A simple and sensitive HPLC-UV method has been developed for the simultaneous determination of quercetin, luteolin, and apigenin in rat plasma after oral administration of
In this study, a simple and sensitive HPLC-UV method was first developed and validated for simultaneous determination of quercetin, luteolin, and apigenin in rat plasma. A compound, kaempferol, was selected as the internal standard (Figure
Chemical structures of quercetin (a), luteolin (b), apigenin (c), and kaempferol (d).
Reference standards of quercetin (batch number 4284, purity > 98.0%), luteolin (batch number 2226, purity > 98.0%), and apigenin (batch number 404, purity > 98.0%) were purchased from Shanghai Standard Biotech Co., Ltd. (Shanghai, China). Kaempferol (batch number 110861-200808, purity > 98.0%) was obtained from National Institutes for Food and Drug Control (Beijing, China) and used as an internal standard (IS). Methanol (HPLC-grade) was obtained from Fisher (USA). HPLC-quality water was obtained using a Cascada™ IX-water Purification System (Pall Co., USA).
All analyses were performed on a Shimadzu HPLC system, equipped with LC-20AT pump, a Shimadzu SCL-10A system controller, and a SPD-20A DAD-UV detector. The HPLC analysis was performed on an Agilent Eclipse XDB-C18 column (250 mm × 4.6 mm, 5
The concentrated stock solutions of quercetin, luteolin, and apigenin were prepared by dissolving the reference standards in methanol to final concentration of 0.11 mg/ml. For the assay of plasma samples, working solutions were prepared by appropriate dilution of the stock solution with methanol. A stock solution of IS was prepared in methanol and then further diluted with methanol to prepare the working internal standard solution containing 22.52
QC samples were prepared in the same way as the calibration samples, representing three different level concentrations (low, medium, and high) of quercetin in plasma at 0.57, 2.84, and 9.09
An aliquot of 100
The method was developed and conducted according to FDA guidelines with respect to specificity, linearity, LLOQ, accuracy, extract recovery, and stability.
The specificity study was to investigate whether endogenous constituents and other substances existing in samples will interfere with the detection of the analytes and IS. The specificity of this method was ascertained by comparatively analyzing blank plasma samples from six different sources of rats, corresponding to blank plasma spiked with the three analytes and IS and the plasma samples from the rats after oral administration of the
The linearity of each calibration curve was determined by plotting the peak area ratio (
The intraday precision and accuracy were assessed during the same day by analyzing six QC replicates at three levels on the same day. The interday precision and accuracy were determined by repeating analysis of QC samples on three consecutive days. The intraday and interday precisions were defined as RSD with criteria of less than 15%. The accuracy was assessed by comparing the observed concentration with its nominal value with a criterion of within ±15% for all QC samples.
The extraction recoveries of quercetin, luteolin, apigenin, and IS were determined by comparing the peak areas from blank plasma samples spiked with QC working solutions and IS before extraction with those from blank plasma samples spiked after extraction.
The stability of the analytes in plasma was investigated by determining QC plasma samples of the three concentration levels under different storage conditions: 24 h at room temperature, three freeze (−20°C) and thaw (room temperature) cycles, stored at −20°C for 30 days. They were considered stable when 85–115% of the initial concentrations were got. All stability testing QC samples were determined by using the calibration curve of freshly prepared standard samples.
Male Sprague-Dawley rats (body weight 160 ± 20 g) were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). Rats were housed in a temperature- and humidity-controlled environment (25°C, 65% RH) and maintained on a 12-hour light/dark cycle for 3 days with free access to food and water before starting the experiment. The animal experiments were performed in accordance with the principles of the International Guide for the Care and Use of Laboratory Animals and were approved by the Committee on Animal Care and Usage of the Beijing University of Chinese Medicine (number SYXK 2011-0024).
The extracts of
The pharmacokinetic parameters of quercetin, luteolin, and apigenin were calculated by Kinetica 4.4 software (Thermo Scientific, USA). Noncompartmental analysis was used to determine standard pharmacokinetic parameters of analytes. All the results were expressed as means ± standard deviation (SD) of six replicates.
An appropriate IS will control variability in extraction and HPLC injection. In this study, several substances, such as chloromycetin, naringenin, genkwanin, and kaempferol were tested as internal standards. Among these, kaempferol has been chosen to be the most appropriate in the present analysis because it is stable and does not exist endogenously in plasma. Moreover, its retention time was suitable and it was well separated from quercetin, luteolin, and apigenin. Therefore, kaempferol was finally selected as the IS due to the relatively equal recovery, and similar polarity and retention time to the analytes.
An Agilent Eclipse XDB-C18 column (250 mm × 4.6 mm, 5
In our experiment, liquid-liquid extraction (LLE) and protein precipitation were compared for the sample preparation. Initially, we used the protein precipitation method; 1 mL methanol (or acetonitrile) was added for protein precipitation. However, the results showed that the aimed compounds could not be isolated completely from the proteins and interfering peaks in rat plasma by protein precipitation, but could be isolated by LLE. Therefore, liquid-liquid extraction by ethyl acetate was chosen, which considerably reduced the sample processing time. This method is rapid, and extraction using ethyl acetate is simple without any loss of analytes.
The developed method was validated according to FDA guidelines for the following parameters: specificity, linearity, precisions, accuracy, recovery, and stability.
This study confirms the specificity of this method. The specificity of the method towards endogenous plasma matrix was evaluated with plasma from six rats. Typical chromatograms obtained from a blank, a spiked plasma sample with the three analytes and IS, and 20 min plasma sample after an oral administration of
Chromatograms of quercetin (1), luteolin (2), IS (3), and apigenin (4) in plasma: (a) blank plasma; (b) blank plasma spiked with the three analytes and IS; (c) plasma sample obtained at 20 min from a rat after oral administration of
The excellent linear relationships are shown in Table
Linearity and LLOQ for the analysis of quercetin, luteolin, and apigenin under standard solutions.
Compound | Linearity ( |
Calibration curve | Correlation coefficient ( |
LLOQ ( |
---|---|---|---|---|
Quercetin | 0.11–11.36 |
|
0.99 | 0.11 |
Luteolin | 0.11–11.20 |
|
0.99 | 0.11 |
Apigenin | 0.11–10.60 |
|
0.99 | 0.11 |
The intra- and interday precision and accuracy are demonstrated in Table
The intra- and interday precision and accuracy of quercetin, luteolin, and apigenin in rat plasma (
Compounds | Spiked conc. ( |
Intraday | Interday | ||||
---|---|---|---|---|---|---|---|
Found conc. ( |
Precision (RSD%) | Accuracy (%) | Found conc. ( |
Precision (RSD%) | Accuracy (%) | ||
Quercetin | 0.57 |
|
|
|
|
2.20 | 102.39 |
2.84 |
|
|
|
|
8.81 | 99.05 | |
9.09 |
|
|
|
|
1.31 | 95.31 | |
Luteolin | 0.56 |
|
|
|
|
1.06 | 109.02 |
2.80 |
|
|
|
|
2.89 | 103.68 | |
8.96 |
|
|
|
|
0.54 | 99.61 | |
Apigenin | 0.53 |
|
|
|
|
2.64 | 93.17 |
2.65 |
|
|
|
|
1.88 | 102.36 | |
8.48 |
|
|
|
|
0.47 | 99.23 |
The mean extraction recoveries and RSD for quercetin, luteolin, and apigenin from rat plasma were determined at low, medium, and high concentrations in Table
Extraction recovery of quercetin, luteolin, and apigenin in rat plasma (
Compounds | Spiked conc. ( |
Recovery (%) | RSD (%) | Average (%) |
---|---|---|---|---|
Quercetin | 0.57 |
|
7.08 | 99.11 |
2.84 |
|
9.01 | ||
9.09 |
|
9.07 | ||
Luteolin | 0.56 |
|
9.58 | 95.62 |
2.80 |
|
4.29 | ||
8.96 |
|
9.97 | ||
Apigenin | 0.53 |
|
10.56 | 95.21 |
2.65 |
|
4.47 | ||
8.48 |
|
10.78 |
Stability study showed that the concentrations of quercetin, luteolin, and apigenin were stable in plasma stored at 20°C for 24 h, −20°C for 30 days, and after three freeze-thaw cycles at low, medium, and high concentrations, respectively. All the data are summarized in Table
Stability of quercetin, luteolin, and apigenin in rat plasma (
Compounds | Spiked conc. ( |
Short-term stability |
Long-term stability |
Freeze-thaw stability | |||
---|---|---|---|---|---|---|---|
Found conc. ( |
RSD (%) | Found conc. ( |
RSD (%) | Found conc. ( |
RSD (%) | ||
Quercetin | 0.57 |
|
1.30 |
|
5.16 |
|
2.08 |
2.84 |
|
5.68 |
|
5.17 |
|
2.78 | |
9.09 |
|
7.52 |
|
5.52 |
|
5.97 | |
Luteolin | 0.56 |
|
1.21 |
|
5.52 |
|
3.01 |
2.80 |
|
4.65 |
|
5.03 |
|
5.23 | |
8.96 |
|
8.53 |
|
7.28 |
|
6.33 | |
Apigenin | 0.53 |
|
8.36 |
|
8.47 |
|
3.21 |
2.65 |
|
6.14 |
|
5.07 |
|
5.31 | |
8.48 |
|
8.61 |
|
7.46 |
|
6.39 |
Pharmacokinetic parameters of quercetin, luteolin, and apigenin in rats after oral administration of
Parameters | Unit | Quercetin | Luteolin | Apigenin |
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h |
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Parameters were estimated using the mean concentration-time profiles obtained from six different rats per time point (
The mean (±SD) plasma concentration-time profiles of quercetin, luteolin, and apigenin after oral administration of
The plasma concentration of quercetin reached a maximum at 0.79 h after administration with an average
Quercetin, luteolin, and apigenin are the main active constituents present in the
The present study described a simple, specific, and reliable HPLC-UV with IS method for the simultaneous determination of three flavonoids in blood samples. This is the first validated HPLC method for analysis of quercetin, luteolin, and apigenin in rat plasma, which was highly sensitive and accurate. The method has been successfully applied to pharmacokinetic studies of quercetin, luteolin, and apigenin in rats after oral administration of
The authors have declared that there is no conflict of interests.
Xiaoxv Dong and Wei Lan contributed equally to this work.
This work was financially supported by the National Natural Science Foundation of China (no. 81173563) and the Collaborative Innovation Construction Plan of Beijing University of Chinese Medicine (no. 2013-XTCX-03).