Jan 07, 2024

Public workspaceAnalysis of products from deconstructed nylon-6 by UHPLC-MS/MS (dMRM) V.1

Nature Communications
DOI
dx.doi.org/10.17504/protocols.io.kxygx331dg8j/v1
  • 1Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA;
  • 2BOTTLE Consortium, Golden, CO, USA
Kelsey J. Ramirez
Open access
DOI: dx.doi.org/10.17504/protocols.io.kxygx331dg8j/v1
External link: https://doi.org/10.1038/s41467-024-45523-5
Protocol Citation: Kelsey J. Ramirez, Morgan A. Ingraham, Elizabeth L. Bell, Gregg T. Beckham 2024. Analysis of products from deconstructed nylon-6 by UHPLC-MS/MS (dMRM). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx331dg8j/v1
Manuscript citation:
Bell, E.L., Rosetto, G., Ingraham, M.A. et al. Natural diversity screening, assay development, and characterization of nylon-6 enzymatic depolymerization. Nat Commun 15, 1217 (2024). https://doi.org/10.1038/s41467-024-45523-5
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: August 21, 2023
Last Modified: January 07, 2024
Protocol Integer ID: 86732
Keywords: mass spectrometry, nylonase, nylon-6 deconstruction, enzymatic deconstruction, UHPLC-MS/MS of nylon-6 products
Funders Acknowledgement:
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. This work was performed as part of the BOTTLE™ Consortium and was supported by Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (BETO), and Advanced Materials and Manufacturing Technologies Office (AMMTO).
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 deconstruction of nylon-6 utilizing ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection. This method employs reverse phase chromatography and dynamic multiple reaction monitoring (dMRM) in positive ion mode using electrospray ionization (ESI).  
Guidelines
This protocol utilizes an ultra-high pressure liquid chromatography tandem mass spectrometer (UHPLC-MS/MS) system manufactured by Agilent Technologies as referenced in 'Materials'. A similar liquid chromatography tandem mass spectrometry system can be utilized however, some parameter nomenclature may deviate depending on the manufacturer.
Materials


Reagents:
ReagentMethanol OptimaFisher ScientificCatalog # A454SK
ReagentFormic acid 98 % pureThermo ScientificCatalog # AC147932500

Reagent6-Aminocaproic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2504
Reagent6-(6-aminohexanamido)hexanoic acidAdvanced ChemBlocksCatalog #P51557
Reagent6-(6-(6-Aminohexanamido)hexanamido)hexanoic AcidToronto Research Chemicals IncCatalog #A618823-100MG

Reagentε-CaprolactamMerck MilliporeSigma (Sigma-Aldrich)Catalog #C2204-500G

Reagent18-Diaza-29-diketocyclotetradecaneToronto Research Chemicals IncCatalog #D417643

Materials:

syringe filters for aqueous matrices-
Equipment
syringe filters, 13mm nylon membrane
NAME
syringe filter
TYPE
Cytiva
BRAND
4550
SKU
https://www.cytivalifesciences.com/en/us/shop/lab-filtration/syringe-filters-non-sterile/nylon-non-sterile-syringe-filters/acrodisc-syringe-filters-with-nylon-membrane-p-36371
LINK
13mm
SPECIFICATIONS
syringe filters for organic matrices-
Equipment
Syringe Filters, 13 mm PTFE
NAME
syringe filter
TYPE
Cytiva
BRAND
2400
SKU
https://www.cytivalifesciences.com/en/us/shop/lab-filtration/syringe-filters-non-sterile/wwptfe-non-sterile-syringe-filters/acrodisc-syringe-filters-with-universal-membranes-p-36635
LINK
13mm
SPECIFICATIONS


guard column holder-
Equipment
SecurityGuard ULTRA holder
NAME
Guard column holder
TYPE
Phenomenex
BRAND
AJ0-9000
SKU
https://www.phenomenex.com/part?partNo=AJ0-9000
LINK
2.1 to 4.6mm ID
SPECIFICATIONS
guard column-
Equipment
UHPLC C18 guard cartridge
NAME
guard column
TYPE
Phenomenex
BRAND
AJ0-8782
SKU
https://www.phenomenex.com/part?partNo=AJ0-8782
LINK
2.1mm ID
SPECIFICATIONS
analytical column-
Equipment
Zorbax Eclipse plus C18 Rapid Resolution HD
NAME
analytical separation column
TYPE
Agilent
BRAND
959757-902
SKU
https://www.agilent.com/store/en_US/Prod-959757-902/959757-902
LINK
2.1 mm x 50 mm, 1.8 µm
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
https://www.agilent.com/en/product/liquid-chromatography/hplc-systems/analytical-hplc-systems
LINK



Safety warnings
Attention
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
Preparation of Standards
  • By weight, create individual 2000 µg/mL stock solutions of all analytes listed below using ultrapure water (18.2MΩ⋅cm)(UPW) as a diluent, except for 6-aminohexanoic acid cyclic-dimer in which methanol is used:
  • ɛ-Caprolactam
  • 6-Aminohexanoic acid
  • 6-Aminohexanoic acid dimer (6-(6-aminohexanamido)hexanoic acid)
  • 6-Aminohexanoic acid trimer (6-(6-(6-aminohexanamido)hexanamido)hexanoic acid)
  • 6-Aminohexanoic acid cyclic-dimer (18-diaza-29-diketocyclotetradecane)

Combine the stock solutions to make a 400 µg/mL mixed standard working solution (mSWS) in UPW.  
  1. This is accomplished by mixing equal volumes of the stock solutions of all 5 analytes
  2. Prior to creating the calibration curve, perform an additional dilution of the mSWS. The final concentration will be 10 µg/mL. This can be prepared by adding 0.5 mL of the mSWS to 19.5 mL of UPW. 
  1. Using the mSWS at 10 µg/mL, create a calibration curve with a minimum of 5 points using UPW as a diluent. 

Table showing an example standard curve preparation. (click to enlarge)

Sample Preparation
Sample Preparation
Ensure sample matrix is compatible with instrumentation. The calibration range for this method is between 0.01 µg/mL and 7 µg/mL. All samples should be 0.2µm filtered prior to injection. Any samples expected to have analyte concentrations that fall outside of this range should be diluted appropriately.
Note: Analyte suppression was observed in high salt containing samples. As a result, a 5x dilution was performed using methanol to precipitate the salts. The samples were then 0.2µm filtered and analyzed.  
UHPLC- MS/MS Analysis
UHPLC- MS/MS 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)

Analyze samples using an Agilent 6470A 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)
Below a table is provided with the optimized dynamic multiple reaction monitoring reactions (dMRM) as well as corresponding fragmentor voltages (V) and collision energies (CE) for the quantifying and qualifying transitions. 


dMRM analyte transitions (click to enlarge)
Note: Standards could not be sourced for 6-aminohexanoic acid cylic-trimer, 6-aminohexanoic  acid tetramer, and 6-aminohexanoic acid pentamer. These were optimized from a sample that verified their presence by high resolution mass spectrometry ESI (HRMS-ESI). 
Data Analysis and Quality Control
Data Analysis and Quality Control
Data analysis completed using Agilent Quantitative Analysis for QQQ version 10.1 
Quality Control
Several criteria are used to ensure instrument stability and reproducibility throughout the analysis.
  • Calibration curves must have a correlation coefficient (r2) of greater than or equal to 0.995 using a quadratic or linear fit.
  • 6-Aminohexanoic acid cyclic-trimer is quantified using the calibration curve of 6-aminohexanoic acid cyclic-dimer
  • 6-Aminohexanoic acid tetramer and 6-aminohexanoic acid pentamer are quantitated using the calibration curve of 6-aminohexanoic acid trimer.   
  • 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, acceptable CVS recovery range is within +/- 15% of the expected amount.  
Example Chromatogram
Example Chromatogram



An example chromatogram of a sample containing deconstruction products of nylon-6. The solid line indicates quantifying MRM transition and dashed lines represent the qualifying MRM transistion (when available). (click to enlarge)