A Rapid and Sensitive UHPLC-MS/MS Method for Determination of 2, 3, 8-Trimethylellagic, a Potent Active Compound from Sanguisorba officinalisL., and Its Application in the Pharmacokinetic Study within Thrombocytopenia Rats

School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, China School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China Institute of Cardiovascular Research, #e Key Laboratory of Medical Electrophysiology, Ministry of Education of China, Medical Key Laboratory for Drug Discovery and Druggability Evaluation of Sichuan Province, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, Luzhou, China


Introduction
Cancer is a disease associated with high global morbidity and mortality rates. It is caused by the abnormal proliferation of cells, a process that is induced by a variety of tumorigenic factors. At present, radiotherapy and chemotherapy are the primary treatment modalities used for malignant tumors. However, as they result in the untargeted killing of cells, such treatments often cause a series of adverse reactions, including nausea, vomiting, diarrhea, skin inflammation, fever, and bone marrow and immunological suppression [1]. In particular, damage to the bone marrow and the suppression of immunologic function lead to the reduction of the leukocyte, erythrocyte, and platelet counts, as well as an increased risk of infection and bleeding.
rombocytopenia is defined as a condition where the platelet count is less than 100 × 10 9 /L, and its clinical manifestations include splenomegaly, ecchymosis, purpura, superficial lymphadenopathy, intracranial hemorrhage, and death [2]. In addition to radiotherapy and chemotherapy, other processes that contribute to thrombocytopenia include the metastatic cancers of the bone marrow, leukemia, myelodysplastic syndrome, decreased thrombocytopoiesis caused by lesions in the hematopoietic cells, the inactivation of thrombocytopoiesis caused by the deficiency of vitamin B or folate, and increased platelet destruction caused by thrombotic thrombocytopenic purpura [3]. e intravenous infusions of the platelets, hormones, cytokines, and immunoglobulins are common treatments for thrombocytopenia [4]. Despite being temporarily palliative, these treatments are expensive and can lead to serious side effects. erefore, there is an urgent need to develop safer and more effective drugs for treating thrombocytopenia and reducing the adverse effects of radiotherapy and chemotherapy during the treatment of cancer.
Traditional Chinese medicine is a natural repository from which new active compounds are often discovered. Although there exist several herbal formulations used to treat thrombocytopenia induced by radiotherapy, chemotherapy, or immune thrombocytopenia, only a few reports exist on single herbs and natural medications, such as Panax ginseng C. A. Mey., Ligustrum lucidum Ait., Lycium barbarum L., Spatholobus suberectus Dunn, Agrimonia pilosa Ledeb., Eclipta prostrata L., Sarcandra glabra ( unb.) Nakai, Dioscorea esculenta (Lour.) Burkill, and Carica papaya L. [5]. Related studies have reported that tannins in Sanguisorba officinalis L. can increase the DNA content and combat myelosuppression in mice with cyclophosphamideinduced thrombocytopenia [6], and the saponins of the herb can enhance the levels of the bone marrow nucleated cells and peripheral blood cells [7]. e mechanism through which these substances promote thrombocytopoiesis is related to the thrombopoietin/thrombopoietin receptor (TPO/ c-Mpl) and the stem cell factor/kit ligand (SCF/C-KIT) pathways. e binding ability of cytokines with their corresponding receptors is thought to be enhanced by the upregulation of TPOR and C-KIT, although it is also possible for pleiotropic cytokines to directly activate TPOR or C-KIT, as well as TPO or SCF [7]. e downstream signal transduction pathway is subsequently activated to promote the megakaryocyte proliferation and differentiation, and the platelet production is further promoted by a series of reactions. e previous experiment is committed to studying the pharmacodynamic foundations by which S. officinalis L. affects platelet production, which has found that the total saponins of S. officinalis L. can increase the white blood cell and platelet counts [7]. e pentacyclic triterpenes, such as ziyuglycoside I, and ellagic acids, such as TMEA, DMAG, and DOMA (Figure 1), can increase platelet production by enhancing the proliferation and differentiation of the megakaryotic progenitor cells. e ellagic acids are known to be much more potent than the pentacyclic triterpenes, especially in promoting the megakaryocyte differentiation, a process related to the upregulation of hTPOR and hC-KIT mRNA [7,8].
is lays an important foundation for the development of candidate drugs to promote platelet production. Pharmacokinetics focuses on the regulation of absorption, distribution, metabolism, and excretion of drugs in vivo. It provides a basis for the route, dosage, interval, and duration of drug administration, thus ensuring the safety and efficacy of drug therapy. However, the pharmacokinetics of TMEA from S. officinalis L. in rats has not been reported, and its compatibility with different drugs may also cause changes in its pharmacokinetic parameters. UHPLC-MS/MS can avoid the complicated and cumbersome sample pretreatment steps to save the time of separation and purification, as well as increase the peak capacity, shorten the analysis time, and reduce the interference of the endogenous substance [9,10]. It has been gradually applied to pharmacokinetics, metabolomics, proteomics, drug residues, and so on [11]. It is now the most efficient method for the analysis and identification of trace components in biological samples because of its high sensitivity and selectivity. For example, Deng et al. [12] established and verified a simple and sensitive UHPLC-MS/MS method for the analysis of furandienone in rat plasma. Huang et al. [13] have reported a UHPLC-MS/MS method for the determination of kurarinone and its preclinical pharmacokinetic study in dogs after oral administration. A UHPLC-MS/MS method has been successfully applied to explore the oral and intravenous pharmacokinetics of skimmin [14]. erefore, for the first time, this study aims to establish an efficient and sensitive UHPLC-MS/MS method to study the pharmacokinetic characteristics of TMEA after its administration in rats. It also aims to compare the pharmacokinetic changes between the normal and thrombocytopenic samples and those among the groups with and without compatibility. At the same time, the efficacy and safety of TMEA for the treatment of thrombocytopenia can be evaluated, which provides a scientific basis for further in-depth research and development of candidate drugs for thrombocytopenia.

Animal
Care. Sprague Dawley rats (certificate number: SCXK2020-030, 8 to 10 weeks old, bodyweight of 180 to 220 g, half male and half female) were obtained from Da-suo Bio-technology Co., Ltd. (Chengdu, China). All animal experiments were performed in strict accordance with the U. S. guidelines [15] that were approved by the ethical committee on Use and Care of Animals of Southwest Medical University (No. 20160276). Prior to the experiment, animals were housed in a temperaturecontrolled facility with a 12-h light/dark cycle and were provided unlimited access to food and water for 7 days. e animals were subsequently fasted for 12 h before dosing, with free access to water.

Establishment of the rombocytopenia Rat Model.
A thrombocytopenic rat model was established according to the method in relevant literature [16]. Following this method, 40 healthy SD rats (half male and half female) were arbitrarily divided into the normal (n � 12) and model groups (n � 28). e model group was intraperitoneally injected with 150 mg/kg cyclophosphamide on the first day and 50 mg/kg cyclophosphamide daily from the days 2 to 3. A solution of 0.9% physiological saline was intraperitoneally injected into the normal group using the same dosage. On the last day, 40-μL blood samples were collected from six normal rats and six model rats. Routine blood tests were subsequently performed using an XT-1800i/2000IV automatic blood cell analyzer (Sysmex Corp., Kobe, Japan). e bone marrow cells were collected, and the total bone marrow cell count, DNA content, and protein content of each sample were calculated with the automatic blood cell analyzer, a NanoDrop OneC microultraviolet-visible spectrophoto meter ( ermo Electron Corp., Waltham, USA), and a BioTek Cytation3 multifunctional cell imaging detection system (BioTek Corp., Vermont, USA), respectively. Simultaneously, the heart, liver, spleen, lung, kidney, and thymus were harvested to measure the visceral index.

Instrument and Chromatographic Conditions.
e analysis was performed on a UHPLC-MS/MS system (Shimadzu Corp., Tokyo, Japan) using an Acquity Shimpack GIST column (2.0 × 75 mm, 2.0 μm, Shimadzu Corp., Japan), consisting of a SIL-30AC automatic sampling device, four LC-30AD infusion pumps, a DGU-20A5 online degasser, an SPD-M30A detector, an LC-MS-8050 triplequadrupole mass spectrometer, a CTO20A column oven, and a CBM-20A system controller. e column temperature was maintained at 40°C with a flow rate of 0.3 mL/min. e mobile phase for this analysis was composed of 0.1% formic acid water (solvent A) and acetonitrile (solvent B). e gradient condition was as follows: a linear gradient elution of 70% to 90% solvent B for the initial 2 min, followed by an isocratic elution of solvent B for the next. e injection volume was 10 μL.
e mass spectrometer was operated in the positive and negative ion mode. Multiple reaction monitoring (MRM) acquisition mode was used to monitor the precursor-toproduct ion transition. e MRM fragmentation transitions were m/z 343.20⟶328.10 for TMEA and m/z 389.15⟶331.15 for IS ( Figure 2). e collision energy for TMEA and IS were set at 17 eV and 35 eV, respectively. e flow rates of atomizing gas, heating gas, and drying gas were 2.0 L/min, 10.0 L/min, and 10.0 L/min, respectively. e interface voltage and temperature were maintained at 4 kV and 300°C. e desolvation temperature was 250°C, heating block temperature was 400°C, and detector voltage was 2.24 kV.

Preparation of Samples.
e stock solutions of TMEA and IS were prepared with acetonitrile at 0.051 and 19.41 mg/ mL, respectively. e working standard solutions of TMEA were prepared by serially diluting a stock solution to achieve a series of concentrations: 5, 10, 50, 100, 500, 1000, 4000 ng/mL and IS stock solutions were diluted into 500 ng/mL. e solutions were stored for no longer than 14 days at −20°C. Standard samples were prepared by spiking appropriate amounts of the stock solutions of TMEA and IS in blank plasma. e quality control samples for assessing the method accuracy and precision were independently prepared at low (1 ng/mL), medium (10 ng/mL), and high (100 ng/mL) concentrations in the same manner. After 100 μL plasma samples were spiked with 10 μL IS solution and mixed for 30 s, 2 mL ethyl acetate was added to each tube. e tubes were vortexed for 5 min and left to stand for 1 min at room temperature. e extracts were centrifuged at 8000 g for 10 min at 4°C. e supernatant was dried under a gentle stream of nitrogen at 37°C. e extract residues were reconstituted, and 10 μL samples were subsequently injected for analysis. Journal of Chemistry

Method Validation.
e method was validated in terms of specificity, linearity, matrix effect, recovery, accuracy, precision, and stability in accordance with Food and Drug Administration (FDA) guidelines [17]. Specificity was evaluated by comparing the chromatograms of blank plasma, blank plasma spiked with TMEA and IS, and plasma collected after the oral administration of TMEA. e calibration curve was generated by plotting the peak-area ratio of TMEA and IS with nominated concentrations. ree concentrations of the quality control (QC) samples (1, 10, and 100 ng/mL) were analyzed in six replicates on the same day and on three consecutive days to evaluate the intraday and interday precision and accuracy. Accuracy was expressed as the relative error between the spiked and determined concentrations, while precision was presented as a relative standard deviation (RSD). e matrix effect and recovery were calculated as the peak area of TMEA with or without plasma and extraction with ethyl acetate. e stability of TMEA in rat plasma was evaluated in different storage and handling conditions with all three QC sample concentrations.

Statistical Analysis.
e pharmacokinetic parameters were calculated using DAS 3.0. e T-test was performed using GraphPad Prism 8.0. e data were expressed as mean ± standard error. A difference at p < 0.05 was considered to be statistically significant.

Establishment of the rombocytopenia Rat Model.
Ideal and appropriate animal models play a key role in drug development. e cyclophosphamide-induced thrombocytopenia rat model used in the study is a classical model in hematological research. Cyclophosphamide is a commonly used antitumor drug that also inhibits hematopoietic and immune functions. e platelet and bone marrow cell counts, DNA and protein content, and spleen and thymus index of the model rats were all found to be significantly decreased compared to normal rats (Figure 3), indicating that the thrombocytopenia rat model was successfully established.

Optimization of Determination Conditions.
Firstly, a rapid, sensitive, and specific method to detect TMEA was established in this study. e use of formic acid contributed to an enhanced ionization efficiency and a stabler baseline (Figure 4). To improve the peak shape and retention time, 0.1% formic acid water and acetonitrile were chosen as the mobile phase. In this experiment, acetone, ethyl ether, dichloromethane, chloroform, ethyl acetate, and other solvents were used to investigate the extraction efficiency of TMEA.
e results showed that the recovery of acetone, ethyl ether, dichloromethane, and chloroform was much lower than that of ethyl acetate. Hence, ethyl acetate was selected as the extraction solvent (Table 1).

Method Validation.
e specificity of the method was investigated by analyzing the rat plasma from different sources. e typical chromatograms of blank plasma, blank plasma spiked with TMEA and IS, and plasma collected after the oral administration of TMEA are shown in Figure 5. e retention times of TMEA and IS were 1.183 and 1.530 min, respectively. e chromatograms showed no endogenous peaks interfering with the detection of TMEA and IS, establishing the excellent specificity of this method. e calibration curve of TMEA was fitted to linear regression in the concentration range of 0.5 to 400.0 ng/mL. e linear equation was y � 0.013752x + 0.006824 (r 2 � 0.9996). e lower limit of quantification (LLOQ) was defined as 0.5 ng/ mL in the study. e intraassay and interassay precision for TMEA is shown in Table 2. e intraday RSD was <9.44%, interday was RSD <9.50%, and its relative error of accuracy was between −5.70% and 2.96% in rat plasma. e recovery and matrix effect of TMEA at three QC concentrations are enumerated in Table 3, the former was between 94.77% and 102.47% and the latter was between 93.16% and 100.15%. As shown in Table 4, the stability of TMEA in different storage conditions contained at 25°C for 12 h, 4°C for 24 h, −80°C for one month, and three freeze-thaw cycles was evaluated at 1, 10, and 100 ng/mL. ese results indicate stability in the rat samples during the preparation and analytical processes, with the stability of RSD between −8.55% and 2.98%. ese findings suggest that the method was sufficiently sensitive for investigating the pharmacokinetics of TMEA in vivo. Table 5 and Figure 6, with a remarkably decreased T max and CL/F, the values of AUC (0 − t) , AUC (0 − ∞), and C max of TMEA in the TMEA group were clearly higher than those in the normal group. e results showed that the pharmacokinetics of TMEA differed between the normal and thrombocytopenia rats, which indicated that the pharmacokinetic changes may be caused by the pathological conditions. A similar phenomenon has also been observed in relevant literature. Zhao et al. [18] have reported that the C max and AUC of genipin 1-gentiobioside, geniposide, naringenine-7-rhamnosidoglucoside, hesperidin, and neohesperidin in the depression rats increased significantly compared to the normal rats after the administration of Zhi-Shi-Zhi-Zi-Chi decoction. Hui et al. [19] found that there were significant differences in AUC, mean residence time (MRT),    Journal of Chemistry CL/F, and the apparent volume of distribution (V z /F) between the normal and arthritis rats for the five active components of Periploca forrestii Schltr (P. forrestii): neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid C, and periplocin. Compared to cerebral ischemia rats, the CL/ F value of tanshinol, hydroxysafflor yellow A, and salvianolic acid A and B was lower in the normal rats, although AUC was increased after the intravenous injection of Salvia miltiorrhiza Bge. and Carthamus tinctorius L. ese findings might be related to the destruction of the blood-brain barrier [20]. Fan et al. [21] initially reported that the pharmacokinetics of thirteen phenolic acids and six triterpenoids were significantly different between the normal and leukopenia rats after the oral administration of S. officinalis L. extract. In chemotherapyinduced thrombocytopenia rats, the C max and terminal half-life (T 1/2 ) values for the H12-(ADP)-liposomes were decreased, however, CL/F was increased compared to the normal rats [22]. e intestinal flora may be altered in pathological conditions, potentially affecting drug metabolism. e intestinal tract is considered to be the largest immune organ in vivo and is the center of numerous immunoregulatory molecules that act to resist an invasion by foreign pathogens and maintain the stability of the human internal environment. A clinical study found that most patients with immune thrombocytopenia who also have intestinal infections or gastrointestinal dysfunction presented improved platelet counts after the alteration of intestinal flora [23]. At the moment, it is generally accepted that the imbalances in the intestinal flora may be highly correlated with the occurrence and development of immune thrombocytopenia. Gallotannins from S. officinalis L. can be hydrolyzed to an ellagic acid, which can be further metabolized by the intestinal flora into urolitins, such as urolitin A, urolitin B, urolitin C, urolitin D, and urolitin E [24]. It is possible that thrombocytopenia can be cured by regulating the intestinal flora after administering TMEA, DMAG, and DOMA. However, further study is needed to test this hypothesis.    Abbreviations: NG, normal group; TG, TMEA group; TMG, TMEA-DMAG group; TOG, TMEA-DOMA group; AUC 0 − t , area under the concentration-time curve from 0 to 72 h; AUC 0 − ∞ , area under the concentration-time curve from 0 to time infinite; MRT, mean residence time; t 1/2 , half-life of elimination; T max , time to achieve maximum concentration; V z /F, volume of distribution; CL/F, clearance; C max , maximum value of concentration; * * P < 0.01, * * * P < 0.01 significant compared with NG, ▲ P < 0.05, ▲▲ P < 0.01 compared with TG.

Pharmacokinetic Study. As shown in
e AUC (0 − t) , AUC (0 − ∞) , and C max values for TMEA in the TMEA-DOMA group were nearly two times those of the TMEA group, while the CL/F value was remarkably reduced compared with the TMEA group. Unlike with DOMA, the AUC (0 − t) and C max values were significantly decreased for TMEA with clearly prolonged T max and increased CL/F after combination with DMAG (Table 5 and Figure 6). e results indicated that the pharmacokinetics of TMEA were altered after compatibility with DMAG or DOMA, which suggested that the pharmacokinetic changes may also be caused by compatibility. is phenomenon has been found in literature. According to the past research, T max was increased while C max and AUC were decreased for albiflorin in the extracts of Danggui Shaoyao powder [25]. Liu et al. found that the pharmacokinetic parameters and brain distribution of magnolol and honokiol from the extracts of Magnolia officinalis Rehd. et Wils. were altered after combination with Gardenia jasminoides Ellis. and Citrus aurantium L. in Zhi-Zi-Hou-Po decoction [26]. For example, V z /F and CL/F of catechin, epicatechin, and ziyuglycoside I were significantly reduced, while the T max value of ziyuglycoside I was lengthened with an accessorial C max alongside an increase in the proportion of tannins [27]. e pharmacokinetic properties of typhylside, vanillic acid, and p-coumaric acid in Typha angustifolia L. were also altered after the compatibility with the components of trogopterus dung [28].
Compatibility may have an impact on the content and activity of the drug metabolizing enzymes. e liver is the primary metabolic organ, and the liver microsomes house most metabolic enzymes, including cytochrome P450 (CYP450), monoamine oxidase (Mao), lipase, glucuronidase (UGT), and glutathione transferase (GST). CYP450 levels have been shown to significantly decrease upon the administration of tannins from Rubus chingii Hu [29]. e GST activity was also found to be inhibited by the ellagic acid in a noncompetitive manner, suggesting that the ellagic acid may affect its active site [30]. ese studies imply that the pharmacokinetics of TMEA in rats could be affected by DOMA, which is a similar kind of ellagic acid, by way of inhibiting CYP450 expression and GST activity. At the same time, DOMA can be converted to TMEA with the help of microsomes by the loss of glycosyl. However, because of the different chemical structure of DMAG, it may quicken the metabolism of TMEA to reduce the absorption.
Compatibility may also affect the function of efflux proteins such as breast cancer resistance protein (BCRP), multidrug resistance-associated protein 2 (MRP2), and especially P-glycoprotein (P-gp). e chemical composition of a herb is complex. ere are several active components in each herb that may interact in vivo and directly affect the efficacy and toxicity. As the transport rates of the gallic acid and ellagic acid were changed with the addition of verapamil, some components of S. officinalis L. may be substrates of P-gp [31]. It suggests that the other components of S. officinalis L., such as TMEA and DOMA, may also be the substrates of P-gp, may competitively bind P-gp, and affect the pharmacokinetic parameters of TMEA. Nevertheless, DMAG may be not the substrate of P-gp, BCRP, and MRP2.
is study found that pathological conditions and compatibility can affect the pharmacokinetics of TMEA. However, the molecular pathways involved remain unclear and need further research. Meanwhile, drug safety is key during the development of new drugs, and zebrafish has become the preferred model for screening and evaluating drug toxicity. A zebrafish model will be used to further investigate drug distribution in various organs in vivo and further evaluate toxicity.

Concluding Remarks
In this paper, a rapid, sensitive, and specific UHPLC-MS/MS method for the determination of TMEA was established and successfully applied in pharmacokinetic research in a rat model for the first time.
Data Availability e data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors have declared no conflicts of interest.