Quality assessment: Qualitative and quantitative analysis of nine active components in Sargentodoxa cuneata by HPLC/Q Exactive Plus MS and UPLC-ESI- MS/MS

Lin Zheng Guizhou Medical University Zu-Ying Zhou Guizhou Medical University Chang-Quan Wang Guizhou Medical University Yong-Lin Wang Guizhou Medical University Zi-Peng Gong Guizhou Medical University Yue-Ting Li Guizhou Medical University Ai-Min Wang Guizhou Medical University Si-Ying Chen Guizhou Medical University Xue Ma Guizhou Medical University Yong-Jun Li Guizhou Medical University Yong Huang (  HUANGY2020@126.com ) Guizhou Medical University


Background
Sargentodoxa cuneata (DXT), also called "DaHuoxue", "HongPiTeng", "HongTeng", etc., was the dried cane of Largentizabalaceae plant Sargentodoxa cuneata (Oliw.) Rehd. Et Wils., which was rst published in "WaiMuMan" class of "Ben Cao Tu Jing· Ben Jing" [1]. It was harvested in the autumn and winter, with side branches removed, cut and dried. And its taste was bitter, and medicinal properties were at, attributed to the large intestine and liver meridian. It had the effects of detoxi cation, promoting blood circulation, removing wind and pain, used for intestinal carbuncle abdominal pain, heat poisoning sores, amenorrhea, dysmenorrhea, tumbling pain and rheumatic arthralgia, which was mainly produced in Anhui, Guizhou, Guangxi, Sichuan, Yunnan, Hubei, and other provinces [2]. DXT was also a traditional medicinal material for the Miao people, which belonged to the "blood circulation" of the "four major blood" of the Miao medicine. DXT was commonly used by Miao nationality to promote blood circulation, remove wind and dehumidify, as well as the treatment of rheumatic bone pain and bruises [3]. The preparations of Compound Xueteng Yaojiu, Guyanning granules, Miao Yaoshengxian decoction, Tongqiaohua suppository decoction, etc. all contained DXT medicinal materials, which had good curative effect [4][5][6]. Modern pharmacological research [7][8][9][10][11] also showed that DXT and its extracts had various physiological activities such as analgesic, anti-in ammatory, antibacterial, anti-oxidation, anti-viral and so on. Current standards and works only required identi cation of its traits, microscopic characteristics [1], moisture, total ash, leachables [2], and thin-layer chromatography [12] to control the quality of medicinal materials, and the requirements for the speci c chemical component contained in medicinal materials were not clear. In the published literature, only salidroside [13,14], chlorogenic acid [9,13,15], total avonoids [16,17], proanthocyanidins [18], volatile oil [19], emodin [20], protocatechuic acid [15], or a few or a class of components were measured. The DXT production areas were widely distributed, and the content of chemical components in different production areas was also different. In the reports of multi-batch multi-component content measurement, Li Hao [21] used spectrophotometry and HPLC to determine the content of total phenols, total saponins, and index components of salidroside, chlorogenic acid, and 3,4dihydroxyphenylethanol glucoside. Li Dihua [22] used HPLC coupled with evaporative light scattering detection to simultaneously determine the content of four active ingredients (3,4-dihydroxyphenylethyl alcohol glycoside, salidroside, chlorogenic acid, and liriodendrin) in thirteen test samples. However, the resolution and sensitivity of these measurement methods were not very high, and the measurement results had certain limitations.
Therefore, in this study, an accurate, e cient, and convenient method for the determination of multiple batches of DXT in different regions was established. HPLC tandem Q Exactive Plus MS was performed to full-component qualitative analysis and the higher-content or characteristic components were screened out.
Subsequently, the contents of the selected components were determined using UPLC-ESI-MS/MS. According to the measured data, further multivariate statistical analysis was carried out to nd the differences in the content of the selected components in DXT in different regions. This might provide a certain basis for improving DXT quality control standards.
Plant materials DXT samples were collected from different provinces in China. These herbs were identi ed by Professor Long Qingde (Pharmacy, School of Pharmacy, Guizhou Medical University). The voucher specimen was deposited in the herbarium of Guizhou Medical University School of Pharmacy.

Preparation of sample solutions
Weighed 0.2 g of DXT medicinal material, added 25 mL of 50% methanol, weighed and re uxed for 2 h, added 50% methanol to make up the weight, took 200 µL, added 50% methanol 800 µL, vortexed, centrifuged at 12, 000 rpm, 10 min. The supernatant sample was taken to inject, and the injection volume was 1 µL. The nal concentration of the DXT extract was 1.0 g/mL.

Results
Identification of constituents in the samples by HPLC and Ultra-high-resolution MS Vanquish horizon HPLC system combined with Q Exactive Plus ultra-high resolution mass spectrometry (Thermo Fisher Scientific, MA, USA) and compound discovery structure identification software was used to detect the main chemical components in DXT extraction solution samples. The precursor ion selectivity of the conjugate hyperboloid quadrupole combined with the high resolution accurate mass detection technology based on orbitrap had the advantages of ultra-high resolution (R = 140, 000 @ m/z 200, could quickly switch between positive and negative at the same time to obtain comprehensive sample information), ultra-high quality accuracy and stability, high sensitivity (ag-fg level) and ultra-wide linear range (10 4 -10 5 ), which could greatly increase the credibility and efficiency of identification.

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In the Q Exactive Plus MS system, an electrospray ion source was used for detection in the positive and negative ion scanning modes, and the conditions are as follows: Spray voltage: The total ion chromatograms in negative ion and positive ion modes were shown in Fig.1 and 2, and the mass spectrum details of the measured component obtained from the detector were shown in Table 1. A total of twenty-one components were tested and identified. Based on preliminary experiments, nine components with high activity or high content were selected for subsequent content determination. The nine ingredients were protocatechuic acid, hydroxytyrosol, caffeic acid, epicatechin, salidroside, chlorogenic acid, desrhamnosyl isoacteoside, rutin, and liriodendrin. And the chemical formulas were shown in Additional file 2. The specificity, calibration curves, limits of detection (LOD), quantitation (LOQ), precision, stability, repeatability, and recovery were investigated.
The specificity was investigated by taking the mixed reference solution and 50% methanol-water blank solvent, and performing sample analysis according to the optimized LC/MS conditions. The calibration curve for each constituent was established by plotting the peak area (y) versus the concentration (x) of each analyte. The LOD and LOQ for nine analytes were estimated at S/N of 3 and 10, respectively, by injecting a series of dilute solutions with known concentration. The intra-day and inter-day precision for each analyte was investigated by determining the nine analytes in six replicates during a single day and three consecutive days. Variations of the peak area were taken as the measures of intra-day and inter-day analysis precision to calculate the RSD. Stability was investigated by analyzing the solution at 0, 2, 4, 8, 12, 24 h, respectively. To assess the repeatability, six solutions prepared from sample were analyzed. Variability was expressed in RSD (%). The recoveries of the analytes were determined by using the method of standard addition within the same day. Three different concentrations of mixed standard solutions (50%, 100%, and 150% of the known amount in sample) were spiked into sample. The recovery results were calculated by comparing the difference between the spiked and the un-spiked sample that were analyzed under the same conditions.
The extracted ion chromatogram obtained from the specificity validation was shown in Fig.3, indicating that the method had good specificity. The calibration curve had good linearity, and LOD and LOQ also met the requirements (as shown in Table 2). During the investigation of the precision, stability, repeatability and sample recovery rate of the nine components, the RSD values were all ≤ 3.2%, which met the requirements. These results were shown in Additional file 3, which showed that the method was stable and reliable. was used for the analyses. The system was also equipped with a Waters VanGuard BEH C18 Capillary ionization voltage, 3 kV; ion source temperature, 120 °C; spray gas and backflush gas, N 2 ; desolvation gas flow rate, 650 L/h; and desolvation gas temperature, 350 °C.
Multiple reaction monitoring (MRM) mode was used for quantification. The optimal parameters for the analytes in the MRM mode are listed in Table 3. All data were obtained using MasslynxTM V4.1 software and processed using the QuanlynaTM V4.1 (Waters Corp., Millford, MA, USA) workstation.

Determination of UPLC-ESI-TQS MS/MS content of nine components in thirty batches of DXT
Nine components with higher content or stronger activity from the tested components were selected, and UPLC-ESI-TQS MS/MS was used to determine the content of different batches of DXT medicinal materials in thirty different regions under the above optimized conditions. The content determination results were shown in Table 4.

Quality assessment
Hierarchical clustering analysis And 2D and 3D plots of loading plot and score plot were obtained as shown in Fig.5.
The loading plot was used to describe data features, it could be known that the relationship between variable attribute features. And the distinction between samples could be got from the score plot. It can be seen from the calculation of the principal component (PC) that the larger the absolute value of the loading, the greater the influence on the PC. Three PCs were extracted (PC 1, 2 and 3), and the cumulative contribution rate was 77.18%.
Combining the distribution characteristics (Additional file 4) and the loading plot ( Fig.5 (a) and (b)) of the content, in the PC1 axis, protocatechuic acid and rutin were negatively correlated and other components were Page 12/28 positively correlated. The contents of the first two component variables in the southwestern samples were higher than that in the eastern samples, while other components in the southwestern samples were less than that in eastern samples, which indicated that the differences in the contents of the component variables in different regions were mainly reflected in this PC. Additionally, hydroxytyrosol and desrhamnosyl isoacteoside were mainly moved on PC1 axis, while not obvious on other axis, indicating that these two components were almost completely affected by PC1, that is, the content of the two components in the eastern region was significantly higher than that in the western region, which was consistent with the quantitative results.
As shown in score plots (Fig.5 (c) and (d)), the samples in the eastern and southwest regions except sample 19 were almost completely separated, which was approximately consistent with HCA result. On the 2D graph, sample 25 from Guizhou Bijie was moved in the second quadrant, and other samples were distributed near by the PC1 axis.
Additionally, the samples derived from eastern regions were shifted in the positive direction of PC1 axis, while the samples in the southwest region were mainly distributed in the negative direction of the axis. Analyzed with 2D load plot, it could be speculated that the medicinal materials from eastern region were mainly affected by the component variables desrhamnosyl isoacteoside, while the medicinal materials from southwestern region were mainly affected by the component variable protocatechuic acid. When the third PC was added, the sample 4 from Jiangxi Jiujiang moved significantly. The quantitative results showed that desrhamnosyl isoacteoside was significantly less than other samples in the same region, while the content of salidroside was significantly higher. Combining the results of loading plots and score plots, the main variable that affects sample 25 was rutin, and the main variable that made sample 4 moved was salidroside.
To sum up, the components that made the quality difference of DXT medicinal materials of the east and the southwest might include desrhamnosyl isoacteoside and protocatechuic acid.

Partial least squares discriminant analysis
In order to further screen the key components that caused the differences in the quality of DXT from various regions, the raw data of the nine measured components of DXT from different regions was input as variable values into SIMCA 14.1 software (Umetrics, Sweden) for partial least squares discriminant analysis (PLS-DA), the variable importance of projection (VIP) value of each index component was obtained. As shown in Fig.6, when VIP > 1 as the screening criterion, it could be concluded that protocatechuic acid, desrhamnosyl isoacteoside, liriodendrin, and hydroxytyrosol had significant differences in the DXT from different regions.

Discussion
The extraction method (re ux method and ultrasonic method), solvent (various concentrations of methanol) and extraction time were studied to optimize the extraction procedure. The results were shown that the ultrasonic method could extract chemical components more effectively than the re ux method. Compared different extraction solvent conditions (25%, 50%, 75%, and 100% methanol), 50% methanol showed complete extraction of all major components. When the pharmaceutical powder was extracted with 50% methanol under re ux for 0.5, 1, 2, and 4 h, re ux was obtained for 2 h to obtain the most complete extraction (Since there was no signi cant difference between 2 and 4 h, 2 h was su cient for extraction.).
Therefore, the preferred extraction method was re uxing in 50% methanol for 2 hours.

Conclusion
Protocatechuic acid, desrhamnosyl isoacteoside, liriodendrin, and hydroxytyrosol were important factors that caused the difference in the quality of Sargentodoxa cuneata from different origins, which may become a new marker for quality assessment of Sargentodoxa cuneata.  Availability of data and materials The research data generated from this study is included within the article.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.