Chromatographia

, 69:1251

LC Method for Analysis of Three Flavonols in Rat Plasma and Urine after Oral Administration of Polygonum aviculare Extract

Authors

  • Fuquan Xu
    • Key Laboratory of Marine Drugs, Ministry of Education, Marine Drug and Food InstituteOcean University of China
    • Key Laboratory of Marine Drugs, Ministry of Education, Marine Drug and Food InstituteOcean University of China
  • Guoqiang Li
    • Key Laboratory of Marine Drugs, Ministry of Education, Marine Drug and Food InstituteOcean University of China
  • Hongbing Liu
    • Key Laboratory of Marine Drugs, Ministry of Education, Marine Drug and Food InstituteOcean University of China
Original

DOI: 10.1365/s10337-009-1088-x

Cite this article as:
Xu, F., Guan, H., Li, G. et al. Chroma (2009) 69: 1251. doi:10.1365/s10337-009-1088-x

Abstract

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of the flavonols myricitrin (1), avicularin (2), and juglanin (3) in rat plasma and urine after oral administration of the total flavonoids from Polygonum aviculare. Samples were prepared by solid-phase extraction then separated on a C18 reversed-phase column by use of a mobile-phase gradient prepared from methanol and aqueous formic acid solution. The flow rate was 1 mL min−1. Detection was performed at 254 nm. The calibration range was 11–1,100 μg mL−1 for both 2 and 3 in plasma; in urine the calibration ranges for 1, 2, and 3 were 32–1,600, 11–1,100, and 22–1,100 μg mL−1, respectively. Intra-day and inter-day RSD were less than 4.33 and 3.62% for 2 and 3, respectively, in plasma, and no more than 4.03 and 2.22% for all the analytes in urine. The analytical sensitivity and selectivity of the assay enabled successful application to pharmacokinetic studies of flavonols 13 in rats.

Keywords

Column liquid chromatographyFlavonolsPlasmaUrinePolygonum aviculare

1 Introduction

The aerial part of Polygonum aviculare L., a traditional Chinese medicine (Bianxu), has diuretic, antidotal, antiphlogistic, antipyretic, antiparasitic, and antidiarrheal effects [1, 2]. It has been used in Chinese folk medicine to treat urinary system infection, nephritis, and gingivitis [13]. Pharmacological tests have revealed that extracts of the whole plant have significant antioxidant and anti-inflammatory activity in vitro [4]. The plant has also attracted particular attention because of its vasorelaxant effect [5] and protective function against DNA damage [6, 7]. In previous phytochemical investigation of this plant [8], myricitrin, avicularin, and juglanin were identified as the major components among the total flavonoids from Polygonum aviculare (TFP). These flavonols had been found to have antibacterial, antioxidative, antimutagenic, hepatoprotective, and anti-HIV activity [912].

Several techniques for analysis of flavonols in complex mixtures have been reported in the literature, for example thin-layer chromatography (TLC) [13, 14], capillary electrophoresis (CE) [1517], high-performance liquid chromatography (LC) [1820], and liquid chromatography–mass spectrometry (LC–MS) [21]. Rapid quantitative study of 1 has been performed by TLC [22], and quantitative analysis of 2 and 3 has been achieved by LC and LC–MS [2326]. Although avicularin and juglanin occur together in many plants [27, 28], few simultaneous analyses in crude plant extracts have been reported [29]. Until now there has been no report of analysis of avicularin and juglanin in plasma and urine.

Although pharmacological activity has been attributed to TFP after oral administration of Polygonum aviculare L. extract, as far as we are aware there has been no published study involving simultaneous analysis of marker constituents of Bianxu in plasma or urine. An investigation to illuminate the multiple bioactive components of this drug is, therefore, urgently required. The objective of this study was to develop a method for simultaneous analysis of the constituents of Bianxu in biological fluids. A simple and reliable method for simultaneous analysis of the flavonols avicularin (2), and juglanin (3) in rat plasma and of myricitrin (1) and 2, and 3 in rat urine has been established. Solid-phase extraction (SPE) was followed by high-performance liquid chromatography (LC) with diode-array detection (DAD). The method was validated for sensitivity, selectivity, precision, and accuracy in rat plasma and urine samples and then used for a pharmacokinetic study of these three flavonols in the rat. As far as we are aware, this is the first study of rat biological fluids after oral administration of TFP.

2 Experimental

2.1 Chemicals, Reagents, and Solutions

Bianxu was purchased from Tongrentang Drugstore (Qingdao, Shandong Province, P. R. China). A voucher specimen (no. 0704012) was deposited in the Marine Drugs and Food Institute, Ocean University of China. Bianxu (5.0 kg) was extracted with 90% EtOH (3 × 3 h) under reflux. After evaporation of the ethanol in vacuo, the concentrated extract was suspended in water and then subjected to polyamide column chromatography with H2O–EtOH mixtures as eluents to furnish four fractions. The TFP was obtained in the fraction eluted with 60% ethanol (fraction II). Myricitrin, avicularin and juglanin standards were isolated from fraction II by semi-preparative LC, and their structures were identified on the basis of spectral data (1H NMR, 13C NMR, and MS) and by comparison with literature values [8]. LC–DAD analysis proved the purity of all the compounds was >98%.

Quercetin, used as internal standard (IS), was provided by Professor Huimin Zhong (Qingdao University of Science and Technology); its purity was >99%. The structures of all the compounds are given in Fig. 1.
https://static-content.springer.com/image/art%3A10.1365%2Fs10337-009-1088-x/MediaObjects/10337_2009_1088_Fig1_HTML.gif
Fig. 1

The chemical structures of flavonols 14

HPLC-grade methanol was purchased from Honeywell International (Burdick and Jackson, Muskegon, MI, USA). Gracepure C18 cartridge columns for SPE were purchased from W. R. Grace (Cambridge, Massachusetts, USA). Deionized water was prepared by use of a Milli-Q Academic water-purification system (Millipore, Milford, MA, USA).

Pure solutions of flavonols 13 were prepared separately in methanol at concentrations of 1,600, 1,100, and 1,100 μg mL−1, respectively. These three solutions were then serially diluted with methanol to furnish working solutions containing 800, 160, 80, 16, 8, and 1.6 μg mL−1 for myricitrin and 550, 110, 55, 11, 5.5, and 1.1 μg mL−1 for avicularin and juglanin. A stock solution containing 320, 220, and 220 μg mL−1, flavonols 13, respectively, was prepared by mixing and diluting the pure solutions with methanol.

Quercetin (4, IS) solution of concentration 275 μg mL−1 was prepared in methanol. All solutions were stored at 4 °C and were shown to be stable for 40 days.

2.2 Animals

Male Wistar rats (200–230 g) were purchased from Qingdao Institute of Drug Control (Qingdao, Shandong Province, P. R. China; SCXK2003010), and were housed with unlimited access to food and water in an animal room for three days before the test. The animals were maintained on a 12 h light–12 h dark cycle (light on at 7.30 a.m.) at ambient temperature (22–25 °C) and 65% relative humidity.

2.3 Instrumentation and Conditions

LC analysis was performed on an Agilent Technologies Series 1100 HPLC equipped with quaternary pump (G1311A), auto-injector (G1313A), column compartment (G1316A), diode array detector (G1315B), and Chemstation software. A Varian Microsorb-MV 300-5 C18 reversed-phase column (5-μm particles, 250 mm × 4.6 mm) was used for separation and quantification of the flavonols. The mobile phase for both plasma and urine samples was a gradient prepared from methanol (component A) and 0.15% aqueous formic acid solution (pH 2.7) (component B). The initial mobile phase composition condition was A–B 10:90 (v/v). This was changed linearly to A–B 20:80 (v/v) at 5 min, then changed linearly to A–B 35:65 (v/v) at 14 min and held at this composition until 25 min. The composition was then changed linearly to A–B 50:50 (v/v) at 30 min and then changed linearly to A–B 60:40 (v/v) at 38 min. The flow rate was 1.0 mL min−1, the DAD detection wavelength was 254 nm, and the column temperature was 35 °C. The injection volume was 30 μL for plasma and 20 μL for urine.

2.4 Calibration

Calibration solutions were prepared by transferring the working solutions (15 μL) to Eppendorf (EP) tubes, evaporating to dryness under a gentle stream of nitrogen at 40 °C, and adding 200 μL blank plasma (i.e. plasma from rats which did not receive TFP), 10 μL quercetin (IS, 275 μg mL−1) and 20 μL 1% phosphoric acid solution for plasma samples (pH 3.0), or 2 mL blank urine (i.e. urine from rats which did not receive TFP) and 10 μL quercetin (IS, 275 μg mL−1) for urine samples. Extraction was then performed as described in the next section.

2.5 Sample Preparation

Immediately after collection of blood samples plasma was separated by centrifugation at 3,000 rpm (10 min) and stored at −20 °C until analysis. Plasma (200 μL) was transferred to a 1.5-mL EP tube and spiked with 5 μL IS solution (275 μg mL−1) and 20 μL 1% phosphoric acid solution (pH 3.0). The mixed solution was vortex mixed for approximately 60 s then applied to a C18 reversed-phase SPE cartridge, which was then successively eluted with water (0.4 mL) and methanol (0.4 mL). The methanol fraction was evaporated to dryness under a stream of nitrogen at 40 °C and the residue was reconstituted in 200 μL methanol. Finally, 30 μL of this sample was injected for LC analysis.

Urine samples were pretreated by polyamide (100–200 mesh) column chromatography then stored at −20 °C until analysis. Samples were extracted on a C18 reversed-phase SPE cartridge (approx. 2 mL urine for each column) which was successively eluted by water (2 mL) and methanol (2 mL). The methanol fraction was evaporated to dryness under the same conditions as for the plasma sample and the residue was reconstituted in 300 μL methanol. Finally, 20 μL of this sample was injected for LC analysis.

2.6 Validation

2.6.1 Recovery, Precision, and Accuracy

Recovery was calculated by comparing the average peak areas obtained from plasma or urine with those obtained from the same quantities of the flavonols added to the mobile phase.

For plasma samples, 15 μL working standard solutions of high, middle, and low concentration were transferred to 1.5-mL EP tubes and evaporated to dryness under a stream of nitrogen at 40 °C. Blank plasma (200 μL) and 20 μL 1% phosphoric acid solution was then added to the residue and extraction was performed as described above for plasma samples. The residue was reconstituted in 200 μL methanol and 30 μL was injected for LC analysis.

For urine samples, 15 μL working standard solutions of high, middle, and low concentration were transferred to 5-mL EP tubes and evaporated to dryness under a stream of nitrogen at 40 °C. Blank urine (2 mL) was added to the residue and extraction was performed as described above for urine samples. The residue was reconstituted in 300 μL methanol and 20 μL was injected for LC analysis.

Five replicate analyses of both plasma and urine samples were performed to calculate intra-day and inter-day accuracy and precision. Inter-day accuracy was assessed on five different days for plasma and urine samples.

2.6.2 Stability

The freeze (12 h)–thaw (3 h) stability of the analytes was assessed for rat plasma and urine through three storage cycles, the results after each cycle being compared with the initial concentration (samples treated immediately after being freshly prepared).

Mixed working solutions (15 μL) of high, middle, and low concentration were transferred to EP tubes and evaporated to dryness under a stream of nitrogen at 40 °C. IS solution (275 μg mL−1, 5 μL) was added to the residue, followed by 200 μL blank plasma or 2 mL blank urine. After one, two, and three cycles the solutions were processed as described in the section “Sample Preparation”.

2.7 Pharmacokinetic Study

Before the experiment fifteen rats were randomly divided into three groups. All rats were deprived of food but had free access to water for 12 h before the experiment. TFP was mixed with 0.5% sodium carboxymethyl cellulose solution as a suspension (30 mg mL−1) and each rat was administered a dose of 600 mg kg−1 TFP orally; this dose contained 100 mg kg−1 myricitrin, 113 mg kg−1 avicularin, and 41.6 mg kg−1 juglanin.

Blood samples were collected from the retrobulbar capillary plexus 5, 10, 15, 20, 30, 40, 60, 120, and 240 min after administration. Each rat was used for three time points: the first group was used for 5, 10, and 15 min, the second group used from 20 to 40 min, and the third group used for 60, 120, and 240 min.

Urine samples were collected 1, 3, 5, 7, 11, 15, 24, 36, 48, and 60 h after administration. The volume of each urine sample was recorded, and five rats were used at each time interval.

Blank plasma and urine were collected from the five rats which did not receive TFP.

3 Results and Discussion

3.1 SPE Method Development

The objective of sample preparation was to remove biological interferences from the samples, with suitable recovery of the compounds of interest, by use of simple working steps. Several methods were tested, including protein precipitation, solid-phase extraction (SPE), and liquid–liquid extraction [3032]. Use of SPE for preparation of plasma and urine sample has been widely reported [3335]. It was believed SPE would be the most efficient method for removing proteins and other interfering components from rat plasma and urine with satisfactory sample recovery. Proteins and interfering compounds could be removed from the cartridge by elution with water and the three analytes retained on the C18 reversed-phase cartridge could then be completely eluted with methanol. The well-defined analyte peaks and the weak signals from interfering compounds (Fig. 2) suggest that SPE was appropriate for our research.
https://static-content.springer.com/image/art%3A10.1365%2Fs10337-009-1088-x/MediaObjects/10337_2009_1088_Fig2_HTML.gif
Fig. 2

Chromatographic profiles of plasma and urine samples: (a) blank plasma; (b) blank plasma spiked with flavonols 24; (c) plasma sample 30 min after oral administration of TFP; (d) blank urine; (e) blank urine spiked with flavonols 14; (f) urine sample 7 h after oral administration of TFP

3.2 Validation

3.2.1 Extraction Recovery

Results from determination of extraction recovery of avicularin and juglanin from rat plasma are listed in Table 1. These results were obtained from five replicate analyses of rat plasma spiked at low, medium, and high concentrations. Recovery of avicularin and juglanin from the samples was >87.7% and the average recovery of the internal standard was not less than 99.0%. These results indicate that extraction recovery of avicularin, juglanin, and the IS from plasma was acceptable.
Table 1

Intra-day and inter-day accuracy and precision of analysis of avicularin and juglanin in rat plasma, and recovery of avicularin, juglanin, and quercetin from rat plasma

Spiked concentration (μg mL−1)

Intra-day (n = 5)

Inter-day (n = 5)

Recovery (%; n = 6)

RSD (%)

Measured concentration (μg mL−1)

RSD (%)

Measured concentration (μg mL−1)

RSD (%)

Avicularin

 550

536.4 ± 4.94

0.921

542.1 ± 10.9

2.01

99.4 ± 1.56

1.57

 110

96.3 ± 2.71

2.81

99.1 ± 2.84

2.87

90.3 ± 1.91

2.11

 11

9.20 ± 0.21

2.28

9.4 ± 0.34

3.62

87.7 ± 4.98

5.68

Juglanin

 550

534.6 ± 8.20

1.53

543.9 ± 8.22

1.51

99.1 ± 1.17

1.18

 110

97.0 ± 2.69

2.77

99.4 ± 1.36

1.37

90.3 ± 1.14

1.26

 11

9.33 ± 0.404

4.33

9.7 ± 0.33

3.40

89.7 ± 2.48

2.77

Quercetin

 1,100

    

99.0 ± 1.22

1.23

 275

    

99.5 ± 1.20

1.21

 13.8

    

99.3 ± 3.72

3.75

Results from determination of extraction recovery of myricitrin, avicularin, and juglanin from rat urine are listed in Table 2. These results were obtained in the same way as for plasma samples. Recovery from the samples was >88.5% and the average extraction recovery of the internal standard was not less than 96.4%. These results again show the method gives acceptable results.
Table 2

Intra-day and inter-day accuracy and precision of analysis of myricitrin, avicularin, and juglanin in rat urine, and recovery of myricitrin, avicularin, juglanin, and quercetin from rat urine

Spiked concentration (μg mL−1)

Inter-day (n = 5)

Intra-day (n = 5)

Recovery (%; n = 6)

RSD (%)

Measured concentration (μg mL−1)

RSD (%)

Measured concentration (μg mL−1)

RSD (%)

Myricitrin

 800

781.1 ± 15.1

1.93

793.2 ± 5.18

0.653

99.3 ± 0.629

0.633

 160

151.2 ± 6.07

4.01

157.6 ± 2.15

1.36

98.7 ± 0.630

0.994

 16

14.6 ± 0.416

2.85

14.9 ± 0.31

2.08

93.8 ± 2.00

2.13

Avicularin

 550

535.0 ± 7.50

1.40

542.9 ± 6.38

1.18

99.0 ± 0.813

0.821

 110

95.0 ± 3.82

4.02

98.6 ± 2.11

2.14

90.0 ± 1.77

1.97

 11

9.21 ± 0.371

4.03

9.6 ± 0.20

2.08

88.5 ± 1.10

1.24

Juglanin

 550

535.4 ± 8.95

1.67

544.4 ± 6.75

1.24

98.9 ± 0.949

0.960

 110

95.3 ± 3.13

3.28

98.7 ± 2.19

2.22

91.1 ± 1.57

1.72

 11

9.43 ± 0.351

3.69

9.8 ± 0.21

2.14

89.5 ± 1.79

2.00

Quercetin

 1,100

    

99.4 ± 0.586

0.590

 275

    

99.0 ± 1.19

1.20

 13.8

    

96.4 ± 3.76

3.90

It was perplexing that myricitrin could not be detected in plasma whereas it could be found in urine. Several different methods were tried but the same results were obtained. There are no literature reports of successful detection of myricitrin in plasma or blood. It is probable that myricitrin participates in biochemical reactions in blood or plasma; further studies will be performed to discover the cause of this interesting phenomenon.

3.2.2 Selectivity

High-performance liquid chromatography is widely used as an economic and convenient method [36]. Retention times of flavonols 13 were determined to assess the selectivity of the method. Figure 2 shows the chromatographic profiles obtained from blank plasma (Fig. 2a), blank plasma spiked with avicularin, juglanin, and the IS (Fig. 2b), plasma obtained 30 min after oral administration of TFP (Fig. 2c), blank urine (Fig. 2d), blank urine spiked with myricitrin, avicularin, juglanin, and the IS (Fig. 2e), and urine collected 7 h after dosing (Fig. 2f). Endogenous substance peaks were separated from the analyte peaks and acceptable selectivity was obtained by use of the method.

3.2.3 Precision and Accuracy

The precision and accuracy of the SPE–LC method were assessed for plasma and urine by performing replicate analysis (n = 5) of spiked samples and comparing the results with those obtained from calibration standards. The results, expressed as mean concentrations and relative standard deviations (RSD), are listed in Tables 1 (for plasma) and 2 (for urine). RSD for plasma and urine were <5.68 and <2.13, respectively. All values of accuracy and precision were within recommended limits.

3.2.4 Linearity and Sensitivity

Quantitative analysis of flavonols 2 and 3 in plasma was performed over the range 11 to 1,100 μg mL−1. The calibration ranges for flavonols 13 in urine were 16–1,600, 11–1,100, and 22–1,100 μg mL−1, respectively. The calibration plots were obtained by linear least-squares regression analysis, and the correlation coefficients (r) were larger than 0.997 and 0.996 for plasma and urine, respectively.

Sensitivity was evaluated by determination of the limits of detection (LOD) and quantitation (LOQ) for myricitrin, avicularin, and juglanin for both plasma and urine; the results are listed in Table 3. LOD and LOQ were defined as the concentrations for which signal-to-noise (S/N) ratios were 3 and 10, respectively. A series of urine and plasma standards of different dilution were investigated by performing at least five replicate analyses. The sensitivity and reproducibility of the results obtained were satisfactory for all three analytes.
Table 3

Calibration data for myricitrin, avicularin, and juglanin in rat plasma and urine

Compound and sample

Regression equation

r

Sa

Sb

Test range (μg mL−1)

LOD (μg mL−1)

LOQ (μg mL−1)

Myricitrin in urine

Y = 0.0015X − 0.0298

0.9984

1.13 × 10−2

1.34 × 10−3

32–1,600

3.2

8.0

Avicularin in plasma

Y = 0.0026X − 0.0517

0.9974

3.52 × 10−2

2.07 × 10−3

11–1,100

0.22

1.1

Avicularin in urine

Y = 0.0026X − 0.0539

0.9969

1.38 × 10−2

8.10 × 10−3

11–1,100

0.44

2.8

Juglanin in plasma

Y = 0.0025X − 0.0121

0.9976

1.45 × 10−2

8.49 × 10−3

11–1,100

0.11

0.82

Juglanin in urine

Y = 0.0025X − 0.0109

0.9992

1.35 × 10−2

8.13 × 10−3

22–1,100

0.55

2.2

Y analyte/IS peak-area ratio, X concentration (μg mL−1), r correlation coefficient, Sa standard deviation of the intercept, Sb standard deviation of the slope, LOQ concentration for which S/N = 10:1, LOD concentration for which S/N = 3:1

The standard deviations of the intercepts (Sa) and slopes (Sb) of the calibration plots are also listed in Table 3. The values indicate the calibration plots were stable.

3.2.5 Stability

Freeze–thaw experiments were used to determine the stability of the analytes. After each cycle the concentrations detected were compared with the initial values. The result indicated that flavonols 13 were stable throughout three freeze (12 h)–thaw (3 h) cycles; Tables 4 and 5 show recoveries were always >88%. All samples were kept frozen for no longer than 24 h before pretreatment; this ensured good stability of analytes and the veracity of the results.
Table 4

Freeze–thaw stability data for avicularin and juglanin in rat plasma

Compound

Concentration added (μg mL−1)

Measured concentration (μg mL−1)

One cycle

Two cycles

Three cycles

Avicularin

550

99.2 ± 2.67

98.6 ± 2.82

98.0 ± 2.45

110

94.5 ± 6.90

90.1 ± 1.89

88.3 ± 2.35

11

90.9 ± 3.45

89.4 ± 1.39

88.5 ± 1.39

Juglanin

550

100.3 ± 1.95

99.5 ± 1.12

99.1 ± 1.28

110

93.8 ± 5.15

90.1 ± 2.24

90.5 ± 2.84

11

88.8 ± 0.524

88.5 ± 0.524

88.2 ± 0.909

Table 5

Freeze–thaw stability data for myricitrin, avicularin, and juglanin in rat urine

Compound

Concentration added (μg mL−1)

Measured concentration (μg mL−1)

One cycle

Two cycles

Three cycles

Myricitrin

800

98.8 ± 0.968

97.9 ± 0.564

97.2 ± 1.56

160

98.2 ± 0.275

97.9 ± 1.84

95.7 ± 4.91

16

94.4 ± 4.16

93.1 ± 3.91

91.9 ± 2.19

Avicularin

550

99.5 ± 0.938

99.1 ± 2.62

98.4 ± 1.04

110

92.7 ± 6.06

89.5 ± 1.51

88.1 ± 2.33

11

89.7 ± 2.29

89.7 ± 2.63

88.2 ± 1.01

Juglanin

550

100.2 ± 0.833

99.4 ± 0.780

98.9 ± 1.62

110

91.2 ± 2.73

89.9 ± 0.345

89.6 ± 2.97

11

90.9 ± 3.67

89.7 ± 2.29

88.5 ± 0.525

3.3 Pharmacokinetic Study

The concentration–time profiles of avicularin and juglanin in rat plasma (n = 5) are shown in Fig. 3. The concentrations of avicularin and juglanin after 5 min were approximately 1 μg mL−1, suggesting the compounds were absorbed very quickly. The concentrations of the compounds remained high for a long time (240 min), not substantially lower than the peak concentrations; this is typical of the lasting characteristics of traditional Chinese medicine (TCM).
https://static-content.springer.com/image/art%3A10.1365%2Fs10337-009-1088-x/MediaObjects/10337_2009_1088_Fig3_HTML.gif
Fig. 3

Concentration–time profiles of avicularin and juglanin in rat plasma after oral administration of TFP (n = 5)

Cumulative excretion of myricitrin, avicularin, and juglanin in the urine (n = 5) after oral administration of TFP is presented in Fig. 4. The maximum concentration of juglanin appeared between 15 and 24 h whereas those of avicularin and myricitrin occurred between 11 and 15 h. In China, TFP is regarded as a diuretic constituent of Polygonum aviculare extract, and avicularin was the most abundant constituent in TFP [8]. We observed that the amount in urine increased from 5 until 24 h after administration. The amount then began to decline gradually and reached the normal level after 36 h. The amounts of avicularin and myricitrin excreted from 5 to 36 h were 77% and 86%, respectively. The amount of urine excreted in this period was 46.4 mL, and the total volume was 69.5 mL. This indicates the diuretic effect of Bianxu may be because of either avicularin and myricitrin or the combined effect of both. The peak plasma concentration of avicularin occurred after approximately 30 min, but excretion in urine increased from 3 to 5 h. This result showed that TCM took effect slowly but lasted for a long time, which is a general characteristic of TCM.
https://static-content.springer.com/image/art%3A10.1365%2Fs10337-009-1088-x/MediaObjects/10337_2009_1088_Fig4_HTML.gif
Fig. 4

Cumulative excretion of myricitrin, avicularin, and juglanin recorded after oral administration of TFP to rats (n = 5)

4 Conclusion

A simple, reliable, and reproducible LC method has been developed and validated for simultaneous analysis of myricitrin, avicularin, and juglanin in rat plasma and urine after oral administration of TFP. Sample pretreatment by solid-phase extraction resulted in excellent recovery. Because of its high sensitivity and good selectivity, the method was successfully applied to a pharmacokinetic study of flavonols 13 in rats. The results from the study confirmed one of the primary characteristics of TCM, that of taking effect slowly and lasting for a long time. As far as we are aware, this is the first report of simultaneous analysis of these flavonols in rat biological fluids after oral administration of TFP, or of pharmacokinetic study of these compounds in such samples.

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