Fn Wu,Yu Zhng,Ynling Qio,Ting Zho,Bojing Zhng,Bngjing Fng,Xiokui Huo,＊,Xiochi M,＊

a Institute of Integrative Medicine,College of Pharmacy,The Second Affiliated Hospital,Dalian Medical University,Dalian 116044,China;

b Department of Emergency,LongHua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China

Abstract Jinhongtang is a traditional Chinese medicine formula composed of Rheum palmatum L.stem,Sargentodoxa cuneata stem,and Taraxacum mongolicum and is used for the treatment of sepsis.However,quality assessment method for Jinhongtang is not available.In present study,we developed a UFLC-MS/MS method to determine 16 analytes in 20 batches of home-made and commercial Jinhongtang.Multivariate statistical analysis revealed the significant differences in the quality of home-made and commercial Jinhongtang and the difference in the quality of home-made samples was more significant.The integrated strategy based on UFLC-MS/MS and multivariate statistical analysis provided a new basis for the overall quality assessment of Jinhongtang.

Keywords:Jinhongtang;quality assessment;UFLC-MS/MS;multivariate statistical analysis

1 Introduction

Traditional Chinese medicine (TCM) is widely used in China and has been accepted as an important complementary medicine by more and more countries and regions in the world.Although significant progress has been made in the study of pharmacological mechanisms of TCM,quality control of TCM is still a challenge due to its chemical complexity [1].Recently,simultaneous determination of multiple hallmark components using high performance liquid chromatography has been recommended by

Chinese Pharmacopoeia

(2020)

to evaluate the quality of TCM.Liu et al.also proposed the new concept of Quality-Marker (Q-Marker) for the quality evaluation of TCM,and believed that Q-Marker should represent both the main chemical constituents and the pharmacologically active ingredients [2,3].Based on these ideas,liquid chromatographytandem mass spectrometry (UFLC-MS/MS) that can simultaneously determine multiple components without requiring long-time chromatographic separation is increasingly being used to develop quality control methods of TCM [4-6].Jinhongtang is a TCM formula composed of

Rheum palmatum

L.stem,

Sargentodoxa cuneata

stem,and

Taraxacum mongolicum

[7].It is used for the treatment of acute abdominal diseases,including acute biliary infection,severe acute pancreatitis,acute appendicitis and sepsis [7-10].Clinical observation revealed that Jinhongtang could reduce fever,relieve abdominal pain,reduce the serum level of inflammatory cytokines,and maintain organisms’balance between inflammation and anti-inflammatory in severe acute biliary infection,pancreatitis and sepsis [7,8,11].Inhibition of excessive inflammatory response has been considered as the key to the treatment of inflammatory acute abdomen,because it can avoid disease progression and reduce the risk of systemic inflammatory response syndrome and multiple organ failure [12,13].It was reported that Jinhongtang improved intestinal mucosal barrier by inhibiting the inflammation and apoptosis of intestinal epithelial cells [7].

Rheum palmatum

L.stem,

Sargentodoxa cuneata

stem,and

Taraxacum mongolicum

in Jinhongtang were commonly used herbs with antimicrobial and anti-inflammatory activities [14-16].The pharmacological activity of Jinhongtang comes from the synergistic effect of the three herbs.Therefore,the quality of Jinhongtang is always affected by the source of crude drugs.Recently,to ensure safety and efficacy,Jinhongtang has been developed into tablets and granules with the optimal standard process adopted.However,systematic quality control method is still not available.To date,there is no report on the quality markers of Jinhongtang.Instead,a totally of 16 components,including Rhein,Emodin,Aloe emodin,Rhein-8-glucoside,Physcion-8-glucoside,Emodin-8-glucoside,Aloe Emodin 8-Glucoside,Catechin,Epicatechin,Gallic acid,Luteolin,4-Hydroxycinnamic acid,Ferulic acid,Chlorogenic acid,Protocatechuic acid and Salidroside,were collected as quality markers of Rheum palmatum,Taraxacum mongolicum,and Sargentodoxa cuneata according to

Chinese pharmacopoeia

(2020)

and previous studies [17-21].In the present study,UFLCMS/MS was employed to simultaneously determine the main active ingredients of Jinhongtang and the difference among batches was further analyzed by multivariate statistical analysis.This is the first report on the quality control method of Jinhongtang,which will provide a new perspective for the quality assessment of other TCM.

2 Material and methods

2.1 Chemicals and reagents

Sixteen reference standards,namely Rhein,Emodin,Aloe emodin,Rhein-8-glucoside,Physcion 8-glucoside,Emodin-8-glucoside,Aloe Emodin 8-Glucoside,Catechin,Epicatechin,Gallic acid,Luteolin,4-Hydroxycinnamic acid,Ferulic acid,Chlorogenic acid,Protocatechuic acid and Salidroside,were purchased from Chengdu Pufei De Biotech Co.,Ltd.(Chengdu,China).Jinhongtang was prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co.,Ltd.(Chengdu,China).

Rheum palmatum

L.stem,

Sargentodoxa cuneata

stem,and

Taraxacum mongolicum

were collected from ten provinces of China,including Hebei,Hunan,Shanxi,Qinghai,Gansu,Anhui,Sichuan,Jiangsu,Yunnan,and Hubei.All of them were deposited at the authors’ laboratory in Dalian Medical University and identified by Professor Xiaochi Ma.All other chemicals were of analytical grade and were obtained from commercial sources.

2.2 Chromatography and MS conditions

The concentration of analysts was determined using AB Sciex Qtrap® 5500 LC-MS/MS system(Foster City,CA,USA).Chromatographic separation was performed on a Phenomenex Kinetex EVO-Ccolumn (100 mm × 2.1 mm,2.6

μ

m) at 30C.The mobile phase consisted of water with 0.1% (

v/v

)formic acid (A) and acetonitrile (B).The flow rate was set to 0.2 mL/min.The following gradient elution program was used:15% B at 0–3 min,30% B at 4–7 min,60% B at 8–9 min,95% B at 10–13 min,and 15% B at 14–16 min.The detection was performed by multiple reaction monitoring(MRM) in negative ionization mode.The source parameters were set as follows:Ion spray voltage,-4.5 kV;Temperature,500 °C;Gas source 1,55 psi;Gas source 2,65 psi;Curtain gas,35 psi;CAD,medium.The MRM transitions and related optimized declustering potential (DP),entrance potential (EP),collision energy (CE),and collision cell exit potential(CXP) for the analytes were listed in Table 1.Analyst 1.6.3 software (Applied Biosystems) was used to control the equipment and collect and analyze data.

2.3 Sample and standard solution preparation

Each reference standard was dissolved in methanol to give stock solutions of 1 mg/mL.Different volumes of the 16 stock solutions were mixed and diluted with mobile phase to obtain standard curve samples,the concentration of which were listed in Table 2.Home-made Jinhongtang was prepared by pulverizing and mixing

Rheum palmatum

L.stem(0.13 g),

Sargentodoxa cuneata

stem (0.43 g),and

Taraxacum mongolicum

(0.43 g).The home-made and commercial Jinhongtang was extracted with methanol to obtain stock solutions of 1 mg/mL.The stock solutions were further diluted 500,1000 and 2000 times with mobile phase to obtain sample solutions.

2.4 Method validation

2.4.1 Specificity,linearity and lower limit of quantification

Selectivity was tested by comparing blank samples,standard curve samples,and sample solutions.Linearity was evaluated by calibration curve using 1/

x

weighted linear least-squares regression model,in which the peak area ratios of analytes were plotted against the corresponding concentrations of standard curve samples.LOD and LOQ were determined by diluting different concentrations of each compound until the signal-tonoise (S/N) ratios reached 3 and 10,respectively.

2.4.2 Precision,reproducibility,stability,and recovery

Precision was expressed as relative standard deviation (RSD) of the results from six determination of the sample solution.Reproducibility was expressed as RSD of the six independent sample solutions prepared as described in Section 2.3.Stability was expressed as RSD of the results of one sample solution examined at 0,2,4,8,12,and 24 h.To test the recovery,three different mixtures of the 16 compounds (50%,100% and 150% of the original content of each compound) were spiked to the sample solutions.Recovery was calculated as the ratio of found concentration and (original +spiked) concentration.The sample solutions were determined in triplicate.

2.5 Multivariate statistical analysis

SIMCAP 14.1 software was used to perform principal component analysis (PCA) and supervise orthogonal partial least squares discriminant analysis(OPLS-DA).

3 Results and discussion

3.1 Optimization of chromatography and mass spectrometric conditions

Chromatography condition was optimized to obtain good separability in a short analysis time.According to

Chinese pharmacopoeia

(2020) and previous studies,totally 16 bioactive components in Jinhongtang including 7 anthraquinones,3 flavonoids,and 6 phenolic acids were collected as the quality markers (Fig.1).

Fig.1 Chemical structures of 16 reference standards

They have similar chemical structures and several of them are isomers,which cannot be distinguished by mass spectrum.Therefore,after comparing with the performance of Xbridge BEH Ccolumn (2.1 mm × 150 mm,2.5

μ

m) column,a Phenomenex Kinetex EVO-Ccolumn (100 mm ×2.10 mm,2.6

μ

m) was used for chromatographic separation.Due to the polarity difference between anthraquinones and phenolic acids,gradient elution with acetonitrile and 0.1% formic acid as the mobile phase was used to reach high resolution and reproducible peak shapes.The column temperature was set to 30C and the flow rate was 0.2 mL/min.Under the chromatography condition,16 analytes were separated in 16 min with good resolution,high detection sensitivity,and symmetric peak shapes.

Mass spectrometric condition was established using reference compounds dissolved in mobile phase.Scanning results of positive and negative ion detection modes of ESI source showed that all analytes had higher response in the negative ion pattern.The deprotonated [M-H]molecular ions were generally produced as precursor ions and their major fragment ions were further obtained for MRM analysis.MRM transitions and optimal compound parameters were listed in Table 1.

Table 1 Mass spectrum parameters of 16 analytes

3.2 Optimization of sample preparation

Different extraction methods (ultrasoundassisted extraction and reflux exaction),extraction solvents (methanol,ethanol,and water) and extraction conditions (volume,temperature,time,and amount of extraction) were optimized to improve the extraction efficiency.The results showed that ultrasound-assisted extraction was comparatively simple and the extraction effect was satisfactory.The optimum conditions were determined as follows:ultrasonic extraction in methanol at 25C for 30 min.

3.3 Method validation

The established method was verified with respect to specificity,linearity,precision,reproducibility,stability,and recovery.Specificity of the method was evaluated by analyzing blank samples,blank solvent spiked with 16 reference standards,and validation samples.None of the 16 analytes were detected in the blank extraction solvent,while the retention time of 16 analytes in the standard samples and the validation samples matched well (Fig.2).

Fig.2 Retention time of 16 analytes in validation samples

All 16 analytes showed good linearity (

R

>0.9992) in an appropriate dynamic range of 128-fold(0.195-7.8 to 25-1000 ng/mL) (Table 2),covering the concentration levels of 16 analytes in all batches.LOD ranged from 0.12 to 3.9 ng/mL (Table 2),indicating the sensitivity of the method.In addition,precisions(RSD%) of 16 analytes were all less than 2.33%(Table 3).In the reproducibility and stability tests,RSD values were below 2.97% and 2.93%,respectively,indicating that precision,reproducibility and stability of the method were acceptable (Table 3).A standard addition test was also conducted to evaluate the recovery of the 16 analytes during sample preparation.Three levels (equal to 50%,100%,and 150% of original concentrations) of the reference standards were spiked into the validation samples and the recovery ranged from 97.8% to 102.9% with RSD values lower than 2.67% (Table 4).The results indicated that the method had high precision,reproducibility,stability,and recovery,and could be used to simultaneously determine the 16 analytes in Jinhongtang.

Table 2 Regression equations,linear ranges,LOD and LOQ of the analytes

Continued table 2

Table 3 Precision,reproducibility,and stability of the analytes

Table 4 Recovery of the analytes

Continued table 4

3.4 Determination of 16 components in crude drugs and formulated products

The validated method was applied to determine the concentration of 16 components for the quality assessment of Jinhongtang.Crude drugs were first analyzed,including

Rheum palmatum

L.stem,

Sargentodoxa cuneata

stem,and

Taraxacum mongolicum

.Among the 16 components in Jinhongtang,anthraquinones,Catechin,L-Epicatechin and Gallic acid were mainly from Rheum

palmatum

L.stem (Fig.3).Most of Luteolin,4-Hydroxycinnamic acid and Ferulic Acid were determined in

Taraxacum mongolicum

,while

Sargentodoxa cuneata

stem was rich in Chlorogenic acid,Protocatechuic acid,and Salidroside (Fig.3A).

Fig.3 (A) Concentrations of 16 components in Crude drugs of Jinhongtang;(B) Concentrations of 16 components in home-made and commercial of Jinhongtang

Anthraquinones,flavonoids and phenolic acids are well known for the antimicrobial and antiinflammatory activities [22-26].The 16 components collectively represented the main chemical constituents and pharmacologically active ingredients,and satisfied the requirements of quality markers for TCM [3].The 16 components could be used as the quality markers for the quality assessment of Jinhongtang.The concentrations of 16 components in home-made and commercial Jinhongtang were further determined to evaluate the potential difference in the preparation process.There was relative difference in the contents of the 16 components in both home-made and commercial Jinhongtang samples.The variance of the ten batches of home-made and commercial Jinhongtang samples were 2.39-82.37-fold and 1.83-3.40-fold,respectively (Fig.3).Gallic acid had the largest variance in commercial Jinhongtang samples(Fig.3),indicating that

Rheum palmatum

L.stem used in the production contained diverse content of Gallic acid.Besides,the contents of the 16 components in the home-made samples varied significantly.The variance of Emodin-8-glucoside was 82.37-fold among the ten batches (Fig.3),indicating significant quality difference among diverse crude drugs.Generally,the chemical constituents in herbal medicine vary significantly due to different cultivation areas and climatic conditions.It has been reported that there exist differences in the chemical compositions and compound contents of

Rheum palmatum

L.stem,

Sargentodoxa cuneata

stem,and

Taraxacum mongolicum

from different sources [27-29].Therefore,some quality markers representing both the main chemical compositions and the pharmacologically active ingredients have recently been recommended for the holistic quality identification of TCM.The UFLC-MS/MS method established in the present study provided a novel,simple and reliable tool for the quality control of Jinhongtang.

In order to further reveal the quality consistency and difference between home-made and commercial samples of Jinhongtang,multivariate statistical analysis was used to compare the contents of 16 components in different batches.Unsupervised principal component analysis (PCA) showed that the first and second principal components with 55.6% and 12.5% of the variability accounted for 68.1% of the total variance (Fig.4A).The results clearly showed two different clusters (Fig.4A),indicating significant quality difference between home-made and commercial Jinhongtang.Besides,the separation between different sources of homemade samples was more significant than that of commercial Jinhongtang,indicating that the difference in the quality of home-made samples was more significant.Similar results were observed in supervised orthogonal partial least squares discriminant (OPLS-DA) analysis,indicating that the quality of commercial Jinhongtang was consistent(Fig.4B).Furthermore,the variable importance in the projection (VIP) scores of compound 4-Hydroxycinnamic acid (12),Protocatechuic acid (15),Aloe Emodin-8-Glucoside (7),Catechin(8),Epicatechin (9),Gallic acid (10),Physcion-8-glucoside (5) and Ferulic acid (6) were higher than 1,indicating that these compounds had significant relationship with the sample variations.

Fig.4 Multivariate statistical analysis of 20 batches of Jinhongtang.(A) PCA score plot.(B) OPLS-DA score plot.

Commercial Jinhongtang was prepared according to the optimal standard process,which effectively reduced errors during mass preparation.Besides,the source of crude drugs might contribute to the quality difference of Jinhongtang.The findings demonstrated the applicability of the established method.

4 Conclusion

In conclusion,the established UFLC-MS/MS method coupled with multivariate statistical analysis provided a powerful tool for monitoring the quality difference of the main active ingredients in Jinhongtang,and could be used for the quality assessment of Jinhongtang.

Acknowledgments

The authors thank National Key Research and Development Program of China (2018YFC1705900),National Natural Science Foundation of China (No.81903706),Distinguished professor of Liaoning Province (XLYC2002008),and Science Foundation of Department of Education of Liaoning Province(LZ2020054) for financial support.

Conflict of interest

The authors declare that they have no conflict of interest.