TITLE: Analysis of products from deconstructed nylon-6 by UHPLC-MS/MS (dMRM)
AUTHORS: Kelsey J. Ramirez, Morgan A Ingraham, Elizabeth L. Bell, Gregg T. Beckham

[DESCRIPTION]
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).  

[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.  




[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. 

[STEPS]
SECTION: Preparation of Standards
1. 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)

SECTION: Preparation of Standards
2. Combine the stock solutions to make a 400 µg/mL mixed standard working solution (mSWS) in UPW.  
This is accomplished by mixing equal volumes of the stock solutions of all 5 analytes 
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. 
SECTION: Preparation of Standards
3. Using the mSWS at 10 µg/mL, create a calibration curve with a minimum of 5 points using UPW as a diluent. 

 

SECTION: Sample Preparation
4. 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.  
SECTION: UHPLC- MS/MS Analysis
5. Prepare an Agilent 1290 UHPLC system according to the following parameters for a total run time of 7.0 minutes: 

 

SECTION: UHPLC- MS/MS Analysis
6. 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.

 
 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. 


 
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). 
SECTION: Data Analysis and Quality Control
7. Data analysis completed using Agilent Quantitative Analysis for QQQ version 10.1 

SECTION: Data Analysis and Quality Control
8. 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.  
SECTION: Example Chromatogram
9.