Measurement of Pharmacokinetics and Tissue Distribution of Four Compounds from Nauclea officinalis in Rat Plasma and Tissues through HPLC-MS/MS

A rapid, sensitive, selective, and accurate HPLC–MS/MS method was developed and validated for the simultaneous determination of chlorogenic acid, naucleactonin C, khaephuoside A 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in rat plasma and tissues after oral administration of Nauclea officinalis extracts. Chloramphenicol was used as an internal standard (IS). The plasma and tissue samples were extracted by protein precipitation with methanol-ethyl acetate (1 : 1, v/v) including 0.1% (v/v) formic acid. The chromatographic separation was achieved by using an C18 column with gradient elution using mobile phase, which consisted of 0.1% formic acid water (A) and acetonitrile (B) and the flow rate of 0.8 mL/min. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode utilizing electrospray ionization (ESI) in negative mode. The developed method exhibited good linearity (determination coefficients, R2 ≥ 0.9849), and the lower limits of quantification were 2, 5, 5, and 25 ng/mL for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside. The intraday and interday precisions (relative standard deviation, RSD) were less than 12.65%, while the accuracy was ranged from 86.31 to 114.17%. The recovery rate were 51.85–97.06%, 75.99–106.68%, 77.46–105.35%, and 68.36–103.75% for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside the matrix effects were 50.17–116.62%, 86.75–115.99%, 45.79–87.44%, and 51.60–92.34% for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in different matrix. The developed method was successfully applied to a pharmacokinetic study and tissue distribution of four compounds in rats after oral administration of Nauclea officinalis extracts.


Introduction
Nauclea ofcinalis Pierre. ex Pitard, is one of the most commonly used traditional medicines in China and is mainly distributed in Hainan, Guangxi, Guangdong, and other provinces in the south of China [1]. Modern pharmacological studies reported that Nauclea ofcinalis exhibits various biological properties such as antibacterial, anti-infammatory, and analgesic activity [2][3][4][5][6]. As a traditional Chinese medicine, the stems and twigs of Nauclea ofcinalis are used for the treatment of colds, fever, acute tonsillitis, sore throat, and other diseases [1,7]. A number of diferent and excretion of bioactive compounds but also reveal the mechanism of action and the cause of toxicity [19]. Several reports have been published on the pharmacokinetics of the alkaloids of Nauclea ofcinalis. For example, the pharmacokinetics of alkaloids of Nauclea ofcinalis extracts and Danmu preparations in rat plasma [20][21][22] and the pharmacokinetics of Strictosamide in dog plasma [23]. However, as one of the main active compounds of Nauclea ofcinalis, the pharmacokinetics of phenolic acid compounds were rarely researched. To our knowledge, the quantitative detection of pharmacokinetics and tissue distribution of chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside of Nauclea ofcinalis extract have not been reported in rats. Hence, it is necessary to establish rapid and accurate approaches to investigate its pharmacokinetics and tissue distribution.
Te aim of the present investigation is to develop a reliable and sensitive method based on HPLC-MS/MS to quantify the four compounds of Nauclea ofcinalis extract in the plasma and tissue distribution of rats. It is expected that the results of this study can provide a useful reference for understanding the mechanism of action, safety evaluation, and clinical application of Nauclea ofcinalis.

Chemicals and Reagents.
Te stems of Nauclea ofcinalis were collected from Qiongzhong County, Hainan Province, and identifed by Prof. Jianping Tian of Hainan Medical University. Standards of naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)β-D-glucopyranoside were isolated from Nauclea ofcinalis in our laboratory by silica gel column, semi-preparative high performance liquid chromatography, which structure was identifed by NMR, and the HPLC purity was over than 98%. Te standards for chlorogenic acid were purchased from Chengdu Pufeide Biological Technology Co., Ltd (HPLC >98%, Sichuan, China). Chloramphenicol was provided by the National Institute for Food and Drug Control (Beijing, China). Methanol and acetonitrile were all chromatographically pure and purchased from Fisher Scientifc (Fair Lawn, NJ, USA); ethyl acetate and formic acid were all chromatographically pure and purchased from Aladdin Industrial Corporation (Shanghai, China); purifed water was prepared by a LabTower EDI system (Termo Scientifc, USA).
Mass spectrometric detection was performed on an AB-SCIEX API 4000 mass spectrometer (AB SCIEX, Singapore) with ESI in negative ion multiple reaction monitoring (MRM) mode. Te optimized instrument parameters were as follows: nebulizer gas: 50 psi, heated by N 2 gas: 55 psi, ion spray voltage: −4500 V and temperature: 550°C; nebulizer, blowback gas, and collision gas were nitrogen; declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP) of the four analytes and chloramphenicol (IS) are shown in Table 1.

Preparation of Nauclea ofcinalis Extract. Powdered
Nauclea ofcinalis stems (1 kg) were accurately weighed and heated and refuxed by water (1 : 10, w/v) for 2 h. Te extractions were combined and concentrated under reduced pressure to obtain the 25 g of crude water extracts.

Preparations of Standard Solutions, IS, and Quality
Control (QC) Samples. Te chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-Oβ-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside and chloramphenicol (IS) were dissolved in methanol at a concentration of 1 mg/mL as the stock solution. A series of mixed standard working solutions were prepared by diluting the primary mixed stock solution with methanol at appropriate ratios. All the solutions were kept away from light at 4°C until analysis.

Animals.
Male Sprague-Dawley (SD) rats (250 ± 20 g weight) were purchased from Tianqin Biotechnology Co. Ltd. (License No. 4307256220100013061), Changsha, China. Te animals were maintained at 22 ± 2°C and 60% ± 10% humidity with a 12 h light/dark cycle and allowed free access to food and water. All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee at the Hainan Medical University (Haikou, China).

Preparation of Biological
Samples. An aliquot of 100 μL of rat plasma and tissue homogenate were transferred to a 2 mL tube. Subsequently, 10 μL IS (500 ng/mL) were added followed by 1 mL of methanol-ethyl acetate (1 : 1, v/v) including 0.1% (v/v) formic acid. Te sample was then vortexed for 1 min and centrifuged at 13,000 rpm for 5 min. Tereafter, the supernatant was dried at 37°C with nitrogen, which the residue was reconstituted with 80 μL methanol, centrifuged at 13,000 rpm for 5 min, and 5 μL of the sample were injected into the HPLC-MS/MS system for analysis.

Method Validation.
Te method validation assays were carried out according to the U.S. Food and Drug Administration (FDA) Bioanalytical Method Validation (Food and Drug Administration, 2018), including specifcity, linearity, recovery, matrix efect, precision, accuracy, and stability [24].

Pharmacokinetic Study.
Six male SD rats were tested in pharmacokinetics studies. Te rats were housed at 22 ± 2°C and fasted for 12 h with free access to water prior to dosing, which were orally administered Nauclea ofcinalis extracts at the dose of 2 g/kg (equivalent to 32.74 mg/kg chlorogenic acid, 0.78 mg/kg naucleactonin C, 14.1 mg/kg khaephuoside A, and 10.39 mg/kg 3,4-dimethoxyphenyl-1-Oβ-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside). Te blood samples (approximately 0.5 mL) were collected via the venous plexus of the eye socket at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 1, 12, and 24 h under anesthesia. Next, each sample was immediately centrifuged at 4000 rpm for 10 min to acquire the plasma. Te plasma was transferred to new tubes and stored at −80°C until further use. Te pharmacokinetics parameters of the four compounds in rat plasma were calculated by the noncompartment model using Drug and Statistics (DAS 3.3.0) software (Beijing, China).

Tissue Distribution.
Tirty rats were divided into fve groups (n � 6) at random, which were orally administered Nauclea ofcinalis extracts at a dose of 2 g/kg for tissue distribution conducted at 0.5, 1, 2, 4, and 6 h. During the collection, the heart, liver, spleen, lung, kidney, stomach, small intestine, and brain were rinsed with physiological saline solution to get rid of the blood or content and blotted on flter paper and then weighed. Each tissue sample was accurately weighed and homogenized by using physiological saline at four times the tissue weight (w/v). Te homogenates were stored at −80°C until analysis.

Optimization of Chromatographic and Mass Spectrometric
Conditions. Optimization of chromatographic conditions: by analyzing the separation efect and peak shape of four analytes and the IS on diferent columns, we found that the Phenomenex Kinete EVO C18 column gave the best separation and peak shape. Subsequently, by exploring diferent mobile phase systems, such as methanol-water and acetonitrile-water, we found that the four analytes had better response values in acetonitrile. Finally, by assessing the pH of the mobile phase (0.1, 0.2, 0.5, and 0.8% formic acid solution), the response values of four analytes were highest when the concentration of formic acid was 0.1%. Terefore, 0.1% formic acid water-acetonitrile was selected for use as the mobile phase.
Optimization of mass spectrometric conditions: the four analytes and the IS had strong [M-H] − peaks in the negative ion mode, which can be easily broken and used for stable fragment analysis and detection. Terefore, the ESI in negative ion mode and Q1, MS2, and MRM scan modes were adopted. Te chemical structures, precursor ion, and product ion of four analytes and IS are shown in Figure 1.

Optimization of the Sample Preparation.
For sample processing, a simple protein precipitation method was frst tried for methanol and acetonitrile, respectively, but the recoveries of chlorogenic acid and naucleactonin C were unsatisfactory. In addition, liquid-liquid extraction (LLE) was tried for various solvents tested, including n-butanol, dichloromethane, isopropanol, methyl tert-butyl ether, and ethyl acetate, but no satisfactory recovery was obtained for khaephuoside A, and 3,4-dimethoxyphenyl-1-Oβ-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside. Te extraction of chlorogenic acid should be in an acidic condition. Terefore, the extraction results of chlorogenic acid at diferent pH are investigated.
Finally, the combination method using mixture solution of methanol-ethyl acetate (1 : 1, v/v) including 0.1% (v/v) formic acid was selected. Consequently, satisfactory and   consistent recovery from plasma and tissues samples was achieved for four analytes and IS.

Method Validation
3.3.1. Selectivity. Te selectivity was assessed by analyzing chromatograms of blank rat plasma and tissue homogenates from diferent batches, blank plasma and tissue homogenates spiked with analytes and IS, and plasma, tissue samples obtained from rats after oral administration of Nauclea ofcinalis extracts (n � 6). Figure 2 shows typical chromatograms of four analytes and IS in rat plasma (Figures S1 and S2 of the Supporting Material show typical chromatograms of four analytes and IS in liver tissue and kidney tissue). Under the given condition, Chlorogenic acid,

Linearity of Calibration Curve and Lower Limit of
Quantifcation. Te calibration curves were constructed by plotting the peak areas ratios of chlorogenic acid/IS, Naucleactonin C/IS, Khaephuoside A/IS, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside/ IS versus theoretical concentrations. Tis method exhibited a good linear response for the range of concentrations from 2 to 2500 ng/mL in plasma and from 2 to 2500 ng/mL in Journal of Analytical Methods in Chemistry tissues; all determination coefcients (R 2 ) were greater than 0.9849. Te lower limit of quantifcation (LLOQ) was defned as the lowest concentration of a signal-noise (S/N) ratios of 10 : 1, respectively, the precision was less than 15% and the accuracy was within ±20%. Te limits were adequate for studies of pharmacokinetics and tissue distribution by oral administration of Nauclea ofcinalis extracts. Data from the determination are shown in Table 2.

Accuracy and Precision.
Accuracy and precision were evaluated by analyzing QC samples at high, medium, low, and LLOQ concentrations (n � 6) on the same day and three consecutive days using the standard curve, respectively. Te acceptable limits of accuracy were required to be within ±15% of the actual value except when at LLOQ, and the intra-and interday precision and accuracy data of the determination were shown in Table 3. In our study, the intraday precision ranged from 0.62 to 12.65%, and intraday accuracy ranged from 86.48 to 114.17%. Te interday precision ranged from 2.42 to 12.61%, and the interday accuracy ranged from 86.31 to 113.99%. Te results showed the accuracy and precision were within the acceptable limits, which proved the method was reproducible, reliable, and accurate for the determination of four analytes in rat plasma and tissue samples.

Extraction Recovery and Matrix Efect.
Six batches of blank plasma from independent sources were used to obtain extracted samples, postextracted spiked samples, and unextracted samples at high, medium, low, and LLOQ concentrations. Te peak areas of the three types of samples were recorded as A, B, and C, respectively. Extraction recovery was evaluated by A/B. Matrix efects were calculated by comparing the A/C of analytes. Te extraction, recovery, and matrix efect of four analytes in rat plasma and tissues are shown in Table 4 Figure 3, and the main pharmacokinetic parameters were estimated of four analytes in Table 6. Tese compounds were detected at 5 min after oral administration of Nauclea ofcinalis extracts, indicating four compounds were rapidly absorbed, exhibiting a therapeutic efect. Chlorogenic acid, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside were rapidly absorbed and peaked at approximately 0.5 h, while naucleactonin C peaked at 1.66 h, indicating that naucleactonin C was absorbed relatively slowly. Te content of naucleactonin C was much lower than that of the other three compounds in the Nauclea ofcinalis extracts, but naucleactonin C was present in a much larger amount than other three compounds for AUC 0−t (area under the plasma concentration-time curve) and AUC 0−∞ , indicating that naucleactonin C may had excellent bioavailability. However, the bioavailability of chlorogenic acid and khaephuoside A may not be satisfactory, the CL of chlorogenic acid and khaephuoside A were 62.93 ± 15.996 and 54.40 ± 6.04 L/h/kg after oral administration of Nauclea ofcinalis extract, respectively. Excessive clearance rate will afect the residence time of the drug in the body, thereby reducing the efcacy. In addition, literature reported that the prototype chlorogenic acid absorbed into the blood by oral chlorogenic acid  Journal of Analytical Methods in Chemistry 9  Journal of Analytical Methods in Chemistry 11  accounts for only 30% [25], which greatly afected the pharmacological activity of chlorogenic acid from Nauclea ofcinalis. Terefore, considering the clinical application of Nauclea ofcinalis [26], we could design Nauclea ofcinalis to diferent dosage forms with advanced technology in order to improve the bioavailability and fnally increase the efcacy.

Tissue Distribution Study.
Tis method was also applied to investigate the tissue distribution of chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in rats. Te result is shown in Figure 4 that naucleactonin C and khaephuoside A could be detected in all studied tissue. However, chlorogenic acid and 3,4-dimethoxyphenyl-1-Oβ-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside were not detected in the spleen. Te concentration orders in eight diferent tissues were ranked as 3,4-dimethoxyphenyl-1-Oβ-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside > Khaephuoside A > Naucleactonin C > Chlorogenic acid in the small intestine, stomach, heart, liver, spleen, lung, and brain tissues, and naucleactonin C > 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl  genic acid in the kidney. Te contents of the four compounds in the liver, kidney, and lung were relatively high within after oral administration of Nauclea ofcinalis extrac, indicating that the liver, kidney, and lung may be the main target organs for the pharmacological efects of Nauclea ofcinalis. Tis result is consistent with existing research on the inhibition of bronchitis by Nauclea ofcinalis [27]. Te results show that all compounds except chlorogenic acid were more abundant in the small intestine than in the stomach, indicating that most of naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside possibly absorbed by the small intestine after oral administration of Nauclea ofcinalis extract for enter the systemic circulation system. With the exception of chlorogenic acid, the peak times and concentrations of the other three compounds in most tissues are nearly identical to those in plasma, which means that the distribution of the other three compounds depends on the blood fow or perfusion rate of the organ [28]. Te peak concentration of chlorogenic acid in most tissues is much lower than in plasma and may be related to the absorption of the compound in the tissue. Drugs enter tissues that receive high blood fow frst, followed by those that receive low blood fow [29]. Our study demonstrated that the four compounds are mainly distributed in organs with relatively large blood fows, such as the liver and kidneys. In addition to this, the frst-pass efect of oral administration might be the main cause of high distribution in the liver.

Conclusion
We report the development and validation of a sensitive, rapid, and reliable HPLC-MS/MS analytical method for the simultaneous determination and quantifcation of chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in the plasma and tissues of rats. Tis validated analytical method was assessed on the basis of the FDA guidelines for bioanalytical method validation and applied the study of pharmacokinetics and tissue distribution of oral administration of Nauclea ofcinalis extract in SD rats. To the best of our knowledge, this is the frst study that determined the pharmacokinetic and tissue distribution of naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside. In addition, the pharmacokinetics and tissue distribution of chlorogenic acid after oral administration of Nauclea ofcinalis extract in rats was also reported for the frst time.

Data Availability
Te data used to support the fndings of this study are included within the article and the supplementary information fles.  Figure 4: Mean concentration of four analytes in the heart, liver, spleen, lung, kidney, small intestine, stomach, and brain at 0.5, 1, 2, 4, and 6 h after oral administration of Nauclea ofcinalis extracts at a dose of 2 g/kg.