Metabolomic Analysis of Two Parmotrema Lichens: P. robustum (Degel.) Hale and P. andinum (Mull. Arg.) Hale Using UHPLC-ESI-OT-MS-MS

Lichens are symbiotic associations of fungi with microalgae and/or cyanobacteria. Lichens belonging to the Parmeliaceae family comprise 2700 species of lichens, including the Parmotrema genus which is composed of 300 species. The metabolites of this genus include depsides, depsidones, phenolics, polysaccharides, lipids, diphenylethers and dibenzofurans, which are responsible for the biological activities reported including antidiabetic, antihelmintic, anticancer, antioxidant, antibacterial, anti-inflammatory, antimitotic, antitumoral, antifungal, and antioxidant enzyme inhibitory. Due to scarce knowledge of metabolomic profiles of Parmotrema species (P. andinum and P. robustum), a full metabolome study based on ultra-high performance liquid chromatography- diode array detector-electrospray ionization-quadrupole-orbitrap-mass-spectrometry (UHPLC-DAD-ESI-Q-orbitrap MS) was performed for a comprehensive characterization of their substances. From the methanolic extracts of these species, a total of 54 metabolites were identified for the first time using this hyphenated technique, including thirty compounds in P. andinum, and thirty-seven in P. robustum. Moreover, two compounds were not identified as known compounds, and could be new structures, according to our data. This report shows that this technique is effective and accurate for rapid chemical identification of lichen substances and the compounds identified could serve as chemotaxonomic markers to differentiate these ruffle lichens.


Introduction
Lichens, by definition, correspond to a symbiotic association between a fungus and one or more photosynthetic autotrophic organisms that may be a green algae or a cyanobacterium, resulting in a morphologically different thallus to each of its components as a totally new morphological entity [1] Recently it was discovered that a third party, such as basidiomicete yeast, can also be a component of flight-mass-mass spectrometry (HPLC-ESI-Qq-TOF-MS-MS) on negative ion mode and fifteen compounds were detected and identified from the organic extracts [23].
The Q-Exactive Focus is a hybrid high-resolution mass spectrometer used to detect and quantify small organic compounds. The hyphenated Q-Exactive Focus instrument is an HRAM instrument (high resolution accurate mass) which combines UHPLC-DAD (UHPLC-diode array detection) with an orbital trap (orbitrap), a quadrupole (Q) and a high-resolution collision cell (HCD), which allows high resolution diagnostic MS fragments [22][23][24][25][26]. In a continuation of our research on the identification of lichen substances [25,26], we have selected two unstudied Parmotrema lichens for chemotaxonomic fingerprinting and describe the full comprehensive phytochemical profile of P. andinum and P. robustum for the first time, based on UHPLC-DAD coupled with high resolution electrospray ionization tandem mass spectrometry (UHPLC-Q-Orbitrap-HRMS). The profiles serve as fingerprints to differentiate these lichens since the ruffle lichens are difficult to differentiate.

Results and Discussion
Electrospray orbitrap emerged as a very fast and versatile tool for the rapid identification of lichens [25,26]. Thus, two Parmotrema species were selected, P. andinum from Ancash, Peru and P. robustum from Colombia, in order to determine their metabolomic profiles and chemical fingerprints in order to differentiate them since the ruffle lichens are similar in appearance and very difficult to differentiate [4,6,8,27]. Below is the detailed explanation of the rapid metabolome analysis of the aforementioned unstudied Parmotrema species using this HRAM technique.

Diphenylethers:
A diphenylether was detected in this species. Peak 36 was identified as β-alectoronic acid [7], which showed an [M − H] − ion at m/z 511.1974. Its major diagnostic daughter ions were at m/z 369.1339, 247.0967 and 163.0396 a.m.u.
Aromatic compounds: An aromatic compound corresponding to peak 1 was identified in this analysis. As indicated above, peak 1 was identified as orsellinic acid [26].
Unknown compounds: Two compounds (peaks 3 and 34) were not identified.

Instrument
A Thermo Scientific Dionex Ultimate 3000 UHPLC system, equipped with a quaternary Series RS pump and Thermo Scientific Dionex Ultimate 3000 Series TCC-3000RS column compartments with a Thermo Fisher Scientific Ultimate 3000 Series WPS-3000RS autosampler and a rapid separations PDA detector controlled by Chromeleon 7.2 Software (Thermo Fisher Scientific, Waltham, MA, USA and Dionex Softron GmbH Part of Thermo Fisher Scientific, Germering, Germany) hyphenated with a Thermo high resolution Q Exactive focus mass spectrometer (Thermo, Bremen, Germany) were used for the analysis. The chromatographic system was coupled to the MS with a Heated Electrospray Ionization Source II (HESI II). Nitrogen (purity > 99.999%) obtained from a Genius NM32LA nitrogen

Instrument
A Thermo Scientific Dionex Ultimate 3000 UHPLC system, equipped with a quaternary Series RS pump and Thermo Scientific Dionex Ultimate 3000 Series TCC-3000RS column compartments with a Thermo Fisher Scientific Ultimate 3000 Series WPS-3000RS autosampler and a rapid separations PDA detector controlled by Chromeleon 7.2 Software (Thermo Fisher Scientific, Waltham, MA, USA and Dionex Softron GmbH Part of Thermo Fisher Scientific, Germering, Germany) hyphenated with a Thermo high resolution Q Exactive focus mass spectrometer (Thermo, Bremen, Germany) were used for the analysis. The chromatographic system was coupled to the MS with a Heated Electrospray Ionization Source II (HESI II). Nitrogen (purity > 99.999%) obtained from a Genius NM32LA nitrogen generator (Peak Scientific, Billerica, MA, USA) was employed as both the collision and damping gas. Mass calibration for Orbitrap was performed once a week, in both negative and positive modes, to ensure a working mass accuracy lower than or equal to 5 ppm. Caffeine and N-butylamine (Sigma Aldrich, Saint Louis, MO, USA) were the calibration standards for positive ions and buspirone hydrochloride, sodium dodecyl sulfate, and taurocholic acid sodium salt (Sigma Aldrich, Saint Louis, MO, USA) were used to calibrate the mass spectrometer. These compounds were dissolved in a mixture of acetic acid, acetonitrile, water and methanol (Merck, Darmstadt, Germany) and were infused using a Chemyx Fusion 100 syringe pump (Thermo Fisher Scientific, Bremen, Germany). XCalibur 2.3 software (Thermo Fisher Scientific, Bremen, Germany) and Trace Finder 3.2 (Thermo Fisher Scientific, San José, CA, USA) were used for UHPLC control and data processing, respectively. Q Exactive 2.0 SP 2 from Thermo Fisher Scientific was used to control the mass spectrometer.

MS Parameters
The HESI parameters were as follows: sheath gas flow rate, 75 units; auxiliary gas unit flow rate, 20; capillary temperature, 400 • C; auxiliary gas heater temperature, 500 • C; spray voltage, 2500 V (for ESI-); and S lens, RF level 30. Full scan data in positive and negative were acquired at a resolving power of 70,000 FWHM (full width half maximum) at m/z 200. For the compounds of interest, a scan range of m/z 100-1000 was chosen; the automatic gain control (AGC) was set at 3 × 10 6 and the injection time was set to 200 ms. The scan-rate was set at 2 scans s −1 . External calibration was performed using a calibration solution in positive and negative modes. For confirmation purposes, a targeted MS-MS analysis was performed using the mass inclusion list, with a 30 s time window, with the Orbitrap spectrometer operating both in positive and negative modes at 17,500 FWHM (m/z 200). The AGC target was set to 2 × 10 5 , with the maximum injection time of 20 ms. The precursor ions were filtered by the quadrupole, which operated at an isolation window of m/z 2. The fore vacuum, high vacuum and ultrahigh vacuum were maintained at approximately 2 mbar, from 105 and below 1010 mbar, respectively. Collision energy (HCD cell) was operated at 30 kv. Detection was based on calculated exact mass and on retention time of target compounds, as shown in Table 1. The mass tolerance window was set to 5 ppm for the two modes.

Conclusions
In the present report, fifty-four metabolites were detected using UHPLC-Q-Orbitrap-ESI-MS-MS for the first time in P. andinum and P. robustum. Our study indicates that lipids, depsides, depsidones, dibenzofurans, diphenylethers and aromatic compounds were the main compounds detected and identified. This report could contribute to the better understanding of the chemistry of Parmotrema genus, and it supports that the HPLC fingerprints are very important for the fast chemical differentiation of these ruffle lichens.
Supplementary Materials: The following are available online, Table S1: Structure of the compounds identified by UHPLC-ESI-MS-MS from Parmotrema species.