Simultaneous Determination of Seven Phenolic Acids, Two Flavonoids, and Tussilagone in Rat Plasma after Administration of Farfarae Flos Extract by LC–MS/MS

A simple LC-MS/MS method was established for the simultaneous quantitative analysis of concentration of seven phenolic acids, two flavonoids, and tussilagone in biological samples. The lower limit of quantitation of each target compound was less than 10 ng·mL−1. The precision of these three types of compounds was less than 15%, and all accuracy was between 85.9% and 115%. The preliminary pharmacokinetics of these three types of compounds in plasma samples were carried out using LC-MS/MS after administration of Farfarae Flos extract (3.90 and 7.80 g·kg−1) to rats, respectively. The results showed that Tmax of all ten compounds varied from 0.21 ± 0.04 h to 0.69 ± 0.19 h. Maximum concentrations and area under concentration-time curves of seven analyzed phenolic acids were higher than those of the two flavonoids and tussilagone. Terminal elimination half-life of tussilagone was the shortest among these three types of compounds. The results showed that the developed LC-MS/MS method was suitable for clarifying the pharmacokinetic characteristics of these three types of compounds in plasma after administration of Farfarae Flos extract in rats.


Introduction
Farfarae Flos has been used as the herbal medicine to treat the diseases for thousands of years in China. It is the dried flower bud of the perennial herbal plant Tussilago farfara L and is named as Kuandonghua in China [1]. Farfarae Flos has been used as a main herbal medicine to relieve bronchitic and asthmatic conditions in clinic. It has many pharmacological effects including antioxidant [2], antimicrobial [3], antitubercular, [4] and inhibitory a-glucosidase activity [5]. Furthermore, some literature studies have reported that Farfarae Flos could be applied to the treatment of pulmonary, obesity, type 2 diabetes, and even hepatitis [3,5]. e compounds in Farfarae Flos include some phenolic acids, flavonoids, terpenoids (tussilagone), and pyrrolizidine alkaloids [6]. ese complex compounds have different biological activities. Phenolic acids and flavonoids from Farfarae Flos have antimicrobial activity [3]. Tussilagone is selected as an index to evaluate its quality of Farfarae Flos in the content determination item in Chinese Pharmacopoeia (2020). It was reported that tussilagone had the anti-inflammatory and antitumor effects [7][8][9]. In order to clarify the pharmacological effect, it is very important to detect the concentration of these three types of compounds in the biosamples after intragastric administration of Farfarae Flos.
At present, there were a great deal of methods to detect the content of active components in the medicinal materials and decoction pieces of Farfarae Flos samples. e contents of phenolic acids, senkirkine, and senecionine were quantitatively assayed by HPLC and LC-MS methods, respectively [10,11]. Meanwhile, GC-MS and NMR spectroscopies were developed and validated to investigate and identify the metabolic fingerprinting of Farfarae Flos [12,13]. However, there is no bioanalysis method to determine these three types of compounds including flavonoids, terpenoids and phenolic acids in the plasma and to study the pharmacokinetics characteristics of three types of compounds after intragastric administration of Farfarae Flos extract.
In present study, the concentrations of seven phenolic acids, two flavonoids, and tussilagone in plasma samples were successfully, quantitatively, and simultaneously analyzed by the newly developed LC-MS/MS method. All pharmacokinetic characteristics of three types of compounds successfully fully illuminated after intragastric administration of Farfarae Flos to rat.

Farfarae Flos Sample Preparation. Farfarae
Flos were purchased from a herbal market of Anguo city (Hebei, China). e plant was identified and authenticated as Farfarae Flos by Dr. Guangying Yu (Department of Traditional Chinese Medicine, Tianjin Hospital). e sample of Farfarae Flos was storied in Tianjin Hospital (Tianjin, China). One kilogram of the raw Farfarae Flos sample was extracted using 10 liters of 95% ethanol for 2 hours by a reflux method, and the supernatant was added into the container. e residue was then extracted by 60% ethanol solution (10 Liters) for another 2 hours. e extract solutions were mixed together and then concentrated at 40°C until they were dry. A final extract of 376 g was obtained and stored at −80°C in a refrigerator.

Preparation of Different Types of Solutions.
Stock solution of 10 analytical compounds (1.00 mg·mL −1 ) and three internal standards (ISs) was dissolved in methanol and placed in the refrigerator before testing. Methanol was used to prepare ferulic acid (IS 1 ), puerarin (IS 2 ), and artemisinin (IS 3 ) solutions to reach a concentration of 1 μg mL −1 , respectively. A specific volume of each analyte stock solution is mixed together to prepare the standard working solution containing 10 analytes with a concentration of 10 times at LLOQ and three levels (high, medium, and low). Quality control (QC) samples for methodological validation were prepared to obtain the final concentration by adding standard working solution (10 μL) into blank plasma samples (100 μL).

LC-MS/MS Conditions.
A high-performance liquid chromatography instrument was used to separate three types of compounds (Agilent 1200 series, USA). e HPLC instrument is composed of four parts; a G1322A degasser, G1312A Bin Pump, G1367B auto-sampler, and G1316A thermostatic column compartment.
ree types of compounds and three internal standards (ISs) were separated on an Eclipse Plus C18 (4.6 × 100 mm, 1.8 μm). e mobile phase was composed of formic acid aqueous solution (A) and formic acid methanol solution (B). e concentration of formic acid in both A and B solution was 0.1% (v/v). e gradient elution was as follows. e proportion of 10% B changes to 40% from 0 min to 3mins. 40% B changes to 90% from 16 to 18 min. B is maintained at 100% from 19 to 28 min.
Quantitative analysis of all samples was performed on Sciex API 3200 with an electrospray ionization (ESI) source. Analyst 1.6.2 workstation for instrument operation, data management, and data analysis were employed to analyze and process data (AB, Sciex). Ten analyzed compounds and three ISs were optimized during multiple reactions monitoring (MRM). e optimized data are shown in Table 1 Phenolic acids (seven acids and IS 1 ) and flavonoids (rutin, isoquercitrin, and IS 2 ) were detected in the negative ionization mode in the first 23 minutes. Tussilagone and IS 3 were detected in the positive ion mode in the next 6 minutes by mass spectrometry. e key source parameters in negative ion mode/in positive ion mode were 4500 V/5000 V ion spray voltage, 500°C/350°C temperature, 25 psi/30 psi curtain Gas, 8 psi/8 psi collision Gas, 30 psi/45 psi ion Source Gas 1, 55 psi/55 psi ion Source Gas 2, respectively. 2.6. Specificity. Biological blank plasma samples were independently taken from 6 rats. e specificity of the newly established LC-MS/MS was investigated by analyzing the chromatograms of blank biological samples, the simulated biological samples with three mixed reference standards at LLOQ, and the chromatograms of real plasma at 30 minutes after intragastric administration of Farfarae Flos extract to exclude endogenous interference. e peak of all analyzed compounds should separate each other. It needs to be found that there were no interferences in blank plasma samples.

Lower Limit of Quantitation, Linearity, Matrix Effect, and Extraction Recovery.
e signal to noise ratio of ten analyzed components should be at least more than 5 at LLOQ. e accuracy should range from 80% to 120% of the nominal concentration while RSDs of determined concentration should be less than 20%. Calibration curves of ten analyzed components were prepared by adding different concentrations of reference standards into blank plasma samples. e stock solutions of ten reference standards were continuously diluted with methanol to achieve the linear concentration of the solutions, as follows: NCLA, CCLA, and IAC (20-60000 ng·mL −1 ); caffeic acid (100-300000 ng·mL −1 ); CLA, isoquercitrin, rutin, and tussilagone (10-30000 ng·mL −1 ); IAB and IAA (40-120000 ng·mL −1 ). e ratio of peak area/IS of 10 analytical components to their actual concentration was used to construct the standard curve by weighted least squares when 1/x 2 was selected as weighting factor. Evidence-Based Complementary and Alternative Medicine e matrix effects of tested compounds were investigated by measuring the percentage of peak areas of 10 compounds added to the blank rat plasma matrix after extraction and the peak areas in the corresponding working solution at three concentration levels.
e extraction recoveries of ten compounds were tested by measuring the percentage of the average peak areas of ten compounds added to blank plasma sample and the  Evidence-Based Complementary and Alternative Medicine 5 average peak areas of ten compounds added to blank matrix after extraction. e matrix effect extract and extraction recoveries at each concentration with RSD <15% were acceptable.

Precision and Accuracy.
e precision and accuracy are tested on the same day and three consecutive days and calculated by QC samples with low, medium, and high concentrations, respectively. RSD of the calculated concentration of each compound was calculated at three concentration of QC samples. e intraday and interday accuracy of the determination of each compound was calculated by the percentage of the measured concentration and the spiked concentration. e RSD value of precision needs to be less than 15%. Accuracy is required to be between 85% and 115%.

Stability.
In the stability test, 24-hour stability ( e samples were placed for 24 hours at 20°C and then measured), freeze-thaw cycles ( e sample was injected after the temperature changing from −20°C to 20°C), and 4 weeks stability ( e sample was determined after stored at −80°C for 4 weeks) of representative QC samples were all tested. All of them were assayed with six replicates.

Pharmacokinetic Study.
Twenty male rats (4 months old, weight 240 ± 20 g) were obtained commercially from Vital River Laboratory Animal Company (Beijing, China). Before the pharmacokinetic experiments, they were free to eat food and drink water for one week for adapting to the environment when the dark light cycle was 12 hours, while the temperature was maintained at 25 ± 5°C. e animal experiments were guided and strictly approved by Tianjin Hospital animal ethics committee. ey were fasted for 12 hours and could only drink freely water before intragastric administration of Farfarae Flos extract. Ten rats in each group were employed for eliminating data variation caused by different animal individuals. All rats in groups 1 and group 2 were orally administrated one time at two dosages of Farfarae Flos extract (carboxymethyl cellulose sodium salt (0.5%) was used to suspend the extract to prepare the administration solution 3.90 g·kg −1 and 7.80 g·kg −1 ), respectively. Rats were anesthetized with ether. e heparinized 1.5 mL polythene tube was used to collect bank plasma samples before administering dose of the extract. About 250 μL plasma samples were collected at each collecting points of 5 min, 10 min, 0.25 h, 0.50 h, 0.75 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 36 h via fossa orbitalis after dosing. All plasma samples from collecting points need to be centrifuged at 4,000 rpm for ten minutes. e supernatant fluid of centrifuged plasma samples was obtained and immediately stored at −20°C in a refrigerator until they were analyzed. e software DAS 1.0 (Anhui, China) was adopted to calculate the main pharmacokinetic parameters of 10 target compounds such as area under concentration-time curve (AUC), elimination half-life (t 1/2 ), and mean residence time (MRT). e maximum concentration (C max ) and the time to reach the maximum concentration (T max ) of ten target compounds were directly calculated by the real concentration-time data in the plasma. e data were expressed as mean ± SD.

Plasma Sample Extraction Method. Ten microliters of
ISs solution (contained puerarin, ferulic acid, and artemisinin) and the plasma sample (100 μL) were added into a clean tube, and then acetonitrile (400 μL) was added to precipitate protein. After centrifugation at 14000 rpm for 10 minutes, a mild nitrogen flow was used to evaporate the liquid of the supernatant to dryness. en, the residue was dissolved in methanol (100 μL). e solution was vortexed for 3 min, mixed for 3 minutes with ultrasound, and Evidence-Based Complementary and Alternative Medicine centrifuged for 10 min. Finally, the solution (10 μL) was used to detect concentration of ten target compounds in rat plasma.

Internal Standards
Selection. e internal standards should have the similar chemical properties and suitable retention time comparable to the analytes. In our experiments, 3 different types of compounds were analyzed. erefore, ferulic acid was selected as the IS 1 for phenolic acid. Puerarin was chosen as the IS 2 for flavonoids. Artemisinin was selected as IS 3 for tussilagone.

Optimization of LC-MS/MS Conditions.
e better compounds separation and peak shape could be obtained when the mobile phase was composed of formic acid aqueous solution and formic acid methanol solution. Ten target compounds and the three ISs were separated well within 29 min. For the mass conditions, the ten analyzed compounds were optimized to obtain a great sensitivity of them. In the first 23 minutes, flavonoids and phenolic acids were detected in the negative ion mode. In the next 6 minutes, tussilagone and artemisinin were detected in positive ion mode (Figures 2   and 3). Retention times of NCLA, CLA, CCLA, caffeic acid, rutin, isoquercitrin, IAB, IAA, IAC, and tussilagone were 8.73 min, 9.75 min, 10.80 min, 11.21 min, 20.52 min, 20.77 min, 15.19 min, 15.51 min, 22.45 min, and 25.61 min, respectively. Retention times of IS 1, IS 2 , and IS 3 were 15.43 min 10.48 min, and 24.69 min, respectively.

Method Validation.
e LC-MS/MS chromatograms of ten target compounds (phenolic acids, flavonoids, and tussilagone) in rat plasma are shown in Figure 3. It was found that there were no endogenous and exogenous substances interfering with the detection of target compounds in the real plasma samples. All compounds showed good linearities according to the correlation coefficients (r 2 > 0.9917). e LLOQs of ten compounds were less than 10 ng ml −1 . e detailed information is listed in Table 2.
e detailed precision and accuracy of ten compounds in QC samples at three different concentration and LLOQ levels are shown in Table 3. RSDs of ten compounds in QC sample at LLOQ were lower than 20%. e accuracy of ten compounds at LLOQ ranged from 80.6% to 112%. Meanwhile, the accuracy of ten compounds in QC sample at other three different concentrations ranged from 85.9% to 116%. All RSDs of ten compounds at three different concentration e recovery and matrix effect of these ten compounds and three ISs are listed in Table 4. It was indicated that the mean recoveries of these ten compounds in rat plasma were more than 80.3% and less than 114%. e matrix effects of   these ten compounds ranged from 85.1% to 115%. Recoveries of ferulic acid, puerarin, and artemisinin were more than 70%. e matrix effect of three ISs ranged from 91.9% to 95.6% with RSD less than 11%. ese data showed that acetonitrile precipitation protein was a reliable method for extracting ten compounds from plasma samples. It was found that ten target compounds were stable in 24 h stability, three freeze-thaw, and four-weeks stability tests (Table 5).

Pharmacokinetics Study.
e pharmacokinetic analysis of seven phenolic acids, two flavonoids, and tussilagone were successfully performed by HPLC-MS/MS. e compartmental analysis was employed to obtain the main key pharmacokinetic parameters of ten bioactive components. e results showed that the pharmacokinetic behavior of seven phenolic acids, two flavonoids, and tussilagone were more consistent with the one compartment model. e concentration-time curves of seven phenolic acids, two flavonoids, and tussilagone in the plasma are shown in Figure 4. It was found that C max of each compound increased with the increase of administration dose ( Table 6). C max of CLA, IAA, and CCLA was more than 1000 ng·mL −1 in the plasma after oral administration of 7.80 g·kg −1 extract while C max of tussilagone was 18.07 ± 12.99 ng·mL −1 . is result showed that three phenolic acids (CLA, CCLA, and IAA) possessed higher exposure concentration in vivo than other seven components. It suggested that these components should be preferentially screened for active ingredients from Farfarae Flos extract to treat some diseases. It was found that T max of ten components was less than 0.69 ± 0.19 h. is result showed that ten components was absorbed rapidly after intragastric administration of Farfarae Flos extract. T max of seven phenolic acids was in the range of 0.21 ± 0.04 to 0.69 ± 0.19 h. e mean residence time (MRT (0-24h) ) (h) values ranged 4.17 ± 1.73 h to 8.80 ± 2.73 h. In previous pharmacokinetic study on chlorogenic acid [15], T max and MRT of chlorogenic acid was 0.70 ± 0.19 h and 5.08 ± 0.89 h, respectively, which is similar to the current research.
Focus on the pharmacokinetic study of flavonoids, T max values of rutin and isoquercitrin were closer at low-dose and high-dose group extract. MRT (0-24h) of rutin were 1.95 ± 1.06 h and 2.79 ± 0.24 h after intragastric administration of extract at low-dose and high-dose groups, respectively. e t 1/2 (h) values of rutin were 0.61 ± 0.46 h and 0.82 ± 0.51 h, respectively. e C max value of tussilagone was 0.83 ± 0.50 ng·mL −1 after intragastric administration of 3.9 g·kg −1 Farfarae Flos, which was closer to the LLOQ of tussilagone. erefore, studying on the high-dose group (7.8 g·kg −1 ) was necessary. After intragastric administration of 7.8 g·kg −1 of Farfarae Flos extract, C max of tussilagone reached 18.07 ± 12.99 ng·mL −1 . Tussilagone was absorbed after intragastric administration and reached T max within 0.40 ± 0.33 h of tussilagone. e value of t 1/2 (h) was 0.82 ± 0.72 h. ese pharmacokinetic parameters will contribute to the clinical application of Farfarae Flos extract.

Conclusion
An accurate, rapid, and reproducible LC-MS/MS method was successfully established to perform the pharmacokinetic analysis of seven phenolic acids, two flavonoids, and tussilagone after intragastric administration of Farfarae Flos. e advantage of this method was that simultaneous detection of ten bioactive components in the plasma can be completed in a run with the relative LLOQs. e pharmacokinetic characteristics of these three types of compounds was obtained in rat plasma after intragastric administration of Farfarae Flos extract. Limitations of this study were that only preliminary pharmacokinetic studies about three types of compounds (ten bioactive components) have been carried out and more information needs to be in depth explored in the future. e pharmacokinetic profiles of ten active compounds were characterized for the first time, which will be useful for future clinical applications of Farfarae Flos.

Data Availability
e data used to support the findings of this study are included in this article.

Conflicts of Interest
All authors declare that there are no conflicts of interest.