High-throughput analysis of products from deconstructed nylon-6 by UHPLC-MS/MS (dMRM)

By weight, create individual 2000 µg/mL stock solutions of all analytes listed below using ultrapure water (18.2MΩ⋅cm)(UPW) water 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 mixed standard working solution (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.

Using the mSWS at 10 µg/mL, create a calibration curve with a minimum of 5 points using UPW as a diluent.

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.  

Prepare an Agilent 1290 UHPLC system according to the following parameters for a total run time of 3.0 minutes: 

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). 

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.