# Identification of the Constituents of Percutaneous Absorption fromDuhaldea nervosa Based on UHPLC-Q-Exactive Orbitrap MS and Microdialysis Technique

Authors: Lianghong Liu; Lian Zhu; Kaiyan Gong; Xinjun Zhi; Ying Guan; Wei Cai
Journal: International Journal of Analytical Chemistry
 (2019)

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## Abstract

Duhaldea nervosa (D. nervosa) has been used for treatment of bone fracture by external use. Thus, the percutaneous absorption was crucial to the effect of D. nervosa, especially the constituents of percutaneous absorption. However, the constituents in vivo were never investigated to date. In this study, an efficient method was developed for the identification of constituents of percutaneous absorption using UHPLC-Q-Exactive Orbitrap MS and microdialysis technique. A total of 20 constituents including 15 chlorogenic acid analogues, 3 amino acids, and 2 organic acids were unambiguously or tentatively identified based on high-resolution mass data including MS and MS2, chromatography retention time, and bibliography data. To the best of our knowledge, this is the first study to report the constituents of percutaneous absorption from D. nervosa, which will be very helpful for understanding the bioactive compounds and quality control.


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## Body

## 1. Introduction

Duhaldea nervosa (Wallich ex Candolle) A. Anderberg (D. nervosa), which belongs to the plant family Compositae, known as Maoxiucai or Xiaoheiyao in China, is a perennial plant widely distributed in the southwestern region of China and Southeast Asia [1]. Traditionally, it has been used as folk medicine in dispelling wind-chill, alleviating pain, promoting the circulation in meridian and collateral for treating migraine, rheumatism, and traumatic injury, especially in accelerating the healing of a fracture by external use or oral administration [2, 3]. Previous studies showed that the main constituents of this plant are steroids, terpenes, flavones, and chlorogenic acid analogues, which possess a variety of biological activities including anti-inflammatory activity [4–6].Considering the usage ofD. nervosa, which has been used as an external drug for the treatment of bone fracture, the percutaneous absorption was crucial to the effect of D. nervosa, especially the constituents of percutaneous absorption [7–9]. As far as we know, the constituent in vivo has not been investigated; therefore, it is worthwhile to identify the constituents of percutaneous absorption after D. nervosa for external use.In recent decades, microdialysis has become a very powerful sampling technique that enables monitoring of the small molecules invivo due to its excellent versatility [10, 11]. However, it is limited by the method of analyzing the resulting dialysate. LC-MS as a new technique was used to analyze and identify the constituent in extract botanical and biological sample including dialysate. Among all exiting platforms, ultra-high performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry (HRMS), including UHPLC-Q-TOF MS, UHPLC LTQ-Orbitrap MS, and UHPLC Q-Exactive Orbitrap MS, is the most powerful technique for the detection and identification of constituents, as UHPLC can provide a fast and effective separation while HRMS can provide accurate mass measurement and fragment ion, which will be very much beneficial for structure elucidation [12–14].The present study was designed to detect and identify the main constituents of percutaneous absorption fromD. nervosa by UHPLC-Q-Exactive Orbitrap MS and microdialysis. Finally, a total of 20 constituents including 15 chlorogenic acid analogues, 3 amino acids, and 2 organic acids were detected and identified based on high-resolution mass data including MS and MS2, chromatography retention time, and bibliography data. To the best of our knowledge, this is the first study to report the constituents of percutaneous absorption from D. nervosa, which will be very helpful for understanding the bioactive compounds and quality control.


## 2. Material and Methods

### 2.1. Chemicals and Materials

Acetonitrile and formic acid of LC-MS grade and methanol of LC grade were obtained from Aladdin Industrial Corporation. The ultrapure water used throughout the experiment was purified by a Milli-Q water purification system (Millipore, Milford, MA, United States). Other reagents of analytical grade were obtained from Aladdin Industrial Corporation.The reference standards of 3-caffeoylquinic acid (3-CQA, neochlorogenic acid, X-014-170309), 4-caffeoylquinic acid (4-CQA, cryptochlorogenic acid, Y-067-180425), 5-caffeoylquinic acid (5-CQA, CGA, L-007-171216), 3,5-dicaffeoylquinic acid (3,5-DiCQA, isochlorogenic acid A, Y-068-170903), 3,4-dicaffeoylquinic acid (3,4-DiCQA, isochlorogenic acid B, Y-069-180105), and 4,5-dicaffeoylquinic acid (4,5-DiCQA, isochlorogenic acid C, Y-070-170515) were purchased from ChengDu Herbpurify Co., Ltd. (ChengDu, China). The purities of all reference standards were no less than 98% based on HPLC-UV analysis.


### 2.2. Sample Preparation

The root ofD. nervosa was extracted by reflux with about eightfold 50% ethanol at 70°C for two hours. Then, the filtrate extracts were concentrated under reduced pressure to yield a black residue. Finally, the residue was redissolved in 50% ethanol to give a sample with a concentration of 0.5 g/mL.


### 2.3. Animal Experiments

Allin vivo microdialysis were performed with CMA 402 syringe pump and MAB 85 refrigerated fraction collector (CMA, Microdialysis AB, Sweden). Four male SD rats (weighing 150–200 g, Hunan SJA Laboratory Animal Company, China) were used in in vivo study. All procedures were performed under the conditions of National Act on the Use of Experimental Animals. Anesthesia was induced by an intraperitoneal injection of 1.2 mL/100 g 20% urethane before each experiment. The leg of the rat was shaved carefully with razor without breaking the cuticle. After shaving, the rats were placed on an animal heat insulator. Body temperature was kept at 36–38°C. A CMA 20 Elite microdialysis probe (4 mm, polyarylethersulfone membrane, MWCO of 20 kDa) was then inserted into the dermis after fixing the introducer needle, parallel to the skin on the leg. Probe was perfused with normal saline before the insertion. The inlet and outlet were sealed to keep air from entering the probes.The probes were perfused with normal saline at a flow rate of 2.0μL/min. A 60 min blank dialysate sample was collected for release of the insertion microtrauma prior to the application of D. nervosa. Then, a dosage of 5 g/mL of the material was applied to an area of 2.0 × 3.0 cm2 and covered with gauze and bandages. Microdialysate samples were obtained every 60 min up to 10 h, which was combined into one sample for each rat. During sampling, the microdialysis vials were cooled to 4°C; afterward, they were stored at −80°C until analysis.


### 2.4. Sample Pretreatment

All the microdialysates were pretreated by a solid-phase extraction (SPE) method. A SPE column (WondaSep C18, 200 mg/3 mL) was activated and equilibrated with 6 mL of methanol and 6 mL of water containing 0.5% formic acid, successively. A total of 1 mL microdialysate was loaded on the column. Then, the column was washed by 3 mL of water containing 0.5% formic acid and 3 mL of methanol, respectively. The methanol elute was collected and concentrated under N2 at room temperature to gain residue, which was redissolved in 100 μL of acetronitrile-water (1 : 1, v/v) and centrifuged at 12000 rpm for 30 min at 4°C. Finally, an aliquot of 5 μL supernatant was injected into the UHPLC-Q-Excative Orbitrap MS.


### 2.5. Instruments and Conditions

An Ultimate 3000 focused (Dionex, Sunnyvale, CA, USA) system equipped with an online vacuum degasser, a quaternary pump, and an auto sampler was used for UHPLC analysis. Separation was performed on an HYPERSIL GOLD C18 column (100 × 2.1 mm, 1.9μm) at 35°C. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B) using a gradient elution at a flow rate of 0.3 mL/min. The flowing gradient was applied: 5%–10% B at 0–2 min; 10%–20% B at 2–5 min; 20%–25% B at 5–10 min; 25%–55% B at 10–12 min; 55%–80% B at 12–15 min; 80%–5% B at 15–16 min; and 5% B at 16–20 min.All ESI-MSn analyses were carried out on a Q-Exactive Focus Orbitrap MS (Thermo Electron, Bremen, Germany) coupled with a heated electrospray ionization source (Thermo Electron, Bremen, Germany) in the negative mode. The tune operating parameters were as follows: the rate of sheath gas flow and auxiliary gas flow was 30 and 10 (arbitrary unit), respectively; spray voltage, 3.0 kV; the temperature of capillary and auxiliary gas heater was 320°C and 350°C, respectively; high-resolution MSn was operated at full scan with a mass range of m/z 100–1200 at a resolution of 35000 and MS2 at a resolution of 17500 triggered by data-dependent MSn scanning; nitrogen served as collision gas; and the energy was set as normalized collision energy 30%.


### 2.6. Data Processing and Analysis

The Xcalibur software version 4, (Thermo Fisher Scientific, San Jose, CA, USA) was used to acquire the raw data including the full-scan MS and MS2 data, which were processed by the Compound Discover version 3 using the metabolomics workflow templates to detect the differential components between the microdialysates before and after application of D. nervosa. The detailed parameters of metabolomics workflow template were as follows: The minimum peak intensity was set as 10000; the maximum element counts were C30 H60 O20 S4 N10 Cl4; the mass tolerance of MS and MS2 was within 5 and 10 ppm, respectively; and differential analysis was selected for postprocessing.


## 2.1. Chemicals and Materials

Acetonitrile and formic acid of LC-MS grade and methanol of LC grade were obtained from Aladdin Industrial Corporation. The ultrapure water used throughout the experiment was purified by a Milli-Q water purification system (Millipore, Milford, MA, United States). Other reagents of analytical grade were obtained from Aladdin Industrial Corporation.The reference standards of 3-caffeoylquinic acid (3-CQA, neochlorogenic acid, X-014-170309), 4-caffeoylquinic acid (4-CQA, cryptochlorogenic acid, Y-067-180425), 5-caffeoylquinic acid (5-CQA, CGA, L-007-171216), 3,5-dicaffeoylquinic acid (3,5-DiCQA, isochlorogenic acid A, Y-068-170903), 3,4-dicaffeoylquinic acid (3,4-DiCQA, isochlorogenic acid B, Y-069-180105), and 4,5-dicaffeoylquinic acid (4,5-DiCQA, isochlorogenic acid C, Y-070-170515) were purchased from ChengDu Herbpurify Co., Ltd. (ChengDu, China). The purities of all reference standards were no less than 98% based on HPLC-UV analysis.


## 2.2. Sample Preparation

The root ofD. nervosa was extracted by reflux with about eightfold 50% ethanol at 70°C for two hours. Then, the filtrate extracts were concentrated under reduced pressure to yield a black residue. Finally, the residue was redissolved in 50% ethanol to give a sample with a concentration of 0.5 g/mL.


## 2.3. Animal Experiments

Allin vivo microdialysis were performed with CMA 402 syringe pump and MAB 85 refrigerated fraction collector (CMA, Microdialysis AB, Sweden). Four male SD rats (weighing 150–200 g, Hunan SJA Laboratory Animal Company, China) were used in in vivo study. All procedures were performed under the conditions of National Act on the Use of Experimental Animals. Anesthesia was induced by an intraperitoneal injection of 1.2 mL/100 g 20% urethane before each experiment. The leg of the rat was shaved carefully with razor without breaking the cuticle. After shaving, the rats were placed on an animal heat insulator. Body temperature was kept at 36–38°C. A CMA 20 Elite microdialysis probe (4 mm, polyarylethersulfone membrane, MWCO of 20 kDa) was then inserted into the dermis after fixing the introducer needle, parallel to the skin on the leg. Probe was perfused with normal saline before the insertion. The inlet and outlet were sealed to keep air from entering the probes.The probes were perfused with normal saline at a flow rate of 2.0μL/min. A 60 min blank dialysate sample was collected for release of the insertion microtrauma prior to the application of D. nervosa. Then, a dosage of 5 g/mL of the material was applied to an area of 2.0 × 3.0 cm2 and covered with gauze and bandages. Microdialysate samples were obtained every 60 min up to 10 h, which was combined into one sample for each rat. During sampling, the microdialysis vials were cooled to 4°C; afterward, they were stored at −80°C until analysis.


## 2.4. Sample Pretreatment

All the microdialysates were pretreated by a solid-phase extraction (SPE) method. A SPE column (WondaSep C18, 200 mg/3 mL) was activated and equilibrated with 6 mL of methanol and 6 mL of water containing 0.5% formic acid, successively. A total of 1 mL microdialysate was loaded on the column. Then, the column was washed by 3 mL of water containing 0.5% formic acid and 3 mL of methanol, respectively. The methanol elute was collected and concentrated under N2 at room temperature to gain residue, which was redissolved in 100 μL of acetronitrile-water (1 : 1, v/v) and centrifuged at 12000 rpm for 30 min at 4°C. Finally, an aliquot of 5 μL supernatant was injected into the UHPLC-Q-Excative Orbitrap MS.


## 2.5. Instruments and Conditions

An Ultimate 3000 focused (Dionex, Sunnyvale, CA, USA) system equipped with an online vacuum degasser, a quaternary pump, and an auto sampler was used for UHPLC analysis. Separation was performed on an HYPERSIL GOLD C18 column (100 × 2.1 mm, 1.9μm) at 35°C. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B) using a gradient elution at a flow rate of 0.3 mL/min. The flowing gradient was applied: 5%–10% B at 0–2 min; 10%–20% B at 2–5 min; 20%–25% B at 5–10 min; 25%–55% B at 10–12 min; 55%–80% B at 12–15 min; 80%–5% B at 15–16 min; and 5% B at 16–20 min.All ESI-MSn analyses were carried out on a Q-Exactive Focus Orbitrap MS (Thermo Electron, Bremen, Germany) coupled with a heated electrospray ionization source (Thermo Electron, Bremen, Germany) in the negative mode. The tune operating parameters were as follows: the rate of sheath gas flow and auxiliary gas flow was 30 and 10 (arbitrary unit), respectively; spray voltage, 3.0 kV; the temperature of capillary and auxiliary gas heater was 320°C and 350°C, respectively; high-resolution MSn was operated at full scan with a mass range of m/z 100–1200 at a resolution of 35000 and MS2 at a resolution of 17500 triggered by data-dependent MSn scanning; nitrogen served as collision gas; and the energy was set as normalized collision energy 30%.


## 2.6. Data Processing and Analysis

The Xcalibur software version 4, (Thermo Fisher Scientific, San Jose, CA, USA) was used to acquire the raw data including the full-scan MS and MS2 data, which were processed by the Compound Discover version 3 using the metabolomics workflow templates to detect the differential components between the microdialysates before and after application of D. nervosa. The detailed parameters of metabolomics workflow template were as follows: The minimum peak intensity was set as 10000; the maximum element counts were C30 H60 O20 S4 N10 Cl4; the mass tolerance of MS and MS2 was within 5 and 10 ppm, respectively; and differential analysis was selected for postprocessing.


## 3. Results and Discussion

### 3.1. Identification of Percutaneous Absorption Constituents ofD. nervosa

A total of 20 constituents were detected and identified based on UHPLC-Q-Exactive Orbitrap MS and microdialysis technique. The retention time and mass spectrometric data of those constituents are listed in Table1. The high-resolution extracted ion chromatography of those compounds is shown in Figure 1.Table 1
The retention time and mass spectrometric data of constituents of percutaneous absorption.
Peak
t
R
Theoretical mass
Experimental mass
Error
Formula [M-H]−
Fragment
Identification
1
0.96
133.01425
133.01361
−4.78
C4H5O5
MS2 [133]: 115.0026 (100), 71.0131 (42), 89.0235 (8)
Malic acid isomer
2
1.11
133.01425
133.01365
−4.49
C4H5O5
MS2 [133]: 115.0025 (100), 71.0131 (43), 89.0236 (6)
Malic acid isomer
3
3.15
353.08781
353.08743
−1.06
C16H17O9
MS2 [353]: 191.0547 (100), 179.0336 (76), 135.0436 (21)
3-CQA
4
3.45
203.08260
203.08188
−3.55
C11H11O2N2
MS2 [203]: 116.0492 (100), 74.0233 (38), 142.0650 (27), 159.0918 (21)
Tryptophan
5
4.12
337.09289
337.09323
1.01
C16H17O8
MS2 [337]: 163.0389 (55), 119.0489 (20), 191.0552 (9)
3-pCoQA
6
4.50
353.08781
353.08771
−0.27
C16H17O9
MS2 [353]: 191.0547 (100)
5-CQA
7
4.67
353.08781
353.08780
−0.02
C16H17O9
MS2 [353]: 173.0441 (100), 179.0336 (82), 191.0547 (52), 135.0435 (30)
4-CQA
8
4.70
367.10345
367.10306
−1.08
C17H19O9
MS2 [367]: 193.0497 (100), 134.0361 (18)
3-FQA
9
5.37
353.08781
353.08783
0.07
C16H17O9
MS2 [353]: 191.0548 (100), 135.0435 (4), 179.0334 (3), 173.0078 (3),
Cis-5-CQA
10
5.59
337.09289
337.09302
0.38
C16H17O8
MS2 [337]: 191.0552 (100), 173.0445 (10), 163.0390 (8)
5-pCoQA
11
5.69
515.11950
515.11969
0.37
C25H23O12
MS2 [515]: 191.0545 (100), 179.0333 (88), 353.0860 (16), 135.0434 (13)
1,3-DiCQA
12
5.70
172.09792
172.09697
−5.50
C8H15NO3
MS2 [172]: 130.0861 (100)
N-Acetyl-leucine
13
6.07
172.09792
172.09691
−5.85
C8H15NO3
MS2 [172]: 130.0861 (100)
N-Acetyl-alloisoleucine
14
6.13
367.10345
367.10355
0.26
C17H19O9
MS2 [367]: 173.0445 (100), 93.0331 (88), 191.0552 (47)
4-FQA
15
6.31
337.09289
337.09286
−0.09
C16H17O8
MS2 [337]: 173.0445 (100)
4-pCoQA
16
6.64
367.10345
367.10294
−1.40
C17H19O9
MS2 [367]: 191.0547 (100)
5-FQA
17
7.69
515.11950
515.11981
0.60
C25H23O12
MS2 [515]: 173.0441 (100), 179.0335 (89), 191.0547 (34), 135.0435 (14), 353.0865 (14)
3,4-DiCQA
18
7.85
515.11950
515.11938
−0.23
C25H23O12
MS2 [515]: 191.0546 (100), 179.0335 (62), 353.0865 (15), 135.0435 (11)
3,5-DiCQA
19
8.16
515.11950
515.11920
−0.58
C25H23O12
MS2 [515]: 191.0546 (100), 179.0335 (72), 353.0865 (18), 173.0445 (11), 135.0435 (11)
1,5-DiCQA
20
8.62
515.11950
515.11902
−0.93
C25H23O12
MS2 [515]: 173.0445 (100), 179.0340 (71), 191.0552 (25), 353.0865 (22), 135.0439 (8)
4,5-DiCQAFigure 1
The high-resolution extracted ion chromatography of multiple constituents at m/z 133.01425, 172.09792, 203.08260, 337.09289, 353.08781, 367.10345, and 515.11950.


### 3.2. Identification of Chlorogenic Acid Analogues

Compounds 3, 6, 7, 17, 18, and 20 with the pseudomolecular ion [M-H]− of m/z 353.08783 (0.07 ppm, C16H17O9), 353.08771 (−0.27 ppm, C16H17O9), 353.08780 (−0.02 ppm, C16H17O9), 515.11981 (0.60 ppm, C25H23O12), 515.11938 (−0.23 ppm, C25H23O12), and 515.11902 (−0.93 ppm, C25H23O12), respectively, were accurately identified as tran-3-CQA, tran-5-CQA, tran-4-CQA, 3,4-DiCQA, 3,5-DiCQA, and 4,5-DiCQA by comparing their chromatography retention times, accurate mass measurement, and fragment pattern with those data of reference compounds.Compounds 9, 11, and 19 were eluted at 5.37, 5.69, and 8.16 min, respectively, with the deprotonation ion [M-H]− of m/z 353.08783 (0.07 ppm, C16H17O9), 515.11969 (0.37 ppm, C25H23O12), and 515.11920 (−0.58 ppm, C25H23O12), suggesting that those are isomer of compounds 3, 6, 7 and 17, 18, 20, respectively, which were confirmed by the MS2 spectrum. Finally, those were tentatively characterized as cis-5-CQA, 1,3-DiCQA, and 1,5-DiCQA according to the literature data [15, 16]. Compounds 5, 10, and 15 possessing a [M-H]− ion of m/z 337.09323 (1.01 ppm, C16H17O8), 337.09302 (0.38 ppm, C16H17O8), and 337.09286 (−0.09 ppm, C16H17O8), respectively, were eluted at 4.12, 5.59, and 6.31 min, respectively, suggesting that they were p-coumaroylquinic acid (pCoQA). Considering the base peak at m/z 163.0389, 191.0552, and 173.0445 of the precursor ion at m/z 337 [17, 18], they were tentatively presumed to be 3-pCoQA, 5-pCoQA, and 4-pCoQA, respectively.Compounds 8, 14, and 16 eluted at 4.70, 6.13, and 6.64 min, respectively. All of them showed a pseudomolecular ion [M-H]− of m/z 367.10306 (−1.08 ppm, C17H19O9), 367.10355 (0.26 ppm, C17H19O9), and 367.10294 (−1.40 ppm, C17H19O9), respectively. The base peak at 193.0497, 173.0445, and 191.0547 in MS2 spectrum were used to discriminate isomers of feruloylquinic acid (FQA) [17, 18]; therefore, they were tentatively inferred to be 3-FQA, 4-FQA, and 5-FQA, respectively.


### 3.3. Identification of Amino Acid

Compounds 4, 12, and 13 were detected at 3.45, 5.70, and 6.07 min with the [M-H]− ion at m/z 203.08181 (−3.55 ppm, C11H11O2N2), 172.09697 (−5.05 ppm, C8H14O3N), and 172.09691 (−5.85 ppm, C8H14O3N), respectively. The MS2 spectrum was used to elucidate the structure by searching various MS/MS databases including massbank, METLIN, and KEGG [19]. Therefore, they were temporarily determined to be tryptophan, N-acetyl-leucine, and N-acetyl-alloisoleucine, respectively.


### 3.4. Identification of Organic Acid

Compounds 1 and 2 with the same pseudomolecular ion [M-H]− of m/z 133.014 (C4H5O5) were detected at 0.96 and 1.11 min, respectively. The fragment ion at 115.0026 (−9.4 ppm, C4H3O4), 71.0131 (−10.6 ppm, C3H3O2) and 89.0235 (−10.3 ppm, C3H5O3) was detected in MS2 spectrum by natural loss of H2O, CH2O3, and CO2, suggesting the presence of carboxyl and hydroxyl. According to the literature [20], they were temporarily identified as malic acid isomers.


## 3.1. Identification of Percutaneous Absorption Constituents ofD. nervosa

A total of 20 constituents were detected and identified based on UHPLC-Q-Exactive Orbitrap MS and microdialysis technique. The retention time and mass spectrometric data of those constituents are listed in Table1. The high-resolution extracted ion chromatography of those compounds is shown in Figure 1.Table 1
The retention time and mass spectrometric data of constituents of percutaneous absorption.
Peak
t
R
Theoretical mass
Experimental mass
Error
Formula [M-H]−
Fragment
Identification
1
0.96
133.01425
133.01361
−4.78
C4H5O5
MS2 [133]: 115.0026 (100), 71.0131 (42), 89.0235 (8)
Malic acid isomer
2
1.11
133.01425
133.01365
−4.49
C4H5O5
MS2 [133]: 115.0025 (100), 71.0131 (43), 89.0236 (6)
Malic acid isomer
3
3.15
353.08781
353.08743
−1.06
C16H17O9
MS2 [353]: 191.0547 (100), 179.0336 (76), 135.0436 (21)
3-CQA
4
3.45
203.08260
203.08188
−3.55
C11H11O2N2
MS2 [203]: 116.0492 (100), 74.0233 (38), 142.0650 (27), 159.0918 (21)
Tryptophan
5
4.12
337.09289
337.09323
1.01
C16H17O8
MS2 [337]: 163.0389 (55), 119.0489 (20), 191.0552 (9)
3-pCoQA
6
4.50
353.08781
353.08771
−0.27
C16H17O9
MS2 [353]: 191.0547 (100)
5-CQA
7
4.67
353.08781
353.08780
−0.02
C16H17O9
MS2 [353]: 173.0441 (100), 179.0336 (82), 191.0547 (52), 135.0435 (30)
4-CQA
8
4.70
367.10345
367.10306
−1.08
C17H19O9
MS2 [367]: 193.0497 (100), 134.0361 (18)
3-FQA
9
5.37
353.08781
353.08783
0.07
C16H17O9
MS2 [353]: 191.0548 (100), 135.0435 (4), 179.0334 (3), 173.0078 (3),
Cis-5-CQA
10
5.59
337.09289
337.09302
0.38
C16H17O8
MS2 [337]: 191.0552 (100), 173.0445 (10), 163.0390 (8)
5-pCoQA
11
5.69
515.11950
515.11969
0.37
C25H23O12
MS2 [515]: 191.0545 (100), 179.0333 (88), 353.0860 (16), 135.0434 (13)
1,3-DiCQA
12
5.70
172.09792
172.09697
−5.50
C8H15NO3
MS2 [172]: 130.0861 (100)
N-Acetyl-leucine
13
6.07
172.09792
172.09691
−5.85
C8H15NO3
MS2 [172]: 130.0861 (100)
N-Acetyl-alloisoleucine
14
6.13
367.10345
367.10355
0.26
C17H19O9
MS2 [367]: 173.0445 (100), 93.0331 (88), 191.0552 (47)
4-FQA
15
6.31
337.09289
337.09286
−0.09
C16H17O8
MS2 [337]: 173.0445 (100)
4-pCoQA
16
6.64
367.10345
367.10294
−1.40
C17H19O9
MS2 [367]: 191.0547 (100)
5-FQA
17
7.69
515.11950
515.11981
0.60
C25H23O12
MS2 [515]: 173.0441 (100), 179.0335 (89), 191.0547 (34), 135.0435 (14), 353.0865 (14)
3,4-DiCQA
18
7.85
515.11950
515.11938
−0.23
C25H23O12
MS2 [515]: 191.0546 (100), 179.0335 (62), 353.0865 (15), 135.0435 (11)
3,5-DiCQA
19
8.16
515.11950
515.11920
−0.58
C25H23O12
MS2 [515]: 191.0546 (100), 179.0335 (72), 353.0865 (18), 173.0445 (11), 135.0435 (11)
1,5-DiCQA
20
8.62
515.11950
515.11902
−0.93
C25H23O12
MS2 [515]: 173.0445 (100), 179.0340 (71), 191.0552 (25), 353.0865 (22), 135.0439 (8)
4,5-DiCQAFigure 1
The high-resolution extracted ion chromatography of multiple constituents at m/z 133.01425, 172.09792, 203.08260, 337.09289, 353.08781, 367.10345, and 515.11950.


## 3.2. Identification of Chlorogenic Acid Analogues

Compounds 3, 6, 7, 17, 18, and 20 with the pseudomolecular ion [M-H]− of m/z 353.08783 (0.07 ppm, C16H17O9), 353.08771 (−0.27 ppm, C16H17O9), 353.08780 (−0.02 ppm, C16H17O9), 515.11981 (0.60 ppm, C25H23O12), 515.11938 (−0.23 ppm, C25H23O12), and 515.11902 (−0.93 ppm, C25H23O12), respectively, were accurately identified as tran-3-CQA, tran-5-CQA, tran-4-CQA, 3,4-DiCQA, 3,5-DiCQA, and 4,5-DiCQA by comparing their chromatography retention times, accurate mass measurement, and fragment pattern with those data of reference compounds.Compounds 9, 11, and 19 were eluted at 5.37, 5.69, and 8.16 min, respectively, with the deprotonation ion [M-H]− of m/z 353.08783 (0.07 ppm, C16H17O9), 515.11969 (0.37 ppm, C25H23O12), and 515.11920 (−0.58 ppm, C25H23O12), suggesting that those are isomer of compounds 3, 6, 7 and 17, 18, 20, respectively, which were confirmed by the MS2 spectrum. Finally, those were tentatively characterized as cis-5-CQA, 1,3-DiCQA, and 1,5-DiCQA according to the literature data [15, 16]. Compounds 5, 10, and 15 possessing a [M-H]− ion of m/z 337.09323 (1.01 ppm, C16H17O8), 337.09302 (0.38 ppm, C16H17O8), and 337.09286 (−0.09 ppm, C16H17O8), respectively, were eluted at 4.12, 5.59, and 6.31 min, respectively, suggesting that they were p-coumaroylquinic acid (pCoQA). Considering the base peak at m/z 163.0389, 191.0552, and 173.0445 of the precursor ion at m/z 337 [17, 18], they were tentatively presumed to be 3-pCoQA, 5-pCoQA, and 4-pCoQA, respectively.Compounds 8, 14, and 16 eluted at 4.70, 6.13, and 6.64 min, respectively. All of them showed a pseudomolecular ion [M-H]− of m/z 367.10306 (−1.08 ppm, C17H19O9), 367.10355 (0.26 ppm, C17H19O9), and 367.10294 (−1.40 ppm, C17H19O9), respectively. The base peak at 193.0497, 173.0445, and 191.0547 in MS2 spectrum were used to discriminate isomers of feruloylquinic acid (FQA) [17, 18]; therefore, they were tentatively inferred to be 3-FQA, 4-FQA, and 5-FQA, respectively.


## 3.3. Identification of Amino Acid

Compounds 4, 12, and 13 were detected at 3.45, 5.70, and 6.07 min with the [M-H]− ion at m/z 203.08181 (−3.55 ppm, C11H11O2N2), 172.09697 (−5.05 ppm, C8H14O3N), and 172.09691 (−5.85 ppm, C8H14O3N), respectively. The MS2 spectrum was used to elucidate the structure by searching various MS/MS databases including massbank, METLIN, and KEGG [19]. Therefore, they were temporarily determined to be tryptophan, N-acetyl-leucine, and N-acetyl-alloisoleucine, respectively.


## 3.4. Identification of Organic Acid

Compounds 1 and 2 with the same pseudomolecular ion [M-H]− of m/z 133.014 (C4H5O5) were detected at 0.96 and 1.11 min, respectively. The fragment ion at 115.0026 (−9.4 ppm, C4H3O4), 71.0131 (−10.6 ppm, C3H3O2) and 89.0235 (−10.3 ppm, C3H5O3) was detected in MS2 spectrum by natural loss of H2O, CH2O3, and CO2, suggesting the presence of carboxyl and hydroxyl. According to the literature [20], they were temporarily identified as malic acid isomers.


## 4. Conclusion

The constituents of percutaneous absorption following the external use ofD. nervosa were investigated using UHPLC-Q-Exactive Orbitrap MS and microdialysis technique. Finally, a total of 20 constituents including 15 chlorogenic acid analogues, 3 amino acids, and 2 organic acids were detected and identified based on high-resolution mass data including MS and MS2, chromatography retention time, and bibliography data. To our best knowledge, this is the first study to investigate the constituents of percutaneous absorption from D. nervosa, which will be very beneficial for understanding the bioactive compounds and quality control.



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*Source: 8328942-2019-11-11.xml*