Pharmacokinetics of Eight Flavonoids in Rats Assayed by UPLC-MS/MS after Oral Administration of Drynariae rhizoma Extract

As a traditional Chinese medicine, Drynariae rhizoma (Kunze ex Mett.) J. Sm. has been used to treat osteoporosis and bone resorption for 2500 years. Based on the previous study and literature references, flavonoids were proved to be the most abundant and main active compounds of Drynariae rhizoma for osteoporosis treatment. In order to make good and rational use of Drynariae rhizoma in future, a rapid, sensitive, and selective ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed to investigate the pharmacokinetics of eight main flavonoids in rat plasma after oral administration of the Drynariae rhizoma extract, including neoeriocitrin, luteolin-7-O-β-D-glucoside, astragalin, naringin, eriodictyol, luteolin, naringenin, and kaempferol. Plasma samples' pretreatment involved a solid-phase extraction column. The separation was performed on an ACQUITY UPLCTM BEH C18 column with a gradient mobile-phase system of acetonitrile and 1% acetic acid in water. The detection was performed using a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization interface (ESI) by multiple reaction monitoring (MRM) in the positive ion mode. All calibration curves exhibited good linearity (r 2 > 0.9990) over the measured ranges. The intraday and interday precisions (RSD) were within 13.87%, and the accuracy (RE) ranged from −14.57% to −0.25% at three quality control levels. Extraction recovery, matrix effect, and stability were satisfactory. The pharmacokinetic characteristics of the eight flavonoids of interest were clearly elucidated.


Introduction
e traditional Chinese medicine Drynariae rhizoma (Kunze ex Mett.) J. Sm., commonly known as Gu-Sui-Bu, is a fern plant widely distributed in southern China. Drynariae rhizoma is effective for the treatment of osteoporosis and bone resorption [1]. Drynariae rhizoma contains various types of chemical constituents, including flavonoids, triterpenes, phenolic acids, and their glycosides. But flavonoids and their glycosides are the most abundant constituents of Drynariae rhizoma [2,3]. Furthermore, flavonoids showed protective activities of osteoporosis, bone fractures, oxidative damage, and inflammation [4][5][6][7][8][9][10]. Total flavonoids in Drynariae rhizoma could activate the estrogen receptors and have the trend of replacing estrogen for clinical use [11,12]. Flavonoids have been regarded as the principle constituents that contribute to the bioactivities of Drynariae rhizoma.
In the bioactive research of Drynariae rhizoma, the key issue is how to study the effective substance of Drynariae rhizoma to play a key role in osteoporosis. Wang suggested a conceptual framework for illuminating the absorbed bioactive compounds in herb medicines [13]. In general, absorbed bioactive compounds more possibly play a part in the therapeutic effect in vivo after oral administration. us, it is necessary to measure the absorbed bioactive compounds in plasma to understand the effective substances in the herb. In our pilot study, eight flavonoids were detected in plasma after oral administration of Drynariae rhizoma extract (DRE), including neoeriocitrin, luteolin-7-O-β-D-glucoside, astragalin, naringin, eriodictyol, luteolin, naringenin, and kaempferol ( Figure 1), which could contribute to the therapeutic effect of the DRE.
As far as we know, no study of the pharmacokinetic characteristics of the eight flavonoids in rats after oral administration of the DRE has been reported. Up to now, only one or two flavonoids or their metabolites of the DRE were determined in rat plasma, which could not fully reflect the drug metabolism process in the body after oral administration of DRE to humans or a model animal [14]. erefore, it is necessary to establish an appropriate analysis method to characterize the pharmacokinetics of DRE in vivo.
In the present study, a rapid, sensitive, and selective UPLC-MS/MS method was developed and validated for the simultaneous determination of eight flavonoids from the DRE in rat plasma. Plasma samples were pretreated with the C 18 SPE column. e detection was performed using a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization interface (ESI) by multiple reaction monitoring (MRM) in the positive ion mode. e newly described UPLC-MS/MS method was validated and successfully applied to pharmacokinetic study of eight flavonoids in rats after oral administration of DRE.
Mass spectrometric detection was performed using a Waters ® Micromass ® Quattro Premier ™ XE triple quadrupole tandem mass spectrometer equipped with an electrospray ionization interface in the positive ion mode. e ESI + source operation optimal parameters were capillary voltage 3.5 kV, source temperature 120°C, and desolvation temperature 300°C. Nitrogen was used as the desolvation and cone gas with flow rates of 600 and 30 L·h −1 , respectively. Argon was used as the collision gas at a pressure of approximately 2.61 × 10 −3 mbar. Quantitative analysis was performed in multiple reaction monitoring (MRM) mode, and the parent ion, daughter ion, cone voltage, and collision energies of eight analytes and the IS were optimized. Table 1

Preparation and Quality Assessment of DRE.
e dried roots of Drynariae rhizoma (1 kg) were powdered and extracted three times under reflux in 15 times volume of methanol for every 2 hours. e solution was filtered and evaporated under reduced pressure in a Rotavapor R-3 rotary evaporator (Buchi Ltd., Labortechnik AG, Switzerland). Subsequently, the concentrated extract was dried in an oven, and the final weight of the DRE was 198.9 g with a yield of 19.9%. e contents of the main flavonoid constituents in DRE were quantitatively determined with the method described above, with the results of neoeriocitrin (5.1 mg/g), luteolin-7-O-β-D-glucoside (1.2 mg/g), astragalin (0.96 mg/ g), naringin (8.5 mg/g), eriodictyol (0.66 mg/g), luteolin (0.11 mg/g), naringenin (2.71 mg/g), and kaempferol (0.14 mg/g), respectively.

Preparation of Plasma.
Aliquots of plasma samples (400 μl) were placed in a 10 ml centrifuge tube; 50 μl of internal standard solution (200 ng/ml) and 50 μl acetic acid were added. e mixture was vortexed for 1.0 min and then loaded onto an activated SPE C 18 column (the SPE C 18 column was activated by 3 ml of methanol before loading the sample, and the excess was washed off with 5 ml of purified water). After rinsing with 2 ml purified water, the SPE column was eluted with 4.5 ml of methanol and the eluate was evaporated to dryness under a gentle stream of nitrogen. e residue was reconstituted in 140 μl methanol and centrifuged at 16000 × g for 15 min; then, a 5 μl aliquot was injected into the UPLC-MS/MS system for analysis.

Method Validation.
e method was validated in terms of specificity, linearity, lower limit of detection (LLOD), lower limit of quantification (LLOQ), accuracy and  precision, extraction recovery, stability, and matrix effects based on the method validation procedure in the previous work [15].

Specificity.
We compared the chromatograms of blank plasma from six individual rats with those of corresponding plasma samples spiked with eight mixed standard samples and IS and plasma samples after oral administration of DRE.

Linearity and Quantification.
Calibration curves for the eight standards were constructed by plotting the peak area ratios of each analyte to that of the IS versus the corresponding concentration. e lower limit of detection (LLOD) was defined as the lowest concentration with a signal-to-noise ratio of 3 : 1. e lower limit of quantification (LLOQ) was determined as the lowest concentration of the calibration curve with a signal-to-noise ratio of 10 : 1.

Precision and Accuracy.
Intraday precision and interday precision and accuracy were assessed for low-, middle-, and high-concentration QC samples in six replicates on the same day and once a day for three consecutive days, respectively. Each tested sample was related to the calibration curve. e precision was calculated as the relative standard deviation (RSD%) and the accuracy as relative error (RE%). ese results indicated that the precision and accuracy of the method were within acceptable limits. e intraday precision and interday precision and accuracy values for lowest acceptable reproducibility concentrations were denied as being within ±15%, and the precision and accuracy were within 80%-120%.

Recovery and Matrix
Effect. e recovery and matrix effects of analytes were determined for low-, middle-, and high-concentration QC samples with six replicates. Extraction recovery was determined at the three QC levels by comparing the peak areas of analytes between plasma samples spiked with analytes before and after extraction. Matrix effects were evaluated at the three QC levels by comparing the peak areas of analytes obtained from plasma samples spiked with analytes after extraction to those of pure standard solutions at the same concentrations; the acceptable range was 80-120%.

Stability.
e stability of determination was assessed by analysis of low-, middle-, and high-concentration QC samples with three replicates. e short-term stability was assessed by analyzing the QC samples kept in the autosampler (4°C) for 36 h. To assess long-term stability, the QC samples were stored at −20°C for 30 days. Freeze-thaw stability was evaluated by subjecting the QC plasma samples to three complete freeze/thaw cycles from −20°C to room temperature.  Heilongjiang). All protocols for animal experiments were approved in accordance with the Regulations of Experimental Animal Administration issued by the State Commission of Science and Technology of the People's Republic of China. e rats were housed at 24 ± 2°C, and relative humidity was 60 ± 5%, with a 12 h-12 h light-dark cycle. Water and food were supplied freely.

Drug Administration and Sampling.
e UPLC-MS/ MS method was successfully applied in a pharmacokinetic study of eight flavonoids in rats after oral administration of the DRE. e dosing solutions were freshly prepared DRE administrated via an oral gavage to the rats at a single dose of 4 g/kg. e animals had free access to water during the experiment. A series of blood samples were collected in 1.5 ml heparinized polythene tubes from the suborbital venous lexus of each rat at 0.08, 0.33, 0.5, 0.67, 1, 2, 4, 6, 8, 12, and 24 h after administration. e blood samples were centrifuged at 4500 × g for 10 min, and the supernatants were collected immediately and stored at −20°C until analysis.

Data Analysis.
Pharmacokinetic parameters were determined using a noncompartmental model and analyzed using pharmacokinetic software WinNonlin Standard Edition, version 1.1. Data are shown as the mean ± standard deviation (SD) for each parameter.

Optimization of Mass Spectrometric Conditions.
To optimize the mass spectrometry conditions, appropriate mixed standard solutions of neoeriocitrin, luteolin-7-Oβ-D-glucoside, astragalin, naringin, eriodictyol, luteolin, naringenin, kaempferol, and quercetin were monitored across a full scan in both positive and negative modes. e signal intensity and fragment stability in the positive mode were better than those in the negative mode for all the analytes. erefore, the positive mode was used for analysis. e parent ion and daughter ion for each compound were obtained (Figure 1), and the cone voltage and collision energy for the eight analytes and the IS (quercetin) were optimized. e molecular weight, parent ion, daughter ion, cone voltage, collision energy, and retention time of the eight flavonoids and the IS are shown in Table 1.

Chromatographic Conditions.
To achieve symmetric peak shape and short running time for the simultaneous analysis of the eight analytes, we tested various mobile phase conditions to achieve good separation of the analytes. e  Journal of Analytical Methods in Chemistry 5 mobile phase we finally optimized as 1% acetic acid-water as the aqueous phase (A) and 100% acetonitrile as the organic phase (B). In the optimized UPLC-MS/MS conditions for simultaneous determination of the eight compounds, all analytes were eluted rapidly with 5.0 min. e representative chromatograms of blank plasma, blank plasma spiked with reference standards and IS, and plasma obtained after the oral administration of DRE are shown in Figure 2. Under the established optimal chromatographic conditions, no significant interfering peaks were observed at the analyte elution times, and no interference occurred between IS and the eight analytes.

Precision and Accuracy.
e precision and accuracy of the UPLC-MS/MS method (Table 3) were within acceptable limits. e intraday precision and interday precision (R.S.D.) were within 13.87%, and the accuracy (RE) ranged from −14.79% to −0.25% at three quality control levels.

Recovery and Matrix Effect.
e matrix effect and recovery results (Table 4) indicated that no endogenous substances from plasma significantly influenced the ionization of analytes. e matrix effects were within an acceptable range (80.23%-98.99%), and the mean extraction recoveries of the eight analytes and IS were greater than 89.45%.

Stability.
e results of stability experiments (Table 5) showed that no significant degradation occurred. e concentrations of the eight analytes measured in the stability study were within −5.66%-3.56%. e data indicated   Journal of Analytical Methods in Chemistry that all analytes in the rat plasma were stable after storage at −20°C for 30 days, after three freeze/thaw cycles, and after storage in the autosampler (4°C) for 36 h.

Pharmacokinetic Studies.
e UPLC-MS/MS method was successfully applied in a pharmacokinetic study of eight flavonoids after oral administration of DRE at a dose of 4 g/ kg body weight. Mean plasma concentration-time plots are shown in Figure 3. e major pharmacokinetic parameters are listed in Table 6.
After oral administration to rats, all the analytes were absorbed from the gastrointestinal tract and detected in plasma at 5 min. However, the two highest abundant flavonoids in the DRE, naringin and neoeriocitrin, showed high maximum plasma concentration (C max ) (4414.18 ± 360.38 ng/ml and 1490.98 ± 124.54 ng/ml) and high area under the plasma concentration-time curve from 0 h to infinity AUC (0-∞) (8760.77 ± 347.83 and 3340.34 ± 237.36). Neoeriocitrin still exhibited a shortest T max to reach the maximum drug concentration (T max � 0.33 h). In the plasma concentration time profile of naringin, another small peak could be seen at 8 h (Figure 3), which is in agreement with the literature where pure naringin was orally administrated to rats [16]. And this phenomenon may be due to the enterohepatic circulation of naringin in rats, which was also reported for other glycosides.
Naringenin, the aglycone of naringin, was absorbed into blood with T max at 6 h and eliminated with T 1/2 at 3.2 h after oral administration of DRE, which was significantly different from the other compounds (T max < 2 h). e slow elimination may be due to the fact that orally administrated naringin can be metabolized into naringenin and naringenin glucuronide [17]. Pharmacokinetic characters of naringenin were very close to a previous report [14]. e plasma concentrations of luteolin-7-O-β-D-glucoside,    eriodictyol, and kaempferol were lower, with small area under curve (AUC) and C max . e T max values for luteolin-7-O-β-D-glucoside and luteolin were 1 h and 0.5 h, respectively, which were in line with a previous report [18,19]. Luteolin is the metabolite of luteolin-7-O-β-Dglucoside, the plasma concentration of the former is greater   Table 6: Pharmacokinetic parameters of eight analytes in rat plasma after oral administration of DRE (mean ± SD, n�6).