TITLE: Aromatic Monomer Analysis by UHPLC-MS/MS
AUTHORS: Stefan J. Haugen, Gregg T. Beckham, Kelsey J. Ramirez

[DESCRIPTION]
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). 

[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. 

[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.

[STEPS]
SECTION: Internal Standard Preparation
1. 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. 
SECTION: Internal Standard Preparation
1.1. 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.
SECTION: Preparation of Standards
2. 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. 

SECTION: Preparation of Standards
3. Combine the stock solutions to create a 100 µg/mL mixed standard working solution in 50:50 Acetone / MilliQ Water. 
SECTION: Preparation of Standards
3.1. 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.
SECTION: Preparation of Standards
4. 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.
SECTION: Preparation of Standards
5. Add 10 µL of the 500 µg/mL d4-4HBA internal standard working solution per every 1000 µL of calibration standard. 

SECTION: Preparation of Standards
5.1.  
  

SECTION: Sample Preparation
6. 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.
SECTION: Sample Preparation
6.1. If dilutions are required, add internal working solution (ISWS) at a 1:100 (ISWS volume / final sample volume) scheme.
SECTION: Sample Preparation
7. 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. 
SECTION: UHPLC-MS/MS Analysis
8. Prepare an Agilent 1290 UHPLC system according to the following parameters:

    
  
 
Use the analytical column listed here:
 

SECTION: UHPLC-MS/MS Analysis
9. 
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: 
  
  
SECTION: Analytical Quality Control
11. Several strategies are utilized when performing this analysis to ensure instrument stability and reproducibility. 
SECTION: Analytical Quality Control
11.1. Calibration Curves
All compounds must have a correlation coefficient (r2) of 0.995 or greater using a quadratic calibration fit. 
SECTION: Analytical Quality Control
11.2. 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.
SECTION: UHPLC-MS/MS Analysis
10. Data analysis utilizes Agilent Quantitative Analysis for QQQ Build Version B.08.