May 07, 2026

Analysis of a lignin derived single product using chemical and biological redox using UHPLC-MS/MS-DAD

DOI
https://dx.doi.org/10.17504/protocols.io.36wgqprjyvk5/v1
Analysis of a lignin derived single product using chemical and biological redox using UHPLC-MS/MS-DAD
  • 1Renewable Resources and Enabling Sciences Center, National Laboratory of the Rockies, Golden, CO, USA
  • Stefan Haugen: Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA
Kelsey J. Ramirez
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgqprjyvk5/v1
Protocol Citation: Kelsey Ramirez, Morgan Ingraham, Stefan Haugen, Gregg T. Beckham 2026. Analysis of a lignin derived single product using chemical and biological redox using UHPLC-MS/MS-DAD. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqprjyvk5/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 30, 2025
Last Modified: May 07, 2026
Protocol  Integer ID: 221318
Keywords: Muconolactone, Lignin conversion, benzene tricarboxylic acids, BTAs, phthalic acid, benzoic acid, ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) , biological funneling, isophthalic acid, biological redox of lignin, lignin, reverse phase chromatography, performance liquid chromatography, biological redox, quantitation of product
Funders Acknowledgements:
This work was authored by the National Laboratory of the Rockies, operated by Alliance for Energy Innovation. Funding was provided by the U.S. Department of Energy's (DOE) Office of Critical Minerals and Energy Innovation (CMEI), Alternative Fuels and Feedstocks Office (AFFO) under Contract No. DE-AC36-08GO28308.
Grant ID: DE-AC36-08GO28308
Disclaimer
This protocol is for research purposes only.
Abstract
An analysis method was developed for the quantitation of products produced by chemical and biological redox of lignin funneling into a single, high-yield product. This method utilizes ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) and diode array detection (DAD). This method employs reverse phase chromatography and multiple reaction monitoring (MRM) in negative ion mode using electrospray ionization (ESI) and quantitation using multiple wavelengths from the DAD.
Guidelines
This protocol utilizes an ultra-high pressure liquid chromatography tandem mass spectrometer (UHPLC-MS/MS) system with a diode array detector (DAD) manufactured by Agilent Technologies as referenced in 'Materials'. A similar chromatography tandem mass spectrometry system can be utilized however, some parameter nomenclature may deviate depending on the manufacturer.
Materials
Dimethyl sulfoxideThermo ScientificCatalog #127790010 Terephthalic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #185361 Phthalic acidTCI ChemicalsCatalog #P0287 Benzoic acidThermo ScientificCatalog #149135000
Isophthalic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #I19209 1,3,5-Benzenetricarboxylic acid (Trimesic acid)AmBeedCatalog #A284402 Biphenyl-3,3-dicarboxylic acidAmBeedCatalog #A136778 Protocatechuic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #37580-25G-F CatecholMerck MilliporeSigma (Sigma-Aldrich)Catalog #PHL82372-100MG cis cis-Muconic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #15992
1,2,4-Benzenetricarboxylic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #B4589
1,2,3-Benzenetricarboxylic acid (Hemimellitic acid)TCI ChemicalsCatalog #H1592
Ethanol 99.5% ACSThermo ScientificCatalog # AC615090010 Potassium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P3786
M9, Minimal Salts, 5XMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6030
Equipment
1290 Infinity UHPLC
NAME
Ultra-high performance liquid chromatography system
TYPE
Agilent Technologies
BRAND
1290 Infinity UHPLC
SKU
https://www.agilent.com/en/product/liquid-chromatography/hplc-systems/analytical-hplc-systems
LINK

Equipment
ACQUITY UPLC Guard column
NAME
VanGuard Pre-column BEH
TYPE
Waters Corporation
BRAND
186003975
SKU
https://www.waters.com/nextgen/us/en/shop/columns/186003975-acquity-uplc-beh-c18-vanguard-pre-column-130a-17--m-21-mm-x-5-mm.html
LINK
ACQUITY UPLC BEH C18 VanGuard Pre-column, 130Å, 1.7 µm, 2.1 mm X 5 mm, 3/pk
SPECIFICATIONS

Equipment
ACQUITY UPLC Analytical Column
NAME
ACQUITY UPLC BEH Analytical Column
TYPE
Waters Corporation
BRAND
186002350
SKU
https://www.waters.com/nextgen/us/en/shop/columns/186002350-acquity-uplc-beh-c18-column-130a-17--m-21-mm-x-50-mm-1-pk.html
LINK
ACQUITY UPLC BEH C18 Column, 130Å, 1.7 µm, 2.1 mm X 50 mm, 1/pk
SPECIFICATIONS

Equipment
6475 Triple Quadrupole LC/MS
NAME
LC-QQQ System
TYPE
Agilent Technologies
BRAND
G6475AA
SKU
https://www.agilent.com/en/product/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-instruments/triple-quadrupole-lc-ms/6475-triple-quadrupole-lc-ms
LINK




Safety warnings
All chemicals used for this procedure are hazardous. Read the Safety Data Sheet (SDS) for all chemicals and follow all applicable chemical handling and waste disposal procedures. Manufacturer specific SDS information can be found by following the CAS numbers of compounds in 'Materials' list.
Before start
All solvents and chemicals used are listed in the ‘Materials’ section. These are excluded from in-line references to maintain clarity and keep the steps concise. 
Preparation of Standards
By weight, create individual stock standards of desired concentration using the appropriate diluent as listed below.

  • Terephthalic acid (See dx.doi.org/10.17504/protocols.io.kqdg3xeb7g25/v1 for preparation)
  • Phthalic acid (18.2MΩ⋅cm ultrapure water (UPW) and Ethanol)
  • Benzoic acid (Ethanol)
  • Isophthalic acid (Dimethylsulfoxide (DMSO))
  • Biphenyl-3,3-dicarboxylic acid (DMSO)
  • Protocatechuic acid (Ethanol)
  • Catechol (Ethanol)
  • cis,cis- muconic acid (0.05% v/v sodium hydroxide)
  • cis,trans- muconic acid (0.05% v/v sodium hydroxide) (For guidance on how to prepare this, reference dx.doi.org/10.17504/protocols.io.36wgqjjxyvk5/v3)
  • 1,2,4-Benzenetricarboxylic acid (Ethanol)
  • 1,2,3-Benzenetricarboxylic acid (Hemimellitic acid) (Ethanol)
  • 1,3,5-Benzenetricarboxylic acid (Trimesic acid)(Ethanol)
  • Muconolactone (M9 minimal media) (Note: this compound is not commercially available and was synthesized in-house. The structure and purity of >99% were verified using NMR)

These standards can be combined to make mixed standard working solutions (MSWS) as highlighted in the table below. TPA, c,c-muconic acid, c,t-muconic acid, and muconolactone should be kept as individual analyte standards due to isomerization of the muconic acid standard, solvent/analyte compatibility, and solubility of analytes.

Table of mixed standard working solutions.

Using the MSWS, create calibration curves with a minimum of 5 calibration points using the appropriate diluent.

An example dilution scheme is given below for a calibration curve of 9 points ranging from 1 to 500 µg/mL


Example calibration curve preparation (click to enlarge)

Sample Preparation
Ensure sample matrix is compatible with instrumentation. The calibration range for this method is between 1 µg/mL and 500 µg/mL. Select the appropriate linear range based on analyte response and instrument performance.

All samples should be 0.2µm filtered prior to injection on instrument. Any samples expected to have analyte concentrations that fall outside of this range should be diluted appropriately to avoid potential carryover or system contamination.
UHPLC- MS/MS-DAD Analysis
Prepare an Agilent 1290 UHPLC system according to the following parameters for a total run time of 7.0 minutes:

Defined UHPLC parameters (click to enlarge)

Defined UHPLC-DAD parameters (click to enlarge)

Defined Multi-wash parameters (click to enlarge)

Analyze samples using an Agilent 6475 triple quadrupole mass spectrometer equipped with dual Agilent jet stream electrospray ionization (AJS ESI) utilizing the source method parameters illustrated below.


Defined MS/MS parameters (click to enlarge)
For muconolactone, a table is provided below with the optimized multiple reaction monitoring (MRM) transitions, corresponding fragmentor voltages (V), and collision energies (CE) for the quantifying and qualifying transitions.

MRM analyte transitions

A table is provided below with the appropriate DAD wavelengths for all other analytes of interest.

Table with analytes and wavelength used for quantitation (click to enlarge)

Data Analysis and Quality Control
Data analysis completed using Agilent Quantitative Analysis for QQQ version 12.1 Update 1.
Quality Control
Several criteria are used to ensure instrument stability and reproducibility throughout the analysis.
  • Calibration curves used have a correlation coefficient (r²) of greater than or equal to 0.995, obtained using either a quadratic or linear fit.
  • A calibration verification standard (CVS) is a standard from the calibration curve that is analyzed every 20 or fewer samples to check for instrument drift. For this analysis method, an acceptable CVS recovery range is within +/- 15% of the expected amount.
Example Chromatograms


Example chromatogram (click to enlarge)


Protocol references
Hannah M. Alt, Kelsey J. Ramirez, Stefan J. Haugen, William E. Michener, Gregg T. Beckham 2025. Polyethylene terephthalic acid deconstruction product analysis by UHPLC-DAD. protocols.iohttps://dx.doi.org/10.17504/protocols.io.kqdg3xeb7g25/v1

Sean P. Woodworth, Stefan J. Haugen, William E. Michener, Kelsey J. Ramirez, Gregg T. Beckham 2024. Muconic acid isomers and aromatic compounds analyzed by UHPLC-DAD. protocols.iohttps://dx.doi.org/10.17504/protocols.io.36wgqjjxyvk5/v3
Acknowledgements
We would like to acknowledge and thank Gloria Rosetto for providing the purified muconolactone for use as a quantitative standard and Kathryn M. Mains for providing samples and media matrix.