Abstract A ultra-performance liquid chromatography tandem mass spectrometry method was initially developed and validated for quantification of sophorabioside in rat plasma using kaempferol-3-O-β-D-rutinoside as the internal standard (IS). Analyte and IS were preparation through a protein precipitation procedure with 1.0 mL of methanol to a 0.1 mL plasma sample. The processed samples were separated by C18 analytical column using methanol/water containing 0.1% formic acid with gradient elution as the mobile phase at a flow rate of 0.3 mL/min. Sophorabioside (m/z 577.15 → 269.45) and kaempferol-3-O-β-D-rutinoside (m/z 593.15 → 285.84) were detected by a triple quadrupole tandem mass spectrometer in negative electrospray ionization mode using multiple reaction monitoring. The calibration curve for sophorabioside was linear in the range of 6–1,200 ng/mL (r2 > 0.995) with a lower limit of quantification of 6 ng/mL. The inter- and intra-day precision and accuracy were well within the acceptable limits. The matrix effects were satisfactory in all of the biological matrices examined. The mean recovery of sophorabioside was always >90%. This method was successfully applied to a pharmacokinetic study of sophorabioside in rats after an oral administration of 90 mg/kg sophorabioside. The main pharmacokinetic parameters: Tmax, Cmax and t1/2 were 6.2 ± 0.8 h, 1430.83 ± 183.25 ng/mL, 7.2 ± 0.5 h, respectively. Introduction The natural isoflavone compounds exist widely in nature, which are the effective components from many Chinese herbal medicines (1). Sophorabioside (Figure 1), a natural isoflavone, which mainly exists in the Sophora japonica L.(Leguminosae), such as fruit (2), seed (3, 4), pericarp (5, 6) and bark (7). Previous pharmacological studies have shown that sophorabioside have various pharmacological activities including anti-allergic, anti-inflammatory, anti-platelet and anti-oxidant effects (8–11). Figure 1. View largeDownload slide The chemical structures of sophorabioside (A) and kaempferol-3-O-β-D-rutinoside (IS, B). Figure 1. View largeDownload slide The chemical structures of sophorabioside (A) and kaempferol-3-O-β-D-rutinoside (IS, B). There is only one HPLC methods exist for quantifying sophorabioside in the plants (12). To our best knowledge, no bioanalytical method has been reported for the quantification of sophorabioside in biological samples. Pharmacokinetic studies of traditional Chinese medicine (TCM) provide necessary theoretical basis for clinical application. Therefore, it is necessary to characterize the pharmacokinetic properties of sophorabioside and develop a sensitive and selective bioanalytical method for the quantitation of sophorabioside in plasma. In the present study, a sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was initially developed and validated to determine sophorabioside in rat plasma using kaempferol-3-O-β-D-rutinoside as the internal standard (IS). This method has been validated for specificity, linearity, lower limit of quantification (LLOQ), accuracy, precision, matrix effect, recovery, stability, dilution integrity and carry-over effect with a total run time of 2.3 min. It has been further and successfully applied to a pharmacokinetic study of sophorabioside after its oral administration in rats. Experimental Chemicals and reagents Sophorabioside (Batch No. 20140721, purity >98%) and kaempferol-3-O-β-D-rutinoside (purity >98%, internal standard, IS) were prepared in College of Pharmacy of Jilin University (Changchun, China). HPLC grade methanol and formic acid were purchased from Merck Company (Darmstadt, Germany). Ultrapure water was obtained by a Milli-Q system (Millipore, Billerica, USA). UPLC-MS/MS conditions Liquid chromatography was performed on an Acquity ultra-performance liquid chromatography (UPLC) unit (Waters Corp., USA) with an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm). The mobile phase comprised of 0.1% formic acid in H2O (mobile phase A) and methanol (mobile phase B). A gradient elution procedure was as follows: 0–0.3 min (15.0–50.0% B), 0.3–1.9 min (50.0–50.0% B) and 1.9–2.0 min (50.0–15.0% B) at a flow rate of 0.30 mL/min. The equilibrated time was 0.3 min and the total run time was 2.3 min per sample. The injection volume was 2.0 μL. The column and sample temperature were maintained at 30°C and 15°C, respectively. An XEVO TQ-S triple quadrupole mass spectrometer equipped with the negative-ion electrospray ionization (ESI-) source (Waters, USA) was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) mode. The dwell time was set to 0.163 s for each MRM transition. The MRM transitions were m/z 577.15 → 269.45 for sophorabioside and m/z 593.15 → 285.84 for IS. The optimized source parameters were set as follows: capillary voltage, 2,300 V; cone voltage, 30 V; source temperature, 150°C; desolvation temperature, 500°C; desolvation gas, 1,000 L/h; cone gas, 150 L/h; collision gas, 0.16 mL/min; nebulizer gas, 7 bar. MassLynx software (Version 4.1) was used to control, acquire and analyze the data and TargetLynx™ program was used to process the data. Working solutions, calibration and quality control samples Standard stock solutions of sophorabioside and IS were prepared in methanol at a concentration of 1.0 mg/mL. The working solutions were prepared in methanol by a serial dilution from the stock solutions. The plasma samples for calibration standards and quality control (QC) samples were prepared by diluting the corresponding working solutions with blank rat plasma. Final concentrations of the calibration standards were 6, 12, 60, 120, 360, 600 and 1,200 ng/mL for sophorabioside in rat plasma. The three levels of QC samples in plasma were 18, 240, 960 ng/mL for sophorabioside. The IS working solution (20 ng/mL) was prepared in a similar manner as previously described. All working solutions, calibration standards and QC samples were stored at 4°C and used within one month after preparation. Sample preparation The plasma samples were thawed to room temperature before analysis. An aliquot of 500 μL of the IS working solution was added to 50 μL of collected plasma sample. The resulting mixtures were vortex mixed for 1.0 min and centrifuged at 15,000 rpm for 10 min. The aliquot of 2 μL of each supernatant was injected into the UPLC-MS/MS system for sophorabioside quantification. Method validation This method has been validated for specificity, linearity, LLOQ, accuracy, precision, matrix effect, recovery, stability, dilution integrity and carry-over effect in compliance with the guidelines set by the United States Food and Drug Administration (USFDA) and European Medicines Agency (EMA) (13, 14). Specificity The specificity of the method was evaluated by analysis of blank plasma samples from six different batches of rats with those of corresponding blank plasma samples spiked with working solutions and IS, and a rat plasma sample 3 h after oral administration of single dosage 90 mg/kg sophorabioside in order to exclude any endogenous interference at retention times of sophorabioside and IS. Each sample was handled by the proposed preparation procedure and instrument conditions. Linearity and LLOQ The calibration curve (y = a + bx) was obtained by plotting the peak area ratio(y) of sophorabioside and IS against concentrations (x) of the analyte with weighted (1/x2) least square linear regression. The LLOQ was defined as the lowest concentration on the calibration curve where sophorabioside signal-to-noise (S/N) ratio was ≥10 when compared with plasma blank. Each LLOQ sample should be obtained with an acceptable accuracy (RE) and precision (RSD) both less then ±20%. Accuracy and precision The accuracy and precision of the method were tested by analyzing six replicate QC samples at three different concentrations of 18, 240 and 960 ng/mL. Accuracy was expressed as the percentage relative error (RE, %) which should be within ±15% at all concentrations. The intra-day (on same day) variation and inter-day (on three consecutive days) variation was expressed as the percentage relative standard deviation (RSD, %) which should not exceed 15% at all concentrations. Matrix effect and recovery The matrix effect and recovery of sophorabioside were evaluated by analyzing six replicates of plasma samples at three different QC concentration levels (18, 240 and 960 ng/mL). The matrix effect was calculated by comparing the peak areas of sophorabioside and IS spiked in post-extracted blank plasma samples with those of the analytes in methanol at equivalent concentrations. The recovery was calculated by comparing the peak area ratio of sophorabioside and IS spiked in the extracted QC samples with those of the analytes added to post-extracted blank plasma at equivalent concentrations. Stability The stability of sophorabioside in plasma was evaluated by analyzing six replicates of plasma samples at three concentration levels (18, 240 and 960 ng/mL) under different conditions. The short-term stability was determined after the exposure of the spiked samples at room temperature for 4 h, and the processed QC samples kept in the UPLC autosampler at 15°C for 24 h. The freeze-thaw stability was evaluated by determining the QC samples after three complete freeze-thaw cycles (−20 to 25°C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at −20°C for 30 days. Dilution integrity In order to analyze the samples at the concentration above the upper limit of quantification (ULOQ), the dilution integrity was tested in six replicates by diluting the blank plasma samples spiked with analytes 2-fold (1,000 ng/mL) and 10-fold (200 ng/mL) with blank plasma. The precision and accuracy from the nominal concentrations after dilution should be less then 15%. Carry-over effect Carry-over effect was assessed by injecting a blank plasma sample following the injection of an ULOQ (1,200 ng/mL) sample during one analytical run. The carry-over was considered negligible when the measured peak areas were less than 20% of the LLOQ level peak area for each analyte and 5% of the IS peak area detected in the same batch. Application to a pharmacokinetic study The pharmacokinetic study in rats was performed according to protocols which had been approved by the Review Committee of Animal Care and Use of Jilin University and were in accordance with the Guide for the Care and Use of Laboratory Animals. Six wistar rats (three males and three females, weighing 200 ± 20 g) were obtained from the Laboratory Animal Center of Jilin University (Changchun, China) used to study the pharmacokinetics of sophorabioside. All animals were housed under a controlled environment (23–25°C, humidity 40–60%, 12 h light/dark cycle) for 5 days acclimation before the experiments, with free access to standard laboratory food and water. The rats were fasted for 12 h but water was freely available prior to the experiments. They received a single dose of sophorabioside (90 mg/kg) suspension by oral administration. Blood samples were collected from the retinal venous plexus into heparinized 1.5 mL polythene tubes at 0.25, 0.5, 1, 1.5, 2, 3, 5, 6, 7, 8, 10, 12, 24, 36 and 48 h after a single oral administration; 100 μL of blood were collected at the first nine time points, and 300 μL were collected at the other time point. All samples were centrifuged at 5,000 rpm for 10 min. The plasma obtained were stored at −20°C until analysis. The plasma profile of sophorabioside was constructed and pharmacokinetic parameters were calculated by using DAS (Data Analysis System) 3.0 statistical software (Pharmacology Institute of China). Results and Discussion Method development and optimization The UPLC-MS/MS method provided a rapid, simple and sensitive assay for sophorabioside in rat plasma. In this study, both the positive and negative ionization modes were tested for the analysis and good response was achieved in negative ionization mode. Kaempferol-3-O-β-D-rutinoside was chosen as the IS because its ionization characteristics and extraction efficiency were similar to those of sophorabioside. The Full-scan product ion spectra of the [M–H]− ions and the fragmentation pathways of sophorabioside and IS are displayed in Figure 2. The transitions m/z 577.15 → 269.45 for sophorabioside and m/z 593.15 → 285.84 for IS were selected for quantitative analysis, respectively. A number of UPLC columns and various mobile phases were evaluated for their chromatographic behavior of sophorabioside and IS. Finally, a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) was selected for the chromatographic separation, and gradient elution using methanol/water containing 0.1% formic acid at a flow rate of 0.3 mL/min gave symmetrical peak shapes for sophorabioside and IS with retention times of 1.22 and 1.30 min, respectively. Formic acid was added to the mobile phase to increase the sensitivity of sophorabioside. In this assay, no significant endogenous interference was found using the current conditions and the whole separation of the sophorabioside and IS was completed within only 2.3 min. Figure 2. View largeDownload slide MS/MS spectra of sophorabioside (A) and kaempferol-3-O-β-D-rutinoside (IS, B) showing prominent precursor to production transitions. Figure 2. View largeDownload slide MS/MS spectra of sophorabioside (A) and kaempferol-3-O-β-D-rutinoside (IS, B) showing prominent precursor to production transitions. In order to maximize recoveries the sample preparation, we investigated two methods, including a protein precipitation procedure and ethyl acetate extraction. Protein precipitation showed extremely high recoveries compared to liquid–liquid extraction for sophorabioside. Initially, acetonitrile, methanol and different ratios of acetonitrile-methanol (from 1:5 to 5:1, v/v) were tested. And, it showed best recoveries of sophorabioside and IS when we used methanol alone as precipitant. Therefore, methanol was chosen as the protein precipitation solvent. Then we tested the volume of methanol to extract sophorabioside. It turned out that 0.1 mL of plasma sample add 1.0 mL of methanol could provide less matrix effect and higher recoveries. Assay validation Specificity UPLC-MS/MS chromatogram of a blank rat plasma sample, a blank rat plasma sample spiked with sophorabioside at LLOQ and IS, a blank rat plasma sample spiked with sophorabioside and IS, and a rat plasma sample obtained at 3.0 h after oral administration of sophorabioside are illustrated in Figure 3. Obviously, there was no endogenous interference at the retention times of sophorabioside (1.22 min) and IS (1.30 min). Figure 3. View largeDownload slide Representative chromatograms of sophorabioside and IS in rat plasma samples. (A) a blank plasma sample; (B) a blank plasma sample spiked with sophorabioside (LLOQ) and IS (20 ng/mL); (C) a blank plasma sample spiked with sophorabioside (240 ng/mL) and IS (20 ng/mL); (D) a rat plasma sample 3 h after oral administration of single dosage 90 mg/kg sophorabioside (IS: 20 ng/mL). Figure 3. View largeDownload slide Representative chromatograms of sophorabioside and IS in rat plasma samples. (A) a blank plasma sample; (B) a blank plasma sample spiked with sophorabioside (LLOQ) and IS (20 ng/mL); (C) a blank plasma sample spiked with sophorabioside (240 ng/mL) and IS (20 ng/mL); (D) a rat plasma sample 3 h after oral administration of single dosage 90 mg/kg sophorabioside (IS: 20 ng/mL). Linearity and LLOQ The linear regressions of the peak area ratios of sophorabioside and IS against concentrations were fitted over the concentration range 6–1200 ng/mL in rat plasma. A typical calibration curve equation was: y = 0.003x + 0.073 (r2 = 0.9959), where y represents the ratio of sophorabioside to IS peak area, and x represents the sophorabioside concentration. The LLOQ for the determination of sophorabioside in rat plasma was 6 ng/mL with the precision and accuracy of 9.80% and 5.22%, respectively. Accuracy and precision The results for intra- and inter-day precision (RSD) were less than 6.09% and the accuracy (RE) were found to be within −5.74% to 2.75% for all the investigated concentrations of sophorabioside in rat plasma. The data are summarized in Table I. The results indicated that both precision and accuracy achieved with this method are acceptable. Table I. Precision and Accuracy for Sophorabioside of QC Sample in Rat Plasma (n = 6) Concentration (ng/mL) RSD(%) RE(%) Intra-day Inter-day 18 6.09 1.50 −5.74 240 2.18 5.35 2.75 960 1.33 3.97 −1.85 Concentration (ng/mL) RSD(%) RE(%) Intra-day Inter-day 18 6.09 1.50 −5.74 240 2.18 5.35 2.75 960 1.33 3.97 −1.85 Matrix effect and recovery The results of matrix effect and recovery studies for sophorabioside at concentrations of 18, 240 and 960 ng/mL were shown in Table II. The recovery of sophorabioside was in the range 90.92–93.74% with matrix effect were within in the range of 98.30–100.74% at the three QC concentration levels. These data suggest that the preparation efficiency for sophorabioside was acceptable and the matrix effect from plasma was considered negligible in this method. Table II. Recoveries and Matrix Effects of Sophorabioside in Rat Plasma (n = 6) Compound Concentration (ng/mL) Recovery (%) Matrix effect (%) Mean ± SD RSD (%) Mean ± SD RSD (%) Sophorabioside 18 92.14 ± 5.30 5.75 98.30 ± 4.92 5.02 240 93.74 ± 4.47 4.77 100.74 ± 4.41 4.38 960 90.92 ± 3.75 4.12 99.07 ± 3.94 3.98 Compound Concentration (ng/mL) Recovery (%) Matrix effect (%) Mean ± SD RSD (%) Mean ± SD RSD (%) Sophorabioside 18 92.14 ± 5.30 5.75 98.30 ± 4.92 5.02 240 93.74 ± 4.47 4.77 100.74 ± 4.41 4.38 960 90.92 ± 3.75 4.12 99.07 ± 3.94 3.98 Stability The results of stability tests of sophorabioside in rat plasma (18, 240 and 960 ng/mL) under different conditions are reported in Table III. The RSDs of the mean test responses were within 15% in all stability tests. Sophorabioside in plasma was stable after being kept at room temperature for 4 h and at 15°C for 24 h, stored at −20°C for 30 days or taken through three freeze-thaw (−20°C to 25°C) cycles. These results showed that the established method was suitable for pharmacokinetic study. Table III. Summary of Stability of Sophorabioside Under Various Storage Conditions (n = 6) Condition Concentration (ng/mL) Precision Accuracy Nominal Found RSD (%) RE (%) Room temperature, 4 h 18 18.48 5.19 2.69 240 244.13 3.93 1.72 960 941.70 3.95 −1.91 Autosampler, 15°C, 24 h 18 18.36 4.73 1.97 240 242.75 2.27 1.14 960 973.82 2.69 1.44 Three freeze-thaw (−20 to 25°C) 18 17.56 5.56 −2.47 240 238.09 3.32 −0.79 960 968.25 4.10 0.86 −20°C, 30 days 18 18.15 6.11 0.82 240 242.71 5.44 1.13 960 949.35 2.16 −1.11 Condition Concentration (ng/mL) Precision Accuracy Nominal Found RSD (%) RE (%) Room temperature, 4 h 18 18.48 5.19 2.69 240 244.13 3.93 1.72 960 941.70 3.95 −1.91 Autosampler, 15°C, 24 h 18 18.36 4.73 1.97 240 242.75 2.27 1.14 960 973.82 2.69 1.44 Three freeze-thaw (−20 to 25°C) 18 17.56 5.56 −2.47 240 238.09 3.32 −0.79 960 968.25 4.10 0.86 −20°C, 30 days 18 18.15 6.11 0.82 240 242.71 5.44 1.13 960 949.35 2.16 −1.11 Dilution integrity The precision (RSD) for dilution integrity of 2-fold and 10-fold dilution were 4.83% and 5.49%, while the accuracy (RE) results were 2.90% and 1.54%, respectively. The results indicated that samples could be diluted by an appropriate dilution when their concentrations exceeding the ULOQ of the standard curve. Carry-over effect No enhancement in the response was observed in blank plasma sample after subsequent injection of ULOQ at the retention time of sophorabioside and IS. The results show that the carry-over observed during carry-over effect experiments was negligible. Application to a pharmacokinetic study Non-compartmental analysis (NCA) does not require the assumption of a specific compartmental model, and the PK parameters can be obtained without the need to define the number of compartments (15). In our study, the primary requirement is to determine the degree of exposure following administration of a drug, and to obtain some pharmacokinetic parameters. So, NCA, requiring fewer assumptions than model-based approaches, was used as the preferred methodology to analyzing PK data. The validated method presented here was successfully applied to a pharmacokinetic study of sophorabioside in rats. The mean plasma concentration-time profile of sophorabioside after an oral (90 mg/kg) administration was shown in Figure 4. The plasma pharmacokinetic profile fitted into non-compartment model and the main pharmacokinetic parameters were listed in Table IV. The mean maximum plasma concentration (Cmax, 1430.83 ± 183.25 ng/mL) was achieved at 6.2 ± 0.8 h (Tmax). The elimination half-life (t1/2) of sophorabioside was 7.2 ± 0.5 h, while the AUC (0−t) in 48 h and AUC (0−∞) were 10697.91 ± 510.92 and 10800.15 ± 526.88 ng h/mL, respectively. This is the first time to characterize the pharmacokinetic profile of sophorabioside in rats and the current method is suitable and sufficient to pharmacokinetic study on sophorabioside. Table IV. The Main Pharmacokinetic Parameters after Oral Administration of Sophorabioside (90 mg/kg) to Rats (n = 6) Pharmacokinetic parameters Unit Mean ± SD Cmax ng/mL 1430.83 ± 183.25 Tmax h 6.2 ± 0.8 t1/2 h 7.2 ± 0.5 MRT(0−∞) h 10.92 ± 0.44 AUC(0−t) ng h/mL 10697.91 ± 510.92 AUC(0−∞) ng h/mL 10800.15 ± 526.88 V/F L/kg 87.00 ± 4.87 CL/F L/h/kg 8.35 ± 0.42 Pharmacokinetic parameters Unit Mean ± SD Cmax ng/mL 1430.83 ± 183.25 Tmax h 6.2 ± 0.8 t1/2 h 7.2 ± 0.5 MRT(0−∞) h 10.92 ± 0.44 AUC(0−t) ng h/mL 10697.91 ± 510.92 AUC(0−∞) ng h/mL 10800.15 ± 526.88 V/F L/kg 87.00 ± 4.87 CL/F L/h/kg 8.35 ± 0.42 Figure 4. View largeDownload slide Mean plasma concentration-time profile after oral (90 mg/kg) administration of sophorabioside in six rats. Figure 4. View largeDownload slide Mean plasma concentration-time profile after oral (90 mg/kg) administration of sophorabioside in six rats. Conclusions A rapid, simple and sensitive UPLC-MS/MS method was firstly developed and validated for the determination of sophorabioside in rat plasma. The assay method offered sample preparation with a simple one-step precipitation of plasma protein by methanol, and shorter run time within 2.3 min. The precision, accuracy, recovery and matrix effect met the acceptance criteria with a linear range of 6–1,200 ng/mL in rat plasma. This validated method was successfully applied to a pharmacokinetic study of sophorabioside following oral administration of 90 mg/kg sophorabioside in rats. There is a double peak phenomenon of sophorabioside in Figure 4, the first peak appeared at about 1 h, and the second peak appeared at about 6 h which is higher than the previous one. The reasons for this phenomenon may be enterohepatic recirculation and variability of absorption (16, 17). And bacterial metabolism in the intestine may also play a significant role (18). These hypothesize need further investigation. Funding This work was supported by Science and Technology Development Program of Jilin Province (Grant No. 20120928). Supplementary Data Supplementary data are available at Journal of Chromatographic Science online. Conflict of interest The authors report that they have no conflicts of interest. The authors alone are responsible for the content and writing of this article. References 1 Li, C., Liu, A.L., Du, G.H.; Research progress in pharmacology of natural isoflavones; Chinese Journal of New Drugs , ( 2013); 22: 1415– 1420. 2 Tang, Y.P., Lou, F.C., Wang, J.H., Zhuang, S.F.; Four new isoflavone triglycosides from Sophora japonica; Journal of Natural Products , ( 2001); 64: 1107– 1110. Google Scholar CrossRef Search ADS PubMed 3 Abdallah, H.M., Al-Abd, A.M., Asaad, G.F., Abdel-Naim, A.B., El-halawany, A.M.; Isolation of antiosteoporotic compounds from seeds of Sophora japonica; PLoS One , ( 2014); 9: e98559. Google Scholar CrossRef Search ADS PubMed 4 Wang, J.H., Wang, Y.L., Lou, F.C.; Studies on chemical constituents from seeds of Sophora japonica; Journal of China Pharmaceutical University , ( 2001); 32: 471– 473. 5 Tang, Y.P., Lou, F.C., Wen, W., Wang, J.H., Li, Y.F.; Isoflavonoid glycosides from the pericarps of Sophora japonica; Journal of China Pharmaceutical University , ( 2001); 32: 187– 189. 6 Liu, R.M., Qi, Y.Y., Sun, A.L., Xie, H.Y.; Isolation and purification of chemical constituents from the pericarp of Sophora japonica L. by chromatography on a 12% cross-linked agarose gel; Journal of Liquid Chromatography , ( 2007); 30: 1870– 1874. 7 Wang, C.T., Tang, Y.P., Zhou, L., Gao, H.X.; Studies on flavonoid constituents from bark of Sophora japonica, Jiangsu; Journal of Traditional Chinese Medicine , ( 2008); 40: 65– 67. 8 Kim, S.J., Kim, Y.J., Lee, J.H., Oh, S.R., Park, C.I., Jeong, J.W., et al. .; Genistein-4′-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside from Sophora japonica (Leguminosae) ameliorates mast cell-mediated allergic inflammation in vivo and in vitro; Oriental Pharmacy and Experimental Medicine , ( 2011); 11: 207– 213. Google Scholar CrossRef Search ADS 9 Park, S.J., Kim, J.Y., Jang, Y.P., Cho, Y.W., Ahn, E.M., Baek, N.I., et al. .; Inhibition of LPS induced iNOS, COX-2 and cytokines expression by genistein-4′-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside through the NF-κB inactivation in RAW 264.7 cells; Korean Journal of Pharmacognosy , ( 2007); 38: 339– 348. 10 Kim, J.M., Yun-Choi, H.S.; Anti-platelet effects of flavonoids and flavonoid-glycosides from Sophora japonica; Archives of Pharmacal Research , ( 2008); 31: 886– 890. Google Scholar CrossRef Search ADS PubMed 11 Kim, H.J., Kim, M.K., Shim, J.G., Yeon, S.H., Kwon, S.H., Lee, M.W.; Anti-oxidative phenolic compounds from Sophorae Fructus; Natural Product Sciences , ( 2004); 10: 330– 334. 12 Chen, X., Zhang, G.P., Yu, Z.G.; Simultaneous assay of isoflavones in Fructus Sophorae by QAMS; Chinese Journal of Pharmaceutical Analysis , ( 2013); 33: 1904– 1909. 13 Guidance for Industry, Bioanalytical Method Validation, FDA., http://www.fda.gov/cder/guidance. 14 European Medicines Agency.; Guideline on bioanalytical method validation . Committee for Medicinal Products for Human Use (CHMP), London, ( 2011). 15 Curry, S.H., Whelpton, R.; Multiple-compartment and non-compartment pharmacokinetic models. In Introduction to Drug Disposition and Pharmacokinetics, John Wiley & Sons, Ltd, Chichester, UK, (2016). doi: 10.1002/9781119261087.ch5. 16 Wang, Y., Yang, G.P., Guo, C.X., Pei, Q., Zhang, R.R., Huang, L.; Plasma double-peak phenomenon following oral administration; Chinese Journal of Clinical Pharmacology and Therapeutics , ( 2014); 19: 341– 345. 17 Godfrey, K.R., Arundel, P.A., Dong, Z.M., Bryant, R.; Modelling the double peak phenomenon in pharmacokinetics; Computer Methods and Programs in Biomedicine , ( 2011); 104: 62– 69. Google Scholar CrossRef Search ADS PubMed 18 Zhi, X.R., Sheng, N., Yuan, L., Zhang, Z.Y., Jia, P.P., Zhang, X.X., et al. .; Pharmacokinetics and excretion study of sophoricoside and its metabolite in rats by liquid chromatography tandem mass spectrometry; Journal of Chromatography B , ( 2014); 945–946: 154– 162. Google Scholar CrossRef Search ADS © The Author 2017. Published by Oxford University Press. All rights reserved. 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Journal of Chromatographic Science – Oxford University Press
Published: Feb 1, 2018
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