For Peer Review · For Peer Review 2 Abstract Rationale: We had recently reported the top-down lignomic analysis of the virgin released lignin (VRL) oligomers obtained from the date

For Peer ReviewStructural elucidation of small lignin oligomers isolated

from the Date Palm Wood completed by Electrospray-LTQ-Orbitrap and Atmospheric Pressure Photoionization

Quadrupole Time-of-Flight Tandem Mass Spectrometry

Journal: Rapid Communications in Mass Spectrometry

Manuscript ID RCM-19-0071

Wiley - Manuscript type: Research Article

Date Submitted by the Author: 08-Mar-2019

Complete List of Authors: Mikhael, Abanoub; Memorial University of NewFoundland, ChemistryFridgen, Travis; Memorial University of Newfoundland, Chemistry DELMAS, MICHEL; University of Toulouse, Chemical Engineering Laboratory Banoub, Joe; Memorial University of Newfoundland Faculty of Humanities and Social Sciences, Chemsitry; Governmnet of Canada, Fisheries and Oceans Canada

Keywords: Low-energy CID-MS/MS analysis, ESI-LTQ-Orbitrap MS/MS, APPI-QqTOF-MS/MS, Low molecular weight VRL

Abstract:

Rationale: We had recently reported the top-down lignomic analysis of the virgin released lignin (VRL) oligomers obtained from the date palm wood (DPW), using MALDI-TOF tandem mass spectrometry. [1] In this manuscript, we are extending our recent work, [1] by the structure elucidation of the low molecular weight VRL dilignols and trilignols, using ESI-LTQ-Orbitrap MS/MS (+ ion mode) and APPI-QqTOF-MS/MS (+ ion mode).

Methods: Low-energy CID-MS/MS analyses conducted with an ESI-LTQ-Orbitrap- and APPI-QqTOF-tandem mass spectrometers allowed the direct analysis of the lignin oligomers mixture without any chromatographic pre-separation. Low-energy CID-MS/MS analyses were used to confirm the structures of the selected precursor ions.

Results: Four lignin protonated molecules were identified from ESI-LTQ-Orbitrap MS/MS (+ ion mode): dilignol [C19H24O7+ H]+ composed of H(8-O-4’)G, dilignol [C19H24O8+ H]+ composed of H(8-O-4’)G; dilignol [C22H24O9+H]+ composed of S(8-O-4’)S and trilignol [C34H42O16+H]+ composed of G(8-O-4’)G(8-O-4’’)S. Furthermore, APPI-QqTOF-MS/MS (+ ion mode) allowed us to identify the novel dilignol [C18H22O10 + H]+ composed of L(8-O-4’)C.

Conclusion: In this study, we obtained similar low-energy CID-MS/MS fragmentation pathways like the one obtained recently using high-energy MALDI-CID-

http://mc.manuscriptcentral.com/rcm

Rapid Communications in Mass Spectrometry

For Peer Review

TOF/TOF-MS/MS.[1] The trilignol G(8-O-4’)G(8-O-4”)S obtained from the positive ion mode ESI-LTQ-Orbitrap originate from the lignin Hexamer at m/z 1201 recently identified using MALDI-CID-TOF/TOF-MS/MS analysis. [1] This proves without any doubt that even a very mild extraction method can degrade the lignin oligomers considerably. Finally, using APPI-QqTOF-MS/MS, the novel lignin dilignol L(8-O-4’)C is identified for the first time in literature.

Page 1 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

1

Structural elucidation of small lignin oligomers isolated from the Date Palm Wood completed by Electrospray-LTQ-Orbitrap and Atmospheric Pressure

Photoionization Quadrupole Time-of-Flight Tandem Mass Spectrometry

Abanoub Mikhael,1 Travis. D. Fridgen,1 Michel Delmas,2 Joseph Banoub.1,3*

1 Department of Chemistry, Memorial, University of Newfoundland, St John's, Newfoundland A1C 5X1, Canada

2 INP‐Ensiacet, Chemical Engineering, Laboratory 4, University of Toulouse, Allée Emile Monso, 31432 Toulouse, France

3 Science Branch, Special Projects, Fisheries and Oceans Canada, St John's, NL A1C 5X1,Canada

CorrespondenceJ. Banoub, Science Branch, Special Projects, Fisheries and Oceans Canada, St John's, NL,A1C 5X1, Canada and Department of Chemistry, Memorial University of Newfoundland, St John's, NL, A1C 5X1, Canada.Email: banoubjo@dfo‐mpo.gc.ca

Page 2 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Fridgen%2C+Travis

https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Delmas%2C+Michel

For Peer Review

2

Abstract

Rationale: We had recently reported the top-down lignomic analysis of the virgin released lignin

(VRL) oligomers obtained from the date palm wood (DPW), using MALDI-TOF tandem mass

spectrometry. [1] In this manuscript, we are extending our recent work, [1] by the structure

elucidation of the low molecular weight VRL dilignols and trilignols, using ESI-LTQ-Orbitrap

MS/MS (+ ion mode) and APPI-QqTOF-MS/MS (+ ion mode).

Methods: Low-energy CID-MS/MS analyses conducted with an ESI-LTQ-Orbitrap- and APPI-

QqTOF-tandem mass spectrometers allowed the direct analysis of the lignin oligomers mixture

without any chromatographic pre-separation. Low-energy CID-MS/MS analyses were used to

confirm the structures of the selected precursor ions.

Results: Four lignin protonated molecules were identified from ESI-LTQ-Orbitrap MS/MS (+ ion

mode): dilignol [C19H24O7+ H]+ composed of H(8-O-4’)G, dilignol [C19H24O8+ H]+ composed of

H(8-O-4’)G; dilignol [C22H24O9+H]+ composed of S(8-O-4’)S and trilignol [C34H42O16+H]+

composed of G(8-O-4’)G(8-O-4’’)S. Furthermore, APPI-QqTOF-MS/MS (+ ion mode) allowed

us to identify the novel dilignol [C18H22O10 + H]+ composed of L(8-O-4’)C.

Conclusion:

In this study, we obtained similar low-energy CID-MS/MS fragmentation pathways like the one

obtained recently using high-energy MALDI-CID-TOF/TOF-MS/MS.[1] The trilignol G(8-O-

4’)G(8-O-4”)S obtained from the positive ion mode ESI-LTQ-Orbitrap originate from the lignin

Hexamer at m/z 1201 recently identified using MALDI-CID-TOF/TOF-MS/MS analysis. [1] This

proves without any doubt that even a very mild extraction method can degrade the lignin oligomers

considerably. Finally, using APPI-QqTOF-MS/MS, the novel lignin dilignol L(8-O-4’)C is

identified for the first time in literature.

INTRODUCTION

Lignin oligomers are an important source of aromatic compounds composed mainly of three

monomers: coniferyl (H), sinapyl (S), and p-coumaryl (G) alcohols linked covalently in different

Page 3 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

3

a fashion, forming the scaffolding of the lignin oligomers. [1-5] The most important type of the

lignin monomer linkages is (β-O-4) or (8-O-4’) which represent 40-60 % of all types of lignin

linkages.[6] In addition, recently some whimsically unique lignol monomers were identified such

as C (caffeyl), F (5-hydroxyguaiacyl) and these were shown to be incorporated sporadically in few

lignin oligomer mixtures. Also, the presence of the L (gallyl) lignol unit has been hypothetically

predicted to exist; there have been no structures, of any gallyl lignin oligomers, reported yet. [7]

We have recently identified the C lignol unit in one of the identified oligomers structures of the

DPW lignin oligomers using MALDI-TOF MS/MS. [1]

Scheme 1 uncommon lignin units

The extraction of lignin without any structural modification or cleavage of large oligomers

to smaller units is still a challenge. [8-11] Even if fine extraction techniques like the CIMV solvolysis

technique were reported, using acetic acid/formic acid/water combination, we have shown that it

could alter the structure of the lignin oligomers. [1,12,13] However, another principal advantage of

the CIMV method that we have established, is by virtue of using acidic solvents

(HCOOH/CH3COOH), we decrease the probability of the formation of sodiated molecular ions

[M+Na] + in the Electrospray Ionization (ESI) and Atmospheric Pressure Photoionization (APPI)

mass spectra. [14]

Mass spectrometry is one of the most important techniques to determine the structure of

the lignin biopolymer. [15,16] Soft ionization methods like ESI and APPI were used in the past few

decades for sequencing lignin oligomers. [14,17,18] ESI-MS was used for the Eucalyptus globulus

lignin structural elucidation, [19] and APPI was used for the investigation of the wheat straw lignin

complex molecular structure. [14] The number of possible proposed molecular formulas for a

specific molecular mass can be reduced by using the high accuracy and resolution in mass

measurement. [20] Accurate determination of molecular formulas using high-resolution instruments

prefer mass accuracy of 1-2 ppm which can be achieved using the last invented mass analyzer

Orbitrap. [20]

In this manuscript, we present the structural elucidation of series of DPW small lignin

oligomers which were extracted by the CIMV solvolysis technique.[12,13] This extraction method

degrades the Vegetable matter by chemical hydrolysis to produce the virgin released Lignin

Page 4 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

4

(VRL).[15] The extracted VRL was used directly for the analysis without any further chemical

modifications or purification which can alter the native lignin structure.[15] The structural

identification of DPW VRLs was performed by using ESI-LTQ-Orbitrap MS/MS (+ ion mode).

For these analyses, the most common LTQ orbitrap operation mode was used. The Orbitrap-MS

was used for measuring the exact masses in the full scans, and the Linear Ion Trap was used for

low-energy CID-MS/MS analyses. [21] Furthermore, low-energy APPI-QqTOF-CID-MS/MS was

used to establish a comparison between the MS/MS analyses obtained using these two soft

ionization techniques.

ExperimentalSamples

The samples date palm wood (Phoenix dactylifera) examined were collected manually from the

Salman Alfarsi garden, Almadinah, Saudi Arabia. All the samples were frozen and dried for seven

days at -48°C and 30 x 10-3 mbar (Freezone 6, model 77530, Labconco Co., Kansas City, MO).

The dried samples were then grounded, vacuum packed and stored in a freezer at -20 ºC.

Lignin Oligomers Isolation

The date palm wood (DPW) lignin was extracted using the CIMV procedure which selectively

separates the cellulose, hemicellulose, and lignin, and allows the destructing of the vegetable

matter at atmospheric pressure (Lignin yield 17%).[12,13] The catalyst-solvent system used was a

mixture of formic acid/acetic acid/water (30/50/20) which produced after precipitation with water

and filtered the Date Palm Wood (DPW) lignin.

ESI-LTQ-Orbitrap MS and Low-Energy CID-MS/MS

ESI-MS was carried by infusing the sample using a syringe pump at 5 ul/min in a heated

Electrospray Ionization (HESI) source connected to an Orbitrap Velos Pro mass spectrometer

(Thermo Fisher, Bremen, Germany). O.3 mg of the CIMV virgin released lignin (VRL) was

dissolved in 1 mL Dioxane/methanol/chloroform (1:1:1) for the preparation of the sample solution.

The oligomers mixture was electrosprayed at a spray voltage of 2.5 kV and Capillary temperature

275 °C. The Orbitrap cell with a resolution of 60,000 acquires a full-scan MS spectrum of intact

lignin dilignols and trilignols (m/z 100–1000) with an automated gain control accumulation target

Page 5 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

5

value of 1,000,0000 ions. In the linear ion trap, the most abundant ions were isolated and

fragmented by applying collision-induced dissociation using an accumulation target value of

10,000 and normalized collision energy of 35%. All the raw files were viewed using mMass

program. [22]

APPI-QqTOF-MS and Low-Energy CID-MS/MS

Mass spectrometry was performed using an Applied Biosystems (Foster City, CA, USA)

API-QSTAR-XL MS/MS quadrupole orthogonal time-of-flight (QqToF)-MS/MS hybrid

instrument. APPI was performed with a PhotoSpray ion source (Applied Biosystems) operated at

1300V at a temperature of 400 ºC, with all acquisitions completed in the positive ion mode.

Samples were infused into the mass spectrometer with an integrated Harvard syringe pump at a

rate of 0.1 mL/min. The auxiliary nebulizer gas pressure setting was fixed at 25 psi, and the

nebulizer gas pressure at 74 psi. The curtain gas pressure was set at 30 psi. The declustering

potential (DP) was set at + 100 eV. The focus potential (FP) was adjusted to +100 V. Toluene was

selected as the dopant for its ability to undergo trouble-free photoionization at 8.83 eV. The eluent

was composed of Dioxane/methanol/chloroform (1:1:1). No modifier was used to enhance ion

production.

The mass calibration of the ToF analyzer in the positive ion mode was performed with

the PhotoSpray ion source, using 1,2,3,5-tetra-O-acetyl-b-D-ribofuranose and checking for the

exact masses of the [M+H]+ ion [C13H19O9] at m/z 320.1107 and the [M+H–AcOH]+ ion

[C12H15O7] at m/z 271.0808. Calibration for higher masses was performed with hexa-O-acetyl-ß-

D-lactopyranose and checking for the [M+H]+ ion [C28H37O19] at m/z 677.1929.

RESULTS AND DISCUSSION

In this work, the direct analysis of the small lignin oligomers mixture was undertaken

without any chromatographic pre-separation. For the determination of the chemical structures of

the precursor ions and their chemical formulae, we have entertained some basic rules described by

Thomas De Vijlder et al., [23] for application in MS and MS/MS analyses. Consequently, the exact

chemical formulae and molecular structures were calculated using the ESI-LTQ-Orbitrap- MSMS

Page 6 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

6

high-resolution instrument. The identification of the lignin oligomers was performed according to

the presence of one heteroatom (Oxygen), error ppm, isotopic distributions of the precursor ions, [23,24], and the possible combinations of the different lignol units. Furthermore, the level of

unsaturation also called the double bond equivalent (DBE) was calculated for either of the

characterized molecules and/or precursor ions, to determine the number of unsaturation. [25]

It should be mentioned that in this study we used a very low concentration of the infused

analyte solution of the lignin oligomers. This was implemented to ensure that the major constituent

ions represented the most abundant lignin oligomers in the sample.

Positive ion mode ESI-Orbitrap MS and Low-Energy CID-MS/MS of CIMV extracts of

DPW

The positive ion mode ESI-Orbitrap-MS of the DPW showed a series of protonated

molecules inter alia at m/z 365.1599 (1); 381.1550 (2); 433.1489 (3) and 707.2537 (4) (Figure 2,

Figure 3 and Table 1).

Figures 2 and 3

Table 1

The protonated molecule 1 at m/z 365.1599 with formula [C19H24O7 + H]+ was tentatively

assigned to the dilignol derivative H(8-O-4’)G (Figure 4 and Scheme 1).

The product ion scan of this protonated molecule 1 at m/z 365.1599 shows three types of

bond cleavage that occurs between C1- C7 of the G unit, C7- C8 of the G unit and the (8-O-4’)

that links the H and G units together, to give the product ions at m/z 305.1, 275.1 and 215.1,

respectively and assigned as [C17H20O5 + H]+, [C16H18O4 + H]+ and [ C10H14O5 + H]+. The last two

product ions at m/z 275.1 and 215.1 can lose one molecule of formaldehyde to afford the secondary

product ions at m/z 245.1 and 185.1, respectively and assigned as [C15H16O3 + H]+ and [C9H12O4

+ H]+. The protonated molecule 1 at m/z 365.1599 afforded the product ions at m/z 347.1 and 333.1

by the loss of one water molecule and one methanol molecule, respectively and assigned as

[C19H22O6 + H]+ and [C18H20O6 + H]+. The primary product ion at m/z 347.1 undergoes an

elimination reaction which leads to the formation of the secondary product ion at m/z 239.1

assigned as [C12H14O5 + H]+. This last product ion at m/z 239.1 loses two molecules of water to

Page 7 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

7

afford the product ion at m/z 203.1 assigned as [C12H10O3 + H]+. The CID-MS/MS fragmentation

patterns of the protonated molecule 1 at m/z 365.1599 are shown in scheme 1.

Figure 4 and Scheme 1

The chemical structure of the protonated molecules 2 at m/z 381.1550 was tentatively

assigned to have the following chemical composition [C19H24O8 + H]+ and was attributed to H(8-

O-4’)G dilignol derivative (Figure 5 and Scheme 2).

The product ion scan of this protonated molecules 2 at m/z 381.1550 gave the product ions

at m/z 349.1 and m/z 317.1 through the loss of two consecutive molecules of methanol and assigned

as [C18H20O7 + H]+ and [C17H16O6 + H]+, respectively. The protonated molecule 2 at m/z 381.1550

lose one molecule of water to afford the product ion at m/z 363.1 assigned as [C19H22O7 + H]+.

This latter product ion at m/z 363.1 afforded the secondary product ions at m/z 343.1 and 323.1

through the consecutive loss of 20 Da (one water molecule and one hydrogen molecule) and

assigned as [C19H18 O6 + H]+ and [C19H14O5 + H]+, respectively. The product ion at m/z 343.1

undergoes an elimination reaction to afford the product ion at m/z 219.1 assigned [C12H10O4 + H]

+. This last product ion at m/z 219.1 loses one molecule of water to afford the product ion at m/z

201.0 assigned as [ C12H8O4 + H] +. Also, this product ion at m/z 219.1 undergoes (8-O-4’) bond

cleavage to yield the product ion at m/z 176.0 assigned as [C10H8O3] •+. This latter product ion at

m/z 176.0 loses one molecule of formaldehyde to afford the product ion at m/z 146.0 assigned as

[C9H6O2]•+. The CID-MS/MS fragmentation patterns of the protonated molecules 2 at m/z

381.1550 are shown in scheme 2.


The protonated molecule 3 at m/z 433.1489 [C22H24O9 + H]+ was assigned to S(8-O-4’)S

dilignol derivative (Figure 6 and Scheme 3).

The product ion scan of this protonated molecules 3 at m/z 433.1489 gave the product ion

at m/z 415.1 by the loss of 18 Da (one methane molecule and one hydrogen molecule) assigned as

[C21H18O9 + H]+. This latter product ion at m/z 415.1 loses one molecule of water to afford the

product ion at m/z 397.1 assigned as [C21H16O8 + H]+. This latter product ion at m/z 397.1 loses

Page 8 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

8

three consecutive molecules of formaldehyde to afford the product ions at m/z 367.1, 337.1 and

307.1, respectively. The protonated molecules 3 at m/z 433.1489 undergoes an elimination reaction

along with the loss of one hydrogen molecule to yield the product ion at m/z 277.1 assigned as

[C14H13O6 + H]+. This latter product ion at m/z 277.1 loses two molecules of formaldehyde to

afford the product ion at m/z 217.0. Also, this product ion at m/z 277.1 undergoes rearrangement

followed by the bond cleavage of the C7-C8 bond located in the second S unit to afford the benzylic

cation at m/z 235.0 assigned as [C12H11O5] +. Furthermore, The protonated molecule 3 at m/z

433.1489 undergoes bond cleavage of the C6-C7 bond located in the second S unit to yield the

product ion at m/z 379.1 assigned as [C19H22O8 + H]+. This latter product ion at m/z 379.1 loses

three consecutive molecules of formaldehyde to afford the product ion at m/z 349.1, 319.1 and

289.1, respectively. This latter product ion at m/z 289.1 loses 18 Da (one methane molecule and

one hydrogen molecule) to give the product ion at m/z 271.1 assigned as [C15H11O5]+. This latter

product ion m/z 271.1 loses one molecule of water to afford the product ion at m/z 253.1 assigned

as [C15H9O4]+. The CID-MS/MS fragmentation patterns of the protonated molecule 3 at m/z

433.1489 are shown in scheme 3.


The protonated molecule 4 at m/z 707.2537 [C34H42O16 + H]+ was assigned to G(8-O-

4’)G(8-O-4’’)S derivative ( Figure 7, Scheme 4A and 4B ).

The product ion scan of the protonated molecule 4 at m/z 707.2537 gave the product ions

at m/z 689.2 and 671.2 formed by the elimination of two consecutive molecules of water, assigned

as [C34H40O15 + H]+ and [C34H38O14 + H]+, respectively. This latter product ion at m/z 689.2

afforded the product ion at m/z 601.2 assigned as [C31H36O12 + H]+ through aryl ether bond

cleavage of the first G unit. The consecutive losses of the fragments C3H6O3 (90 Da) and C3H4O2

(72 Da) from both extremities of the protonated molecule 4 at m/z 707.2537 afforded the product

ions at m/z 617.2 and 545.2, respectively and assigned as [C31H36O12 + H]+ and [C28H32O11+H]+.

This latter product ion at m/z 617.2 loses one molecule of formaldehyde to afford the product ion

at m/z 587.2 assigned as [C30H34O11 + H]+. Also, the latter product ion at m/z 545.2 loses two

consecutive molecules of water to afford the product ions at m/z 527.2 and 509.2, respectively and

assigned as [C28H30O10+H]+ and [C28H28O9+H]+(Scheme 4A).

Page 9 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

9

Figure 7, Scheme 4A

Both the protonated molecule 4 at m/z 707.2537 and its product ion at m/z 587.1 undergoes

C7-C8 of the first G unit (between the two contiguous G units) to yield the product ions at m/z

485.2 and 365.2, respectively and assigned as [C23H32O11 + H]+ and [C19H24O12 + H]+. This last

product ion at m/z 485.2 loses three consecutive molecules of formaldehyde to afford the product

ions at m/z 455.2, 425.2 and 395.2, respectively. Furthermore, both product ions at m/z 455.2 and

425.2 lose one molecule of water to afford the product ions at m/z 407.2 and 437.2, respectively.

In addition, the product ion at m/z 365.2 can lose one molecule of formaldehyde to afford the

product ion at m/z 335.1 or one molecule of water to afford the product ion at m/z 347.1. This last

product ion at m/z 347.1 undergoes an aryl ether bond cleavage to afford the product ion at m/z

305.1 assigned as [C17H20O5 + H]+. This latter product ion at m/z 305.1 loses two consecutive

molecules of formaldehyde to afford the product ions at m/z 275.1 and 245.1, respectively (Scheme

4B).

Scheme 4B

It is crucial to understand that the protonated molecules at m/z 707.2537 (4) most probably

originated from the lignin oligomer identified in our recent work using MALDI-TOF MS/MS.[1]

This small oligomer 4 appeared to be one of the hydrolysis products of the large oligomers

(Hexamer at m/z 1201) during the mild acidic extraction techniques of the CIMV solvolysis

technique using acetic acid/formic acid/water combination (Figure 8). [11,12]

Figure 8

APPI-QqTOF-MS and Low-Energy CID-MS/MS of CIMV extracts of the Date Palm Wood (DPW)

The APPI-QqTOf-MS (+ ion mode) showed a series of protonated molecules inter-alia at

m/z 707.2568; 399.1279; 381.1542, 365.1587; 219.0649 and 203.0716 (Figure 9). It should be

highlighted that the most important difference between the ESI- LTQ-Orbitrap-MS and APPI-

Page 10 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

10

QqTOF-MS is the presence of the protonated dilignol at m/z 399.1279 which is not seen in the

ESI-LTQ-Orbitrap-MS. In addition, it is important to notice the two diagnostic ions at m/z 219 and

203 can be formed from the two protonated dilignols at m/z 365 and 381 and this was proven by

conducting low-energy CID-MS/MS of both protonated molecules at m/z 365 and 381 (Scheme 1

and 2).

Please note that the product ion scans of the protonated molecules at m/z 707; 381 and 365

have already been explained in the low-energy CID-MS/MS analyses using the ESI-LTQ-

Orbitrap-MS. Both ESI- and APPI-MS/MS analyses were identical.

Figure 9

In this section, we will focus only on the protonated molecule at m/z 399.1279 [C18H22O10

+ H ]+ ( error ppm = -3) assigned as L(8-O-4’)C derivative (Figure 10, Schemes 5A and 5B). It is

primordial to realize that we were the first to observe the C unit in one of the lignin oligomers of

the DPW identified by MALDI-TOF-MS. [1] Most importantly, it should be pointed out that the

gallyl unit L has been “hypothetically” proposed to exist as a one of the possible lignin monomers

by Vanholme et al. [7] The product ion scan of this protonated molecule at m/z 399.1279 gave the

product ion at m/z 381.1 by the loss of one molecule of water, and assigned as [C18H20O9 + H]+.

This latter product ion at m/z 381.1 loses one molecule of carbon monoxide through ring

contraction to afford the product ion at m/z 353.1 assigned as [C17H20O8 + H ]+. This latter product

ion at m/z 353.1 undergoes 8-O-4’ bond cleavage to afford the product ion at m/z 173.1 assigned

as [C8H12O4 + H ] +. Also, this product ion at m/z 353.1 experience the cleavage of C7 – C8 bond

of the L unit to afford the product ion at m/z 124.0 assigned as [C6H4O3]+. The protonated molecule

at m/z 399.1279 undergoes double cleavage to afford the product ion at m/z 111.0 assigned as

[C6H6O2 + H]+. Moreover, the protonated molecule at m/z 399.1279 experience the cleavage of

C7–C8 bond of the L unit to afford two product ions at m/z 157.0 and 243.1 assigned as [C7H8O4

+ H ]+ and [C11H14O6 + H ]+, respectively. This latter product ion at m/z 243.1 loses one molecule

of water along with two molecules of hydrogen molecules to afford the product ion at m/z 221.0

assigned as [C11H8O5 + H]+. This latter product ion at m/z 221.0 can lose one molecule of hydrogen

to afford the product ion at m/z 219.0 assigned as [C11H6O5 + H ]+ or one molecule of water to

afford the product ion at m/z 203.0 assigned as [C11H6O4 + H ]+( Scheme 5A).

Page 11 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

11

Scheme 5A

Furthermore, the protonated molecule at m/z 399.1279 loses one molecule of methanol to

afford the product ion at m/z 367.1 assigned as [C17H18O9 + H]+. This latter product ion at m/z

367.1 loses one molecule of carbon monoxide through ring contraction to afford the product ion

at m/z 339.1 assigned as [C16H18O8 + H]+. This latter product ion at m/z 339.1 undergoes bond

cleavage of the C7-C8 located in the C unit to afford the product ion at m/z 279.1 assigned as

[C14H14O6 + H ]+. In addition, the product ion at product ion at m/z 339.1 loses two molecules of

water to afford the product ion at m/z 303.1 assigned as [C16H14O6 + H]+. This latter product ion

at m/z 303.1 undergoes bond cleavage of the C1-C7 bond located in the C unit along with the loss

of hydrogen radical to afford the product ion at m/z 228.0 assigned as [C13H8O4 + H ]+( Scheme

5B).

Scheme 5B

Once more we would like to state that the ESI- and APPI-MS/MS gas-phase fragmentation

of the dilignol, and trilignol are different than those conducted by APCI-MS/MS with a QIT

instrument.[26,27] This difference can be attributed to the fact that APPI-QqTOF-MS/MS

fragmentations were measured by MS/MS conducted in space” and as such, these were expected

to be more energetic. [26,27]

CONCLUSION

In this manuscript, we are extending the work that we had initiated recently by the structural

investigation of the VRL obtained from the DPW using MALDI-TOF MS/MS, [1] that exhibited

the date palm wood (Phoenix dactylifera) as a rich source of lignin.

The high-resolution ESI-LTQ-Orbitrap-MS (+ ion mode) of the VRL of DPW showed the

presence of the protonated molecules at m/z 365.1599, 381.1550, 433.1489 and 707.2537. The

protonated molecule at m/z 707.2537 was attributed to being formed by degradation occurring

during the mild CIMV solvolysis of VRL of the date palm wood, and most probably originates

from a higher mass oligomer proposed in our recent work using MALDI- TOF MS/MS. [1].

Page 12 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

12

The APPI-QqTOF-MS (+ ion mode) of the VRL obtained from DPW confirmed the

presence of the same protonated molecules at m/z 365, 381 and 707 obtained from the ESI-

Orbitrap-MS. However, the most exciting and novel compound was found by unraveling the

structure of the protonated molecules at m/z 399.1279, assigned as [C18H22O10 + H]+, which was

considered to be derived from L(8-O-4’)C dilignol structure.

The low-energy CID-MS/MS analyses conducted with both the ESI-LTQ-Orbitrap-MS

and APPI-QqTOF-MS/MS instruments displayed similar MS/MS fragmentation patterns for the

protonated molecules at m/z 365, 381 and 707.

This manuscript extends the structural work performed on the Date Palm Wood lignin.

Both the presence of the C and L units in this series of small lignin oligomers allowed us to reveal

novel dilignol lignin that did not concur with the current knowledge of any lignin structures

proposed.

References

1. Albishi, T., Mikhael, A., Shahidi, F., Fridgen, T., Delmas, M., Banoub J., Top-down

Lignomic MALDI-TOF-Tandem mass spectrometry analysis of Lignin oligomers

extracted from date palm wood, Rapid Commun. Mass Spectrom, 2018 (Accepted

Manuscript)

2. Dolk, M., Pla, F., Yan, J.F., McCarthy, J.L., Lignin. 22. Macromolecular characteristics of

alkali lignin from western hemlock wood. Macromolecules, 1986; 19(5): 1464-1470.

3. Campbell M.M., Sederoff. R.R., Variation in lignin content and composition. Mechanisms

of control and implications for genetic improvement of plants. Plant Physiol., 1996; 110

(1): 3-13.

4. MacKay, J., Dimmel, D.R., Boon. J.J., Pyrolysis mass spectral characterization of wood

from CAD-deficient pine., J. Wood Chem. Technol., 2001, 21, 19-29.

5. Vanholme, R., Demedts, B., Morreel, K., Ralph, J., Boerjan, W., Lignin biosynthesis and

structure. Plant physiol., 2010; 153(3): 895-905.

6. Stevanovic, T., in Lignocellulosic Fibers and Wood Handbook: Renewable Materials for

Today’s Environment (Eds.: Belgacem, M.N., Pizzi, A.), Scrivener Publishing, Salem,

Massachusetts, USA, 2016, pp. 49-110.

Page 13 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Albishi%2C+Tasahil

https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Mikhael%2C+Abanoub

https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Shahidi%2C+Fereidoon

https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Fridgen%2C+Travis

https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Delmas%2C+Michel

For Peer Review

13

7. Vanholme, R., Morreel, K., Darrah, C., Oyarce, P., Grabber, J. H., Ralph, J., & Boerjan,

W. (2012). Metabolic engineering of novel lignin in biomass crops. New

Phytologist, 196(4), 978-1000

8. Glasser, W.G., Lignin. In Pulp and Paper. Chemistry and Chemical Technology, Vol.1, 3rd

ed., J. P. Casey (Ed.). John Wiley & Sons: New York, 1980; 39–111.

9. Lu, F., Ralph, J., Derivatization followed by reductive cleavage (DFRC Method), a new

method for lignin analysis: protocol for analysis of DFRC monomers., J. Agric. Food

Chem., 1997, 7, 2590-2592 and references cited therein.

10. Guerra, A., Filpponen, I., Lucia, L.A., Saquing, C., Baumberger, S., Argyropoulos, D.S.,

Toward a better understanding of the lignin isolation process from wood., J. Agric. Food

Chem., 2006, 54(16), 5939-5947

11. Perlack, R.D., Wright, L.L., Turhollow, A.F., Graham, R.L., Stokes, B.J., Erbach, D.C.,

Biomass as a feedstock for a bioenergy and bioproducts industry: the technical feasibility

of a billion-ton annual supply, Oak Ridge National Laboratory, Tennessee, 2005.

12. Delmas, M., Avignon, G., Brevet Fr., 1997; 97: 13658.

13. Lam, H.Q., Le Bigot, Y., Delmas, M., Avignon, G., A new procedure for the destructuring

of vegetable matter at atmospheric pressure by a catalyst/solvent system of formic

acid/acetic acid. Applied to the pulping of triticale straw, Ind. Crops Prod., 2001; 14: 139-

144.

14. Banoub, J., Benjelloun-Mlayah, B., Ziarelli, F., Joly, N., Delmas, M., Elucidation of the

complex molecular structure of wheat straw lignin polymer by atmospheric pressure

photoionization quadrupole time-of-flight tandem mass spectrometry. Rapid Commun.

Mass Spectrom., 2007, 21, 2867-2888.

15. Banoub, J., Delmas, G., Joly, N., Mackenzie, G., Cachet, N., Delmas, M., A critique on the

structural analysis of lignins and application of novel tandem mass spectrometric strategies

to determine lignin sequencing. J. Mass Spectrom., 2015; 50: 5-48.

16. Brunow. G., Methods to reveal the structure of lignin. In: Biopolymers: Lignin, Humic

Substances and Coal, Vol.1, Hofrichter, M., Steinbüchel, A. (Eds). Wiley Blackwell:

Michigan, USA, 2001; 89–116.

Page 14 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

14

17. Evtuguin, D.V., Domingues, P.M., Amado, F.M.L., Pascoal Neto, C., Ferrer Correia, A.J.,

Electrospray ionization mass spectrometry as a tool for lignins molecular weight and

structural characterisation. Holzforschung, 1999, 53, 525-528.

18. Evtuguin, D.V.; Amado, F.M.L., Application of Electrospray Ionization Mass

Spectrometry to the Elucidation of the Primary Structure of Lignin, Macromol. Biosci.,

2003, 3, 339-343.

19. Evtuguin, D.V., Pascoal Neto, C., Silva, A.M., Domingues, P.M., Amado, F.M.L, Robert,

D., Faix, O., Comprehensive Study on the Chemical Structure of Dioxane Lignin from

Plantation Eucalyptus globulus Wood, J. Agric. Food Chem., 2001, 49, 4252-4261.

20. Hu, Q., Noll, R.G., Li, H., Makarov, A., Hardman, M., Cooks, R.G., The Orbitrap: a new

mass spectrometer, J. Mass Spectrom., 2005, 40, 430–443.

21. Eliuk, S., Makarov, A., Evolution of Orbitrap Mass Spectrometry Instrumentation, Annu.

Rev. Anal. Chem., 2015, 8, 61–80

22. Strohalm, M., Hassman, M., Košata, B., Kodíček, M., mMass data miner: an open source

alternative for mass spectrometric data analysis. Rapid Commun. Mass Spectrom, 2008,

22(6), 905-908.

23. Vijlder, T.D., Valkenborg, D., Lemière, F., Romijn, E.P., Laukens, K., & Cuyckens, F., A

tutorial in small molecule identification via electrospray ionization‐mass spectrometry:

The practical art of structural elucidation, Mass spectrom. rev., 2018; 37: 607–629.

24. Schymanski, E.L., Meinert, C., Meringer, M., Brack, W. The use of MS classifiers and

structure generation to assist in the identification of unknowns in effect-directed analysis,

Anal. Chim. Acta, 2008; 615: 136-147.

25. Bae, E., Yeo, I.J., Jeong, B., Shin, Y., Shin, K-H., Kim, S., Study of double bond

equivalents and the numbers of carbon and oxygen atom distribution of dissolved organic

matter with negative-mode FT-ICR MS., Anal Chem., 2011;83(11):4193–4199.

Page 15 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

15

26. Morreel, K., Kim, H., Lu, F., Dima, O., Akiyama, T., Vanholme, R., Niculaes, C.,

Goeminne, G., Inze´, D., Messens, E., Ralph, J., Boerjan, W., Mass spectrometry-based

fragmentation as an identification tool in lignomics, Anal. Chem., 2010, 82, 8095-8105.

27. Morreel, K., Dima, O., Kim, H., Lu, F., Niculaes, C., Vanholme, R., Dauwe, R., Goeminne,

G., Inze´, D., Messens, E., Ralph, J., Boerjan, W., Mass Spectrometry-Based Sequencing

of Lignin Oligomers, Plant Physiol., 2010; 153: 1464-1478

Page 16 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

16

Table 1. Identification of small lignin oligomers peaks in ESI-Orbitrap Mass Spectrum of the DPW Wood VRL Lignin.

PrecursorIons

AssignedFormula DBE

Calculated[M+H] +

(m/z)

Observed[M+H] +

(m/z)

Error ppm CID-MS/MS Product ions

1 [C19H24O7 + H] + 7.5 365.1600 365.1599 - 0.2 347.1, 333.1, 305.1, 275.1, 245.1, 239.1, 215.1, 185.1, 203.0

2 [C19H24O8 + H] + 7.5 381.1549 381.1550 + 0.2 363.1, 349.1, 343.1, 323.1, 317.1, 219.1, 201.0, 176.0, 146.0

3 [C22H24O9 + H] + 10.5 433.1498 433.1489 - 2.0415.1, 397.1, 379.1, 367.1, 349.1, 337.1, 319.1, 307.1, 289.1, 277.1, 271.1, 253.1, 235.0, 217.0

4 [C34H42O16 + H] + 12.5 707.2551 707.2537 - 1.9

689.2, 671.2, 617.1, 601.2, 587.2, 545.2, 527.2, 509.2, 485.2, 455.2, 437.2, 425.2, 407.2, 395.2, 365.2, 347.1, 335.1, 305.1, 275.1, 245.1

Page 17 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

LIST OF LEGENDS

FIGURES

Figure 1: Uncommon Lignin monomer units

Figure 2: ESI-Orbitrap MS (+ ion mode) of the extracted VRL Date Palm Wood

Figure 3: Structures of the identified small VRL oligomers extracted from Date Palm Wood

Figure 4: CID-MS/MS of the protonated dilignol [C19H24O7 + H]+ at m/z 365.1599



Figure 7: CID-MS/MS of the protonated trilignol [C34H42O16 + H]+ at m/z 707.2537

Figure 8: The proposed formation of the lignin trilignol at m/z 707 formed during original CIMV

extraction of the DPW.[1]

Figure 9: APPI-QqTOF-MS (+ ion mode) of the extracted VRL Date Palm Wood


SCHEMES

Scheme 1: Proposed fragmentation pattern for the product ion scan of the protonated dilignol at

m/z 365.1599


m/z 381.1550


m/z 433.1489

Scheme 4A: Proposed fragmentation pattern for the product ion scan of the protonated dilignol at

m/z 707.253

Scheme 4B: Propose fragmentation pattern of the product ion scan of the protonated trilignol at

m/z 707.2537

Scheme 5A: Proposed fragmentation pattern of the product ion scan of the protonated dilignol at

m/z 399.1279

Scheme 5B: Proposed fragmentation pattern of the product ion scan of the protonated dilignol at

m/z 399.1279

Page 18 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Page 19 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

HO

OH

OH

HO

OMe

OH

HO

HO

OH

OH

HO

C F L

Figure 1: Uncommon Lignin monomer units

Figure 2: ESI-Orbitrap MS (+ ion mode) of the extracted VRL Date Palm Wood

Page 20 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

HO

OMe

O

HO

HO

HO

+ H

[ C19H24O7 + H ]+

m/z 365.1599(1)

HO

OMe

O

HO

HO

OH

HO

+ H

OH

[ C19H24O8 + H ]+

m/z 381.1549(2)

[ C22H24O9 + H ]+

m/z 433.1489(3)

OMe

O

HO

OMe

O

HO

OMe

O

HO

HO+ H

HO

OHHO

MeO

HO

[ C34H42O16 + H ]+

m/z 707.2537(4)

HO

OH

O

O

OH

HO

OMe

OMe

+ H

MeO

MeO

O

Figure 3: Structures of the identified small VRL oligomers extracted from Date Palm Wood

Page 21 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review


Page 22 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

HO

OMeO

HO

HO

HO

+ H

[ C19H24O7 + H ]+

m/z 365.1599

OH

OMeO

HO

HO

HO

+ H

[ C17H20O5 + H ]+

m/z 305.1

OMeO

HO

HO

+ H

[C16H18O4+ H ]+

m/z 275.1

- CH2OO

HO

HO

+ H

[C15H16O3+ H ]+

m/z 245.1

HO

OMeOH

HO+ H

OH

[C10H14O5+ H ]+

m/z 215.1

HO

OH

HO+ H

OH

[C9H12O4+ H ]+

m/z 185.1

- CH2O- CH

3 OH

OMeO

HO

HO

+ H

OH

[ C18H20O6 + H ]+

m/z 333.1

- H2O

HO

OMeO

HO

HO

HO

+ H

[ C19H22O6 + H ]+

m/z 347.1

HO

OMeO

HO

HO

+ H

- 2 x H2O

C

HO

OMeO

+ H

[ C12H10O3+ H ]+

m/z 203.1

[ C12H14O5+ H ]+

m/z 239.1

HO

Scheme 1: Proposed fragmentation pattern for the product ion scan of the protonated dilignol at m/z

365.1599

Page 23 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review


Page 24 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

HO

OMeO

HO

HO

OH

HO

+ H

OH

[ C19H24O8 + H ]+

m/z 381.1550

HO

OMeO

HO

HO

OH

HO

+ H

[ C19H22O7 + H ]+

m/z 363.1

- H2O - H2O

- H2

O

OMeO

HO

OH

HO

+ H

O

OMeO

HO

+ H

- H2O

O

OMeO

+ H

[C12H8O3+ H ]+

m/z 201.0

O

OMeOH

[ C10H8O3]m/z 176.0

O

OH

[C9H6O2]m/z 146.0

O

OMeO

O

OH

+ H

- H2O- H2

HO

OMeO

HO

OH

HO

+ H

[ C18H18O7 + H ]+

m/z 349.1

[ C19H14O5 + H ]+

m/z 323.1

[ C19H18O6 + H ]+

m/z 343.1

[C12H10O4+ H ]+

m/z 219.1

OH - CH2O

OMeO

HO

OH

HO

+ H

[ C17H14O6 + H ]+

m/z 317.1

OH

- CH3OH

- CH3OH

Scheme 2: Proposed fragmentation pattern for the product ion scan of the protonated dilignol at m/z 381.1550

Page 25 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review


Page 26 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

O

O

OH

HO

OMe

OMe

+ H

MeO

MeO

O

O

O

O

HO O

OMe

[C21H19O9]+

m/z 415.1

MeO

MeO

O

O

C

O

O

O

OMeMeO

MeO

O

[ C21H17O8]+

m/z 397.1

- H2O- CH4

- H2

- H2

O

C

O

O

OMe

+ H

MeO

O

[C14H13O6]+

m/z 277.1

the loss of three consecutivemolecules of Formaldehydegives peaks at m/z 367, 337,307

The loss of twoFormaldehydemolecules givesthe peak at m/z217

O

O

OH

HO

OMe

OMe

+ H

MeO

MeO

the loss of three consecutivemolecules of Formaldehydegives peaks at m/z 349, 319,289

O

O

OH

HO

OMe

+ H

[C16H16O5 + H]+

m/z 289.1- CH4- H2

O

O

O

HO O

O

C

O

O

O[ C15H11O5] +

m/z 271.1

O

C

O

O

OMeMeO

[ C12H11O5]+

m/z 235.0

[C22H24O9+ H]+m/z 433.1489

- 3 x CH2 O

H

[C19H22O8+ H]+m/z 379.1

- C8H10O3

[ C15H9O4] +

m/z 253.1

- H2O

O

C

O

O

OMeMeO

OH

[C14H13O6]+

m/z 277.1

Scheme 3: Proposed fragmentation pattern for the product ion scan of the protonated dilignol at m/z

433.1489

Page 27 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Figure 7: CID-MS/MS of the protonated trilignol [C34H42O16 + H]+ at m/z 707.2537

Page 28 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

OMe

O

HO

OMe

O

HO

OMe

O

HO

HO+ H

HO

OHHO

MeO

HO

[ C34H42O16 + H ]+

m/z 707.2537

OMe

O

HO

OMe

O

OMe

O

+ H

HO

OHHO

MeO

[ C34H40O15 + H ]+

m/z 689.2

- CH2O

HOHO

HO

O

[ C31H36O12 + H ]+

m/z 601.2

OMe

O

HO

OMe

O

OMe

+ H

HO

OHHO

MeO

HO

O

OMe

O

HO

OMe

O

HO

OMe

O

HO

HO+ H

MeO

HO

[ C31H36O13 + H ]+

m/z 617.2

HO

O

HO

OMe

O

HO

OMe

O

HO

HO+ H

MeO

HO

HO

[ C30H34O12 + H ]+

m/z 587.2

OMe

O

HO

OMe

O

HO

OMe

OH

HO

HO

+ H

MeO

[ C28H32O11 + H ]+

m/z 545.2

OMe

O

HO

OMe

O

OMe

O

HO

+ H

MeO

[ C28H28O9 + H ]+

m/z 509.2

- H2O - H2O

OMe

O

HO

OMe

O

O

HO

HO

+

MeO

[ C28H30O10 + H ]+

m/z 527.2

- H2O OMe

O

HO

OMe

O

OMe

O

+ H

HO

OHHO

MeO

[ C34H38O14 + H ]+

m/z 671.2

O

HO

O

H

HO

- H2O

Scheme 4A: Proposed fragmentation pattern for the product ion scan of the protonated dilignol at m/z

707.2537

Page 29 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

OMe

O

HO

OMe

O

HO

OMe

O

HO

HO+ H

HO

OHHO

MeO

HO

[ C34H42O16 + H ]+

m/z 707.2

HO

OMe

O

HO

OMe

O

HO

OMe

O

HO

HO+ H

MeO

HO

[ C31H36O13 + H ]+

m/z 617.2

HO

OMe

O

HO

OMe

O

HO

HO+ H

HO

OHHO

MeO

- CH2O

O

HO

OMe

O

HO

OMe

O

HO

HO+ H

MeO

HO

HO

[ C30H34O12 + H ]+

m/z 587.2

O

HO

OMe

O

HO

HO+ H

MeO

[ C19H24O7+ H ]+

m/z 365.2

O

HO

OMe

HO+ H

MeO

[ C17H20O5+ H ]+

m/z 305.1

OMe

O

HO

OMe

O

HO

HO

+ H

HO

OHHO

O

HO

OMe

O

HO

HO+ H

HO

OHHO

[ C21H28O9 + H ]+

m/z 425.2

O

HO

O

HO

HO+ H

HO

OHHO

[ C20H26O8 + H ]+

m/z 395.2

O

HO

OMe

HO+ H

[ C16H18O4+ H ]+

m/z 275.1

OMe

O

HO

OMe

O

HO

HO

+ H

HO

HO

[ C22H28O9 + H ]+

m/z 437.2

O

HO

OMe

O

HO

HO+ H

HO

HO

[ C21H26O8 + H ]+

m/z 407.2

O

HO

HO+ H

[ C15H16O4+ H ]+

m/z 245.1

O

HO

O

HO

HO+ H

MeO

[ C18H22O6+ H ]+

m/z 335.1

- CH2O

- CH 2O

- CH2O

- CH2O

O

HO

OMe

O

HO+ H

MeO

- H2O

[ C19H22O6+ H ]+

m/z 347.1

- CH2O

[C23H32O11+ H ]+

m/z 485.2

- CH2O

[ C22H30O10 + H ]+

m/z 455.2

- H2O

- H2O

Scheme 4B: Propose fragmentation pattern of the product ion scan of the protonated trilignol at m/z

707.2537

Page 30 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

MeO

O

HO

MeO

O

MeO

HO

MeO

O

HO

MeO

O

HOHO

MeO

O

HOHO

HO

HO

COOH

HOHOMeO

+OH

HO

MeO

O

HO

MeO

O

HOHO

MeO

O

HOHO

HOHOMeO

+H

[ C34H42O16 + H ]+

m/z 707

Lignin Oligomeridentif ied using

MALDI-TOF MS[1]

[ C61H68O25 + H ]+

m/z 1201

H

Figure 8: The proposed formation of the lignin trilignol at m/z 707 formed during original CIMV

extraction of the DPW.[1]

Page 31 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Figure 9: APPI-QqTOF-MS (+ ion mode) of the extracted VRL Date Palm Wood

Page 32 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review Figure 10: CID-MS/MS of the protonated dilignol [C18H22O10 + H]+ at m/z 399.1279

Page 33 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer ReviewO

HO

OH

HO

OH

+ H

HO

HO

OH

OH

O

HO

OH

HO

+ H

HO

HO

OH

OH

- H2O

[C18H22O10 + H]+

m/z 399.1279[C18H20O9 + H]+

m/z 381.1

HO HO

O

OH

HO

OHHO

HO

[C11H14O6+ H]+

m/z 243.1

O

OH

O

CO

C

O

+ H

[ C11H6O5 + H]+

m/z 219.0

- H2O

- 2 x H2

O

OH

O+ H

CO

HO

[C11H8O5+ H]+

m/z 221.0

- H2O

O

OH

O

CO

[C11H6O4+ H]+

m/z 203.0

- COOH

OH

+ H

[C6H6O2+ H]+

111.0

+ H

+ H

HO

OH

OHHO

+ H

[ C7H8O4 + H]+

m/z 157.1

- H2

O

HO

OH

HO

+ H

HO

HO

OH

[C17H20O8 + H]+

m/z 353.1

HO

HO

HO

OH

[C8H12O4+ H]+

m/z 173.1

HO

+ H

C

OHHO124.0

[C6H4O3]m/z 124.0

O

Scheme 5A: Proposed fragmentation pattern of the product ion scan of the protonated dilignol at m/z

399.1279

Page 34 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer ReviewO

HO

OH

HO

OH

+ H

HO

HO

OH

OH

- CH3OH

[C18H22O10 + H]+

m/z 399.1279

HO

O

HO

OH

HO

OH

+ H

HO

OH

OH

- CO

[C17H18O9+ H]+

m/z 367.1

HO

O

HO

OH

HO

OH

+ H

HO

OH

[C16H18O8+ H]+

m/z 339.1

HO

- 2 x H2O

O

OH

+ H

OH

[C16H14O6+ H]+

m/z 303.1

HO

O

HO

OH

HO

+ H

OH

[C14H14O6+ H]+

m/z 279.1

HO

HO

HO

O

OH

HO OH[ C13H8O4]m/z 228.0

- H

Scheme 5B: Proposed fragmentation pattern of the product ion scan of the protonated dilignol at m/z

399.1279

Page 35 of 34



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

Documents

For Peer Review · For Peer Review 2 Abstract Rationale: We had recently reported the top-down lignomic analysis of the virgin released lignin (VRL) oligomers obtained from the date