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

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.


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][5][6].
Considering the usage of D. 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][8][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 in vivo 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][13][14]. e present study was designed to detect and identify the main constituents of percutaneous absorption from D. 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 MS 2 , 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.

Sample Preparation.
e root of D. nervosa was extracted by reflux with about eightfold 50% ethanol at 70°C for two hours. en, 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.

Animal Experiments.
All in 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. e 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. e inlet and outlet were sealed to keep air from entering the probes.
e probes were perfused with normal saline at a flow rate of 2.0 μL/min. A 60 min blank dialysate sample was collected for release of the insertion microtrauma prior to the application of D. nervosa. en, a dosage of 5 g/mL of the material was applied to an area of 2.0 × 3.0 cm 2 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.

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. en, the column was washed by 3 mL of water containing 0.5% formic acid and 3 mL of methanol, respectively. e methanol elute was collected and concentrated under N 2 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.
All ESI-MS n analyses were carried out on a Q-Exactive Focus Orbitrap MS ( ermo Electron, Bremen, Germany) coupled with a heated electrospray ionization source ( ermo Electron, Bremen, Germany) in the negative mode. e 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 MS n was operated at full scan with a mass range of m/z 100-1200 at a resolution of 35000 and MS 2 at a resolution of 17500 triggered by data-dependent MS n scanning; nitrogen served as collision gas; and the energy was set as normalized collision energy 30%.

Data Processing and Analysis.
e Xcalibur software version 4, ( ermo Fisher Scientific, San Jose, CA, USA) was used to acquire the raw data including the full-scan MS and MS 2 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. e detailed parameters of metabolomics workflow template were as follows: e minimum peak intensity was set as 10000; the maximum element counts were C30 H60 O20 S4 N10 Cl4; the mass tolerance of MS and MS 2 was within 5 and 10 ppm, respectively; and differential analysis was selected for postprocessing.

Identification of Percutaneous Absorption Constituents of D. nervosa.
A total of 20 constituents were detected and identified based on UHPLC-Q-Exactive Orbitrap MS and microdialysis technique. e retention time and mass spectrometric data of those constituents are listed in Table 1. e high-resolution extracted ion chromatography of those compounds is shown in Figure 1.   [17,18]; therefore, they were tentatively inferred to be 3-FQA, 4-FQA, and 5-FQA, respectively.  [19]. erefore, they were temporarily determined to be tryptophan, N-acetyl-leucine, and N-acetyl-alloisoleucine, respectively.  [20], they were temporarily identified as malic acid isomers.

Conclusion
e constituents of percutaneous absorption following the external use of D. 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 MS 2 , 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.

Data Availability
e data used to support the finding of this study are available from the corresponding author upon request.

Disclosure
is work was presented as an oral presentation at Consortium for Globalization of Chinese Medicine (CGCM2019) in Shanghai.

Conflicts of Interest
e authors have declared no conflicts of interest.