Apr 06, 2023
Aromatic Monomer Analysis by UHPLC-MS/MS
- Stefan J. Haugen1,
- Gregg T. Beckham1,
- Kelsey J. Ramirez1
- 1Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory
- NRELTech. support email: ftlb_analysis@nrel.gov
Protocol Citation: Stefan J. Haugen, Gregg T. Beckham, Kelsey J. Ramirez 2023. Aromatic Monomer Analysis by UHPLC-MS/MS. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7rzwlx9/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: April 05, 2023
Last Modified: August 21, 2023
Protocol Integer ID: 80071
Keywords: electrospray ionization, tandem mass spectrometry, aromatic monomers, lignin monomers
Funders Acknowledgements:
U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy and Bioenergy Technologies Office (BETO)
Grant ID: DE-AC36-08GO28308
Abstract
An analysis method was developed to allow for quantitation of aromatic compounds by ultra high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection. This was achieved by using reverse phase chromatography and multiple reaction monitoring (MRM) in negative ion mode using electrospray ionization (ESI).
Guidelines
NOTICE
This work was authored by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Funding provided by U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The views expressed herein do not necessarily represent the views of the DOE or the U.S. Government.
Materials
Working Solution Preparations:
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #270725
Milli-Q Water
Internal Standard:
4-Hydroxybenzoic-2356-d4 acidContributed by usersCatalog #662763
Aromatic Analytes:
4-Hydroxybenzoic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #240141
4-HydroxybenzaldehydeContributed by usersCatalog #91554
3-5-Dimethoxy-4-hydroxyacetophenoneContributed by users
Ferulic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #1270311
Vanillic AcidMerck MilliporeSigma (Sigma-Aldrich)
SyringaldehydeMerck MilliporeSigma (Sigma-Aldrich)Catalog #08319
Syringic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #63627
VanillinMerck MilliporeSigma (Sigma-Aldrich)
AcetovanilloneMerck MilliporeSigma (Sigma-Aldrich)
p-Coumaric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #55823
Analytical Column:
Equipment
Kinetex Phenyl-Hexyl
NAME
Analytical Column
TYPE
Phenomenex
BRAND
00D-4500-AN
SKU
LINK
2.1 mm x 100mm (1.7µm pore size)
SPECIFICATIONS
Instrumentation:
Equipment
6470 Triple Quad LC/MS
NAME
LC-QQQ System
TYPE
Agilent Technologies
BRAND
G6470A
SKU
Equipment
1290 Infinity UHPLC
NAME
Ultra-high performance liquid chromatography system
TYPE
Agilent Technologies
BRAND
1290 Infinity UHPLC
SKU
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, analytes, and chemicals used in this protocol are listed in the 'Materials' section. They are excluded from in-line referencing to keep steps clear and concise.
Internal Standard Preparation
Internal Standard Preparation
This analysis uses 4-Hydroxybenzoic-2,3,5,6-d4 acid (d4-4HBA) as an internal standard. Prepare a 500 µg/mL internal standard working solution into 50:50 Acetone / MilliQ Water. Plan to prepare enough solution volume to allow for addition of d4-4HBA to each sample or standard volume at a 1:100 volumetric ratio.
For example, a 500 µg/mL concentration of internal standard working solution was used for a 10 µL spike into 1000 µL sample volume. In this scheme, the 5 µg/mL concentration added will need to be accounted for in data processing.
Preparation of Standards
Preparation of Standards
By weight, create individual 20,000 µg/mL stock solutions of all monomers (listed in Materials section) and use 50:50 Acetone / MilliQ Water as a diluent.
Combine the stock solutions to create a 100 µg/mL mixed standard working solution in 50:50 Acetone / MilliQ Water.
For example, to prepare 10 mLs of mixed standard working solution of aromatic monomers (distinguished in Materials section), add 50 µL of each of the 20,000 µg/mL stock solutions (10 analytes) and add 9500 mL 50:50 Acetone / MilliQ Water.
Using the mixed standard working solution at 100 µg/mL, create a calibration curve with a minimum of five points using 50:50 Acetone / MilliQ Water as the diluent.
Add 10 µL of the 500 µg/mL d4-4HBA internal standard working solution per every 1000 µL of calibration standard.
example calibration level preparation
Sample Preparation
Sample Preparation
Ensure samples to be analyzed are suspended in a matrix compatible with your instrumentation. Analytes of interest in samples should be expected between 1 µg/mL and 100 µg/mL concentrations, otherwise dilution should be carefully performed to achieve concentrations in this calibration range.
If dilutions are required, add internal working solution (ISWS) at a 1:100 (ISWS volume / final sample volume) scheme.
Add 10 µL of the 500 µg/mL d4-4HBA internal standard working solution per every 1000 µL of sample volume. This is best done when sample volume is normalized, either following dilution or by pulling precise volumes of sample into a new sample analysis vial.
UHPLC-MS/MS Analysis
UHPLC-MS/MS Analysis
Prepare an Agilent 1290 UHPLC system according to the following parameters:
Use the analytical column listed here:
Equipment
Kinetex Phenyl-Hexyl
NAME
Analytical Column
TYPE
Phenomenex
BRAND
00D-4500-AN
SKU
LINK
2.1 mm x 100mm (1.7µm pore size)
SPECIFICATIONS
Analyze samples using an Agilent 6470A Triple Quadrupole Mass Spectrometer equipped with dual Agilent jet stream electrospray ionization (AJS ESI) per the method parameters listed below:
Optimized multiple reaction monitoring (MRM) transitions for quantification of aromatic monomers as listed in 'Materials'. Fragmentor voltages (V) and corresponding collision energies (CE) for LC-MS/MS analysis are supplied for quantifier and qualifier transitions respectively.
Data analysis utilizes Agilent Quantitative Analysis for QQQ Build Version B.08.
Analytical Quality Control
Analytical Quality Control
Several strategies are utilized when performing this analysis to ensure instrument stability and reproducibility.
Calibration Curves
All compounds must have a correlation coefficient (r2) of 0.995 or greater using a quadratic calibration fit.
Calibration Verification Standards (CVS)
A calibration verification standard (CVS) is a standard from the calibration curve that is re-analyzed every 20 or fewer samples to ensure instrument drift remains within the determined acceptance criteria. Acceptable CVS recoveries for this analysis are within 15% of the expected amount. Acceptance criteria may differ between instruments and should be determined experimentally.