Journal of Chromatography B, 945–946 (2014) 154–162
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Pharmacokinetics and excretion study of sophoricoside and its metabolite in rats by liquid chromatography tandem mass spectrometry Xuran Zhi, Ning Sheng, Lin Yuan, Zhiyong Zhang, Peipei Jia, Xiaoxu Zhang, Lantong Zhang ∗ Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang, PR China
a r t i c l e
i n f o
Article history: Received 17 September 2013 Received in revised form 18 November 2013 Accepted 25 November 2013 Available online 1 December 2013 Keywords: Pharmacokinetics Excretion Sophoricoside Metabolite Genistein LC–MS/MS
a b s t r a c t In this study, a new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of sophoricoside and its metabolite genistein in rat plasma, bile, urine and feces after oral administration of sophoricoside, using sulfamethalazole as internal standard (IS). The separation was performed on a reverse phase C18 column with gradient elution consisting of 0.2‰ aqueous formic acid and methanol (containing 0.2‰ formic acid). The detection was accomplished by multiple-reaction monitoring (MRM) scanning after electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 431.2/268.2 for sophoricoside, 268.7/133.0 for genistein and 252.0/156.0 for IS. This developed method provides good linearity (r > 0.9983), intra- and inter-day precisions (RSD < 8.31%) with accuracies (RE, −6.91 to 6.66%), stability (RE, −7.45 to 6.59%), extract recovery (76.24 to 93.30%) and matrix effect (81.06–106.2%) of the analytes in plasma, bile, urine and feces. The mean elimination half-life (t1/2 ) of sophoricoside and genistein were 59.78 ± 7.19 and 103.14 ± 16.97 min, respectively. The results showed that sophoricoside was rapidly absorbed and then eliminated from rat plasma. The total recoveries of sophoricoside in bile, urine and feces were about 0.0111%, 1.76% and 11.13%. The amounts excreted of genistein were 0.42 ± 0.02 g in bile, 10.15 ± 0.22 g in urine and 2.92 ± 0.13 g in feces. This is the first report to evaluate the pharmacokinetics and excretion of sophoricoside and its metabolite in rats after oral administration of sophoricoside monomer. The results provided a meaningful basis for the clinical application of sophoricoside. © 2013 Published by Elsevier B.V.
1. Introduction Traditional Chinese Medicine, commonly referred to as “Chinese Medicine” or simply “TCM”, is one of the oldest forms of medical treatment and one of the most commonly used in the world. Fructus Sophorae (common name in Chinese Huai Jiao), the dried ripe fruits of Styphnolobium japonicum (L.) Schott (Leguminosae), is a herbal ingredient and has been used for thousands of years [1–3]. Modern pharmacological and clinical studies have shown that Fructus Sophorae possessed hemostatic properties, anticancer, anti-tumor, anti-obesity, antifertility action, anti-oxidation and it has been commonly used for the treatment of hypertension and hemorrhoids [4–8]. Sophoricoside is a flavonoid compound in the highest
∗ Corresponding author at: Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, No. 361, Zhongshan East Road, Shijiazhuang 050017, PR China. Tel.: +86 311 86266419; fax: +86 311 86266419. E-mail address: [email protected] (L. Zhang). 1570-0232/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jchromb.2013.11.049
concentration in Fructus Sophora [9,10] and can be hydrolyzed to 4,5,7-trihydroxyisoflavone (genistein), which has three OH groups at C-5, C-7, and C-4, respectively [11,12]. Monomer composition on the metabolism of traditional Chinese medicine, especially for the metabolism of the active ingredient of Chinese medicine chemistry, has drawn increasing attention [13,14]. Pharmacokinetics study on active ingredients in natural products and TCM are important to illustrate their mechanism of action. Studying the excretion of medicine is useful to determine the effect, side effects and toxicity of medicine, which can provide the suitable dosage and manner of administration of the medicine [15,16]. Until now, even with the comprehensive research on bioactivity, there was little knowledge about pharmacokinetics and excretion study of sophoricoside and its metabolite. The rate at which sophoricoside undergoes biotransformation to genistein has been shown to directly influence the activity of sophoricoside. For a better usage of sophoricoside, it is important to learn the pharmacokinetics of sophoricoside and its metabolite. Due to the high sensitivity and selectivity of the advanced method, liquid chromatography–mass
X. Zhi et al. / J. Chromatogr. B 945–946 (2014) 154–162
spectrometry (LC–MS) has been widely used in vivo studies [17,18]. These considerations underscore the potential utility of a rapid and convenient assay for the measurement of sophoricoside and its metabolite. Q TRAPTM System, which combines a linear ion trap with the third quadrupole, retained the excellent quantitative capabilities of the triple quadrupole with the addition of a broad range of informative fragment ions. Thus both quantitative and qualitative information could be concomitantly obtained. In the present paper, we studied the pharmacokinetics of sophoricoside and its metabolite, and biliary, urinary and fecal excretion, after a single oral administration of sophoricoside monomer in rats, for exploring its fate in the body and further better understanding its in vivo pharmacological activities. 2. Material and methods 2.1. Chemicals and reagents HPLC-grade methanol (J.T. Baker, USA) was used for HPLC analysis. HPLC-grade formic acid was purchased from Diamond Technology (Dikma Technology Corporation, USA). Purified water was obtained from Wahaha (Hangzhou Wahaha Group Co., Ltd.). Analytical-grade ethyl acetate (Tianjin Chemical, Tianjin, China) was used for sample preparation. Sophoricoside (11061521) and genistein (11012521) (98.0%, purity) were obtained from National Institute for the Control of Pharmaceutical and Biological Products. Sulfamethoxazole [internal standard (IS)] was obtained from National Institute for the Control of Pharmaceutical and Biological Products. Heparin (Liquemine, 125,000 IU, The First Biochemical Pharmaceutical Co. Ltd., Shanghai, China) was used as an anticoagulant in all samples plasma. 2.2. Instrumentation and analytical conditions The LC–MS/MS system was used for all analytes. They include “system” of an Agilent Series 1200 (Agilent, USA) liquid chromatograph equipped with a vacuum degasser, a binary pump and an autosampler, connected to an Agilent ChemStation software and a 3200 QTRAPTM system from Applied Biosystems/MDS Sciex (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion trap mass spectrometer equipped with TurboV sources and TurboIonspray interface. The chromatographic separation was achieved at 30 ◦ C on a Diamonsil C18 column (150 mm × 4.6 mm, 5 m). The mobile phase consisted of methanol containing 0.2‰ formic acid (A) and water containing 0.2‰ formic acid (B). The elution program was optimized as follows: 0–1.5 min, 45%A → 75%A; 1.5–5 min, 75%A → 95%A, 5–8 min, 95%A. This was followed by the equilibration period of 6 min prior to the injection of each sample. The flow rate of mobile phase was set at 0.8 mL/min and the injection volume was 20 L. After chromatographic separation, the mobile phase was directly introduced into the mass spectrometer via an electrospray ionization (ESI) source operating in the negative mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z 431.2–m/z 268.2 for sophoricoside, m/z 268.7–m/z 133.0 for genistein, m/z 252.0–m/z 156.0 for IS, respectively, with a dwell time of 200 ms. The ion spray voltage was set to −4500 V; the turbo spray temperature was 650 ◦ C; nebulizer gas (gas 1) and heater gas (gas 2) were set at 60 and 65 arbitrary units, respectively; the curtain gas was kept at 30 arbitrary units and the interface heater was on. Collision cell exit potential (CXP) and entrance potential (EP) were set at −5.0 V and −10.0 V, respectively. Nitrogen was used in all cases. The declustering potential (DP) were set at −85, −65 and −21 V for sophoricoside, genistein and
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IS, respectively. The values of the collision energy (CE) were −45, −44 and −23 eV for sophoricoside, genistein and IS, respectively. The product ion scan spectra and chemical structure of sophoricoside, genistein and sulfamethoxazole (IS) were shown in Fig. 1. Other parameters were also optimized for maximum abundance of the ion of interest by the automatic tuning procedure of the instrument. All data was controlled and synchronized by Analyst software (Versions 1.5.2) from Applied Biosystems/MDS Sciex. 2.3. Animals, dosing and sampling Male Sprague–Dawley rats, weighing 250 ± 20 g, were supplied by Experimental Animal Research Center, Hebei Medical University, China. The rats were kept at a temperature of 22 ± 2 ◦ C and a relative humidity of 55 ± 5%, and had access to standard laboratory food and water. This project all animal experiments were carried out according to guide lines for experimental animal management committee of Hebei Medical University, China. Rats were fasted for 12 h before oral administration, and water was freely available. All sophoricoside dosing drug were freshly prepared at the concentration of 5 mg/mL before experiment. For oral drug administration, sophoricoside was homogenously suspended in 0.5% sodium carboxyl methyl cellulose water solution by sonication for 10 min. 2.4. Preparation of standard solution and quality control solutions The appropriate amounts of sophoricoside and genistein were separately weighed and dissolved as the stock solutions. Then, the stock solutions were mixed and diluted with methanol to prepare a final mixed standard solution. A series of working solutions of these analytes were obtained by diluting mixed standard solution with methanol at appropriate concentrations. A quantity of sulfamethoxazole was dissolved in methanol to produce the IS solution with a concentration of 200 ng/mL for plasma and bile (IS1), 400 ng/mL for urine and feces (IS2). Calibration curves were prepared by spiking the appropriate standard solution into a certain volume of the four blank biological substrates. Effective concentrations of sophoricoside in samples were 1.8, 3.6, 18, 90, 180, 270, 360 ng/mL for plasma, 0.70, 3.50, 35.32, 70.64, 141.27, 176.60, 353.18 ng/mL for bile, 48.25, 241.25, 482.5, 2412.5, 7237.5, 12,062.5, 24,125 ng/mL for urine and 4.78, 47.80, 478, 1195, 2390, 4780, 9560 ng/mL for feces. Effective concentrations of genistein in samples were 10.75, 21.49, 107.45, 537.27, 1074.53, 1611.80, 2149.07 ng/mL for plasma, 0.58, 2.90, 29.03, 58.07, 116.13, 145.17, 290.33 ng/mL for bile, 6.36, 31.79, 63.58, 317.9, 953.68, 1589.48, 3178.95 ng/mL for urine and 0.147, 0.74, 3.68, 18.38, 36.75, 73.5, 110.25 ng/mL for feces. The quality control (QC) samples containing low, medium and high concentrations were prepared at the concentrations of: sophoricoside (3.6, 90, 270 ng/mL), genistein (21.49, 537.27, 1611.80 ng/mL) for plasma; sophoricoside (3.53, 70.64, 176.6 ng/mL), genistein (2.9, 58.07, 145.17 ng/mL) for bile; sophoricoside (241.25, 2412.5, 12,062.5 ng/mL), genistein (31.79, 317.9, 1589.48 ng/mL) for urine, sophoricoside (47.8, 1195, 4780 ng/mL), genistein (0.74, 18.38, 73.5 ng/mL) for feces. 2.5. The collection of samples 2.5.1. For plasma collection For pharmacokinetics study, six rats received the above sophoricoside dosing drugs, at 20 mg/kg by gastric gavages. Blood samples of approximately 0.3 mL were collected from the epicanthic veins of rats at 5, 15, 30, 45, 60, 90, 120, 180, 270 and 360 min (sophoricoside) and 1.5, 3, 4.5, 5.5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 and 72 h (genistein) after a single oral administration. The blood samples were immediately transferred to heparinized tubes and centrifuged
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X. Zhi et al. / J. Chromatogr. B 945–946 (2014) 154–162
Fig. 1. The product ion spectra and the chemical structures of sophoricoside, genistein and sulfamethoxazole (IS).
at 4000 rpm for 5 min. Then the plasma layer was transferred to clean tubes and stored at −20 ◦ C. Blank plasma was obtained from the rat without oral administration and was used to investigate the assay development and validation. 2.5.2. For bile collection Before bile sample collection, six rats were given the prepared suspensions at a dose of 20 mL/kg and the rats received a surgery under anesthesia condition for cannulation of a polyethylene catheter with the bile fistulas. The bile was collected at 0–2, 2–4, 4–6, 6–8, 8–12, 12–24, 24–30 and 30–36 h, the volume determined and stored at −20 ◦ C. The blank bile was prepared from a rat orally administered 0.5% CMC-Na. 2.5.3. For urine and feces collection The rats (n = 6) were placed in metabolite cages individually and received sophoricoside by gastric gavages at 20 mg/kg as described above. After oral administered, the urine was collected at 0–2, 2–4, 4–8, 8–12, 12–24, 24–36, 36–48, 48–72 h, the volume determined and stored at -20◦ C. The feces were collected at 0–4, 4–8, 8–10, 10–12, 12–24, 24–36, 36–48 h, dried at room temperature. The blank urine and feces were prepared using a rat orally administered 0.5% CMC-Na. 2.6. The preparation of samples 2.6.1. For plasma A simple liquid–liquid extraction (LLE) method was applied to extract the two flavonoids and IS1 from rat plasma. To a 100 L of the rat plasma, 20 L of the IS1 and 20 L of methanol (volume of the corresponding working solution for calibration curve and QC samples). Then the mixture was vortexed for 1 min and extracted with 1 mL of ethyl acetate by shaking on a vortex-mixer for 5 min at room temperature. The upper layer was transferred to a clean tube after centrifugation at 4000 rpm for 5 min. The upper organic phase was evaporated to dryness under a gentle stream of nitrogen. The obtained residue was reconstituted in 100 L of 80% methanol and centrifuged at 12,000 rpm for another 10 min.
Subsequently, aliquots of 20 L were injected into the HPLC–MS system for analysis. 2.6.2. For bile and urine The samples of bile or urine (300 L) were mixed with 20 L of IS solution (IS1 for bile and IS2 for urine) and 20 L of methanol (20 L of the volume of the corresponding working solution for the calibration curve and QC samples). Then the mixture was vortexed for 1 min and extracted with 600 L of ethyl acetate by shaking on a vortex-mixer for 5 min at room temperature. After centrifugation at 12,000 rpm for 10 min in a centrifuge, the clear supernatant fluid was then transferred into another centrifuge tube and evaporated to dryness under a gentle stream of nitrogen. Then the following processing was the same as described above. 2.6.3. For feces The samples of feces (0.2 g) were mixed with 20 L of IS2 solution and 20 L of methanol (the volume of the corresponding working solution for the calibration curve and QC samples). After adding 5 mL methanol as extraction solvent, the mixture was ultrasounded for 45 min. After the centrifugation at 12,000 rpm for 10 min, the supernatant was evaporated to dryness under a gentle nitrogen stream. The residue was finally reconstituted in 500 L of methanol. After the centrifugation at 12,000 rpm for 10 min, 20 L of the supernatant was injected into the HPLC–MS system. 2.7. Method validation The method was validated in terms of specificity, linearity, the lowest limit of quantification (LLOQ), the limit of detection (LOD), intraday precision, interday precision, accuracy, extraction recoveries, matrix effect, and stability. The specificity of the method was evaluated by comparing the blank plasma, bile, urine and feces with the corresponding spiked plasma, bile, urine and feces samples, the plasma, bile, urine and feces samples collected after oral administration. There should be no obvious interference observed at the retention times of the analytes and IS in blank samples. The retention times of analytes and IS
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in spiked plasma, bile, urine and feces should correspond to those of the actual biological samples. The calibration samples were prepared by adding 20 L of working solutions and 20 L of IS solution to 100 L of blank plasma, 300 L of blank bile and blank urine, 0.2 g of blank feces, respectively, and then extracted as described above. Calibration curves were constructed using the analytes and IS peak-area ratios via a weighted (1/2 ) least-squares linear regression in the form of y = ax + b. The LLOQ of the assay was defined as the lowest concentration of the standard curve that could be quantitated (LLOQ, S/N = 10). The LOD was defined as the amount that could be detected (LOD, S/N = 3). The intra- and inter-day precision and accuracy were evaluated by replicate analysis of five sets of samples spiked with QC samples at three concentration levels of sophoricoside and genistein within a day or on three consecutive days. The precision was denoted by the relative standard deviation (RSD) and the accuracy was expressed by relative error (RE). Each concentration was calculated using a calibration curve constructed on the same day. The extraction recoveries of analytes at three QC levels were evaluated by determining the peak area ratios of the analytes in the post-extraction spiked samples to that acquired from preextraction spiked samples. The matrix effects were measured by comparing the peak areas of the analytes dissolved in the pretreated blank plasma with that of pure standard solution containing equivalent amounts of the analytes. The stability of the analytes in plasma, bile, urine and feces were assessed by analyzing QC samples at three concentration levels through three freeze–thaw cycles (−20 ◦ C to room temperature as one cycle), on the bench at room temperature for 24 h (short-term stability), at −20 ◦ C in the freezer for 30 days (long-term stability). Each analyte was determined at least six times. 2.8. Pharmacokinetics study The concentration versus time profiles were obtained for each individual rat, and non-compartmental pharmacokinetic modeling and pharmacokinetic parameters calculation were performed using Excel software. The pharmacokinetic parameters, such as maximum plasma concentration (Cmax ) and time of maximum concentration (Tmax ), were obtained directly from the plasma concentration–time plots [19]. The elimination rate constants (k) were determined by linear regression analysis of the logarithmic transformation of the last four data points of the curve. The elimination half-life (t1/2 ) was calculated using the half-life of a first-order rate equation: t1/2 = 0.693/k. The area under the plasma concentration–time curve up to the last time (t) (AUC0–t ) was determined using the trapezoidal rule. The AUC0–∞ values were calculated by adding the value of Ct × k−1 to AUC0−t .
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suitable and gave good peak separation, sharp peaks and short analysis time. Furthermore, other chromatographic variables were also optimized, including column temperatures (25 ◦ C and 30 ◦ C), and flow rates (0.8 and 1.0 mL/min). Eventually, the optimal condition of the Diamonsil C18 column (150 mm × 4.6 mm, 5 m) at the column temperature of 30 ◦ C with the flow rate of 0.8 mL/min was beneficial for enhancing the ionization of compounds detected in negative electrospray interface mode. In the mobile phase test, the addition of formic acid increased the ionization efficiency of coumarins [15]. In this study, the chromatographic separation was carried out with gradient elution, so formic acid added to both aqueous and organic phase kept the ratio of acid in the mobile phase in the process of analysis, which is why methanol containing 0.2‰ formic acid–0.2‰ formic acid aqueous solution was chosen the optimal mobile phase. In this study, it was found that sophoricoside, genistein and IS could be well ionized under ESI in negative electrospray ionization conditions. The declustering potential and collision energy were optimized in order to obtain the maximum sensitivity of analytes. 3.2. Method validation For specificity, sophoricoside, genistein and IS could be detected on their own selected ion chromatograms without any significant interference (Figs. 2 and 3). The regression equation of calibration curves, their correlation coefficients (r), linear ranges, LODs and LLOQs obtained from typical calibration curves of plasma, bile, urine and feces were shown in Table 1. Over the concentration range for sophoricoside and genistein in rat plasma, bile, urine and feces, the standard curves showed good linearity. The results of the inter- and intra-day precision (
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Pharmacokinetics and excretion study of sophoricoside and its metabolite in rats by liquid chromatography tandem mass spectrometry Xuran Zhi, Ning Sheng, Lin Yuan, Zhiyong Zhang, Peipei Jia, Xiaoxu Zhang, Lantong Zhang ∗ Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang, PR China
a r t i c l e
i n f o
Article history: Received 17 September 2013 Received in revised form 18 November 2013 Accepted 25 November 2013 Available online 1 December 2013 Keywords: Pharmacokinetics Excretion Sophoricoside Metabolite Genistein LC–MS/MS
a b s t r a c t In this study, a new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of sophoricoside and its metabolite genistein in rat plasma, bile, urine and feces after oral administration of sophoricoside, using sulfamethalazole as internal standard (IS). The separation was performed on a reverse phase C18 column with gradient elution consisting of 0.2‰ aqueous formic acid and methanol (containing 0.2‰ formic acid). The detection was accomplished by multiple-reaction monitoring (MRM) scanning after electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 431.2/268.2 for sophoricoside, 268.7/133.0 for genistein and 252.0/156.0 for IS. This developed method provides good linearity (r > 0.9983), intra- and inter-day precisions (RSD < 8.31%) with accuracies (RE, −6.91 to 6.66%), stability (RE, −7.45 to 6.59%), extract recovery (76.24 to 93.30%) and matrix effect (81.06–106.2%) of the analytes in plasma, bile, urine and feces. The mean elimination half-life (t1/2 ) of sophoricoside and genistein were 59.78 ± 7.19 and 103.14 ± 16.97 min, respectively. The results showed that sophoricoside was rapidly absorbed and then eliminated from rat plasma. The total recoveries of sophoricoside in bile, urine and feces were about 0.0111%, 1.76% and 11.13%. The amounts excreted of genistein were 0.42 ± 0.02 g in bile, 10.15 ± 0.22 g in urine and 2.92 ± 0.13 g in feces. This is the first report to evaluate the pharmacokinetics and excretion of sophoricoside and its metabolite in rats after oral administration of sophoricoside monomer. The results provided a meaningful basis for the clinical application of sophoricoside. © 2013 Published by Elsevier B.V.
1. Introduction Traditional Chinese Medicine, commonly referred to as “Chinese Medicine” or simply “TCM”, is one of the oldest forms of medical treatment and one of the most commonly used in the world. Fructus Sophorae (common name in Chinese Huai Jiao), the dried ripe fruits of Styphnolobium japonicum (L.) Schott (Leguminosae), is a herbal ingredient and has been used for thousands of years [1–3]. Modern pharmacological and clinical studies have shown that Fructus Sophorae possessed hemostatic properties, anticancer, anti-tumor, anti-obesity, antifertility action, anti-oxidation and it has been commonly used for the treatment of hypertension and hemorrhoids [4–8]. Sophoricoside is a flavonoid compound in the highest
∗ Corresponding author at: Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, No. 361, Zhongshan East Road, Shijiazhuang 050017, PR China. Tel.: +86 311 86266419; fax: +86 311 86266419. E-mail address: [email protected] (L. Zhang). 1570-0232/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jchromb.2013.11.049
concentration in Fructus Sophora [9,10] and can be hydrolyzed to 4,5,7-trihydroxyisoflavone (genistein), which has three OH groups at C-5, C-7, and C-4, respectively [11,12]. Monomer composition on the metabolism of traditional Chinese medicine, especially for the metabolism of the active ingredient of Chinese medicine chemistry, has drawn increasing attention [13,14]. Pharmacokinetics study on active ingredients in natural products and TCM are important to illustrate their mechanism of action. Studying the excretion of medicine is useful to determine the effect, side effects and toxicity of medicine, which can provide the suitable dosage and manner of administration of the medicine [15,16]. Until now, even with the comprehensive research on bioactivity, there was little knowledge about pharmacokinetics and excretion study of sophoricoside and its metabolite. The rate at which sophoricoside undergoes biotransformation to genistein has been shown to directly influence the activity of sophoricoside. For a better usage of sophoricoside, it is important to learn the pharmacokinetics of sophoricoside and its metabolite. Due to the high sensitivity and selectivity of the advanced method, liquid chromatography–mass
X. Zhi et al. / J. Chromatogr. B 945–946 (2014) 154–162
spectrometry (LC–MS) has been widely used in vivo studies [17,18]. These considerations underscore the potential utility of a rapid and convenient assay for the measurement of sophoricoside and its metabolite. Q TRAPTM System, which combines a linear ion trap with the third quadrupole, retained the excellent quantitative capabilities of the triple quadrupole with the addition of a broad range of informative fragment ions. Thus both quantitative and qualitative information could be concomitantly obtained. In the present paper, we studied the pharmacokinetics of sophoricoside and its metabolite, and biliary, urinary and fecal excretion, after a single oral administration of sophoricoside monomer in rats, for exploring its fate in the body and further better understanding its in vivo pharmacological activities. 2. Material and methods 2.1. Chemicals and reagents HPLC-grade methanol (J.T. Baker, USA) was used for HPLC analysis. HPLC-grade formic acid was purchased from Diamond Technology (Dikma Technology Corporation, USA). Purified water was obtained from Wahaha (Hangzhou Wahaha Group Co., Ltd.). Analytical-grade ethyl acetate (Tianjin Chemical, Tianjin, China) was used for sample preparation. Sophoricoside (11061521) and genistein (11012521) (98.0%, purity) were obtained from National Institute for the Control of Pharmaceutical and Biological Products. Sulfamethoxazole [internal standard (IS)] was obtained from National Institute for the Control of Pharmaceutical and Biological Products. Heparin (Liquemine, 125,000 IU, The First Biochemical Pharmaceutical Co. Ltd., Shanghai, China) was used as an anticoagulant in all samples plasma. 2.2. Instrumentation and analytical conditions The LC–MS/MS system was used for all analytes. They include “system” of an Agilent Series 1200 (Agilent, USA) liquid chromatograph equipped with a vacuum degasser, a binary pump and an autosampler, connected to an Agilent ChemStation software and a 3200 QTRAPTM system from Applied Biosystems/MDS Sciex (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion trap mass spectrometer equipped with TurboV sources and TurboIonspray interface. The chromatographic separation was achieved at 30 ◦ C on a Diamonsil C18 column (150 mm × 4.6 mm, 5 m). The mobile phase consisted of methanol containing 0.2‰ formic acid (A) and water containing 0.2‰ formic acid (B). The elution program was optimized as follows: 0–1.5 min, 45%A → 75%A; 1.5–5 min, 75%A → 95%A, 5–8 min, 95%A. This was followed by the equilibration period of 6 min prior to the injection of each sample. The flow rate of mobile phase was set at 0.8 mL/min and the injection volume was 20 L. After chromatographic separation, the mobile phase was directly introduced into the mass spectrometer via an electrospray ionization (ESI) source operating in the negative mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z 431.2–m/z 268.2 for sophoricoside, m/z 268.7–m/z 133.0 for genistein, m/z 252.0–m/z 156.0 for IS, respectively, with a dwell time of 200 ms. The ion spray voltage was set to −4500 V; the turbo spray temperature was 650 ◦ C; nebulizer gas (gas 1) and heater gas (gas 2) were set at 60 and 65 arbitrary units, respectively; the curtain gas was kept at 30 arbitrary units and the interface heater was on. Collision cell exit potential (CXP) and entrance potential (EP) were set at −5.0 V and −10.0 V, respectively. Nitrogen was used in all cases. The declustering potential (DP) were set at −85, −65 and −21 V for sophoricoside, genistein and
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IS, respectively. The values of the collision energy (CE) were −45, −44 and −23 eV for sophoricoside, genistein and IS, respectively. The product ion scan spectra and chemical structure of sophoricoside, genistein and sulfamethoxazole (IS) were shown in Fig. 1. Other parameters were also optimized for maximum abundance of the ion of interest by the automatic tuning procedure of the instrument. All data was controlled and synchronized by Analyst software (Versions 1.5.2) from Applied Biosystems/MDS Sciex. 2.3. Animals, dosing and sampling Male Sprague–Dawley rats, weighing 250 ± 20 g, were supplied by Experimental Animal Research Center, Hebei Medical University, China. The rats were kept at a temperature of 22 ± 2 ◦ C and a relative humidity of 55 ± 5%, and had access to standard laboratory food and water. This project all animal experiments were carried out according to guide lines for experimental animal management committee of Hebei Medical University, China. Rats were fasted for 12 h before oral administration, and water was freely available. All sophoricoside dosing drug were freshly prepared at the concentration of 5 mg/mL before experiment. For oral drug administration, sophoricoside was homogenously suspended in 0.5% sodium carboxyl methyl cellulose water solution by sonication for 10 min. 2.4. Preparation of standard solution and quality control solutions The appropriate amounts of sophoricoside and genistein were separately weighed and dissolved as the stock solutions. Then, the stock solutions were mixed and diluted with methanol to prepare a final mixed standard solution. A series of working solutions of these analytes were obtained by diluting mixed standard solution with methanol at appropriate concentrations. A quantity of sulfamethoxazole was dissolved in methanol to produce the IS solution with a concentration of 200 ng/mL for plasma and bile (IS1), 400 ng/mL for urine and feces (IS2). Calibration curves were prepared by spiking the appropriate standard solution into a certain volume of the four blank biological substrates. Effective concentrations of sophoricoside in samples were 1.8, 3.6, 18, 90, 180, 270, 360 ng/mL for plasma, 0.70, 3.50, 35.32, 70.64, 141.27, 176.60, 353.18 ng/mL for bile, 48.25, 241.25, 482.5, 2412.5, 7237.5, 12,062.5, 24,125 ng/mL for urine and 4.78, 47.80, 478, 1195, 2390, 4780, 9560 ng/mL for feces. Effective concentrations of genistein in samples were 10.75, 21.49, 107.45, 537.27, 1074.53, 1611.80, 2149.07 ng/mL for plasma, 0.58, 2.90, 29.03, 58.07, 116.13, 145.17, 290.33 ng/mL for bile, 6.36, 31.79, 63.58, 317.9, 953.68, 1589.48, 3178.95 ng/mL for urine and 0.147, 0.74, 3.68, 18.38, 36.75, 73.5, 110.25 ng/mL for feces. The quality control (QC) samples containing low, medium and high concentrations were prepared at the concentrations of: sophoricoside (3.6, 90, 270 ng/mL), genistein (21.49, 537.27, 1611.80 ng/mL) for plasma; sophoricoside (3.53, 70.64, 176.6 ng/mL), genistein (2.9, 58.07, 145.17 ng/mL) for bile; sophoricoside (241.25, 2412.5, 12,062.5 ng/mL), genistein (31.79, 317.9, 1589.48 ng/mL) for urine, sophoricoside (47.8, 1195, 4780 ng/mL), genistein (0.74, 18.38, 73.5 ng/mL) for feces. 2.5. The collection of samples 2.5.1. For plasma collection For pharmacokinetics study, six rats received the above sophoricoside dosing drugs, at 20 mg/kg by gastric gavages. Blood samples of approximately 0.3 mL were collected from the epicanthic veins of rats at 5, 15, 30, 45, 60, 90, 120, 180, 270 and 360 min (sophoricoside) and 1.5, 3, 4.5, 5.5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 and 72 h (genistein) after a single oral administration. The blood samples were immediately transferred to heparinized tubes and centrifuged
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Fig. 1. The product ion spectra and the chemical structures of sophoricoside, genistein and sulfamethoxazole (IS).
at 4000 rpm for 5 min. Then the plasma layer was transferred to clean tubes and stored at −20 ◦ C. Blank plasma was obtained from the rat without oral administration and was used to investigate the assay development and validation. 2.5.2. For bile collection Before bile sample collection, six rats were given the prepared suspensions at a dose of 20 mL/kg and the rats received a surgery under anesthesia condition for cannulation of a polyethylene catheter with the bile fistulas. The bile was collected at 0–2, 2–4, 4–6, 6–8, 8–12, 12–24, 24–30 and 30–36 h, the volume determined and stored at −20 ◦ C. The blank bile was prepared from a rat orally administered 0.5% CMC-Na. 2.5.3. For urine and feces collection The rats (n = 6) were placed in metabolite cages individually and received sophoricoside by gastric gavages at 20 mg/kg as described above. After oral administered, the urine was collected at 0–2, 2–4, 4–8, 8–12, 12–24, 24–36, 36–48, 48–72 h, the volume determined and stored at -20◦ C. The feces were collected at 0–4, 4–8, 8–10, 10–12, 12–24, 24–36, 36–48 h, dried at room temperature. The blank urine and feces were prepared using a rat orally administered 0.5% CMC-Na. 2.6. The preparation of samples 2.6.1. For plasma A simple liquid–liquid extraction (LLE) method was applied to extract the two flavonoids and IS1 from rat plasma. To a 100 L of the rat plasma, 20 L of the IS1 and 20 L of methanol (volume of the corresponding working solution for calibration curve and QC samples). Then the mixture was vortexed for 1 min and extracted with 1 mL of ethyl acetate by shaking on a vortex-mixer for 5 min at room temperature. The upper layer was transferred to a clean tube after centrifugation at 4000 rpm for 5 min. The upper organic phase was evaporated to dryness under a gentle stream of nitrogen. The obtained residue was reconstituted in 100 L of 80% methanol and centrifuged at 12,000 rpm for another 10 min.
Subsequently, aliquots of 20 L were injected into the HPLC–MS system for analysis. 2.6.2. For bile and urine The samples of bile or urine (300 L) were mixed with 20 L of IS solution (IS1 for bile and IS2 for urine) and 20 L of methanol (20 L of the volume of the corresponding working solution for the calibration curve and QC samples). Then the mixture was vortexed for 1 min and extracted with 600 L of ethyl acetate by shaking on a vortex-mixer for 5 min at room temperature. After centrifugation at 12,000 rpm for 10 min in a centrifuge, the clear supernatant fluid was then transferred into another centrifuge tube and evaporated to dryness under a gentle stream of nitrogen. Then the following processing was the same as described above. 2.6.3. For feces The samples of feces (0.2 g) were mixed with 20 L of IS2 solution and 20 L of methanol (the volume of the corresponding working solution for the calibration curve and QC samples). After adding 5 mL methanol as extraction solvent, the mixture was ultrasounded for 45 min. After the centrifugation at 12,000 rpm for 10 min, the supernatant was evaporated to dryness under a gentle nitrogen stream. The residue was finally reconstituted in 500 L of methanol. After the centrifugation at 12,000 rpm for 10 min, 20 L of the supernatant was injected into the HPLC–MS system. 2.7. Method validation The method was validated in terms of specificity, linearity, the lowest limit of quantification (LLOQ), the limit of detection (LOD), intraday precision, interday precision, accuracy, extraction recoveries, matrix effect, and stability. The specificity of the method was evaluated by comparing the blank plasma, bile, urine and feces with the corresponding spiked plasma, bile, urine and feces samples, the plasma, bile, urine and feces samples collected after oral administration. There should be no obvious interference observed at the retention times of the analytes and IS in blank samples. The retention times of analytes and IS
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in spiked plasma, bile, urine and feces should correspond to those of the actual biological samples. The calibration samples were prepared by adding 20 L of working solutions and 20 L of IS solution to 100 L of blank plasma, 300 L of blank bile and blank urine, 0.2 g of blank feces, respectively, and then extracted as described above. Calibration curves were constructed using the analytes and IS peak-area ratios via a weighted (1/2 ) least-squares linear regression in the form of y = ax + b. The LLOQ of the assay was defined as the lowest concentration of the standard curve that could be quantitated (LLOQ, S/N = 10). The LOD was defined as the amount that could be detected (LOD, S/N = 3). The intra- and inter-day precision and accuracy were evaluated by replicate analysis of five sets of samples spiked with QC samples at three concentration levels of sophoricoside and genistein within a day or on three consecutive days. The precision was denoted by the relative standard deviation (RSD) and the accuracy was expressed by relative error (RE). Each concentration was calculated using a calibration curve constructed on the same day. The extraction recoveries of analytes at three QC levels were evaluated by determining the peak area ratios of the analytes in the post-extraction spiked samples to that acquired from preextraction spiked samples. The matrix effects were measured by comparing the peak areas of the analytes dissolved in the pretreated blank plasma with that of pure standard solution containing equivalent amounts of the analytes. The stability of the analytes in plasma, bile, urine and feces were assessed by analyzing QC samples at three concentration levels through three freeze–thaw cycles (−20 ◦ C to room temperature as one cycle), on the bench at room temperature for 24 h (short-term stability), at −20 ◦ C in the freezer for 30 days (long-term stability). Each analyte was determined at least six times. 2.8. Pharmacokinetics study The concentration versus time profiles were obtained for each individual rat, and non-compartmental pharmacokinetic modeling and pharmacokinetic parameters calculation were performed using Excel software. The pharmacokinetic parameters, such as maximum plasma concentration (Cmax ) and time of maximum concentration (Tmax ), were obtained directly from the plasma concentration–time plots [19]. The elimination rate constants (k) were determined by linear regression analysis of the logarithmic transformation of the last four data points of the curve. The elimination half-life (t1/2 ) was calculated using the half-life of a first-order rate equation: t1/2 = 0.693/k. The area under the plasma concentration–time curve up to the last time (t) (AUC0–t ) was determined using the trapezoidal rule. The AUC0–∞ values were calculated by adding the value of Ct × k−1 to AUC0−t .
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suitable and gave good peak separation, sharp peaks and short analysis time. Furthermore, other chromatographic variables were also optimized, including column temperatures (25 ◦ C and 30 ◦ C), and flow rates (0.8 and 1.0 mL/min). Eventually, the optimal condition of the Diamonsil C18 column (150 mm × 4.6 mm, 5 m) at the column temperature of 30 ◦ C with the flow rate of 0.8 mL/min was beneficial for enhancing the ionization of compounds detected in negative electrospray interface mode. In the mobile phase test, the addition of formic acid increased the ionization efficiency of coumarins [15]. In this study, the chromatographic separation was carried out with gradient elution, so formic acid added to both aqueous and organic phase kept the ratio of acid in the mobile phase in the process of analysis, which is why methanol containing 0.2‰ formic acid–0.2‰ formic acid aqueous solution was chosen the optimal mobile phase. In this study, it was found that sophoricoside, genistein and IS could be well ionized under ESI in negative electrospray ionization conditions. The declustering potential and collision energy were optimized in order to obtain the maximum sensitivity of analytes. 3.2. Method validation For specificity, sophoricoside, genistein and IS could be detected on their own selected ion chromatograms without any significant interference (Figs. 2 and 3). The regression equation of calibration curves, their correlation coefficients (r), linear ranges, LODs and LLOQs obtained from typical calibration curves of plasma, bile, urine and feces were shown in Table 1. Over the concentration range for sophoricoside and genistein in rat plasma, bile, urine and feces, the standard curves showed good linearity. The results of the inter- and intra-day precision (
