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Preclinical pharmacokinetics of M10 after intragastrical administration of M10-H and M10-Na in Wistar rats
Journal Pre-proofs Preclinical pharmacokinetics of M10 after intragastrical administration of M10-H and M10-Na in Wistar rats Jiarui Gao, Guifang Dou, Xiaoxia Zhu, Hui Gan, Ruolan Gu, Zhuona Wu, Taoyun Liu, Suxiang Feng, Zhiyun Meng PII: DOI: Reference:
S1570-0232(19)31087-6 https://doi.org/10.1016/j.jchromb.2019.121905 CHROMB 121905
To appear in:
Journal of Chromatography B
Received Date: Revised Date: Accepted Date:
18 July 2019 22 November 2019 23 November 2019
Please cite this article as: J. Gao, G. Dou, X. Zhu, H. Gan, R. Gu, Z. Wu, T. Liu, S. Feng, Z. Meng, Preclinical pharmacokinetics of M10 after intragastrical administration of M10-H and M10-Na in Wistar rats, Journal of Chromatography B (2019), doi: https://doi.org/10.1016/j.jchromb.2019.121905
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© 2019 Published by Elsevier B.V.
Preclinical pharmacokinetics of M10 after intragastrical administration of M10-H and M10-Na in Wistar rats Jiarui Gao
a, b
, Guifang Dou b, Xiaoxia Zhu b, Hui Gan b, Ruolan Gu b, Zhuona Wu b,
Taoyun Liu b, Suxiang Feng a, *, Zhiyun Meng b, * a
Department of Pharmaceutical Analysis, Henan University of Chinese Medicine,
Zhengzhou 450046, China b
Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine,
Beijing 100850, China Jiarui Gao
E-Mail: [email protected] TEL: 010-66931993
Address: No. 156 Jinshui Road, Jinshui District, Zhengzhou, China 450046 No. 27 Taiping Road, Haidian District, Beijing, China 100850
* Correspondence authors: Corresponding author: Zhiyun Meng E-Mail: [email protected] TEL: 010-66931993 Address: No. 27 Taiping Road, Haidian District, Beijing, China 100850 Suxiang Feng E-Mail: [email protected] TEL: 0371-65962746 Address: No. 156 Jinshui Road, Jinshui District, Zhengzhou, China 450046
Abstract: As a myricetin derivative, M10 is a potent agent of anti-chronic colonic inflammation.
It
has
better
activity
than
myricetin
in
preventing
azoxymethane/dextran sulfate sodium - induced ulcerative colitis. Here, we introduce a
sensitive
quantification
method
based
on
ultra
performance
liquid
chromatography-tandem mass spectrometry for the determination of M10-H and M10-Na in Wistar rat plasma. Samples were treated with L - ascorbic acid and phosphate buffer solution to maintain stability and with acetonitrile to remove the proteins in the plasma. The supernatant was separated with BEH C18 column and eluted with ultrapure water and acetonitrile both containing 0.1% formic acid. The detection was performed by a triple quadrupole mass spectrometer with positive electrospray ionization mode in multiple reactive monitoring. This method was validated for the carryover effect, selectivity, accuracy, precision, matrix effect, stability, and recovery. A linear correlation was established between concentration and response by the calibration curves over 10–2000 ng·mL-1 (r > 0.99). This method was applied to a pharmacokinetic study of intragastrical administration of M10-H and M10-Na in Wistar rats. In addition, the relative bioavailability of M10-H to M10-Na in Wistar rats was 60±19%, calculated by the ratio of area under concentration (AUC) of M10-H to M10-Na after intragastrical administration of a single dose (100 mg·kg-1 for M10-H and M10-Na, respectively) in Wistar rats. Key words: M10; pharmacokinetics; UPLC-MS/MS; myricetin derivates 1. Introduction Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a complex, chronic, and relapsing disease characterized by symptoms of diarrhea, bloody stools and abdominal pain [1]. With the aggravation of colonic inflammation and prolongation disease, the risk of colitis-associated colorectal cancer (CAC) increased [2]. So, drugs with good efficacy and low toxicity for the treatment of colonic chronic inflammation and chemoprevention of colorectal tumorigenesis are in high demand [19]. Myricetin is a natural polyphenolic flavonoid [3-5] with biological activities [6-8], such as anti-oxidant [9-12] and anti-inflammatory [13-14]. Reportedly, myricetin exerts anti-inflammatory activity by reducing the expression levels of IL-6, TNF-α, IL-1β [15-18], and inhibits the intestinal tumorigenesis through inhibition of the Wnt/β-catenin pathway in the adenomatous polyposis coli multiple intestinal
neoplasia (APCMin/+) mice [19-22]. However, myricetin shows several deficiencies, such as complex isolation procedures, poor water solubility (< 100 ng·mL-1) and low stability when administrated orally [19-22]. Thus, the synthesis of derivatives of myricetin is needed. As shown in Fig. 1, based on myricetin, M10-H was designed by adding a hydrophilic glycosylation group, and M10-Na was formed as a sodium salt derivative with excellent water-solubility (> 0.1g·mL-1) and good safety which median lethal dose (LD50) is > 5g·kg-1. Moreover, as myricetin derivatives, M10-H and M10-Na inhibit the intestinal tumorigenesis via strong inhibition of the NF-B/IL-6/STAT3 pathway in a mice model with ulcerative colitis induced by azoxymethane (AOM)/dextran sulfate sodium (DSS). Both of them exhibited higher efficacy of prevention in the AOM/DSS - induced UC than myricetin and mesalazine, a clinically used drug for the treatment of ulcerative colitis [19-22]. M10 appears to have a good safety profile as an oral chemoprevention agent, and before entering clinical trials, in vivo preclinical pharmacokinetics is needed. The radio of relative bioavailability of M10-H to M10-Na is not only a critical parameter to estimate the differences between M10-H and M10-Na exposure after oral administration, but also an important factor to assess the differences of their pharmacokinetics characteristic, such as absorption, metabolism and excretion, all of which were taken into considerations about which one is more suitable for the further researches. Several methods have been reported for the detection of flavone and flavone glycoside, such as high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection [24] and liquid chromatography-tandem mass spectrometry (LC-MS/MS) [25]. To our knowledge, there is still no method reported for the determination of M10 in biological matrices. For the development of M10, it is essential to establish an analytical method sensitive enough to detect M10 in biological fluids. A structural analog, kaempferol, was used as internal standard (IS). Fig.1 shows the chemical structures of myricetin, M10-H, M10-Na and IS, respectively. In this study, we developed a sensitive UPLC–MS/MS method for the measurement of M10-H and M10-Na in rat plasma. This method was applied to study the pharmacokinetics of M10-H and M10-Na after oral administration in Wistar rats. This work describes the details of method establishment, validations and
pharmacokinetic profile of M10 in rat plasma. 2. Materials and methods 2.1 Reagents and instruments HPLC-grade acetonitrile, HPLC-grade methanol, and HPLC-grade formic acid (FA) were purchased from Fisher, USA. L - Ascorbic acid (Batch No. 2081206) and sodium carboxymethylcellulose (CMC-Na) (Batch No. F20110915) were purchased from Sinopharm Chemical Reagent Company (Beijing, China). Deionized water was produced by a Milli-Q Reagent Water System (Millipore, Bedford, MA, USA). The UPLC–MS/MS system consisted of a Waters ACQUITY I-Class UPLC system connected to a Waters (Milford, MA, USA) Xevo TQ-S triple quadrupole mass spectrometer with flow-through-needle sample manager, a cooling autosampler, a column oven with temperature control, a degasser, and a binary pump. The derivatives of myricetin (M10-H and M10-Na, purity ≥ 98%) were provided by Dr Li in Marine Biomedical Research Institute of Qingdao. Myricetin (purity ≥ 98%) was purchased from Sigma–Aldrich (St. Louis, MO). 2.2 Chromatography All samples were analyzed on UPLC® I-Class and Xevo® TQS combined system. Separations were carried out with a mobile phase of water (A) and acetonitrile (B), both containing 0.1% formic acid, at the flow rate of 0.5 mL·min-1 using a Waters ACQUITY UPLC® BEH C18 (2.1 mm × 50 mm, 1.7 μm) column. The over run time was 4 min and the gradient elution was adopted as follows: 0–0.5 min, 10% B; 0.5–2 min, 10–90% B; 2–3.5 min, 90% B; 3.5–3.51 min, 90-10% B; 3.51–4 min, 10% B. The Sample Manager-Flow through Needle (SM-FTN) was cleaned after each sample introduction by washing with a methanol/water mixture (4/1, v/v) and a seal wash with water/acetonitrile (9/1, v/v). The volume of injection was 1 μL, the sample temperature was maintained at 10 °C, and the column temperature was maintained at 45 °C. 2.3 Mass spectrometry The detection was performed by a triple quadrupole mass spectrometer with
electrospray ionization (ESI). The mass spectrometer detector was set as multiple reactive monitoring (MRM) in the positive mode. The MRM transitions and correlative parameters were as follows: capillary voltage: 3 kV; cone gas flow: 150 L·h-1; desolvation gas flow: 650 L·h-1; source temperature: 150 °C; desolvation temperature: 350 °C. The analytes were detected at m/z 319.0/153.0 and 287.0/153.0 with the collision energy 28 V and 30 V for M10 and IS (kaempferol), respectively. 2.4 Working and stock solutions The stock solutions of M10-H and M10-Na were prepared in 60% acetonitrile to the concentration of 1.0 mg·mL-1 and maintained at -20 °C. Then working solutions and quality control solutions were obtained by serial dilutions of the stock solutions with 60% acetonitrile. The internal standard solution of kaempferol was prepared in acetonitrile at a concentration of 10 ng·mL-1 and stored at -20 °C. To eliminate systematic error in samples quantification, kaempferol was used as an internal standard (IS) because its functional groups were similar to those of myricetin and M10 and its non-plasma source character. 2.5 Calibration standards and quality control samples preparations In order to prepare the plasma matrix samples with serial concentrations, 10 μL of a 10-fold concentration of the calibrators was added to 90 μL of blank plasma from Wistar rat. Then, the mixture was added to 200 μL of pH 4.0 phosphate-buffered saline (PBS) and 10 μL of 5% L - ascorbic acid dissolved in water. Then the mixture was precipitated with 200 μL of acetonitrile containing IS (10 ng·mL-1), vortex-mixed for 3 min and centrifuged at 12000 rpm for 10 min at 4 °C [26]. Then, 200 μL of supernatant was pipetted out into the autosampler vials and analyzed by UPLC–MS/MS with 1 μL injection volume. Lower limit of quantification (LLOQ) was defined as the lowest quantitative concentration. According to Chinese pharmacopeia, the relative standard deviation [RSD (%)] and the relative error [RE (%)] of LLOQ were set less than 20%. The range of the calibration of M10-H and M10-Na was in the linear range from 10–2000 ng·mL-1 (10, 20, 50, 100, 200, 500, 1000, and 2000 ng·mL-1). Four levels of quality control (QC) samples including LLOQ, low QC (25 ng·mL-1), mid QC (250 ng·mL-1),
and high QC (1600 ng·mL-1) were prepared by spiking the corresponding level of QC working solutions into blank plasma. Standard solutions and six replicates for each QC samples were prepared for every validation assay. 2.6 Sample pre-treatment 100 μL of plasma samples, 200 μL of pH 4.0 phosphate buffered saline (PBS) and 10 μL of 5% L - ascorbic acid were added individually. The mixture was vortex-mixed for 1 min before 200 μL of acetonitrile containing IS (10 ng·mL-1) was added. Then, samples were vortex-mixed for an additional 2 min and centrifuged at a speed of 12000×g for 10 min at 4 °C to remove the plasma proteins. Finally, 200 μL of supernatant were collected for UPLC-MS/MS analysis. 2.7 Analytical method validation According to the recently published guidelines of the National Medical Products Administration (NMPA) for bioanalytical methods validation [23], the method was validated in terms of selectivity, carryover effect, linearity, sensitivity, accuracy, precision, recovery, matrix effects and stability, respectively. 2.7.1 Selectivity and carryover effect The selectivity of the M10-H and M10-Na was determined using blank plasma from six different Wistar rats. 100 μL of blank plasma from six different Wistar rats was added to 200 μL of phosphate buffered saline (PBS) and 10 μL of 5% L - ascorbic acid then precipitation with 200 μL of acetonitrile and centrifugation, respectively. Then, 200 μL of supernatant was pipetted out into the autosampler vials and 1 μL of supernatant was injected into the analytical column for analysis immediately to ensure that there is no endogenous compound with M10. To assess the carryover effect, a blank plasma sample was injected after the injection of the upper limit of quantification (ULOQ) standard, and the peak areas at the same retention time with M10 should be < 20% of the peak areas of the LLOQ sample. 2.7.2 Linearity and sensitivity
Linearity was assessed by peak areas of the known amounts of eight-point work solutions, which were added into the blank plasma to achieve calibration standards. The calibration curves range from 10 to 2000 ng·mL-1 including a zero-level spiked into blank rat plasma samples. All the standard solutions were corrected with the internal standard (kaempferol) and calculated using weighted least squares linear regression. Three quality control samples at 25 ng·mL-1 (low), 250 ng·mL-1 (medium) and 1600 ng·mL-1 (high) were prepared independently of another M10 stock solution used for adjusting the calibration curves. Each batch of the standard curve solutions was freshly prepared in every validation assay and sample test. The sensitivity of the method was assessed by the peak areas from six parallel LLOQ samples in rat plasma, and the deviation should not exceed 20%. 2.7.3 Accuracy and precision Accuracy and precision of the proposed assay were determined by LLOQ and QC samples at low, medium, and high concentrations in intra- and inter-day batch. The intra-day accuracy and precision need be investigated by three batches for a minimum of 2 days. Every single assay needs six replicate analyses of LLOQ and QC samples. The accuracy [expressed as relative error, RE (%)] and precision [expressed as relative standard deviation, RSD (%)] for samples at LLOQ should not exceed 20% while for QC samples, should not exceed 15%. 2.7.4 Stability The stability was assessed by analyzing the QC samples at low and high levels with six replicates under different storage conditions. For instance, the stability of M10-H and M10-Na in plasma was analyzed the samples in plasma at room temperature for 6 h. The stability of freeze-thaw cycles was investigated by analyzing samples in plasma after three freeze (-80 °C)-thaw cycles. The long-term stability was determined by evaluating the QC samples in blank plasma at −80 °C for 60 days. The short-term stability was examined by analyzing the processed M10-H and M10-Na at 4 °C for 24 h. To test the stability of samples into autosampler, the samples were processed and maintained into the autosampler for 24 h. In addition, stock solution stability was also investigated after storage at -20 ℃ for 2 weeks. The RE (%) for any stability test condition was compared to the standard curve on the detection day wand as required to be within ±15%.
2.7.5 Matrix effects and recovery The matrix effect of M10-H and M10-Na was evaluated by comparing the difference of response between samples of 60% acetonitrile and plasma from six individual rats with the addition of low and high QC samples of M10-H and M10-Na respectively. The matrix effects were calculated by the ratio of peak areas of M10-H and M10-Na in the plasma and 60% acetonitrile at low and high QC concentrations, respectively. The RSD (%) of the ratio of the peak areas ratio at each QC sample should be less than 15%. The recovery was examined by comparing the difference of peak areas between samples of blank plasma and the supernatants of the precipitated blank plasma by acetonitrile with the addition of QC samples at low and high levels with six replicates. The recovery was calculated by the ratio of peak areas of M10-H and M10-Na in the supernatants of the processed plasma by acetonitrile and blank plasma at low and high QC concentrations, respectively. The RSD (%) of the peak area ratio should be < 15%. 2.8 Pharmacokinetic study Sixteen Wistar rats (eight males and eight females, weight 180±20 g) were used in this pharmaceutical experiment. The animals were maintained under standard laboratory conditions (relative humidity, 40–70%; temperature, 20–26 °C; 12/12 h light/darkness) and fed separately. All animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (National Research Council of the USA, 1996), and approved by the Association for Assessment and Accreditation of Laboratory Animal Care. The license number of experimental animal production was SCXK (Jing) 2016-0006. The sixteen animals were randomly divided into two groups (M10-H group and M10-Na group, each group comprised of half males and females) and jejunitis overnight. Then, a single dose of 100 mg·kg-1 of M10-H or M10-Na was administered to each rat intragastrically. An equivalent of 0.15 mL blood samples was withdrawn from the rats’ orbital venous plexus at the following time points after dosing: 0.083, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 24 h (i.g.). The blood samples were collected into sodium citrate tubes and cleared by centrifugation at 8000 ×g for 10 min at 4 °C and
stored at −80 °C until further analysis. 2.9 Data analysis Microsoft Office Excel was used for data calculation. Origin 8.0 was used to draw graphs. ChemDraw Ultra 8.0 was used to generate the chemical structures of the M10, myricetin and kaempferol. And the pharmacokinetic parameters were calculated by using WinNonlin 6.3 software (Pharsight Corp., MO, USA). Concentration–time data that are below the lower limit of quantification were treated as zero in summary statistics. The following variables were calculated for both M10-H and M10-Na: time to observed maximum plasma concentration after drug administration (Tmax) and observed maximum plasma concentration (Cmax). Other major pharmacokinetic parameters including the terminal elimination half-life (T1/2), the plasma exposure [area under the plasma concentration–time curve from time 0 to time of last measurable concentration (AUC0-last)], mean residence time (MRT), and clearance (Cl) were all calculated by non-compartmental method. 3. Results 3.1 Method development As to ionization mode, the response of M10 in positive mode was 20 times higher than that in negative mode, so the positive-ion mode was used. When optimizing the mass spectrometry conditions, 2mL of 500 ng·mL-1 M10-H, M10-Na and kaempferol were separately infused into the mass spectrometer in order to obtain a Q1 scan of the parent compound. The mass spectrometric parameters, such as the desolvation gas flow, source temperature, desolvation temperature, collision energy and cone energy, were adjusted to maximal the intensity of the product ions. The analytes were easily cleaved and then generate positive product ions, M10-H mostly consisted of [M+Na]+ ions and M10-Na mostly consisted of [M+H]+ ions, so the transitions for M10-H and M10-Na at the early stage of the method development were both at m/z 665.1 ＞ 347.0. However, in-source fragmentation into aglycones （myricetin）of M10-H and M10-Na were found after injection into UPLC-MS/MS.
So, we finally chose transitions at m/z 319.0 ＞ 153.0 to identify and quantify M10, which showed a stronger response and better sensitivity than transitions at m/z 665.1 ＞ 347.0. 3.2 Selectivity and carryover effect The typical chromatograms were obtained from blank plasma samples, blank plasma after injected ULOQ samples, spiked plasma samples with analytes at the LLOQ (Fig. 2A), spiked plasma samples with analytes at the ULOQ, internal standard (Fig. 2B) and plasma samples after dosing of M10-H and M10-Na are presented in Fig. 2. The method of selectivity was determined by comparing the blank plasma from six individual rats with M10-H and M10-Na in the plasma. The results displayed a lack of interfering peaks with M10. The carryover effect of the method was evaluated by comparing the blank plasma after injecting the ULOQ standard with M10-H and M10-Na in the rat plasma at LLOQ concentration. The peak areas of the blank plasma samples should not exceed 20% of those measured in the LLOQ sample. Fig. 2 demonstrated that there is no carryover peak at the same retention times. 3.3 Linearity and sensitivity Sensitivity was assessed based on the six spiked samples at LLOQ concentration. Linearity was assessed by peak areas of eight-point on calibration curves after adjusting with the internal standard (kaempferol) and calculated by the weighted least squares linear regression. Table 1 demonstrated that the RSD (%) at LLOQs was ≤ 12.3% (i.e., < 20%), conforming to the NMPA standard. The typical calibration equations were as follows: M10-H, y = 0.00599 x + 0.0126 (r2 = 0.995); M10-Na, y = 0.00586 x + 0.0162 (r2 = 0.998). The LLOQ was 10 ng·mL-1, and the calibration standards of M10-H and M10-Na ranged from 10–2000 ng·mL-1, with optimal linearity over the concentration range. 3.4 Accuracy and precision The data of precision and accuracy are summarized in Table 2. The intraday precision [relative standard deviation, RSD (%)] of the M10-H at LLOQs concentration was 17.1%. The other intra- and inter-day precision---- RSD (%) of the M10-H and M10-Na was < 12.3%. Moreover, the intra- and inter-day accuracy ---RE (%) of the M10-H and M10-Na ranged from 0.2–10.9%, demonstrating an optimal
accuracy and reproducibility of the method. 3.5 Stability The data of stability are displayed in Table 3. The results showed that the RE (%) at different conditions varied from -8.5 to 9.0%, i.e., within ±15%. It was illustrated that the M10-H and M10-Na samples were stable at different conditions, such as in the blank plasma at room temperature for 6 h, after three freeze-thaw cycles, and at −80 °C for 60 days. Next, the processed samples were placed at 4 °C for 24 h or loaded onto the auto-sampler for 24 h. Furthermore, the stock solutions of M10-H and M10-Na were stable at -20 °C for 2 weeks. 3.6 Matrix effects and recovery The data of matrix effect and recovery are presented in Table 4. The matrix effects M10-H and M10-Na ranged from 154.1–162.8% and 148.4–180.8%, respectively, and the overall recoveries of M10-H and M10-Na ranged from 93.8–103.2% and 96.8–107.3%, respectively. The RSD (%) values of matrix effect and recovery at two QC concentrations were ≤ 7.3%, which fulfilled the criteria. These findings demonstrated an enhanced but stable and reproducible effect from the plasma matrix in this approach. 3.7 Pharmacokinetic assessment When applied to the preclinical pharmacokinetic study, the method was proved suitable for quantitating the M10 in Wistar rat plasma with an oral dose of 100mg·kg-1 of M10. According to the measured plasma concentrations, in the current comparative pharmacokinetic study of intragastrical administration of M10-H and M10-Na (100 mg·kg-1), the mean plasma concentration-time profiles of M10-H and M10-Na are illustrated in Fig. 3 and the main pharmacokinetic parameters were obtained. The results are demonstrated in Table 5 as follows: for M10-H and M10-Na, respectively, maximum concentration (Cmax), 23.2±9.9 and 129.9±52.7 ng·mL-1; time to reach maximum concentration (Tmax), 4.4±2.3 and 0.2±0.1 h; area under the curve from time 0 to 24 h (AUC0–24), 93.2±29.9 and 155.6±147.3 h·ng·mL-1; elimination half-life (T1/2), 5.4 and 1.8±0.8 h. In addition, the relative oral bioavailability of the M10-H and M10-Na could be calculated as follows: [AUClast (M10-H) / AUClast (M10-Na)] x 100% and the relative bioavailability of the M10-H to M10-Na in the Wistar rat
samples was 60±19% [27].
Relative bioavailability (%) = [AUClast (M10-H) / AUClast (M10-Na)] x 100%
4. Discussion As glycosides derivatized from flavonoids, M10-H and M10-Na were easily proposed in-source fragmentation into aglycones（myricetin）after injection into UPLC-MS/MS. However, the degree of the crack is unstable, and was affected by pH, exhibiting low response and poor linearity during the development of the method. This problem has been solved after the addition of ascorbic acid and PBS (pH 4.0). The results of the pharmacokinetic study showed that the maximum concentration (Cmax) of M10-H and M10-Na were 23.2±9.9 ng·mL-1 and 129.9±52.7 ng·mL-1 respectively, and time to reach maximum concentration (Tmax) of them were 4 h and 15 min, exhibiting the faster and better absorption of M10-Na than M10-H. The elimination half-life (T1/2) of M10-H and M10-Na were 5.4 and 1.8±0.8 h, showing that the elimination of M10-Na was faster than M10-H. The area under the plasma concentration-time curve (AUC) of M10-H and M10-Na were 93.2±29.9 and 155.6±147.3 h·ng·mL-1 respectively, suggesting that M10-Na showed better absorption in vivo than M10-H. The volume of distribution (V/F) of M10-H and M10-Na were 3483348 and 1474438±602798 L·kg-1, demonstrating that M10-H and M10-Na were distributed into a wide range of organs and tissues. The results of the pharmacokinetics study illustrating that M10-Na can be quickly absorbed and eliminate, and M10-H is hardly absorbed relatively. And the relative bioavailability of the M10-H to M10-Na in the Wistar rat samples was 60±19%. We presumed the significant differences in the pharmacokinetics of M10-H and M10-Na were resulted from the better water-solubility of M10-Na than M10-H. 5. Conclusion As a myricetin derivative, M10 is a new compound and potent agent of anti-chronic colonic inflammation. The comparative studies showed that the efficacy of M10 was higher than myricetin and mesalazine, a clinical used drug for the treatment of ulcerative colitis in the DSS-induced chronic colonic inflammation in
mice [20]. Herein, we have developed and validated a sensitively analytical method for M10-H and M10-Na. In addition, this simple, sensitive, reproducible and robust method has been successfully employed to the pharmacokinetics study of M10-H and M10-Na in Wistar rats after a single oral administration, and their pharmacokinetics was achieved. The pharmacokinetics data could provide useful information for the further preclinical study. Considering the excellent performance of this method, it will be utilized for future preclinical developments of M10. Acknowledgments We thank Professor Wenbao Li from Ocean University of China for providing M10-H and M10-Na. And we appreciate the reviewers and editors for helpful suggestions. Reference [1] R.J. Xavier, D.K. Podolsky, Unravelling the pathogenesis of inflammatory bowel disease, Nature 448 (2007): 427-434. [2] Y. Yamada, H. Mori, Multistep carcinogenesis of the colon in Apc (Min/+) mouse, Cancer Science. 98 (2007): 6-10. [3] Bingxu Huang, Juxiong Liu, Dongxu Ma, Guangxin Chen, Wei Wang, Shoupeng Fu. Myricetin prevents dopaminergic neurons from undergoing neuroin flammation-mediated degeneration in a lipopolysaccharide-induced Parkinson’s disease model, Journal of Functional Foods, 45(2018): 452–461. [4] Eshan Khan, Arpita Tawani, Subodh Kumar Mishra, Arun Kumar Verma, Arun Upadhyay, et al., Myricetin Reduces Toxic Level of CAG Repeats RNA in Huntington’s Disease (HD) and Spino Cerebellar Ataxia (SCAs), ACS chemical biology 13 (2018): 180−188. [5] Hua-Fu Zhao, Gang Wang, Chang-Peng Wu, Xiu-Ming Zhou, Jing Wang, et al., A Multi-targeted Natural
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[11] Hyang-Yu Kima, Sun-Young Parka, Se-Young Choung, Enhancing effects of myricetin on the osteogenic differentiation of human periodontal ligament stem cells via BMP-2/Smad and ERK/JNK/p38 mitogen-activated protein kinase signaling pathway, European Journal of Pharmacology, 834(2018): 84–91. [12] Yingqian Ci, Yubo Zhang, Yanjie Liu2, Shuai Lu, et al., Myricetin suppresses breast cancer metastasis through down-regulating the activity of matrix metalloproteinase (MMP)-2/9, Phytotherapy Research, 32(2018):1373–1381. [13] Hahyun Park, Sunwoo Park, Fuller W. Bazer, Whasun Lim3, Gwonhwa Song Myricetin treatment induces apoptosis in canine osteosarcoma cells by inducing DNA fragmentation, disrupting redox homeostasis, and mediating loss of mitochondrial membrane potential, Journal of Cellular Physiology, 233(2018):7457–7466. [14] Allison Knicklea, Wasundara Fernandob, Anna L. Greenshieldsb, H.P. Vasantha Rupasingheb,c, David W. Hoskin, Myricetin-induced apoptosis of triple-negative breast cancer cells is mediated by the iron-dependent generation of reactive oxygen species from hydrogen peroxide, Food and Chemical Toxicology, 118(2018): 154–167. [15] Takahiro Yamashiro, Tomoya Yasujima, Kinya Ohta, Katsuhisa Inoue, Hiroaki Yuasa, Specific inhibitory effects of myricetin on human proton-coupled folate transporter: Comparison with its effects on rat proton-coupled folate transporter and human riboflavin transporter 3, Drug Metabolism and Pharmacokinetics, 32(2017): 311-314. [16] Anjun Yao, Yingzhuo Shen, Zhuangwei Zhang, Zuquan Zou, Anshi Wang, Sulforaphane and myricetin act synergistically to induce apoptosis in 3T3‑ L1 adipocytes, Molecular Medicine Reports, 17(2018): 2945-2951. [17] Chen Ye, Chao Zhang, Hai Huang, Bo Yanga, Guangan Xiao, et al., The Natural Compound Myricetin Effectively Represses the Malignant Progression of Prostate Cancer by Inhibiting PIM1 and Disrupting the PIM1/CXCR4 Interaction, Cell Physiology and Biochemistry; 48(2018):1230-1244. [18] Nan Zhang, Hong Feng4, Hai‐ Han Liao, Si Chen, et al., Myricetin attenuated LPS induced cardiac injury in vivo and in vitro, Phytotherapy Research, 32(2018):459–470. [19] Sifeng Zhu, Chong Yang, Liang Zhang, Shixiao Wang, Mingxu Ma, et al., Development of M10, myricetin-3-O-b-D-lactose sodium salt, a derivative of myricetin as a potent agent of anti-chronic colonic inflammation, European Journal of Medicinal Chemistry, 174(2019): 9-15. [20] Feng Wang, Zhi-Yu Song, Xian-Jun Qu, Feng Li, Liang Zhang, et al., M10, a novel derivative of Myricetin, prevents ulcerative colitis and colorectal tumor through attenuating robust endoplasmic reticulum stress, Carcinogenesis, 39(2018): 889–899. [21] Jeong Hyun Lee, Yong Jun Choi, See-Hyoung Park, Myeong Jin Nam, Potential role of nucleoside diphosphate kinase in myricetin-induced selective apoptosis in colon cancer HCT-15 cells, Food and Chemical Toxicology, 116 (2018): 315–322. [22] Yuchao Sun, Mengqiao Lian, Yuan Lin, Bin Xu, Role of p-MKK7 in myricetin-induced protection against intestinal ischemia/reperfusion injury, Pharmacological Research, 129(2018): 432–442. [23] Guidance for Industry: Bioanalytical Method Validation. Pharmacopoeia of People’S Republic of China, National Pharmacopoeia Committee [M].Part 4, Beijing, 2015. [24] Mai Gamal Elhennawy, Hai-Shu Lin, Determination of Tangeretin in Rat Plasma: Assessment of Its Clearance and Absolute Oral Bioavailability, Pharmaceutics, 10(2018): 1-10. [25] Shunjun Xu, Jiejing Yu, Jingjing Zhan, Pharmacokinetics, Tissue Distribution, and Metabolism Study of Icariin in Rat, BioMed Research International 11(2017): 1-17.
[26] Dong Xiang, Chen-guang Wang, Wen-qing Wang, Chun-yang Shi, Wei Xiong, et al., Gastrointestinal stability of dihydromyricetin, myricetin, and myricitrin: an in vitro investigation, International Journal of Food Sciences and Nutrition, (2017): 1-8. [27] Jennifer Moczarnik, DVM, Darren J. Berger, DVM, DACVD, et al., Relative Oral Bioavailability of Two Amoxicillin–Clavulanic Acid Formulations in Healthy Dogs: A Pilot Study, Journal of the American Animal Hospital Association, 55 (2019): 14-22.
Table 1 The typical standard curve of the M10-H and M10-Na in Wistar plasma
M10-H
M10-Na
Batch
Intercept
Slope
1 2 3 1 2 3
0.0126 0.0154 0.0196 0.0162 0.0176 0.0287
0.00599 0.0252 0.0169 0.00586 0.0106 0.0139
correlation coefficient (R2) 0.9951 0.9981 0.9960 0.9979 0.9970 0.9931
Table 2 Precision and accuracy for the M10-H and M10-Na in Wistar plasma Spiked
Overall Measured Concentration (ng·mL-1) (Mean ± SD, n = 6)
Concentration (ng·mL-1)
Intra-Day
Inter-Day
RSD (%)
RSD (%)
RE
Mean Batch 1
Batch 2
Batch 3
(ng·mL-1)
(%)
LLOQ
10
11.9±2.0
11.1±0.7
10.3±1.1
11.1
17.0
12.3
10.9
LQC
25
26.4±1.8
25.7±2.1
25.6±1.3
25.9
4.5
6.9
3.5
MQC
250
251.3±23.6
253.0±16.7
249.2±8.3
251.2
1.9
6.9
0.5
HQC
1600
1609.1±47.3
1740.5±90.6
1667.3±64.8
1672.3
9.6
4.2
4.5
LLOQ
10
10.6±0.9
10.8±0.8
10.5±0.7
10.6
2.7
7.6
6.4
LQC
25
25.5±2.3
25.6±2.5
25.5±2.6
25.5
0.7
9.7
2.1
MQC
250
246.3±15.0
250.1±9.2
254.7±10.3
250.4
4.1
4.7
0.2
HQC
1600
1524.7±53.0
1647.1±73.5
1641.3±114.1
1613.3
11.9
5.2
0.8
M10-H
M10-Na
Table 3 Stability data of the M10-H and M10-Na under different storage situation M10-H
M10-Na
Concentration Storage Conditions
（ng·mL-1）
Mean ± SD
RSD
Mean ± SD
RSD
(ng·mL-1)
(%)
RE (%) (ng·mL-1)
(%)
RE (%)
Room temperature
25
24.6±1.1
4.3
-1.8
24.0±1.8
7.5
-3.8
for 6 h
1600
1682.2±78.0
4.6
5.1
1652.9±143.5
8.7
3.3
Three freeze-thaw
25
26.4±1.4
5.4
5.7
26.6±1.9
7.3
6.2
cycles
1600
1669.9±44.1
2.6
4.4
1744.7±121.4
7.0
9.0
Long term for 60
25
28.1±1.0
3.5
12.6
24.9±1.6
6.2
-0.2
days (-80 °C)
1600
1605.1±42.2
2.6
0.3
1687.5±71.3
4.2
5.5
Processed sample
25
25.7±1.8
7.1
2.8
24.2±1.4
5.7
-3.3
for 24 h (-20 °C)
1600
1674.5±118.1
7.1
4.7
1560.8±112.4
7.2
-2.5
Autosampler
25
23.2±1.9
8.2
-7.1
22.9±1.4
6.2
-8.5
for 24 h
1600
1624.3±90.7
5.6
1.5
1479.8±99.0
6.7
-7.5
Stock solution for 2
25
25.8±0.9
3.6
3.0
23.4±2.2
11.7
-6.5
weeks (-20 °C)
1600
1614.7±65.8
4.1
0.9
1633.8±54.9
3.4
2.1
Table 4 Matrix effects and Recovery rates of the M10-H and M10-Na in Wistar rat plasma（n=6） concentration （ng·mL-1）
Matrix Effect (n = 6)
Recovery (n = 6)
Mean±SD（%）
RSD%
Mean±SD（%）
RSD%
25
162.8±4.4
2.7
93.8±6.8
7.3
1600
154.1±5.9
3.9
103.2±4.7
4.6
25
148.4±10.8
7.3
107.3±5.5
5.1
1600
180.8±8.3
4.6
96.8±3.9
4.0
M10-H
M10-Na
Table 5 Pharmacokinetic parameters of intragastrical administration the M10-H and M10-Na (100 mg·kg−1) (n = 8) Mean±SD Parameter(unit) M10-H
M10-Na
t1/2 (h)
5.4
1.8±0.8
Tmax (h)
4.4±2.3
0.2±0.1
23.2±9.9
129.9±52.7
AUC0-24h (h·ng·mL )
93.2±29.9
155.6±147.3
V (L·kg-1)
3483348
1474438±602798
CL (L·h ·kg )
445550
658926±302816
MRT (h)
4.0±1.1
1.1±0.4
Cmax (ng·mL-1) -1
-1
-1
Highlights
A sensitive UPLC-MS/MS method was successfully developed and validated for M10-H and M10-Na.
It was reported for the first time that M10-H and M10-Na is determined by UPLC-MS/MS.
This method was successfully applied for pharmacokinetics study of M10-H and M10-Na.
Figure legends Fig. 1 The chemical structure of the myricetin, M10-H, M10-Na and kaempferol.
Fig. 2A The typical chromatograms obtained from M10-H and M10-Na in Wistar rat blank plasma (A) Wistar rat blank plasma；(B) blank plasma after injected ULOQ of M10-H and M10-Na (C) 1. LLOQ of M10-H；2. LLOQ of M10-Na；
Fig. 2B The typical chromatograms obtained from M10-H and M10-Na in Wistar rat blank plasma (D) 1. ULOQ of M10-H；2 ULOQ of.M10-Na；(E) internal standard； (F) plasma samples after dosing of M10-H and M10-Na；
Fig. 3 The drug concentration-time curves of intragastrical administration of the M10-H and M10-Na (100 mg·kg−1) in Wistar plasma (Mean±SD, n = 8).
Conflicts of interest:
All authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare.
Author statement Jiarui Gao: Methodology, Software, Investigation, Formal analysis, Data Curation, Writing - Original Draft. Guifang Dou: Conceptualization, Funding acquisition. Xiaoxia Zhu: Resources, Validation. Hui Gan: Data Curation. Ruolan Gu: Writing - Review & Editing. Zhuona Wu: Visualization. Taoyun Liu: Supervision. Suxiang Feng: Project administration. Zhiyun Meng: Conceptualization, Funding acquisition.
Abstract: As a myricetin derivative, M10 is a potent agent of anti-chronic colonic inflammation.
It
has
better
activity
than
myricetin
in
preventing
azoxymethane/dextran sulfate sodium - induced ulcerative colitis. Here, we introduce a sensitive quantification method based on ultra performance liquid chromatography-tandem mass spectrometry for the determination of M10-H and M10-Na in Wistar rat plasma. Samples were treated with L - ascorbic acid and phosphate buffer solution to maintain stability and with acetonitrile to remove the proteins in the plasma. The supernatant was separated with BEH C18 column and eluted with ultrapure water and acetonitrile both containing 0.1% formic acid. The detection was performed by a triple quadrupole mass spectrometer with positive electrospray ionization mode in multiple reactive monitoring. This method was validated for the carryover effect, selectivity, accuracy, precision, matrix effect, stability, and recovery. A linear correlation was established between concentration and response by the calibration curves over 10–2000 ng·mL-1 (r > 0.99). This method was applied to a pharmacokinetic study of intragastrical administration of M10-H and M10-Na in Wistar rats. In addition, the relative bioavailability of M10-H to M10-Na in Wistar rats was 60±19%, calculated by the ratio of area under concentration (AUC) of M10-H to M10-Na after intragastrical administration of a single dose (100 mg·kg-1 for M10-H and M10-Na, respectively) in Wistar rats.
S1570-0232(19)31087-6 https://doi.org/10.1016/j.jchromb.2019.121905 CHROMB 121905
To appear in:
Journal of Chromatography B
Received Date: Revised Date: Accepted Date:
18 July 2019 22 November 2019 23 November 2019
Please cite this article as: J. Gao, G. Dou, X. Zhu, H. Gan, R. Gu, Z. Wu, T. Liu, S. Feng, Z. Meng, Preclinical pharmacokinetics of M10 after intragastrical administration of M10-H and M10-Na in Wistar rats, Journal of Chromatography B (2019), doi: https://doi.org/10.1016/j.jchromb.2019.121905
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© 2019 Published by Elsevier B.V.
Preclinical pharmacokinetics of M10 after intragastrical administration of M10-H and M10-Na in Wistar rats Jiarui Gao
a, b
, Guifang Dou b, Xiaoxia Zhu b, Hui Gan b, Ruolan Gu b, Zhuona Wu b,
Taoyun Liu b, Suxiang Feng a, *, Zhiyun Meng b, * a
Department of Pharmaceutical Analysis, Henan University of Chinese Medicine,
Zhengzhou 450046, China b
Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine,
Beijing 100850, China Jiarui Gao
E-Mail: [email protected] TEL: 010-66931993
Address: No. 156 Jinshui Road, Jinshui District, Zhengzhou, China 450046 No. 27 Taiping Road, Haidian District, Beijing, China 100850
* Correspondence authors: Corresponding author: Zhiyun Meng E-Mail: [email protected] TEL: 010-66931993 Address: No. 27 Taiping Road, Haidian District, Beijing, China 100850 Suxiang Feng E-Mail: [email protected] TEL: 0371-65962746 Address: No. 156 Jinshui Road, Jinshui District, Zhengzhou, China 450046
Abstract: As a myricetin derivative, M10 is a potent agent of anti-chronic colonic inflammation.
It
has
better
activity
than
myricetin
in
preventing
azoxymethane/dextran sulfate sodium - induced ulcerative colitis. Here, we introduce a
sensitive
quantification
method
based
on
ultra
performance
liquid
chromatography-tandem mass spectrometry for the determination of M10-H and M10-Na in Wistar rat plasma. Samples were treated with L - ascorbic acid and phosphate buffer solution to maintain stability and with acetonitrile to remove the proteins in the plasma. The supernatant was separated with BEH C18 column and eluted with ultrapure water and acetonitrile both containing 0.1% formic acid. The detection was performed by a triple quadrupole mass spectrometer with positive electrospray ionization mode in multiple reactive monitoring. This method was validated for the carryover effect, selectivity, accuracy, precision, matrix effect, stability, and recovery. A linear correlation was established between concentration and response by the calibration curves over 10–2000 ng·mL-1 (r > 0.99). This method was applied to a pharmacokinetic study of intragastrical administration of M10-H and M10-Na in Wistar rats. In addition, the relative bioavailability of M10-H to M10-Na in Wistar rats was 60±19%, calculated by the ratio of area under concentration (AUC) of M10-H to M10-Na after intragastrical administration of a single dose (100 mg·kg-1 for M10-H and M10-Na, respectively) in Wistar rats. Key words: M10; pharmacokinetics; UPLC-MS/MS; myricetin derivates 1. Introduction Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a complex, chronic, and relapsing disease characterized by symptoms of diarrhea, bloody stools and abdominal pain [1]. With the aggravation of colonic inflammation and prolongation disease, the risk of colitis-associated colorectal cancer (CAC) increased [2]. So, drugs with good efficacy and low toxicity for the treatment of colonic chronic inflammation and chemoprevention of colorectal tumorigenesis are in high demand [19]. Myricetin is a natural polyphenolic flavonoid [3-5] with biological activities [6-8], such as anti-oxidant [9-12] and anti-inflammatory [13-14]. Reportedly, myricetin exerts anti-inflammatory activity by reducing the expression levels of IL-6, TNF-α, IL-1β [15-18], and inhibits the intestinal tumorigenesis through inhibition of the Wnt/β-catenin pathway in the adenomatous polyposis coli multiple intestinal
neoplasia (APCMin/+) mice [19-22]. However, myricetin shows several deficiencies, such as complex isolation procedures, poor water solubility (< 100 ng·mL-1) and low stability when administrated orally [19-22]. Thus, the synthesis of derivatives of myricetin is needed. As shown in Fig. 1, based on myricetin, M10-H was designed by adding a hydrophilic glycosylation group, and M10-Na was formed as a sodium salt derivative with excellent water-solubility (> 0.1g·mL-1) and good safety which median lethal dose (LD50) is > 5g·kg-1. Moreover, as myricetin derivatives, M10-H and M10-Na inhibit the intestinal tumorigenesis via strong inhibition of the NF-B/IL-6/STAT3 pathway in a mice model with ulcerative colitis induced by azoxymethane (AOM)/dextran sulfate sodium (DSS). Both of them exhibited higher efficacy of prevention in the AOM/DSS - induced UC than myricetin and mesalazine, a clinically used drug for the treatment of ulcerative colitis [19-22]. M10 appears to have a good safety profile as an oral chemoprevention agent, and before entering clinical trials, in vivo preclinical pharmacokinetics is needed. The radio of relative bioavailability of M10-H to M10-Na is not only a critical parameter to estimate the differences between M10-H and M10-Na exposure after oral administration, but also an important factor to assess the differences of their pharmacokinetics characteristic, such as absorption, metabolism and excretion, all of which were taken into considerations about which one is more suitable for the further researches. Several methods have been reported for the detection of flavone and flavone glycoside, such as high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection [24] and liquid chromatography-tandem mass spectrometry (LC-MS/MS) [25]. To our knowledge, there is still no method reported for the determination of M10 in biological matrices. For the development of M10, it is essential to establish an analytical method sensitive enough to detect M10 in biological fluids. A structural analog, kaempferol, was used as internal standard (IS). Fig.1 shows the chemical structures of myricetin, M10-H, M10-Na and IS, respectively. In this study, we developed a sensitive UPLC–MS/MS method for the measurement of M10-H and M10-Na in rat plasma. This method was applied to study the pharmacokinetics of M10-H and M10-Na after oral administration in Wistar rats. This work describes the details of method establishment, validations and
pharmacokinetic profile of M10 in rat plasma. 2. Materials and methods 2.1 Reagents and instruments HPLC-grade acetonitrile, HPLC-grade methanol, and HPLC-grade formic acid (FA) were purchased from Fisher, USA. L - Ascorbic acid (Batch No. 2081206) and sodium carboxymethylcellulose (CMC-Na) (Batch No. F20110915) were purchased from Sinopharm Chemical Reagent Company (Beijing, China). Deionized water was produced by a Milli-Q Reagent Water System (Millipore, Bedford, MA, USA). The UPLC–MS/MS system consisted of a Waters ACQUITY I-Class UPLC system connected to a Waters (Milford, MA, USA) Xevo TQ-S triple quadrupole mass spectrometer with flow-through-needle sample manager, a cooling autosampler, a column oven with temperature control, a degasser, and a binary pump. The derivatives of myricetin (M10-H and M10-Na, purity ≥ 98%) were provided by Dr Li in Marine Biomedical Research Institute of Qingdao. Myricetin (purity ≥ 98%) was purchased from Sigma–Aldrich (St. Louis, MO). 2.2 Chromatography All samples were analyzed on UPLC® I-Class and Xevo® TQS combined system. Separations were carried out with a mobile phase of water (A) and acetonitrile (B), both containing 0.1% formic acid, at the flow rate of 0.5 mL·min-1 using a Waters ACQUITY UPLC® BEH C18 (2.1 mm × 50 mm, 1.7 μm) column. The over run time was 4 min and the gradient elution was adopted as follows: 0–0.5 min, 10% B; 0.5–2 min, 10–90% B; 2–3.5 min, 90% B; 3.5–3.51 min, 90-10% B; 3.51–4 min, 10% B. The Sample Manager-Flow through Needle (SM-FTN) was cleaned after each sample introduction by washing with a methanol/water mixture (4/1, v/v) and a seal wash with water/acetonitrile (9/1, v/v). The volume of injection was 1 μL, the sample temperature was maintained at 10 °C, and the column temperature was maintained at 45 °C. 2.3 Mass spectrometry The detection was performed by a triple quadrupole mass spectrometer with
electrospray ionization (ESI). The mass spectrometer detector was set as multiple reactive monitoring (MRM) in the positive mode. The MRM transitions and correlative parameters were as follows: capillary voltage: 3 kV; cone gas flow: 150 L·h-1; desolvation gas flow: 650 L·h-1; source temperature: 150 °C; desolvation temperature: 350 °C. The analytes were detected at m/z 319.0/153.0 and 287.0/153.0 with the collision energy 28 V and 30 V for M10 and IS (kaempferol), respectively. 2.4 Working and stock solutions The stock solutions of M10-H and M10-Na were prepared in 60% acetonitrile to the concentration of 1.0 mg·mL-1 and maintained at -20 °C. Then working solutions and quality control solutions were obtained by serial dilutions of the stock solutions with 60% acetonitrile. The internal standard solution of kaempferol was prepared in acetonitrile at a concentration of 10 ng·mL-1 and stored at -20 °C. To eliminate systematic error in samples quantification, kaempferol was used as an internal standard (IS) because its functional groups were similar to those of myricetin and M10 and its non-plasma source character. 2.5 Calibration standards and quality control samples preparations In order to prepare the plasma matrix samples with serial concentrations, 10 μL of a 10-fold concentration of the calibrators was added to 90 μL of blank plasma from Wistar rat. Then, the mixture was added to 200 μL of pH 4.0 phosphate-buffered saline (PBS) and 10 μL of 5% L - ascorbic acid dissolved in water. Then the mixture was precipitated with 200 μL of acetonitrile containing IS (10 ng·mL-1), vortex-mixed for 3 min and centrifuged at 12000 rpm for 10 min at 4 °C [26]. Then, 200 μL of supernatant was pipetted out into the autosampler vials and analyzed by UPLC–MS/MS with 1 μL injection volume. Lower limit of quantification (LLOQ) was defined as the lowest quantitative concentration. According to Chinese pharmacopeia, the relative standard deviation [RSD (%)] and the relative error [RE (%)] of LLOQ were set less than 20%. The range of the calibration of M10-H and M10-Na was in the linear range from 10–2000 ng·mL-1 (10, 20, 50, 100, 200, 500, 1000, and 2000 ng·mL-1). Four levels of quality control (QC) samples including LLOQ, low QC (25 ng·mL-1), mid QC (250 ng·mL-1),
and high QC (1600 ng·mL-1) were prepared by spiking the corresponding level of QC working solutions into blank plasma. Standard solutions and six replicates for each QC samples were prepared for every validation assay. 2.6 Sample pre-treatment 100 μL of plasma samples, 200 μL of pH 4.0 phosphate buffered saline (PBS) and 10 μL of 5% L - ascorbic acid were added individually. The mixture was vortex-mixed for 1 min before 200 μL of acetonitrile containing IS (10 ng·mL-1) was added. Then, samples were vortex-mixed for an additional 2 min and centrifuged at a speed of 12000×g for 10 min at 4 °C to remove the plasma proteins. Finally, 200 μL of supernatant were collected for UPLC-MS/MS analysis. 2.7 Analytical method validation According to the recently published guidelines of the National Medical Products Administration (NMPA) for bioanalytical methods validation [23], the method was validated in terms of selectivity, carryover effect, linearity, sensitivity, accuracy, precision, recovery, matrix effects and stability, respectively. 2.7.1 Selectivity and carryover effect The selectivity of the M10-H and M10-Na was determined using blank plasma from six different Wistar rats. 100 μL of blank plasma from six different Wistar rats was added to 200 μL of phosphate buffered saline (PBS) and 10 μL of 5% L - ascorbic acid then precipitation with 200 μL of acetonitrile and centrifugation, respectively. Then, 200 μL of supernatant was pipetted out into the autosampler vials and 1 μL of supernatant was injected into the analytical column for analysis immediately to ensure that there is no endogenous compound with M10. To assess the carryover effect, a blank plasma sample was injected after the injection of the upper limit of quantification (ULOQ) standard, and the peak areas at the same retention time with M10 should be < 20% of the peak areas of the LLOQ sample. 2.7.2 Linearity and sensitivity
Linearity was assessed by peak areas of the known amounts of eight-point work solutions, which were added into the blank plasma to achieve calibration standards. The calibration curves range from 10 to 2000 ng·mL-1 including a zero-level spiked into blank rat plasma samples. All the standard solutions were corrected with the internal standard (kaempferol) and calculated using weighted least squares linear regression. Three quality control samples at 25 ng·mL-1 (low), 250 ng·mL-1 (medium) and 1600 ng·mL-1 (high) were prepared independently of another M10 stock solution used for adjusting the calibration curves. Each batch of the standard curve solutions was freshly prepared in every validation assay and sample test. The sensitivity of the method was assessed by the peak areas from six parallel LLOQ samples in rat plasma, and the deviation should not exceed 20%. 2.7.3 Accuracy and precision Accuracy and precision of the proposed assay were determined by LLOQ and QC samples at low, medium, and high concentrations in intra- and inter-day batch. The intra-day accuracy and precision need be investigated by three batches for a minimum of 2 days. Every single assay needs six replicate analyses of LLOQ and QC samples. The accuracy [expressed as relative error, RE (%)] and precision [expressed as relative standard deviation, RSD (%)] for samples at LLOQ should not exceed 20% while for QC samples, should not exceed 15%. 2.7.4 Stability The stability was assessed by analyzing the QC samples at low and high levels with six replicates under different storage conditions. For instance, the stability of M10-H and M10-Na in plasma was analyzed the samples in plasma at room temperature for 6 h. The stability of freeze-thaw cycles was investigated by analyzing samples in plasma after three freeze (-80 °C)-thaw cycles. The long-term stability was determined by evaluating the QC samples in blank plasma at −80 °C for 60 days. The short-term stability was examined by analyzing the processed M10-H and M10-Na at 4 °C for 24 h. To test the stability of samples into autosampler, the samples were processed and maintained into the autosampler for 24 h. In addition, stock solution stability was also investigated after storage at -20 ℃ for 2 weeks. The RE (%) for any stability test condition was compared to the standard curve on the detection day wand as required to be within ±15%.
2.7.5 Matrix effects and recovery The matrix effect of M10-H and M10-Na was evaluated by comparing the difference of response between samples of 60% acetonitrile and plasma from six individual rats with the addition of low and high QC samples of M10-H and M10-Na respectively. The matrix effects were calculated by the ratio of peak areas of M10-H and M10-Na in the plasma and 60% acetonitrile at low and high QC concentrations, respectively. The RSD (%) of the ratio of the peak areas ratio at each QC sample should be less than 15%. The recovery was examined by comparing the difference of peak areas between samples of blank plasma and the supernatants of the precipitated blank plasma by acetonitrile with the addition of QC samples at low and high levels with six replicates. The recovery was calculated by the ratio of peak areas of M10-H and M10-Na in the supernatants of the processed plasma by acetonitrile and blank plasma at low and high QC concentrations, respectively. The RSD (%) of the peak area ratio should be < 15%. 2.8 Pharmacokinetic study Sixteen Wistar rats (eight males and eight females, weight 180±20 g) were used in this pharmaceutical experiment. The animals were maintained under standard laboratory conditions (relative humidity, 40–70%; temperature, 20–26 °C; 12/12 h light/darkness) and fed separately. All animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (National Research Council of the USA, 1996), and approved by the Association for Assessment and Accreditation of Laboratory Animal Care. The license number of experimental animal production was SCXK (Jing) 2016-0006. The sixteen animals were randomly divided into two groups (M10-H group and M10-Na group, each group comprised of half males and females) and jejunitis overnight. Then, a single dose of 100 mg·kg-1 of M10-H or M10-Na was administered to each rat intragastrically. An equivalent of 0.15 mL blood samples was withdrawn from the rats’ orbital venous plexus at the following time points after dosing: 0.083, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 24 h (i.g.). The blood samples were collected into sodium citrate tubes and cleared by centrifugation at 8000 ×g for 10 min at 4 °C and
stored at −80 °C until further analysis. 2.9 Data analysis Microsoft Office Excel was used for data calculation. Origin 8.0 was used to draw graphs. ChemDraw Ultra 8.0 was used to generate the chemical structures of the M10, myricetin and kaempferol. And the pharmacokinetic parameters were calculated by using WinNonlin 6.3 software (Pharsight Corp., MO, USA). Concentration–time data that are below the lower limit of quantification were treated as zero in summary statistics. The following variables were calculated for both M10-H and M10-Na: time to observed maximum plasma concentration after drug administration (Tmax) and observed maximum plasma concentration (Cmax). Other major pharmacokinetic parameters including the terminal elimination half-life (T1/2), the plasma exposure [area under the plasma concentration–time curve from time 0 to time of last measurable concentration (AUC0-last)], mean residence time (MRT), and clearance (Cl) were all calculated by non-compartmental method. 3. Results 3.1 Method development As to ionization mode, the response of M10 in positive mode was 20 times higher than that in negative mode, so the positive-ion mode was used. When optimizing the mass spectrometry conditions, 2mL of 500 ng·mL-1 M10-H, M10-Na and kaempferol were separately infused into the mass spectrometer in order to obtain a Q1 scan of the parent compound. The mass spectrometric parameters, such as the desolvation gas flow, source temperature, desolvation temperature, collision energy and cone energy, were adjusted to maximal the intensity of the product ions. The analytes were easily cleaved and then generate positive product ions, M10-H mostly consisted of [M+Na]+ ions and M10-Na mostly consisted of [M+H]+ ions, so the transitions for M10-H and M10-Na at the early stage of the method development were both at m/z 665.1 ＞ 347.0. However, in-source fragmentation into aglycones （myricetin）of M10-H and M10-Na were found after injection into UPLC-MS/MS.
So, we finally chose transitions at m/z 319.0 ＞ 153.0 to identify and quantify M10, which showed a stronger response and better sensitivity than transitions at m/z 665.1 ＞ 347.0. 3.2 Selectivity and carryover effect The typical chromatograms were obtained from blank plasma samples, blank plasma after injected ULOQ samples, spiked plasma samples with analytes at the LLOQ (Fig. 2A), spiked plasma samples with analytes at the ULOQ, internal standard (Fig. 2B) and plasma samples after dosing of M10-H and M10-Na are presented in Fig. 2. The method of selectivity was determined by comparing the blank plasma from six individual rats with M10-H and M10-Na in the plasma. The results displayed a lack of interfering peaks with M10. The carryover effect of the method was evaluated by comparing the blank plasma after injecting the ULOQ standard with M10-H and M10-Na in the rat plasma at LLOQ concentration. The peak areas of the blank plasma samples should not exceed 20% of those measured in the LLOQ sample. Fig. 2 demonstrated that there is no carryover peak at the same retention times. 3.3 Linearity and sensitivity Sensitivity was assessed based on the six spiked samples at LLOQ concentration. Linearity was assessed by peak areas of eight-point on calibration curves after adjusting with the internal standard (kaempferol) and calculated by the weighted least squares linear regression. Table 1 demonstrated that the RSD (%) at LLOQs was ≤ 12.3% (i.e., < 20%), conforming to the NMPA standard. The typical calibration equations were as follows: M10-H, y = 0.00599 x + 0.0126 (r2 = 0.995); M10-Na, y = 0.00586 x + 0.0162 (r2 = 0.998). The LLOQ was 10 ng·mL-1, and the calibration standards of M10-H and M10-Na ranged from 10–2000 ng·mL-1, with optimal linearity over the concentration range. 3.4 Accuracy and precision The data of precision and accuracy are summarized in Table 2. The intraday precision [relative standard deviation, RSD (%)] of the M10-H at LLOQs concentration was 17.1%. The other intra- and inter-day precision---- RSD (%) of the M10-H and M10-Na was < 12.3%. Moreover, the intra- and inter-day accuracy ---RE (%) of the M10-H and M10-Na ranged from 0.2–10.9%, demonstrating an optimal
accuracy and reproducibility of the method. 3.5 Stability The data of stability are displayed in Table 3. The results showed that the RE (%) at different conditions varied from -8.5 to 9.0%, i.e., within ±15%. It was illustrated that the M10-H and M10-Na samples were stable at different conditions, such as in the blank plasma at room temperature for 6 h, after three freeze-thaw cycles, and at −80 °C for 60 days. Next, the processed samples were placed at 4 °C for 24 h or loaded onto the auto-sampler for 24 h. Furthermore, the stock solutions of M10-H and M10-Na were stable at -20 °C for 2 weeks. 3.6 Matrix effects and recovery The data of matrix effect and recovery are presented in Table 4. The matrix effects M10-H and M10-Na ranged from 154.1–162.8% and 148.4–180.8%, respectively, and the overall recoveries of M10-H and M10-Na ranged from 93.8–103.2% and 96.8–107.3%, respectively. The RSD (%) values of matrix effect and recovery at two QC concentrations were ≤ 7.3%, which fulfilled the criteria. These findings demonstrated an enhanced but stable and reproducible effect from the plasma matrix in this approach. 3.7 Pharmacokinetic assessment When applied to the preclinical pharmacokinetic study, the method was proved suitable for quantitating the M10 in Wistar rat plasma with an oral dose of 100mg·kg-1 of M10. According to the measured plasma concentrations, in the current comparative pharmacokinetic study of intragastrical administration of M10-H and M10-Na (100 mg·kg-1), the mean plasma concentration-time profiles of M10-H and M10-Na are illustrated in Fig. 3 and the main pharmacokinetic parameters were obtained. The results are demonstrated in Table 5 as follows: for M10-H and M10-Na, respectively, maximum concentration (Cmax), 23.2±9.9 and 129.9±52.7 ng·mL-1; time to reach maximum concentration (Tmax), 4.4±2.3 and 0.2±0.1 h; area under the curve from time 0 to 24 h (AUC0–24), 93.2±29.9 and 155.6±147.3 h·ng·mL-1; elimination half-life (T1/2), 5.4 and 1.8±0.8 h. In addition, the relative oral bioavailability of the M10-H and M10-Na could be calculated as follows: [AUClast (M10-H) / AUClast (M10-Na)] x 100% and the relative bioavailability of the M10-H to M10-Na in the Wistar rat
samples was 60±19% [27].
Relative bioavailability (%) = [AUClast (M10-H) / AUClast (M10-Na)] x 100%
4. Discussion As glycosides derivatized from flavonoids, M10-H and M10-Na were easily proposed in-source fragmentation into aglycones（myricetin）after injection into UPLC-MS/MS. However, the degree of the crack is unstable, and was affected by pH, exhibiting low response and poor linearity during the development of the method. This problem has been solved after the addition of ascorbic acid and PBS (pH 4.0). The results of the pharmacokinetic study showed that the maximum concentration (Cmax) of M10-H and M10-Na were 23.2±9.9 ng·mL-1 and 129.9±52.7 ng·mL-1 respectively, and time to reach maximum concentration (Tmax) of them were 4 h and 15 min, exhibiting the faster and better absorption of M10-Na than M10-H. The elimination half-life (T1/2) of M10-H and M10-Na were 5.4 and 1.8±0.8 h, showing that the elimination of M10-Na was faster than M10-H. The area under the plasma concentration-time curve (AUC) of M10-H and M10-Na were 93.2±29.9 and 155.6±147.3 h·ng·mL-1 respectively, suggesting that M10-Na showed better absorption in vivo than M10-H. The volume of distribution (V/F) of M10-H and M10-Na were 3483348 and 1474438±602798 L·kg-1, demonstrating that M10-H and M10-Na were distributed into a wide range of organs and tissues. The results of the pharmacokinetics study illustrating that M10-Na can be quickly absorbed and eliminate, and M10-H is hardly absorbed relatively. And the relative bioavailability of the M10-H to M10-Na in the Wistar rat samples was 60±19%. We presumed the significant differences in the pharmacokinetics of M10-H and M10-Na were resulted from the better water-solubility of M10-Na than M10-H. 5. Conclusion As a myricetin derivative, M10 is a new compound and potent agent of anti-chronic colonic inflammation. The comparative studies showed that the efficacy of M10 was higher than myricetin and mesalazine, a clinical used drug for the treatment of ulcerative colitis in the DSS-induced chronic colonic inflammation in
mice [20]. Herein, we have developed and validated a sensitively analytical method for M10-H and M10-Na. In addition, this simple, sensitive, reproducible and robust method has been successfully employed to the pharmacokinetics study of M10-H and M10-Na in Wistar rats after a single oral administration, and their pharmacokinetics was achieved. The pharmacokinetics data could provide useful information for the further preclinical study. Considering the excellent performance of this method, it will be utilized for future preclinical developments of M10. Acknowledgments We thank Professor Wenbao Li from Ocean University of China for providing M10-H and M10-Na. And we appreciate the reviewers and editors for helpful suggestions. Reference [1] R.J. Xavier, D.K. Podolsky, Unravelling the pathogenesis of inflammatory bowel disease, Nature 448 (2007): 427-434. [2] Y. Yamada, H. Mori, Multistep carcinogenesis of the colon in Apc (Min/+) mouse, Cancer Science. 98 (2007): 6-10. [3] Bingxu Huang, Juxiong Liu, Dongxu Ma, Guangxin Chen, Wei Wang, Shoupeng Fu. Myricetin prevents dopaminergic neurons from undergoing neuroin flammation-mediated degeneration in a lipopolysaccharide-induced Parkinson’s disease model, Journal of Functional Foods, 45(2018): 452–461. [4] Eshan Khan, Arpita Tawani, Subodh Kumar Mishra, Arun Kumar Verma, Arun Upadhyay, et al., Myricetin Reduces Toxic Level of CAG Repeats RNA in Huntington’s Disease (HD) and Spino Cerebellar Ataxia (SCAs), ACS chemical biology 13 (2018): 180−188. [5] Hua-Fu Zhao, Gang Wang, Chang-Peng Wu, Xiu-Ming Zhou, Jing Wang, et al., A Multi-targeted Natural
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[11] Hyang-Yu Kima, Sun-Young Parka, Se-Young Choung, Enhancing effects of myricetin on the osteogenic differentiation of human periodontal ligament stem cells via BMP-2/Smad and ERK/JNK/p38 mitogen-activated protein kinase signaling pathway, European Journal of Pharmacology, 834(2018): 84–91. [12] Yingqian Ci, Yubo Zhang, Yanjie Liu2, Shuai Lu, et al., Myricetin suppresses breast cancer metastasis through down-regulating the activity of matrix metalloproteinase (MMP)-2/9, Phytotherapy Research, 32(2018):1373–1381. [13] Hahyun Park, Sunwoo Park, Fuller W. Bazer, Whasun Lim3, Gwonhwa Song Myricetin treatment induces apoptosis in canine osteosarcoma cells by inducing DNA fragmentation, disrupting redox homeostasis, and mediating loss of mitochondrial membrane potential, Journal of Cellular Physiology, 233(2018):7457–7466. [14] Allison Knicklea, Wasundara Fernandob, Anna L. Greenshieldsb, H.P. Vasantha Rupasingheb,c, David W. Hoskin, Myricetin-induced apoptosis of triple-negative breast cancer cells is mediated by the iron-dependent generation of reactive oxygen species from hydrogen peroxide, Food and Chemical Toxicology, 118(2018): 154–167. [15] Takahiro Yamashiro, Tomoya Yasujima, Kinya Ohta, Katsuhisa Inoue, Hiroaki Yuasa, Specific inhibitory effects of myricetin on human proton-coupled folate transporter: Comparison with its effects on rat proton-coupled folate transporter and human riboflavin transporter 3, Drug Metabolism and Pharmacokinetics, 32(2017): 311-314. [16] Anjun Yao, Yingzhuo Shen, Zhuangwei Zhang, Zuquan Zou, Anshi Wang, Sulforaphane and myricetin act synergistically to induce apoptosis in 3T3‑ L1 adipocytes, Molecular Medicine Reports, 17(2018): 2945-2951. [17] Chen Ye, Chao Zhang, Hai Huang, Bo Yanga, Guangan Xiao, et al., The Natural Compound Myricetin Effectively Represses the Malignant Progression of Prostate Cancer by Inhibiting PIM1 and Disrupting the PIM1/CXCR4 Interaction, Cell Physiology and Biochemistry; 48(2018):1230-1244. [18] Nan Zhang, Hong Feng4, Hai‐ Han Liao, Si Chen, et al., Myricetin attenuated LPS induced cardiac injury in vivo and in vitro, Phytotherapy Research, 32(2018):459–470. [19] Sifeng Zhu, Chong Yang, Liang Zhang, Shixiao Wang, Mingxu Ma, et al., Development of M10, myricetin-3-O-b-D-lactose sodium salt, a derivative of myricetin as a potent agent of anti-chronic colonic inflammation, European Journal of Medicinal Chemistry, 174(2019): 9-15. [20] Feng Wang, Zhi-Yu Song, Xian-Jun Qu, Feng Li, Liang Zhang, et al., M10, a novel derivative of Myricetin, prevents ulcerative colitis and colorectal tumor through attenuating robust endoplasmic reticulum stress, Carcinogenesis, 39(2018): 889–899. [21] Jeong Hyun Lee, Yong Jun Choi, See-Hyoung Park, Myeong Jin Nam, Potential role of nucleoside diphosphate kinase in myricetin-induced selective apoptosis in colon cancer HCT-15 cells, Food and Chemical Toxicology, 116 (2018): 315–322. [22] Yuchao Sun, Mengqiao Lian, Yuan Lin, Bin Xu, Role of p-MKK7 in myricetin-induced protection against intestinal ischemia/reperfusion injury, Pharmacological Research, 129(2018): 432–442. [23] Guidance for Industry: Bioanalytical Method Validation. Pharmacopoeia of People’S Republic of China, National Pharmacopoeia Committee [M].Part 4, Beijing, 2015. [24] Mai Gamal Elhennawy, Hai-Shu Lin, Determination of Tangeretin in Rat Plasma: Assessment of Its Clearance and Absolute Oral Bioavailability, Pharmaceutics, 10(2018): 1-10. [25] Shunjun Xu, Jiejing Yu, Jingjing Zhan, Pharmacokinetics, Tissue Distribution, and Metabolism Study of Icariin in Rat, BioMed Research International 11(2017): 1-17.
[26] Dong Xiang, Chen-guang Wang, Wen-qing Wang, Chun-yang Shi, Wei Xiong, et al., Gastrointestinal stability of dihydromyricetin, myricetin, and myricitrin: an in vitro investigation, International Journal of Food Sciences and Nutrition, (2017): 1-8. [27] Jennifer Moczarnik, DVM, Darren J. Berger, DVM, DACVD, et al., Relative Oral Bioavailability of Two Amoxicillin–Clavulanic Acid Formulations in Healthy Dogs: A Pilot Study, Journal of the American Animal Hospital Association, 55 (2019): 14-22.
Table 1 The typical standard curve of the M10-H and M10-Na in Wistar plasma
M10-H
M10-Na
Batch
Intercept
Slope
1 2 3 1 2 3
0.0126 0.0154 0.0196 0.0162 0.0176 0.0287
0.00599 0.0252 0.0169 0.00586 0.0106 0.0139
correlation coefficient (R2) 0.9951 0.9981 0.9960 0.9979 0.9970 0.9931
Table 2 Precision and accuracy for the M10-H and M10-Na in Wistar plasma Spiked
Overall Measured Concentration (ng·mL-1) (Mean ± SD, n = 6)
Concentration (ng·mL-1)
Intra-Day
Inter-Day
RSD (%)
RSD (%)
RE
Mean Batch 1
Batch 2
Batch 3
(ng·mL-1)
(%)
LLOQ
10
11.9±2.0
11.1±0.7
10.3±1.1
11.1
17.0
12.3
10.9
LQC
25
26.4±1.8
25.7±2.1
25.6±1.3
25.9
4.5
6.9
3.5
MQC
250
251.3±23.6
253.0±16.7
249.2±8.3
251.2
1.9
6.9
0.5
HQC
1600
1609.1±47.3
1740.5±90.6
1667.3±64.8
1672.3
9.6
4.2
4.5
LLOQ
10
10.6±0.9
10.8±0.8
10.5±0.7
10.6
2.7
7.6
6.4
LQC
25
25.5±2.3
25.6±2.5
25.5±2.6
25.5
0.7
9.7
2.1
MQC
250
246.3±15.0
250.1±9.2
254.7±10.3
250.4
4.1
4.7
0.2
HQC
1600
1524.7±53.0
1647.1±73.5
1641.3±114.1
1613.3
11.9
5.2
0.8
M10-H
M10-Na
Table 3 Stability data of the M10-H and M10-Na under different storage situation M10-H
M10-Na
Concentration Storage Conditions
（ng·mL-1）
Mean ± SD
RSD
Mean ± SD
RSD
(ng·mL-1)
(%)
RE (%) (ng·mL-1)
(%)
RE (%)
Room temperature
25
24.6±1.1
4.3
-1.8
24.0±1.8
7.5
-3.8
for 6 h
1600
1682.2±78.0
4.6
5.1
1652.9±143.5
8.7
3.3
Three freeze-thaw
25
26.4±1.4
5.4
5.7
26.6±1.9
7.3
6.2
cycles
1600
1669.9±44.1
2.6
4.4
1744.7±121.4
7.0
9.0
Long term for 60
25
28.1±1.0
3.5
12.6
24.9±1.6
6.2
-0.2
days (-80 °C)
1600
1605.1±42.2
2.6
0.3
1687.5±71.3
4.2
5.5
Processed sample
25
25.7±1.8
7.1
2.8
24.2±1.4
5.7
-3.3
for 24 h (-20 °C)
1600
1674.5±118.1
7.1
4.7
1560.8±112.4
7.2
-2.5
Autosampler
25
23.2±1.9
8.2
-7.1
22.9±1.4
6.2
-8.5
for 24 h
1600
1624.3±90.7
5.6
1.5
1479.8±99.0
6.7
-7.5
Stock solution for 2
25
25.8±0.9
3.6
3.0
23.4±2.2
11.7
-6.5
weeks (-20 °C)
1600
1614.7±65.8
4.1
0.9
1633.8±54.9
3.4
2.1
Table 4 Matrix effects and Recovery rates of the M10-H and M10-Na in Wistar rat plasma（n=6） concentration （ng·mL-1）
Matrix Effect (n = 6)
Recovery (n = 6)
Mean±SD（%）
RSD%
Mean±SD（%）
RSD%
25
162.8±4.4
2.7
93.8±6.8
7.3
1600
154.1±5.9
3.9
103.2±4.7
4.6
25
148.4±10.8
7.3
107.3±5.5
5.1
1600
180.8±8.3
4.6
96.8±3.9
4.0
M10-H
M10-Na
Table 5 Pharmacokinetic parameters of intragastrical administration the M10-H and M10-Na (100 mg·kg−1) (n = 8) Mean±SD Parameter(unit) M10-H
M10-Na
t1/2 (h)
5.4
1.8±0.8
Tmax (h)
4.4±2.3
0.2±0.1
23.2±9.9
129.9±52.7
AUC0-24h (h·ng·mL )
93.2±29.9
155.6±147.3
V (L·kg-1)
3483348
1474438±602798
CL (L·h ·kg )
445550
658926±302816
MRT (h)
4.0±1.1
1.1±0.4
Cmax (ng·mL-1) -1
-1
-1
Highlights
A sensitive UPLC-MS/MS method was successfully developed and validated for M10-H and M10-Na.
It was reported for the first time that M10-H and M10-Na is determined by UPLC-MS/MS.
This method was successfully applied for pharmacokinetics study of M10-H and M10-Na.
Figure legends Fig. 1 The chemical structure of the myricetin, M10-H, M10-Na and kaempferol.
Fig. 2A The typical chromatograms obtained from M10-H and M10-Na in Wistar rat blank plasma (A) Wistar rat blank plasma；(B) blank plasma after injected ULOQ of M10-H and M10-Na (C) 1. LLOQ of M10-H；2. LLOQ of M10-Na；
Fig. 2B The typical chromatograms obtained from M10-H and M10-Na in Wistar rat blank plasma (D) 1. ULOQ of M10-H；2 ULOQ of.M10-Na；(E) internal standard； (F) plasma samples after dosing of M10-H and M10-Na；
Fig. 3 The drug concentration-time curves of intragastrical administration of the M10-H and M10-Na (100 mg·kg−1) in Wistar plasma (Mean±SD, n = 8).
Conflicts of interest:
All authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare.
Author statement Jiarui Gao: Methodology, Software, Investigation, Formal analysis, Data Curation, Writing - Original Draft. Guifang Dou: Conceptualization, Funding acquisition. Xiaoxia Zhu: Resources, Validation. Hui Gan: Data Curation. Ruolan Gu: Writing - Review & Editing. Zhuona Wu: Visualization. Taoyun Liu: Supervision. Suxiang Feng: Project administration. Zhiyun Meng: Conceptualization, Funding acquisition.
Abstract: As a myricetin derivative, M10 is a potent agent of anti-chronic colonic inflammation.
It
has
better
activity
than
myricetin
in
preventing
azoxymethane/dextran sulfate sodium - induced ulcerative colitis. Here, we introduce a sensitive quantification method based on ultra performance liquid chromatography-tandem mass spectrometry for the determination of M10-H and M10-Na in Wistar rat plasma. Samples were treated with L - ascorbic acid and phosphate buffer solution to maintain stability and with acetonitrile to remove the proteins in the plasma. The supernatant was separated with BEH C18 column and eluted with ultrapure water and acetonitrile both containing 0.1% formic acid. The detection was performed by a triple quadrupole mass spectrometer with positive electrospray ionization mode in multiple reactive monitoring. This method was validated for the carryover effect, selectivity, accuracy, precision, matrix effect, stability, and recovery. A linear correlation was established between concentration and response by the calibration curves over 10–2000 ng·mL-1 (r > 0.99). This method was applied to a pharmacokinetic study of intragastrical administration of M10-H and M10-Na in Wistar rats. In addition, the relative bioavailability of M10-H to M10-Na in Wistar rats was 60±19%, calculated by the ratio of area under concentration (AUC) of M10-H to M10-Na after intragastrical administration of a single dose (100 mg·kg-1 for M10-H and M10-Na, respectively) in Wistar rats.