted for 12 h with free access to water, prior to the experiments. A polyethylene tube (0.28 mm, I.D., 0.61 mm, O.D.) was inserted into the right femoral artery of the rat while the animal was under anesthesia with ether. The rat was placed in a Bollman cage and allowed to recover from anesthesia for more than 1 h. Matrine aqueous solution was then orally administered to the rat at a dose of 40 mg kg 1 or was injected through the cannulated tube into the blood at a single dose of 40 or 4 mg kg 1. Blood samples (0.25 ml) were
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collected through the cannulated tube at times 0, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360 and 480 min after oral administration, or 0, 5, 10, 20, 30, 45, 60, 90, 120, 180, 240 min after bolus injection. After each sampling, the reduced blood volume was supplemented with an equal volume of saline containing 100 IU ml 1 heparin. Water was supplied to each rat 1 h after dosing. 2.4. Preparation of plasma samples Each collected blood sample was immediately transferred to a heparinized microcentrifuge tube and centrifuged at 8000 rpm for 5 min at 4 8C (Himac CT 13R, Hitachi, Japan). The resulting plasma (0.1 ml) was then vortex-mixed with 0.2 ml of acetonitrile containing phenacetin (5 mg ml 1) as internal standard for 30 s. After 15 min the mixture was centrifuged at 8000 rpm for 2 min at 4 8C to separate precipitated proteins. The supernatant was transferred into 1.5-ml tubes containing 50 /60 mg sodium chloride. The suspension was vortex-mixed briefly and incubated at room temperature for 6 min. After vortex-mixing and centrifuging once again at 8000 rpm for 5 min at 4 8C, 20 ml of the upper organic layer that was filtered through a 0.45-mm Millipore filter was directly injected into the chromatography. The same sample handling process was used for recovery and precision determinations in plasma.
Fig. 2. Chromatrograms of matrine in rat plasma: (a) blank plasma, (b) plasma spiked with matrine (12 mg ml 1) and phenacetin (internal standard), (c) plasma sample 30 min after oral administration of matrine aqueous solution at a dose of 40 mg kg 1. Retention times of phenacetin (internal standard) and matrine were approximately 4.8 and 6.4 min, respectively.
2.5. Calibration curve Calibration curves in the concentration range of 0.5 /50.0 mg ml 1 for matrine was constructed by plotting the peak /area ratios of analyte/internal standard versus matrine concentration in rat plasma. In order to avoid undue bias to the low concentrations of the standard curve by the high concentrations, the calibration curve was split into two ranges: 0.5 /6.0 and 6.0 /50.0 mg ml 1. Leastsquares linear regression analysis was used to determine the slope, intercept and correlation coefficient. The concentration of matrine in plasma was determined from the peak /area ratios by using the equations of linear regression obtained from the calibration curves. To determine the limit of quantification dilutions of 0.1, 0.2, 0.3,
0.4 mg ml 1 matrine in plasma were prepared using a solution of 2 mg ml 1 matrine in plasma for calibration.
2.6. Recovery Plasma samples were spiked with matrine at concentrations of 2.0, 24.0 and 50.0 mg ml 1. The resulting peak /area ratios (analyte: internal standard) were compared with that of the standards prepared in acetonitrile to provide the recovery values.
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Table 1 Recovery of the matrine assay Spiked concentration (mg ml 1)
2.0 24.0 50.0
Peak /area ratio Untreated
Treated
0.079/0.00 1.099/0.02 2.389/0.01
0.069/0.00 1.019/0.01 2.129/0.03
Recovery (%)
CV (%)
93.569/2.45 92.479/1.01 88.969/1.14
2.61 1.09 1.28
Each value represents the mean9/S.D. (n /5).
2.7. Precision Precision over the entire working dose range was determined by analyses of plasma samples (n /5) spiked with matrine at concentrations of 1.0, 2.0, 6.0, 12.0, 24.0 and 50.0 mg ml 1. To determine intra-day variance, the assays were carried out on the same samples at different times during the day. Inter-day variance was determined by assaying the spiked samples over three consecutive days. Coefficients of variation (CVs) were calculated from these values. 2.8. Assay application The present method was used to determine the plasma concentrations of matrine in rat plasma after oral administration or bolus injection of matrine aqueous solution from which the fractions of matrine reaching the systemic circulation were estimated by a deconvolution method.
3. Results and discussion 3.1. HPLC chromatograms Under the condition described above, the HPLC chromatograms of blank, plasma spiked with matrine at concentration of 12.0 mg ml 1 and the plasma obtained 30 min after oral administration of matrine aqueous solution at a dose 40 mg kg 1, were shown in Fig. 2. The retention times for phenacetin (internal standard) and matrine
were approximately 4.8 and 6.4 min, respectively. The peaks were sharp and symmetrical with good baseline resolution and minimal tailings, thus facilitating accurate measurements of the peak / area ratios. No endogenous plasma components elute at the retention time of matrine or internal standard. For the separation of matrine, phenacetin (I.S.) and endogenous plasma components by reverse phase HPLC, the following experimental conditions, namely, choice of mobile phase, phase flow rate, amounts of modifier have been investigate. A mixture of standard matrine and I.S. dissolved in acetonitrile was injected into a C18 column at flow rate of 0.7 ml min 1. Subsequently, the mobile phase flow rate was varied between 0.7 and 1.2 ml min 1. The optimum mobile phase flow rate was found to be 0.9 ml min 1, which was used for further optimization of other parameters. Several mobile phases were investigated using an isocratic system in all cases. The mobile phase with the composition of acetonitrile /water/triethylamine (40:60:0.035) gave sharper peak shapes and approximately four times peak /area as compared with methanol /water/triethylamine (60:40:0.035). The results showed that it was lack of sensitivity by using methanol /water/triethylamine (60:40:0.035) as mobile phase. In the other hand, good resolution of blank or standard samples spiked with drug-free plasma could not obtained by using acetonitrile /water/triethylamine (40:60:0.035) because of poor baseline. To improve baseline stability, instead of water in mobile phase, 0.02 mol l 1 ammonium acetate buffer was
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Table 2 Validation of the intra-day assay Spiked concentration (mg ml 1)
Measured concentration (mg ml 1)
Accuracy (%)
CV (%)
1.0 2.0 6.0 12.0 24.0 50.0
1.009/0.04 1.939/0.04 6.099/0.18 11.799/0.23 24.329/0.25 49.949/0.60
99.98 96.40 101.58 98.29 101.35 99.87
4.35 2.05 2.90 1.99 1.02 1.20
Each value represents the mean9/S.D. (n /5). Table 3 Validation of the inter-day assay Spiked concentration (mg ml 1)
Measured concentration (mg ml 1)
Accuracy (%)
CV (%)
1.0 2.0 6.0 12.0 24.0 50.0
0.929/0.06 1.939/0.12 6.239/0.22 11.949/0.30 24.069/0.48 50.319/0.58
91.75 96.37 103.85 99.46 100.26 100.63
6.30 6.15 3.47 2.52 1.99 1.16
Each value represents the mean9/S.D. (n /3).
adopted throughout the HPLC analysis. Although the addition of triethylamine improved the tailing of matrine peak, the baseline began to drift when its amount exceeded out 0.05 in the proportions of mobile phase. Combining the baseline and the peak tailing of the matrine, the optimum amount of triethylamine was found to be 0.035 in the proportions of mobile phase.
3.2. Calibration curves Matrine was dissolved in acetonitrile and diluted to give a series of standard solutions (0.5, 1.0, 2.0, 6.0, 12.0, 24.0, 50.0 mg ml 1) for the calibration curves of the drug in rat plasma. The linear regression analysis of matrine was constructed by plotting the peak /area ratio of matrine to the internal standard (y) versus analyte concentration (mg ml 1) in spiked plasma samples (x ). The calibration curves for matrine were linear both the low (0.5 /6.0 mg ml 1) and high concentration
ranges (6.0 /50.0 mg ml 1) with r2 values of 0.9991 and 0.9999, respectively. 3.3. Recovery The recovery was assessed by comparing the peak /area ratios (analyte: internal standard) obtained from spiked plasma samples of different analyte concentrations to the peak/area ratios for the samples containing the equivalent amounts of the analyte and internal standard directly dissolved in acetonitrile. The recoveries of matrine from rat plasma were shown in Table 1. 3.4. Accuracy, reproducibility and limit of quantitation The reproducibility of the method was defined by examining both intra- and inter-day variance. Analytical accuracy and precision data were shown in Tables 2 and 3 and expressed as mean detected concentration and CV. The following
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Fig. 3. Plasma concentration profiles of matrine after bolus injection at a dose of 40 mg kg 1 (j) and 4 mg kg 1 ("). Data was shown as mean9/S.D. of three experiments.
validations assess the suitability of the method: the mean value should be within 9/15% of the actual value except at the limit of quantitation where it should be within 9/20%, relative standard deviation (R.S.D.) around the mean value should not exceed 15% except at the limit of quantitation where it should be within 9/20% [11]. The assay precision (CVs) of matrine at low to high concentrations was better than 5 and 7% for intra-day and inter-day assays, respectively. Assay accuracy, assessed by calculating the measured concentrations as a percent of the spiked concentrations, was better than 91% (Tables 2 and 3). The limit of quantitation, defined as the lowest concentration on the calibration curve at which both accuracy and precision should be within 20% [11], was deemed to be 200 ng ml 1 for matrine. The sensitivity of this method for determining matrine in rat plasma is higher than that of the reported methods [9,10]. In addition, a common pretreatment method of biological fluids is as follows: matrine is extracted into organic solvents such as ethyl acetate, dichloromethane or chloroform. The organic solvent is evaporated and the residue is dissolved in eluent. The method described above is also outstanding with respect to simplicity in the samples pretreatment. These results indicate that
Fig. 4. Plasma concentration /time curve of matrine in rat after oral administration of matrine aqueous solution at a dose of 40 mg kg 1. Data was shown as mean9/S.E.M. of five experiments.
the assay was simple, accurate, sensitive and reproducible. 3.5. Application In order to estimate the absorption rate of matrine in gastrointestinal tract, the plasma concentrations of matrine was analyzed in rat plasma,
Fig. 5. Time-courses of fraction of matrine reaching systemic circulation after oral administration of matrine aqueous solution. Data was shown as mean9/S.E.M. of five experiments.
X. Wu et al. / Talanta 59 (2003) 965 /971
following oral administration of matrine aqueous solution or bolus injection. The plasma concentration of matrine was determined at 0, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360 and 480 min after oral dosing or 0, 5, 10, 20, 30, 45, 60, 90, 120, 180, 240 min after bolus injection. Fig. 3 showed the mean9/S.D. plasma concentration /time profile of matrine after bolus injection at a dose of 40 and 4 mg kg 1. When the bolus injection dose of matrine was increased from 4 to 40 mg kg 1, there were no significant differences in the apparent total body clearance, the elimination half-life and volume of distribution. The area under the curve of matrine appears to increase proportionally from 4 to 40 mg kg 1 (data not shown). These results suggest that the pharmacokinetic of matrine is a linear process. Fig. 4 showed the mean9/standard error of the mean (S.E.M.) plasma concentration / time profile of matrine after oral administration. The mean maximum concentration of matrine in plasma was 3.89 mg ml 1 at 30 min after oral dosing. The fractions of matrine absorbed were estimated by a deconvolution method (Fig. 5). The fraction of matrine reaching the systemic circulation rapidly increases to 0.39 within the first 2 h and then slowly to 0.44 by 8 h. In conclusion, this paper describes a simple, rapid, sensitive, accurate and precise procedure for the determination of matrine, suitable for the analysis of large numbers of plasma samples. The assay was validated to meet the requirements of pharmacokinetic studies. The fractions of matrine reaching the systemic circulation following oral
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dosing would be used as a suitable reference in clinical application.
Acknowledgements The project was supported by the National Natural Science Foundation of China (No. 20275014).
References [1] Jiangsu New Medicine College, Zhong yao da ci dian (Dictionary of Chinese Materia Medica), Shanghai Scientific and Technological Publishers, Shanghai, 1985, p. 595. [2] B.G. Zhao, Yao Xue Xue Bao 15 (1980) 182. [3] W. Lin, J.P. Zhang, Z.L. Hu, D.H. Qian, Yao Xue Xue Bao 32 (1997) 93 /96. [4] M. Yamazaki, A. Arai, S. Suzuki, T. Takeuchi, Yakugaku Zasshi 104 (1984) 293 /301. [5] H.R. Tan, B.H. Zhang, Zhong Xi Yi Jie He Za Zhi 5 (1985) 108 /110. [6] L.P. Zhang, J.K. Jiang, J.W.O. Tam, Y. Zhang, X.S. Liu, X.R. Xu, B.Z. Liu, Y.J. He, Leuk. Res. 25 (2001) 793 /800. [7] B.H. Zhang, N.S. Wang, X.J. Li, X.J. Kong, Y.L. Cai, Zhongguo Yao Li Xue Bao 11 (1990) 253 /257. [8] H.M. Zhang, H.Q. Li, Zhongguo Yao Li Xue Bao 20 (1999) 517 /520. [9] P.Q. Wang, G.H. Lu, X.B. Zhou, J.F. Shen, S.X. Chen, S.W. Mei, M.F. Chen, Yao Xue Xue Bao 29 (1994) 326 / 329. [10] X.D. Liu, S.K. Huang, Najin Yao Xue Yuan Xue Bao 17 (1986) 222 /223. [11] V.P. Shah, K.K. Midha, I.J. McGiveray, J.P. Skelly, A. Yacobi, T. Layloff, C.T. Viswanathan, C.E. Cook, R.D. McDowall, K.A. Pittman, S. Spector, J. Pharm. Sci. 81 (1992) 309 /312.
Determination of matrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies Xinan Wu a,b, Fumiyoshi Yamashita b, Mitsuru Hashida b, Xingguo Chen a,*, Zhide Hu a b
a Department of Chemistry, Lanzhou University, Lanzhou 730000, People’s Republic of China Department of Drug Delivery research, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
Received 23 August 2002; received in revised form 20 November 2002; accepted 9 December 2002
Abstract A simple high-performance liquid chromatography (HPLC) method is described for the determination of matrine in rat plasma. The plasma was deproteinized with acetonitrile that contained an internal standard (phenacetin) and was separated from the aqueous layer by adding sodium chloride. Matrine was extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC (column: YMC-pack ODS-A, 5 mm, 150 /4.6 mm, I.D.; eluent: acetonitrile /0.02 mol ammonium acetate buffer /triethylamine (35:65:0.035, v/v/v) and ultraviolet detection (220 nm). The limit of quantitation for matrine was 200 ng ml 1 in plasma, and the recovery was greater than 89%. The assay was linear from 0.5 to 50.0 mg ml 1. Variation over the range of the standard curve was less than 6%. The method was used to determine the concentration /time profiles of matrine in the plasma following oral administration of matrine aqueous solution or bolus injection from which the fractions of matrine reaching the systemic circulation were estimated by a deconvolution method for the first time. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Matrine; HPLC /UV; Rat plasma
1. Introduction The Chinese have used herbs for a wide variety of medical treatments for several thousand years.
* Corresponding author. Fax: /86-931-891-2582. E-mail address: [email protected] (X. Chen).
Ku-dou-zi is the dried roots of Sophora alopecuroides L. and a commonly used Chinese herbal drug. It possesses antipyretic, anti-inflammatory, analgesic effects and is used to treat acute or chronic gastroenteritis [1]. One of the major components of S. alopecuroides is matrine [2] (structure shown in Fig. 1), which has been shown to have protective effect on the lipopolysacchride-
0039-9140/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0039-9140(03)00009-2
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2. Experiment 2.1. Chemicals and reagents Fig. 1. Structure of matrine.
reduced liver injury [3], and on restraint and water immersion stress ulcer in mice [4]. Matrine has been also shown to possess an anti-inflammatory property [5], a significant effect on the inhibition of proliferation cells and inducing differentiation in K-562 cells [6], and to have anti-arrhythmic effect [7]. The discovery of matrine’s various pharmacological effects has been well described, as well as their body fates [8,9]. However, these pharmacokinetics studies have been focused only on the processes after intravenous injection. In spite that Ku-dou-zi is usually administered orally, there is a lack of information concerning the extent to which matrine is absorbed following oral administration. Because most Chinese medicines are administered orally in the form of a crude extract in clinical use, the absorption rate of the active components in gastrointestinal tract would be used as a suitable reference in clinical application. Several methods for the determination of matrine in Plasma have been described in the literature. This includes gas chromatography [10] high-performance liquid chromatography (HPLC) [9]. However, these methods either lack sensitivity or are too complex in the samples pretreatment. In addition, they are not applicable to the quantitation of matrine in rat plasma because only a small amount of blood can been sampled at given interval time in pharmacokinetic study. To obtain the available pharmacokinetic parameters of matrine, a simple, sensitive high performance liquid chromatographic method was developed for the determination of matrine in rat plasma after oral administration or bolus injection of matrine aqueous solution from which the fractions of matrine reaching the systemic circulation were estimated by a deconvolution method.
Matrine was of analytical grade for using as analysis and of drug grade as animal experiment, purchased from the Chinese Medicine Control Institute (Beijing, China) and the Yanchi Pharmaceutical Factory (Ningxia, China), respectively. Acetonitrile and water were of HPLC grade, obtained from Kanto Chemical Co. Inc. (Tokyo, Japan). Ammonium acetate and phenacetin (special grade) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Triethylamine (special grade) was purchased from Sigma / Aldrich Co. (Tokyo, Japan). 2.2. HPLC system The HPLC analysis was carried out using a Shimadzu LC-6A HPLC system (Kyoto, Japan), equipped with SPD-A UV /VIS detector and SIL6B autoinjector. A YMC-pack ODS-A column, 4.6 /150 mm, 5 mm (YMC Co, Ltd, Kyoto, Japan) was used. The mobile phase was acetonitrile /0.02 mol l 1 ammonium acetate buffer / triethylamine (35:65:0.035, v/v/v), filtered through a 0.45-mm Millipore filter and degassed prior to use. The flow rate was 0.9 ml min 1. Detection was performed at a wavelength of 220 nm at room temperature. The sample injection volume was 20 ml. 2.3. Animals and blood sampling Male Wistar-strain rats (180 /210 g) were obtained from SLC Inc. (Shizuoka, Japan), and fasted for 12 h with free access to water, prior to the experiments. A polyethylene tube (0.28 mm, I.D., 0.61 mm, O.D.) was inserted into the right femoral artery of the rat while the animal was under anesthesia with ether. The rat was placed in a Bollman cage and allowed to recover from anesthesia for more than 1 h. Matrine aqueous solution was then orally administered to the rat at a dose of 40 mg kg 1 or was injected through the cannulated tube into the blood at a single dose of 40 or 4 mg kg 1. Blood samples (0.25 ml) were
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collected through the cannulated tube at times 0, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360 and 480 min after oral administration, or 0, 5, 10, 20, 30, 45, 60, 90, 120, 180, 240 min after bolus injection. After each sampling, the reduced blood volume was supplemented with an equal volume of saline containing 100 IU ml 1 heparin. Water was supplied to each rat 1 h after dosing. 2.4. Preparation of plasma samples Each collected blood sample was immediately transferred to a heparinized microcentrifuge tube and centrifuged at 8000 rpm for 5 min at 4 8C (Himac CT 13R, Hitachi, Japan). The resulting plasma (0.1 ml) was then vortex-mixed with 0.2 ml of acetonitrile containing phenacetin (5 mg ml 1) as internal standard for 30 s. After 15 min the mixture was centrifuged at 8000 rpm for 2 min at 4 8C to separate precipitated proteins. The supernatant was transferred into 1.5-ml tubes containing 50 /60 mg sodium chloride. The suspension was vortex-mixed briefly and incubated at room temperature for 6 min. After vortex-mixing and centrifuging once again at 8000 rpm for 5 min at 4 8C, 20 ml of the upper organic layer that was filtered through a 0.45-mm Millipore filter was directly injected into the chromatography. The same sample handling process was used for recovery and precision determinations in plasma.
Fig. 2. Chromatrograms of matrine in rat plasma: (a) blank plasma, (b) plasma spiked with matrine (12 mg ml 1) and phenacetin (internal standard), (c) plasma sample 30 min after oral administration of matrine aqueous solution at a dose of 40 mg kg 1. Retention times of phenacetin (internal standard) and matrine were approximately 4.8 and 6.4 min, respectively.
2.5. Calibration curve Calibration curves in the concentration range of 0.5 /50.0 mg ml 1 for matrine was constructed by plotting the peak /area ratios of analyte/internal standard versus matrine concentration in rat plasma. In order to avoid undue bias to the low concentrations of the standard curve by the high concentrations, the calibration curve was split into two ranges: 0.5 /6.0 and 6.0 /50.0 mg ml 1. Leastsquares linear regression analysis was used to determine the slope, intercept and correlation coefficient. The concentration of matrine in plasma was determined from the peak /area ratios by using the equations of linear regression obtained from the calibration curves. To determine the limit of quantification dilutions of 0.1, 0.2, 0.3,
0.4 mg ml 1 matrine in plasma were prepared using a solution of 2 mg ml 1 matrine in plasma for calibration.
2.6. Recovery Plasma samples were spiked with matrine at concentrations of 2.0, 24.0 and 50.0 mg ml 1. The resulting peak /area ratios (analyte: internal standard) were compared with that of the standards prepared in acetonitrile to provide the recovery values.
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Table 1 Recovery of the matrine assay Spiked concentration (mg ml 1)
2.0 24.0 50.0
Peak /area ratio Untreated
Treated
0.079/0.00 1.099/0.02 2.389/0.01
0.069/0.00 1.019/0.01 2.129/0.03
Recovery (%)
CV (%)
93.569/2.45 92.479/1.01 88.969/1.14
2.61 1.09 1.28
Each value represents the mean9/S.D. (n /5).
2.7. Precision Precision over the entire working dose range was determined by analyses of plasma samples (n /5) spiked with matrine at concentrations of 1.0, 2.0, 6.0, 12.0, 24.0 and 50.0 mg ml 1. To determine intra-day variance, the assays were carried out on the same samples at different times during the day. Inter-day variance was determined by assaying the spiked samples over three consecutive days. Coefficients of variation (CVs) were calculated from these values. 2.8. Assay application The present method was used to determine the plasma concentrations of matrine in rat plasma after oral administration or bolus injection of matrine aqueous solution from which the fractions of matrine reaching the systemic circulation were estimated by a deconvolution method.
3. Results and discussion 3.1. HPLC chromatograms Under the condition described above, the HPLC chromatograms of blank, plasma spiked with matrine at concentration of 12.0 mg ml 1 and the plasma obtained 30 min after oral administration of matrine aqueous solution at a dose 40 mg kg 1, were shown in Fig. 2. The retention times for phenacetin (internal standard) and matrine
were approximately 4.8 and 6.4 min, respectively. The peaks were sharp and symmetrical with good baseline resolution and minimal tailings, thus facilitating accurate measurements of the peak / area ratios. No endogenous plasma components elute at the retention time of matrine or internal standard. For the separation of matrine, phenacetin (I.S.) and endogenous plasma components by reverse phase HPLC, the following experimental conditions, namely, choice of mobile phase, phase flow rate, amounts of modifier have been investigate. A mixture of standard matrine and I.S. dissolved in acetonitrile was injected into a C18 column at flow rate of 0.7 ml min 1. Subsequently, the mobile phase flow rate was varied between 0.7 and 1.2 ml min 1. The optimum mobile phase flow rate was found to be 0.9 ml min 1, which was used for further optimization of other parameters. Several mobile phases were investigated using an isocratic system in all cases. The mobile phase with the composition of acetonitrile /water/triethylamine (40:60:0.035) gave sharper peak shapes and approximately four times peak /area as compared with methanol /water/triethylamine (60:40:0.035). The results showed that it was lack of sensitivity by using methanol /water/triethylamine (60:40:0.035) as mobile phase. In the other hand, good resolution of blank or standard samples spiked with drug-free plasma could not obtained by using acetonitrile /water/triethylamine (40:60:0.035) because of poor baseline. To improve baseline stability, instead of water in mobile phase, 0.02 mol l 1 ammonium acetate buffer was
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Table 2 Validation of the intra-day assay Spiked concentration (mg ml 1)
Measured concentration (mg ml 1)
Accuracy (%)
CV (%)
1.0 2.0 6.0 12.0 24.0 50.0
1.009/0.04 1.939/0.04 6.099/0.18 11.799/0.23 24.329/0.25 49.949/0.60
99.98 96.40 101.58 98.29 101.35 99.87
4.35 2.05 2.90 1.99 1.02 1.20
Each value represents the mean9/S.D. (n /5). Table 3 Validation of the inter-day assay Spiked concentration (mg ml 1)
Measured concentration (mg ml 1)
Accuracy (%)
CV (%)
1.0 2.0 6.0 12.0 24.0 50.0
0.929/0.06 1.939/0.12 6.239/0.22 11.949/0.30 24.069/0.48 50.319/0.58
91.75 96.37 103.85 99.46 100.26 100.63
6.30 6.15 3.47 2.52 1.99 1.16
Each value represents the mean9/S.D. (n /3).
adopted throughout the HPLC analysis. Although the addition of triethylamine improved the tailing of matrine peak, the baseline began to drift when its amount exceeded out 0.05 in the proportions of mobile phase. Combining the baseline and the peak tailing of the matrine, the optimum amount of triethylamine was found to be 0.035 in the proportions of mobile phase.
3.2. Calibration curves Matrine was dissolved in acetonitrile and diluted to give a series of standard solutions (0.5, 1.0, 2.0, 6.0, 12.0, 24.0, 50.0 mg ml 1) for the calibration curves of the drug in rat plasma. The linear regression analysis of matrine was constructed by plotting the peak /area ratio of matrine to the internal standard (y) versus analyte concentration (mg ml 1) in spiked plasma samples (x ). The calibration curves for matrine were linear both the low (0.5 /6.0 mg ml 1) and high concentration
ranges (6.0 /50.0 mg ml 1) with r2 values of 0.9991 and 0.9999, respectively. 3.3. Recovery The recovery was assessed by comparing the peak /area ratios (analyte: internal standard) obtained from spiked plasma samples of different analyte concentrations to the peak/area ratios for the samples containing the equivalent amounts of the analyte and internal standard directly dissolved in acetonitrile. The recoveries of matrine from rat plasma were shown in Table 1. 3.4. Accuracy, reproducibility and limit of quantitation The reproducibility of the method was defined by examining both intra- and inter-day variance. Analytical accuracy and precision data were shown in Tables 2 and 3 and expressed as mean detected concentration and CV. The following
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X. Wu et al. / Talanta 59 (2003) 965 /971
Fig. 3. Plasma concentration profiles of matrine after bolus injection at a dose of 40 mg kg 1 (j) and 4 mg kg 1 ("). Data was shown as mean9/S.D. of three experiments.
validations assess the suitability of the method: the mean value should be within 9/15% of the actual value except at the limit of quantitation where it should be within 9/20%, relative standard deviation (R.S.D.) around the mean value should not exceed 15% except at the limit of quantitation where it should be within 9/20% [11]. The assay precision (CVs) of matrine at low to high concentrations was better than 5 and 7% for intra-day and inter-day assays, respectively. Assay accuracy, assessed by calculating the measured concentrations as a percent of the spiked concentrations, was better than 91% (Tables 2 and 3). The limit of quantitation, defined as the lowest concentration on the calibration curve at which both accuracy and precision should be within 20% [11], was deemed to be 200 ng ml 1 for matrine. The sensitivity of this method for determining matrine in rat plasma is higher than that of the reported methods [9,10]. In addition, a common pretreatment method of biological fluids is as follows: matrine is extracted into organic solvents such as ethyl acetate, dichloromethane or chloroform. The organic solvent is evaporated and the residue is dissolved in eluent. The method described above is also outstanding with respect to simplicity in the samples pretreatment. These results indicate that
Fig. 4. Plasma concentration /time curve of matrine in rat after oral administration of matrine aqueous solution at a dose of 40 mg kg 1. Data was shown as mean9/S.E.M. of five experiments.
the assay was simple, accurate, sensitive and reproducible. 3.5. Application In order to estimate the absorption rate of matrine in gastrointestinal tract, the plasma concentrations of matrine was analyzed in rat plasma,
Fig. 5. Time-courses of fraction of matrine reaching systemic circulation after oral administration of matrine aqueous solution. Data was shown as mean9/S.E.M. of five experiments.
X. Wu et al. / Talanta 59 (2003) 965 /971
following oral administration of matrine aqueous solution or bolus injection. The plasma concentration of matrine was determined at 0, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360 and 480 min after oral dosing or 0, 5, 10, 20, 30, 45, 60, 90, 120, 180, 240 min after bolus injection. Fig. 3 showed the mean9/S.D. plasma concentration /time profile of matrine after bolus injection at a dose of 40 and 4 mg kg 1. When the bolus injection dose of matrine was increased from 4 to 40 mg kg 1, there were no significant differences in the apparent total body clearance, the elimination half-life and volume of distribution. The area under the curve of matrine appears to increase proportionally from 4 to 40 mg kg 1 (data not shown). These results suggest that the pharmacokinetic of matrine is a linear process. Fig. 4 showed the mean9/standard error of the mean (S.E.M.) plasma concentration / time profile of matrine after oral administration. The mean maximum concentration of matrine in plasma was 3.89 mg ml 1 at 30 min after oral dosing. The fractions of matrine absorbed were estimated by a deconvolution method (Fig. 5). The fraction of matrine reaching the systemic circulation rapidly increases to 0.39 within the first 2 h and then slowly to 0.44 by 8 h. In conclusion, this paper describes a simple, rapid, sensitive, accurate and precise procedure for the determination of matrine, suitable for the analysis of large numbers of plasma samples. The assay was validated to meet the requirements of pharmacokinetic studies. The fractions of matrine reaching the systemic circulation following oral
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dosing would be used as a suitable reference in clinical application.
Acknowledgements The project was supported by the National Natural Science Foundation of China (No. 20275014).
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