Identification and Quantification of Chlorogenic Acids from the Root Bark of Acanthopanax gracilistylus by UHPLC-Q-Exactive Orbitrap Mass Spectrometry

The purpose of this study is to identify and quantify the chlorogenic acids (CGAs) from the root bark of Acanthopanax gracilistylus, which is conventionally regarded as a tonic in folk Chinese Traditional medicine. The effective methods for identification and quantification analysis of CGAs were developed based on ultra high performance liquid chromatography-Q-exactive orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) in parallel reaction monitoring (PRM) and selected reaction monitoring (SIM), which showed high sensitivity and resolution for screening and quantifying compounds. The root bark of A. gracilistylus was extracted under ultrasonication with 70% methanol. Ultimately, a for total of 70 CGAs, 64 of these were tentatively identified for the first time. Moreover, a methodological study of seven kinds of CGAs was carried out. The proposed procedure was optimized and validated in terms of selectivity, linearity of analytical curves (r2 > 0.990), accuracy (recovery range from 96.7 to 105%), and repeatability (relative standard deviation <5%). Then it was applied to determine the content of the CGAs in A. gracilistylus roots from 66 of different batches. The total CGAs was quantified in a range between 2.150 and 33.51 mg/g, which could be considered as excellent source of natural bioactive compound. The result was extremely useful for understanding the bioactive substance and quality control of A. gracilistylus in depth.


INTRODUCTION
Acanthopanax gracilistylus W. W. Smith (AGS), belonging to the genus Araliaceae, is generally distributed in the Hubei and Anhui provinces of China as a tonic and folk medicine that plays a crucial role in treating paralysis, bone pains, arthritis, rheumatism, and liver disease. 1−3 In addition, A. gracilistylus combined with several other kinds of traditional Chinese medicines (TCMs) have been made into Wujiapi liquor, which is a famous Chinese medicinal liquor that has been as a sort of TCM health food product for hundreds of years. It not only is not only a potable spirit that has a mellow taste and a long aftertaste but also has the functions of promoting blood circulation and enhancing human immunity as an ingredient of Chinese herba preparations. 4−6 In addition, previous phytochemical investigations on the root bark of A. gracilistylus indicated that volatile oils, terpenoids, and phenolic acids are the primary chemical components that are responsible for its biological and pharmacological activities of anti-inflammatory, antifatigue, antiaging, and antidiabetic. 7 In general, it is recorded that the biological and pharmacological functions of herbs are extremely dependent on the composition of active ingredients, offering powerful assistance on reducing the probability of many chronic diseases. 8,9 In particular, chlorogenic acids (CGAs) play a vital role in the total dietary intake of phenols in the daily human diet and have been classified into a family of polyphenolic compound and esters formed between cinnamic acid derivatives (such as caffeic, ferulic, and coumaric acid) and quinic acid are also a kind of plant defense that has been shown to reduce the definite risk of type 2 diabetes, obesity, Alzheimer's disease, eclampsia, and stroke and shown to possess the active effects of promoting cell proliferation and differentiation as well as anti-inflammatory, antineoplastic, and antioxidant properties. 10−13 To our knowledge, a majority of reports draw strong attention to the pharmacological activities of A. gracilistylus, but there is no adequate relative literature that systematically illustrates the effective constituents and content of A. gracilistylus, especially CGAs. In addition, it is essential to thoroughly investigate the content difference of CGAs. 14 Thus, it is necessary to develop a sensitive, effective, and rapid method for identification and quantification of CGAs from the root bark of A. gracilistylus.
A few methods have been applied to identify the structure of CGAs with the goal of discovering important information that sufficiently explains or takes advantage of available plants. With the recent progress of mass spectrometry and separation methods, liquid chromatography coupled with tandem mass spectrometry, particularly, UHPLC-Q-exactive Orbitrap mass spectrometry, has acquired considerable attraction for the qualitative and quantitative analysis of phenolic acid compounds, which reveals its remarkable high resolution and separation capability in chemical characterization and affords accurate mass measurement (<5 ppm) for providing evidence in trace analytes in complex matrices and detailed mass spectral information. It also shows higher sensitivity in full scan mode and a higher intensity range than triple quadrupole mass spectrometry and time-of-flight mass spectrometry (TOF MS). 15−18 Finally, a rapid and sensitive UHPLC−MS method was considered as one of the most available detection techniques for determination of CGAs.
The aim of this study is to develop an effective UHPLC-Q-Exactive Orbitrap MS method for simultaneous determination of CGAs from the root bark of A. gracilistylus.

Identification of Chemical Compositions.
A total of 70 CGAs were explicitly identified using UHPLC-Q-exactive Orbitrap MS based on comparison of retention time and detailed mass spectrometric data in Table 1. The proposed fragmentation pathways for CGAs in the root bark of A. gracilistylus have been speculated, taking isochlorogenic acid A as an example in Figure 1. High-resolution extracted ion chromatography in negative mode is shown in Figure 2     linearity, and the values of linear ranges (r 2 ) of CGAs calculated from analytical curves both were >0.990 and linearity equations were listed in Table 2. RSDs of the repeatability test of seven kinds of CGA ranged from 2.10% to 3.32%. In addition, the accuracy of the proposed method was assessed in which 0.25 g of the A. gracilistylus roots powder was mixed with a known amount of seven CGAs reference substances and then extracted by the "4.2 Sample preparation" method. Ultimately, the results indicated that the UHPLC-Qorbitrap method possessed good accuracy with recoveries ranging from 96.7% to 105%, while all of the RSDs were less than 5% (Table 2).

Quantification of the CGAs from the Root Bark of A. gracilistylus from Different Batches.
Sixty-six different batches of A. gracilistylus root were extracted by the "4.2" method, and four batches were parallel. The optimized and validated UHPLC-Q-Orbitrap method was used for analysis. The contents and total contents of seven kinds of CGA S in different batches of the root bark of A. gracilistylus are shown in Table 3. The results are expressed as the average content, which range from 2.150 to 33.51 mg/g.

CONCLUSIONS
In conclusion, the qualitative and quantitative methods using UHPLC-Q-Exactive Orbitrap MS combined with PRM mode and SIM mode were successfully established in this study. Finally, a total of 70 CGAs (64 of them for the first time) and 7 CGAs were identified and quantified from the root bark of A. gracilistylus, which suggested that A. gracilistylus is an excellent    Other reagents were of analytical grade.

Sample Preparation.
A stock standard solution of each standard at 1 mg/mL was prepared by accurately weighing solid standards and being dissolved in methanol. The individual solutions of 7 reference standards were mixed and diluted in methanol at 10 μg/mL to prepare standard working mixture solutions.
The root bark of A. gracilistylus samples was ground into powder, accurately weighed to 0.5 g, and extracted under ultrasonication with about 5 and 75 mL of 70% methanol for 1 h, respectively; afterward, the extracted solution was filtered by a 0.45 μm microfiltration membrane. All working solutions were stored at (4°C) until qualitative and quantitative analysis. MS analysis was performed on a Thermo Scientific Q-Exactive Focus Orbitrap MS (Thermo Electron, Bremen, Germany) operated with a heated electrospray ionization (HESI) in negative ion mode. The mass spectra were acquired with full MS mode in a mass range from m/z 100−1200 at a resolution of 70000, combined with the data dependent scan (dd-MS 2 ) at a resolution of 35000 and isolation window at m/ z 3.0. Other Q-Exactive general parameters were nebulizer pressure at 10 arb, sheath gas and auxiliary gas at the flow rate of 30 arb, capillary temperature at 320°C, auxiliary gas heater temperature at 350°C, spray voltage at 3.2 kV, and S-lens level at 50. The method for quantitative analysis of CGAs was validated with regard to its selectivity, linearity, sensibility, accuracy, and precision following the 2020 edition of Chinese Pharmacopoeia guidance document on analytical quality control and method validation procedures. The selectivity of the method was ascertained by analyzing the standards of seven kinds of CGAs and the samples. The peaks for the studied compounds in the samples were confirmed by comparing the retention times of the peaks with those of standards as well as by recognizing both the full MS precursor and product ions MS 2 with an mass error below 5 ppm. The linearity of the methods of isochlorogenic acid C, isochlorogenic acid A, 1,5-dicaffeoylquinic acid, 1,3-dicaffeoylquinic acid, cryptochlorogenic acid, neochlorogenic acid, and chlorogenic acid were assessed using six concentration ranges, respectively. Repeatability is a measure of repeatability of the analytical method in the normal operating conditions and expressed as the percentage relative standard deviation (% RSD). The accuracy is based on recovery studies in the present work. Stability studies of the method including short-term stability (room temperature, 4 h), and postpreparative stability (storage in the autosampler, 10°C, 24 h) were achieved by the test of sample with six replicates. 4.5. Data Processing and Analysis. Xcalibur 4.1 (Thermo Scientific, CA) was applied in the acquisition of raw data in full-scan/dd-MS 2 mode. Compound Discoverer version 3.0 (Thermo Scientific, CA) was used to dispose the data which passed the workflow templates to predict some expected compounds.

Quantification of CGAs in
The data were input into Excel for statistical analysis. Seven kinds of chlorogenic acids were determined from 66 different producing areas of the root bark of A. gracilistylus. All analyses were conducted in triplicate. The data is presented as a mean.