Quality Evaluation of Artemisia capillaris Thunb. Based on Qualitative Analysis of the HPLC Fingerprint and UFLC-Q-TOF-MS/MS Combined with Quantitative Analysis of Multicomponents

In this study, a new method was developed for the comprehensive quality evaluation (QE) of Artemisia capillaris Thunb. (A. capillaris, named Yinchenhao in Chinese), which is one of the most commonly used herbal medicines (HMs). First, fingerprints of 31 batch samples of A. capillaris were determined by HPLC, the reference fingerprint was established, and the common peaks were assigned. Second, the components of common peaks in the HPLC fingerprints were identified by ultrafast liquid chromatography- (UFLC-) Q-TOF-MS/MS. Finally, the contents of the components unambiguously confirmed by reference substances were determined, and the correlation between the contents of chlorogenic acid and the contents of others was analyzed. The results showed that there were 20 common peaks in the HPLC fingerprints of 31 batch samples. The components of these 20 common peaks were identified as ten organic acids, eight flavonoids, and two others. Among nine organic acids such as 1-caffeoylquinic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, 1,3-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid, three flavonoids such as rutin, hyperoside, and isoquercetin, and one other p-hydroxyacetophenone, a total of 13 ones were unambiguously identified by comparison with reference substances; one caffeoylquinic acid glucoside and one flavone di-C-glucoside were detected in A. capillaris for the first time. There were some differences in the contents of 13 components in different samples; chlorogenic acid could be regarded as the quality marker of A. capillaris. The current established method in this study can be used for the comprehensive QE of A. capillaris and can also provide reference for the QE of the other HMs.


Introduction
e quality and quality evaluation (QE) method is crucial in the effectiveness and safety assessment of herbal medicines (HMs) [1]. e method for the QE of TCMs must be based on the holistic principle, and fingerprint describes integral characterization and reflects interactive aspects of complex components; therefore, it can offer the possibility of evaluating quality of HMs following the overall principle [2]. HPLC fingerprint has become the most widely used method due to its high reproducibility and sensitivity [3]. e HPLC fingerprint method applied to QE of HMs is mainly based on the similarity of fingerprints and the presence or absence of chromatographic peaks among samples [4,5]; however, this method cannot identify what these peaks are. Q-TOF-MS/ MS is a kind of tandem mass spectrometry providing a high mass resolution and accurate mass measurement for the structural elucidation of unknown chemicals [6] and can be used in the identification of common peaks in HPLC fingerprints. Based on the qualitative analysis of fingerprint, the quantitative analysis of multiple components is the key step of QE of HMs [5].
Artemisia capillaris unb. (A. capillaris, named Yinchenhao in Chinese) is one of the most commonly used HMs [7], which has been used in China, Korea, and Japan for a long time to treat liver and choleretic disorders, such as cholestasis, jaundice, liver fibrosis, and hepatitis [7][8][9]. e major components contained in A. capillaris include organic acids, flavonoids, coumarins, essential oil, and others, such as p-hydroxyacetophenone [7]. A characteristic fingerprint was developed to determine the volatile constituents in essential oil of A. capillaris by GC-MS [10], but the systematic study on the HPLC fingerprint of A. capillaris and the identification of common peaks by Q-TOF-MS have not been reported so far. In recent years, QE of A. capillaris based on multicomponents quantitative analysis has made some progress. Yu et al. developed a method to determine eight organic acids in A. capillaris extract by HPLC, including chlorogenic acid (CA), neochlorogenic acid (NCA), cryptochlorogenic acid (CCA), 1,3-dicaffeoylquinic acid (1,3-diCQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), 3,5-dicaffeoylquinic acid (3,5-diCQA), 4,5-dicaffeoylquinic acid (4,, and caffeic acid [11]. Tian et al. established a quantitative analysis method of six organic acids of NCA, CA, CCA, 1,3-diCQA, 3,4-diCQA, and 4,5-diCQA in A. capillaris and its decoction by HPLC [12]. irteen components including four organic acids, four flavonoids, four coumarins, and one other of p-hydroxyacetophenone and ten components including four organic acids, five flavonoids, and one coumarin of scoparone in A. capillaris were determined by the same method, respectively [13,14]. However, a comprehensive QE method for A. capillaris has not been established so far. erefore, the aim of this work was to establish a new method for the QE of A. capillaris comprehensively based on qualitative analysis of the HPLC fingerprint and ultrafast liquid chromatography-(UFLC-) Q-TOF-MS/MS combined with quantitative analysis of multicomponents.

Apparatus.
Determination of the HPLC fingerprint and the contents of 13 components were performed on a HPLC system (Waters Corp., Milford, MA, USA), equipped with a Waters e2695 separation unit, a Waters 2998 PDA detector, and an Empower 3 data processing system. Chromatographic separation was performed on a Symmetry C 18 column (4.6 mm × 250 mm, 5 μm, Waters Corp., USA). Acetonitrile (A) and 0.1% (v/v) formic acid (B) were used as mobile phases with the following gradient elution: 0−35 min, 5−10% A; 35−65 min, 10−25% A; 65−67 min, 25−90% A; and 67−80 min, 90% A. e flow rate was set at 1.0 mL/min, and the column temperature was maintained at 30°C. e injection volume of sample solution was 30 µL. e detection wavelength of fingerprint and content of p-hydroxyacetophenone, rutin, hyperoside, and isoquercetin was set at 254 nm and that of content of nine organic acids was set at 324 nm.
Identification of the common peaks in the HPLC fingerprint was performed on a UFLC-Q-TOF-MS/MS system. Separation was performed on a UFLC system (Shimadzu, Kyoto, Japan) by using a Symmetry C 18 column (250 mm × 4.6 mm, 5 μm); with the same mobile phases and the same gradient conditions abovementioned, the injection volume of the mixed reference substances solution for qualitative analysis and sample solution was all 20 µL. After separation, mass spectra were acquired on the AB Triple TOF 4600 plus system (AB SCIEX, Framingham, USA) with the following mass spectrometric parameters: ion source, DuoSpray; ESI mode, negative; ion source temperature, 550°C; ion spray voltage, −4500 V; nebulizer gas (gas 1), 60 psi; heater gas (gas 2), 60 psi; and curtain gas (CUR), 35 psi. e TOFMS-IDA-10MS/MS information acquisition method was used to obtain mass spectrometry information, and the parameters were set as follows: decluster potential (DP) of −80 V, collision energy (CE) of −10 eV, accumulation time of 250 ms, mass range of 105-1500 Da for the TOF-MS scan, collision energy (CE) of −35 eV, collision energy spread (CES) of 15 eV, and mass range of 50-1500 Da for the TOF-MS/MS detection. LC-MS/MS data were analyzed using PeakView mass spectrometry analysis software (Version 1.6, AB SCIEX, USA).

Samples and Sample Preparation.
Information on all 31 batches of samples is given in Table 1, among which, 30 batches of A. capillaris (S1-S30) were purchased from different large TCM hospitals in China and authenticated as the dried aerial part of A. capillaris by the chief Chinese pharmacist Xudong Gong, the director of the Nantong Food and Drug Supervision and Inspection Centre. Herbal reference substance of A. capillaris (S31) was purchased from the National Institutes for Food and Drug Control (Beijing, China) in 2018. e samples were dried at 40°C, ground into powder, and then sieved through a 40-mesh screen. Approximately 0.2 g of sample powder was accurately weighed and placed in a 50 mL dark brown volumetric flask. Approximately 49 mL of 50% (v/v) methanol was added and extracted by ultrasonication (200 W, 53 kHz) for 30 min. After cooling to room temperature, 50% (v/v) methanol was added for calibration of the volumetric flask and shaken well. e extract was filtered through a 0.22 μm filter membrane, and the filtrate was taken as the sample solution.

Preparation of Reference Substance Solutions.
Appropriate amounts (5-20 mg) of 13 reference substances were accurately weighted, dissolved with 50% (v/v) methanol, respectively, and 13 reference substance stock solutions were prepared. e mixed reference substances solution for qualitative analysis with a concentration range of 0.4-50 μg/mL of each compound was prepared by accurately absorbing appropriate volume of 13 reference substance stock solutions, mixing them, and diluting the mixture with 50% (v/v) methanol.
Working solution A for quantitative analysis was prepared by the same method as the mixed reference substances solution for qualitative analysis, and the final concentrations of 13 reference substances were at the range of 3.1-193 μg/ mL. Working solution A was diluted two, five, and ten times with 50% (v/v) methanol to prepare working solutions B, C, and D, respectively.

Method Validation of the HPLC Fingerprint Analysis.
e method of HPLC fingerprint determination was validated with precision, stability, and repeatability tests, by using peak 5 (CA) as the reference peak and the relative standard deviation (RSD) value of the average relative retention time (RRT) and relative peak area (RPA) of the 20 common peaks as measure values. In the precision test, six consecutive injections of the same sample (S1) solution were analyzed. Stability was examined by analyzing the sample solution (S1) at 0, 6, 12, 18, 24, and 36 h after preparation. Repeatability was examined by determination of six sample solutions prepared in parallel from S1.

Method Validation of the Quantitative Analysis.
e method of quantitative analysis was validated with investigation of linear relationships, limit of quantitation (LOQ), limit of detection (LOD), precision, stability, repeatability, and the recovery test of 13 components. Investigation of linear relationships was performed by precisely injecting working solutions B, C, and D 10 μL e LOQ and LOD values were determined by using signal-to-noise ratios of 3 : 1 and 10 : 1 and injecting 10 μL of above different concentrations of reference substance solutions. By using the RSDs of the peak areas of the 13 components as the measurement values, intraday precision, interday precision, and stability tests were performed, respectively. In the intraday precision test, six consecutive injections of 30 μL working solution A were analyzed, and in the interday precision test, six injections of 30 μL working solution A were analyzed twice a day over three consecutive days. Stability was examined by analyzing the peak areas of nine organic acids at 324 nm and four others at 254 nm detected in Section 2.5 of the stability test. Repeatability was examined by calculating the contents of 13 components according to the peak areas of nine organic acids at 324 nm and four others at 254 nm detected in Section 2.5 of the repeatability test and using the RSDs of the contents as the measured values. In the recovery test, approximately 0.1 g of S1 powder was weighed precisely, and then, 13 reference substance stock solutions were added to the sample in a certain volume according to the approximate proportion of the sample content to the reference substance (1 : 1) to prepare six sample solutions in parallel. e six sample solutions were injected into HPLC, and the average recovery rates and RSDs of the 13 components were calculated.
We followed the methods of Author links open overlay panel [15].

Method Validation of the HPLC Fingerprint Analysis.
e RSDs of RRTand RPA for precision were less than 0.10% and 4.4%, those of stability were no more than 0.08% and 4.7%, and those of repeatability did not exceed 0.08% and 4.8%, respectively. e results met the national standards of TCM fingerprinting [16].

Method
Validation of the Quantitative Analysis. As given in Table 2, the high correlation coefficient values (R 2 ＞ 0.9998) displayed good linearity over a wide range of injected amounts, and as given in Table 3, the RSDs of the intraday precision, interday precision, stability, and repeatability were all less than 5%, and the average recovery rates were in the range of 95.49%-103.20% with RSD values ranging 0.90-4.95%. e above results met the requirements of drug quality standard analysis method in Chinese Pharmacopoeia [17].

Establishment of the HPLC Fingerprint and Similarity
Analysis. 31 batches of A. capillaris samples were determined (chromatograms are shown in Figure 1). e chromatographic data of the samples were imported into the software of Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (version 2012, Chinese Pharmacopoeia Commission, Beijing, China). Using the chromatogram of S1 as a reference, the reference chromatogram was generated, and 20 peaks were extracted to be the common peaks (shown in Figure 1). e similarities between sample chromatograms and reference chromatogram were calculated by the above software, and the results showed that the similarities of 31 batches of A. capillaris were all greater than 0.9 (Table 3).

Identification of the Common Peaks by UFLC-Q-TOF-MS/
MS. Since more information and higher identifiability of fragmentation was observed in the negative ion mode, it was chosen for MS analysis rather than the positive ion mode. First, total ion chromatograms of the A. capillaris sample and mixed reference substances ( Figure 2) were extracted using PeakView mass spectrometry analysis software. Second, the mass spectral data and dissociative rules of the reference substances were summarized, and the law of the quasimolecular ion [M − H] − and/or [M + Cl] − that could be selected as the precursor ion for collision-induced dissociation fragmentation to produce MS/MS product ions spectra was revealed. Finally, the components of the total 20 common peaks in the HPLC fingerprint were identified by comparing the retention time, m/z of [M − H] − and/or  Table 4.
Galactosy1 Rhamnosy1 Rutinosesy1  [20], and its possible dissociation pathway is shown in Figure 3. To the best of our knowledge, this component was also first detected in A. capillaris. e component of peak 11 was identified as the isomer of rutin according to literature [21].

Wavelength Selection for Quantitative Analysis of 13
Components. It was found that all 13 components could be detected at 254 nm, but the peak of 3,4-diCQA (peak 18) has not been completely separated from the nearby ones; organic acids had strong absorption near 324 nm, but there was almost no absorption of p-hydroxyacetophenone (peak 8) at this wavelength. erefore, 324 nm was selected as the detection wavelength for nine organic acids, and 254 nm was selected for other four components. e chromatograms of the mixed reference substances and sample are shown in Figure 5.

Contents of 13 Components in 31 Batches of A. capillaris.
As given in Table 5 and Figure 6, there were some differences in the contents of 13 components in different samples, which is basically consistent with the previous literature reports [12][13][14]; however, the content of CA seems to have a certain correlation with the other 12 components and the total of 13 components. erefore, the bivariate correlation analysis method in SPSS 20 statistical software was used to analyze the correlation between the contents of CA and the contents of 12 other components and the total content of all 13 components in 31 batches of A. capillaris. As the results given in Table 6, the contents of 10 components and the total content of 13 components were significantly correlated with the content of CA (P < 0.01 or P < 0.05), except for the content of caffeic acid and 1,3-diCQA, which had poor correlation with the content of CA (P > 0.05). According to the data in Table 5 Figure 5: HPLC chromatograms of the mixed reference substances (a) and A. capillaris sample (b). e number of peaks is the same as Table 4. respectively, so these two components can be ignored to a certain extent. According to the above analysis, the content of other components in different batches of A. capillaris is obviously related to the content of CA, that is to say, the content of CA largely reflects the quality of A. capillaris. erefore, CA can be regard as the quality marker of A. capillaris. CA is the content determination component of A. capillaris in Chinese Pharmacopoeia. It is suggested that hospitals, pharmacies, and pharmaceutical manufacturers purchase multiple batches of A. capillaris and mix the high and low CA content batches before use according to the CA detection report provided by the supplier, so as to ensure clinical safety and effectiveness.

Conclusion
In this study, a new method was developed for the comprehensive QE of A. capillaris based on qualitative analysis of the HPLC fingerprint and UFLC-Q-TOF-MS/MS combined with quantitative analysis of multicomponents. e results showed that there were 20 common peaks in the HPLC fingerprints of A. capillaris.

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

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Rongrong Zhou and Zhihua Dou contributed equally to this work.