516 Original Article

Quantitative Determination of Myricetin in Rat Plasma by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry and its Absolute Bioavailability

Affiliations

Key words ▶ UPLC-MS/MS ● ▶ myricetin ● ▶ rat plasma ● ▶ pharmacokinetics ● ▶ absolute bioavailability ●

received 29.09.2013 accepted 25.11.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1363220 Published online: December 19, 2013 Drug Res 2014; 64: 516–522 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Y. Xie, PhD Associate Professor Research Center for Health and Nutrition Shanghai University of Traditional Chinese Medicine 1200 Cailun Road 201203 Shanghai China Tel.: + 86/21/51322 407 Fax: + 86/21/51322 407 [email protected]

Y. Dang1, G. Lin1, Y. Xie1, J. Duan2, P. Ma3, G. Li4, G. Ji2 1

Research Center for Health and Nutrition, Shanghai University of Traditional Chinese Medicine, Shanghai, China Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China 3 Global Pharmaceutical Research and Development, Hospira Inc., McPherson, KS, USA 4 Pharmacy Department, Shanghai TCM-integrated Hospital, Shanghai, China 2

Abstract



Myricetin is a widely distributed bioactive flavonoid with scientific interest attributed to its anti-oxidant, antitumor, and anti-inflammatory properties. A specific and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for identification and quantification of myricetin in rat plasma after oral and intravenous administrations. Kaempferol was used as an internal standard. Followed by β-glucuronidase and sulfatase hydrolysis and liquid-liquid extraction with ethyl acetate, the analytes were separated on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) and analyzed in the selected ion recording with a negative elec-

Introduction



▶ Fig. 1) is a naturally existing flavoMyricetin (● nol which can be found in many herbs, such as Myrica rubra Sieb. Et Zucc. [1], Vitis vinifera Linn. [2], Dioscorea bulbifera L. [3], Carissa opaca [4], Ardisia colorata Roxb. [5], and Cudrania tricuspidata [6], etc. It also is a major flavonoid distributed in several foods including onions, berries, grapes, as well as red wine [7–9]. In recent years a great deal of research has been revealed that myricetin has diverse therapeutic effects and pharmacological activities, such as anti-oxidative [10], anti-inflammatory [11], anti-cancer [12], anti-microbial [13], cardio-protective [14, 15], neuro-protective [16], anti-diabetic [17], and hepato-protective [18]. All these indicate that myricetin is a valuable natural active flavonol component and deserved to be further investigated as a new drug candidate. As we all known, preclinical pharmacokinetic data on active ingredients are important to the design of drug delivery systems, which could offer a useful guideline for clinical applications. Unfortunately, to the

Dang Y et al. Myricetin Pharmacokinetics in Rats … Drug Res 2014; 64: 516–522

trospray ionization mode. The developed method was validated for selectivity, accuracy, precision, linearity, recovery, stability and matrix effect. The assay was validated over a wide concentration range of 2–4 000 ng/mL. Intra- and inter-day precisions were all less than 13.49 % and accuracy ranged from 95.75 to 109.80 %. The present method was successfully applied to investigate a pharmacokinetic study of myricetin following intravenous and oral administrations to rats. The absolute bioavailability was found to be 9.62 % and 9.74 % at 2 oral doses (50 mg/kg and 100 mg/ kg, respectively), which indicated myricetin was poorly absorbed after oral administration. To our knowledge, this is the first pharmacokinetic evaluation of myricetin as a single active pharmaceutical ingredient in preclinical studies.

best of our knowledge, so far there have been no pharmacokinetic studies reported on myricetin as a single active pharmaceutical ingredient (API) in any dosage forms. In recent years, several analytical techniques have been applied to determine myricetin in herb extracts, phytomedicines, plasma and other biological samples, such as high-performance liquid chromatography (HPLC) [19–21], liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) [22, 23], ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry with collision energy (UPLC-QTOF-MSE) [24]. However, these methods had some disadvantages, such as long run times ( > 11 min) and complex sample preparation procedures (solid phase extraction, multiple liquid-liquid extraction steps, etc.), which were unsuitable for pharmacokinetic and bioavailability studies of this unstable flavonol component. Even though most of these analytical methods were focused on the simultaneous determination of multiple components including myricetin in samples, the meth-

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Authors

Original Article 517

ods were not sensitive enough to determine myricetin alone. Therefore, it is necessary to establish a fast, selective and sensitive analytical method for the quantitative determination of myricetin in preclinical biopharmaceutical and pharmacological studies. In this paper, an ultra performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS/MS) method was successfully developed and validated to quantify ▶ Fig. 1) was used as the myricetin in rat plasma. Kaempferol (● internal standard (IS). The in vivo pharmacokinetics and bioavailability studies of myricetin in rats following intravenous and oral administrations were performed by using this established UPLC-ESI-MS/MS method.

Preparation of calibration standards, quality control and internal standard A 100 μg/mL myricetin stock solution in methanol was prepared. This solution was further diluted with methanol to obtain serial working solutions at concentrations from 20 to 40 000 ng/mL. Low, medium, and high concentrations of quality control (QC) samples were set to 40, 8 000, 32 000 ng/mL, respectively. Kaempferol was dissolved in methanol at a concentration of 100 μg/mL as an IS stock solution, which was further diluted with acetonitrile to obtain the working solution of 5 000 ng/mL. All solutions were stored at − 20 °C.

Animals Materials and Methods



Materials Myricetin and kaempferol with purities greater than 98 % were purchased from the Shanghai Tauto Biotech Co., Ltd. (Shanghai, China) and the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), respectively. β-glucuronidase and sulfatase were purchased from SigmaAldrich (St. Louis, MO, USA). HPLC grade acetonitrile was purchased from Honeywell Burdick & Jackson (Ulsan, Korea) and formic acid was obtained from Fluka (Burchs, Switzerland). Ultra-pure deionized water was generated from a Millipore Milli-Q Gradient system (Millipore, Bedford, MA). Ascorbic acid, carboxymethylcellulose sodium (CMC-Na), anhydrous ethanol, polyethylene glycol-400 (PEG-400), dimethyl sulfoxide (DMSO) and Tween-80 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other reagents used were of analytical grade.

Instrumentation and conditions The concentration of myricetin was determined by an UPLC-ESIMS/MS method. The UPLC system comprised Agilent 1 290 series with a G4220A binary pump, a G4226A autosampler, a G1330B thermostat for ALS/FC/Spotter, and a G1316 thermostatted column compartment. Chromatographic separations were achieved using a Waters UPLC column (Acquity UPLC BEH C18 1.7 μm, 2.1 × 50 mm, Waters, Ireland). The mobile phase consisted of the mixtures of acetonitrile (A) and 0.1 % formic acid aqueous solution (B) with a gradient program as follows: 27–60 % A (0–2 min), 60–90 % A (2–2.8 min), 90–27 % A (2.8–3.5 min), and 27 % A (3.5– 4.5 min). The flow rate was 0.3 mL/min. The autosampler and column were maintained at 4 and 40 °C, respectively, and the injection volume was 5 μL. Mass spectrometric analysis was per-

Male Sprague-Dawley rats weighing 280–300 g were supplied by the Laboratory Animal Center, Shanghai University of Traditional Chinese Medicine, China. All rats were fed and maintained under constant conditions at a temperature of 20–25 °C, a humidity of 55 ± 5 % and with 12 h light/dark cycles. Water and food were accessible to the rats ad libitum. All animal experiments were carried out accordance with the local institutional guidelines for animal care of Shanghai University of Traditional Chinese Medicine.

Sample preparation A liquid-liquid extraction method was used for rat plasma samples. After thawed to room temperature, 100 μL of plasma samples was mixed with 10 μL of methanol (volume of the corresponding working solution for calibration curve and QC samples), 10 μL 10 % ascorbic acid (v/v) and 50 μL IS. Followed by the addition of β-glucuronidase (30 μL, 4110 U/mL) and sulfatase (30 μL, 81.93 U/mL), the samples were incubated in a water bath at 37 °C for 2 h, which would result in complete release of myricetin. After hydrolysis, 200 μL acetonitrile and 200 μL ethyl acetate were added to remove proteins and extract analytes by vortex-mixing for 2 min. Next, the samples were centrifuged at 13 000 rpm for 10 min. The supernatant was transferred into new tubes and evaporated to dryness at 35 °C under a gentle stream of nitrogen. The residue was then reconstituted with 100 μL of mobile phase. After centrifugation at 13 000 rpm for 10 min, 5 μL supernatant was injected into the UPLC-MS/MS system for analysis.

Assay validation The validation process was carried out according to Guidance for Industry-Bioanalytical Method Validation, recommended by US Food and Drug Administration [25]. The specificity of myricetin in plasma samples was evaluated via the analysis of blank samples obtained from 6 rats. They were Dang Y et al. Myricetin Pharmacokinetics in Rats … Drug Res 2014; 64: 516–522

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Fig. 1 Chemical structures of both myricetin and kaempferol (internal standard, IS).

formed by an Agilent 6 410 B triple-quadrupole mass spectrometer (Agilent Technologies, USA) with an electrospray ionization (ESI) interface operating in a negative ionization mode. The working parameters of the MS were as follows: nebulizer temperature, 325 °C; nebulizer pressure (N2), 35 psi; drying gas flow (N2), 10 L/min; fragmentor voltage, 150 V for myricetin and 170 V for kaempferol, respectively; capillary voltage, 4 kV. The quantification was performed using multiple reaction monitoring (MRM) method with the transitions of (m/z) 317.1 → (m/z) 151.1 (179.1) for myricetin and (m/z) 2 85.2 → (m/z) 117 (159.2) for kaempferol, respectively. The Agilent MassHunter software package (B.05.00) was used for data acquisition and analysis (Agilent Technologies, USA).

518 Original Article

Pharmacokinetic study The animals were acclimated at the animal facility for 5 days, and then fasted with free access to water for 12 h prior to each experiment. The rats were randomized into the following 3 groups (n = 6): animals in group A and B were orally administered with myricetin (suspended in 0.5 % CMC-Na) at a dose of 50 mg/kg and 100 mg/kg, respectively, and animals in group C were intravenous (i. v.) injected with myricetin (dissolved in anhydrous ethanol/PEG-400/DMSO/Tween-80 = 1.1:0.5:0.1:0.1, v/v/v/v) at a dose of 0.5 mg/kg. Blood samples (0.25 mL) were collected into a heparinized tube via the oculi chorioideae vein at 0, 15, 30, 60, 120, 240, 360, 480, 600, 720, 960, 1 440, 2 880 min after oral administration and at 0, 1, 5, 10, 20, 40, 60, 120, 240, 480, 720, 1 440, 2 880 min after intravenous injection administration, respectively. Plasma samples (100 μL) were obtained Dang Y et al. Myricetin Pharmacokinetics in Rats … Drug Res 2014; 64: 516–522

centrifuging at 13 000 rpm for 10 min and stored at − 80 °C until analysis.

Statistical analysis Plasma-concentration data for individual rats were analyzed by non-compartmental analysis using the DAS software (ver.2.1.1, Mathematical Pharmacology Professional Committee of China, Shanghai, China). Data were reported as mean ± standard deviation (SD). Non-parametric test was performed to demonstrate statistical differences. For all tests, p-values less than 0.05 were considered significant.

Results and Discussion



Method development In order to develop a sensitive and specific UPLC-MS/MS method for quantification of myricetin in rat plasma, some potential factors were investigated and optimized. For examples, different mobile phase compositions, such as methanol, acetonitrile, 0.1 % formic acid and water were investigated as potential mobile phases. In addition, various mobile phase conditions were tested to obtain optimal responses, suitable retention times, and symmetrical or gaussian peak shapes for the analytes (data not shown). The total run time of this gradient program was only 2 min with the retention time of myricetin at 1.14 min, which was much shorter than analytical time of 30 min reported before [7, 15]. Therefore, it was a fast and high-efficiency analytical UPLC-MS/MS method for myricetin determination. The response of myricetin to ESI was evaluated by recording the full-scan mass spectra in both positive and negative ionization modes, and the results demonstrated that myricetin had a better mass spectrometric response in negative ESI mode. The mass ▶ Fig. 2. scan spectra of myricetin and kaempferol are shown in ● In preliminary experiments, the responses of myricetin in MRM mode and selected ion monitoring (SIM) mode were compared, and the results showed that myricetin achieved higher sensitivity in MRM mode due to its lower background noise in this mode. Kaempferol was selected as the internal standard because of its similarity to myricetin in terms of chemical structure, chromatographic behavior, ionization efficiency, extraction efficiency and relative stability during sample preparation. Liquid-liquid extraction was used for sample clean-up in this study because it produced cleaner extracts, had considerably lower cost and higher efficiency compared to those applied with solid phase extraction (SPE) methods. Since myricetin was an unstable compound due to 6 hydroxyl groups in its chemical structure [22], especially in neutral or slightly basic condition observed in our in vitro experiments, ascorbic acid were added in plasma samples in order to avoid the degradation and obtain the high extraction efficiency. As it reported [26–29], flavonols underwent serious phase II metabolism, which led to the formation of glucuronidated and sulfated conjugates extensively after oral administration, and its free form could not be found in the body. In our study, the free form of myricetin was lower than LLOQ thus could not be quantified (date not shown). Generally, enzymatic and acid hydrolyses are 2 methods to obtain the free and conjugated forms of flavonol. Unfortunately, for the acid hydrolysis method, the sample processing is under drastic conditions (heating at 70 °C for 1 h in a thermostatic water bath) thus unstable target analytes cannot always be quantified accurately and completely, which is consistent with our preliminary

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processed with and without the analyte of myricetin and the IS to check for any interference peaks. Plasma samples were quantified by using the ratio of the peak area of analyte to that of IS as the assay parameter. Linearity was tested at 8 concentrations in the range of 2–4000 ng/mL (2, 4, 8, 40, 200, 800, 3 200, 4 000 ng/mL). The calibration curves were fitted by linear least-squares regression with a weighed factor of 1/ x2. The lower limit of quantitation (LLOQ) was defined as the lowest concentration of analyte in the matrix-analyte mixture giving a signal-to-noise ratio of at least 10-fold with an acceptable accuracy within 20 % deviation of the nominal concentration and a precision below 20 % relative standard deviation. To evaluate the precision and accuracy of the biosample assay, several QC samples were prepared as described above. The QC samples were at the 3 concentrations described in Section “Preparation of calibration standards, quality control and internal standard”. The intra-day precision and accuracy were evaluated by the analysis of QC samples in quintuplicate. Inter-day accuracy and precision were assessed with the same QC samples on 3 consecutive days. The RSD should not exceed 15 % and the bias should be within ± 15 %. The extraction recovery of myricetin was determined at 3 concentration levels by comparing the detector response obtained from plasma samples with the analyte spiked before and after extraction. The extraction recovery of the IS was simultaneously determined. The matrix effect was evaluated to verify whether the potential ion suppression or enhancement occurred due to the co-eluted matrix components of myricetin and kaempferol. The peak areas of myricetin and the IS from the spike-after-extraction samples were compared to those of the standard solutions in the mobile phase at the same concentrations. An endogenous matrix effect was implied if the ratio was beyond 85–115 %. Stability of myricetin in plasma was assessed by analyzing QC samples of 3 levels during the sample storage and processing procedures. Short-term stability was assessed by analyzing QC samples kept at 4 °C for 24 h. Long-term stability was evaluated by keeping QC samples at − 80 °C for 30 days. Freeze-thaw stability was investigated after 3 freeze ( − 80 °C) and thaw (room temperature) cycles. Post-preparation stability was assessed by analyzing the extracted QC samples kept in the autosampler at 4 °C for 24 h. All stability testing QC samples were determined using freshly prepared calibration curves. The analyte was considered stable if the response variation of stored samples and fresh samples, or the measured analyte concentration and its corresponding theoretical value, was less than 15 %.

Original Article 519

study (data not shown) [30]. Therefore, enzymatic hydrolysis method was used to convert the conjugated metabolites into their free forms so that the total myricetin could be quantified. To achieve a complete hydrolyzed condition, the concentrations and volume of β-glucuronidase and sulfatase, and hydrolysis time were investigated. In addition, the following 3 critical factors were optimized in plasma sample preparation to eliminate the interference in blank plasma and achieve the maximum recoveries of the analyte: the type of protein precipitator, the type of extraction solvent, and the volume of extraction solvent (data not shown). As a result, the optimized procedure was obtained as described in the above Sample preparation section.

Assay validation Typical MRM chromatograms of blank plasma, blank plasma spiked with both myricetin and the IS, blank plasma spiked with the IS, a plasma sample from a rat 6 h after oral administration of myricetin at a dose of 50 mg/kg, a plasma sample from a rat 1 min after intravenous injection of myricetin at a dose of 0.5 mg/ ▶ Fig. 3. The retention times of myricetin and kg, are shown in ● the IS were approximately 1.14 and 1.85 min, respectively. The results indicated that the endogenous ingredients did not interfere with myricetin and the IS in the biosamples. A typical equation of the calibration curve was y = 5.7913x + 2.92 × 10− 4 (r = 0.9997) in the range of 2–4 000 ng/mL. The LLOQ of myricetin was determined to be 2 ng/mL with a signal-tonoise ratio of 10.3 and the data were sufficient for quantitative detection of myricetin in the pharmacokinetic studies. The LLOD of myricetin was obtained by diluting the solution to 0.67 ng/mL, and the signal-to-noise ratio was 3-fold. The results of the intra- and inter-day precision and accuracy ▶ Table 1. For all of the QC samples, the studies are shown in ● RSD was less than 13.49 %, and the accuracy ranged from 95.75– 109.80 %. The results were within the acceptance criterion, which indicated that the analytical method was reproducible and reliable. The extraction recovery of myricetin was 89.45–107.42 % from ▶ Table 1. The recovery of the the plasma samples, as shown in ● IS from the plasma was 93.71 %. Collectively, these observations indicated that the current sample processing conditions pro-

vided adequate and stable recoveries for both the analyte and the IS. The matrix effects of myricetin at concentrations of 4, 800 and 3 200 ng/mL were 103.91, 104.87 and 100.80 %, respectively. The results showed that there was no significant interference on the quantitation of analytes from the plasma matrix. ▶ Table 2. The The results of the stability study are listed in ● biases were − 8.25–11.51 % in the post-preparative stability study, − 13.25–10.30 % in the freeze-thaw stability study, − 10.39 to − 2.00 % in the long-term stability study, and − 11.75–10.37 % in the short-term stability study, respectively. Therefore, these observations indicated that myricetin was stable under the handling and storage conditions used in the study and that typical processing and storage conditions did not affect the determination of myricetin concentrations in rat plasma samples.

Pharmacokinetic study The presented method was successfully applied to quantify myricetin in rat plasma following a single oral dose of 50 mg/kg and 100 mg/kg (n = 6) or intravenous injection at the dose of 0.5 mg/kg (n = 6). The main pharmacokinetic parameters of myricetin and the mean plasma concentrations vs. time profiles after oral and intravenous injection administrations are showed ▶ Table 3 and ● ▶ Fig. 4, respectively. The absolute bioavailabilin ● ity was found to be 9.62 % and 9.74 % at 2 oral administered doses (50 mg/kg and 100 mg/kg, respectively), which indicated myricetin might be hard to be absorbed in the gastrointestinal tract [31]. The reasons for its poor absorption might be due to its poor water solubility, poor stability in gastrointestinal fluid and its fast biotransformations occurring in vivo [23]. Specifically, dosedependent increasing in Cmax (p < 0.05) and AUC of myricetin were observed in the dose range of 50–100 mg/kg, which might result from the passive diffusion of myricetin in rats [32]. The analysis of variance of MRT values showed no significant difference among the 2 doses of treatment (p = 0.251). After i. v. administration, the plasma concentration reached 2 232.16 ± 856.36 ng/mL at 1 min and then dropped to 7 % in approximately 2 h followed by a steady decline up to 48 h. For oral administration of 50 mg/kg, myricetin was slowly absorbed and reached Cmax of 1 488.75 ± 200.78 ng/mL at approximately Dang Y et al. Myricetin Pharmacokinetics in Rats … Drug Res 2014; 64: 516–522

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Fig. 2 I Full scan mass spectra by MS: myricetin a and kaempferol b. II Full scan product ion mass spectra by MS/MS: myricetin a and kaempferol b.

520 Original Article

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Fig. 3 Representative MRM chromatograms of myricetin I and kaempferol II in rat plasma. a a blank plasma sample, b a blank plasma sample spiked with myricetin and kaempferol, c a blank plasma sample spiked with kaempferol, d a plasma sample from a rat 6 h after oral administration of myricetin at a dose of 50 mg/kg, e a plasma sample from a rat 1 min after intravenous injection of myricetin at a dose of 0.5 mg/kg.

Table 1 Intra- and inter-day precision, accuracy and extraction recovery of myricetin in rat plasma (3 days, 5 replicates per-day). Analytes

Spiked

Intra-day (n = 5)

Accuracy

Precision

Inter-day (n = 15)

Accuracy

Precision

Extraction

(ng/mL)

Found (mean ±

( %)

RSD ( %)

Found (mean ±

( %)

RSD ( %)

Recovery (n = 3,

95.75 108.07 99.93 –

1.57 3.79 3.05 –

98.25 109.80 100.49 –

13.49 2.88 2.46 –

SD, ng/mL) Myricetin

Kaempferol

4 800 3 200 2 500

3.83 ± 0.06 864.52 ± 32.80 3197.65 ± 97.40 –

SD, ng/mL)

6.4 h. The relatively longer time (Tmax) taken to reach its maximum peak concentration (Cmax) might due to its poor aqueous solubility. In addition, the elimination half-life (t1/2) and mean residence time (MRT) of myricetin following oral administration (50 mg/kg) were 2.78 ± 0.81 h and 7.88 ± 1.80 h, respectively,

Dang Y et al. Myricetin Pharmacokinetics in Rats … Drug Res 2014; 64: 516–522

3.93 ± 0.53 878.37 ± 25.33 3215.80 ± 78.97 –

RSD ( %)

mean ± SD, %) 89.45 ± 3.58 107.42 ± 5.66 99.38 ± 2.59 93.71 ± 3.99

4.00 5.27 2.61 4.26

which were in sharp contrast to the data (978.8 ± 379.3 min and 152.0 ± 0.9 min for t1/2 and MRT, respectively) obtained after oral administration of Ampelopsis grossedentata decoction reported by Zhang et al. [21]. This suggested that the pharmacokinetic properties of myricetin were altered when it was administered

Original Article 521

Spiked in plasma

Found in plasma

RSD

Deviation

(storage condition)

(ng/mL)

(mean ± SD, ng/mL)

( %)

( %)

Short-term stability (24 h at 4 °C)

4 800 3200 4 800 3 200 4 800 3 200 4 800 3 200

1.98 1.93 2.66 4.34 6.87 2.90 5.19 3.16 2.10 1.36 1.20 2.94

− 11.75 10.37 − 0.37 − 2.00 − 6.22 − 10.39 − 13.25 10.30 0.75 − 8.25 11.51 − 5.15

Long-term stability (30 days at − 80 °C) Freeze-thaw stability (after 3rd cycle at − 80 °C) Post-preparation stability (24 h at 4 °C)

Parameters

Administration Routes Oral (50 mg/kg)

Cmax (ng/mL) Tmax (h) t1/2 (h) AUC0-t (ng h/mL) AUC0-∞ (ng h/mL) MRT (h) CL (L/h/kg) Vd (L/kg)

3.53 ± 0.07 882.93 ± 17.04 3 188.15 ± 84.68 3.92 ± 0.17 750.28 ± 51.58 2 867.66 ± 83.21 3.47 ± 0.18 882.36 ± 27.85 3 223.92 ± 67.64 3.67 ± 0.05 892.07 ± 10.67 3 035.25 ± 89.20

1 488.75 ± 200.78 6.4 ± 0.89 2.78 ± 0.81 13 722.05 ± 5574.36 13 731.13 ± 5574.58 7.88 ± 1.80 – –

Oral (100 mg/kg)

Intravenous (0.5 mg/kg)

2 611.76 ± 1019.58 5.2 ± 3.03 3.79 ± 0.75 27 794.42 ± 10722.25 28 081.62 ± 10982.45 8.87 ± 1.42 – –

2 232.16 ± 856.36 0 (pre-dose) 6.99 ± 1.94 1 427.06 ± 157.49 1 452.85 ± 170.30 6.64 ± 1.40 0.35 ± 0.05 4.12 ± 1.52

Table 2 Stability of myricetin in rat plasma (n = 3).

Table 3 The main pharmacokinetic parameters of myricetin after oral (50 mg/kg and 100 mg/ kg) and i. v. (0.5 mg/kg) administrations to 6 rats (mean ± SD, n = 6).

*p < 0.05 as compared to low dose group of oral administration (50 mg/kg)

as a single compound compared to that in complex herb decoction. The reason is unclear and worth to be further investigated.

Conclusion



A simple, rapid and specific UPLC-MS/MS method has been established to investigate pharmacokinetics of myricetin in rats. The method resulted in high sensitivity with a low quantitation limit of 2 ng/mL, wide linearity range (2–4 000 ng/mL), specificity and no interferences from endogenous substances. The present method was then successfully applied to a pharmacokinetic study of myricetin following intravenous and oral administrations to rats. As it is well known, the therapeutic effects of APIs are closely related to their oral bioavailability. Hence, this research indicated that the poor oral absorption of myricetin should be considered when development of myricetin delivery systems.

Acknowledgements



Fig. 4 Mean concentration-time profiles of myricetin in rat plasma after a an intravenous injection at a dose of 0.5 mg/kg and b an oral administration at a dose of 50 mg/kg and 100 mg/kg. Each point represents mean ± SD (n = 6).

This study was sponsored by the Nano-specific Project of Shanghai Science and Technology Commission (12nm0502400), Shanghai Rising-Star Program (12QB1405100), Innovation Program of Shanghai Municipal Education Commission (12YZ061) and Shanghai Natural Science Foundation (12ZR1431300).

Conflict of Interest



The authors declare no conflict of interest.

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Stability experiment

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Quantitative determination of myricetin in rat plasma by ultra performance liquid chromatography tandem mass spectrometry and its absolute bioavailability.

Myricetin is a widely distributed bioactive flavonoid with scientific interest attributed to its anti-oxidant, antitumor, and anti-inflammatory proper...
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