High-resolution quadrupole time-of-flight (QTOF) gas chromatography–mass spectrometry (GC-QTOF-MS) provides a powerful combination of high mass accuracy with trace level detection capabilities. Applying the technique to extractables and leachables testing (E&L) semi-volatiles analysis results in increased identification abilities utilizing accurate masses while reducing the reliance on spectral compound databases.
E&L testing is used to generate chemical information that can support a toxicological risk assessment (TRA). Chemical characterization can yield hundreds, sometimes thousands, of chemicals for toxicologists to assess—many of which are present in very low levels and only present a toxicological risk if they are highly potent. Accurate and complete identification of compounds in the chemical characterization study is critical for completing a TRA that is useful for evaluating the l safety of the materials as well as any unexpected constituents related to manufacturing processes.
Profiling of materials generally includes analysis for volatile organic compounds (VOCs), semi-volatile organic compounds (SVOCs), non-volatile organic compounds (NVOCs) and elemental impurities. Although some SVOCs can be determined based on liquid chromatography–mass spectrometry (LC-MS), GC-MS is the primary tool used for their detection, identification and semi-quantitation. The current norm for E&L testing uses low-resolution GC-MS (i.e., single quadrupole) instruments. This article explores the advantages of utilizing high-resolution GC-QTOF-MS instrumentation for the analysis of SVOCs.
Low-Resolution GC-MS SVOC Profiling
In current practice, after compounds have been detected and extracted for spectra, those that are above a pre-defined threshold (i.e., the analytical evaluation threshold or AET) are then searched against in-house and commercially available nominal mass spectral databases. Combined, the compounds in these databases number in the hundreds of thousands. A significant percentage of compounds detected above the AET in a typical E&L study will be identified based on database searching. The remaining compounds will be identified manually and rely heavily on analyst expertise in structural elucidation and may result in either a complete or partial identification (e.g., compound class or sub-structure).
Perhaps the greatest limitation that an analyst faces when attempting to determine the structure of an unknown based on low-resolution GC-MS data is that the mass measurements in mass-to-charge (m/z) lack the precision and accuracy required to discriminate between potential element combinations of the same nominal mass. As a result, the elemental composition of ions measured with a low-resolution instrument cannot be determined based on their mass measurements (e.g., the elemental composition of an ion at 44 m/z could be one of several different combinations, including C2H6N, C3H8, C2H4O, CH2NO, and CO2).
Shown below is a spectrum for N,N-dimethylpropanamide acquired using a low-resolution instrument. The elemental composition of the ions, including 44 m/z, cannot be determined based on the mass measurements. Although incredibly useful for database searching, low-resolution spectra present significant limitations when manual structural elucidation is required.
High-Resolution GC-QTOF-MS SVOC Profiling
Unlike low-resolution instruments, high-resolution QTOF systems provide mass measurements to the fourth decimal place with low parts per million (ppm) mass error, enabling the elemental composition of ions to be determined with high confidence. Furthermore, acquiring data using low energy ionization (around 10-15 electron volts (eV) vs. 70 eV for high energy) increases the likelihood that the intact molecular ion will be detected. The spectrum below shows the results obtained when analyzing N,N-dimethylpropanamide with a high-resolution system.
Using the accurate mass measurement of the 44 m/z ion the elemental composition is unequivocally determined to be C2H6N. With such information, an analyst is better equipped to determine the structure of what could otherwise be an unknown, partially identified compound, or incorrectly identified compound.
Low & High-Resolution Combination Profiling
At first blush, it may seem that low-resolution GC-MS systems will be replaced in the E&L laboratory with high-resolution ones, but that may not be the case. The main reason is how extensive high-energy, nominal mass, GC-MS compound databases are. Although GC-QTOF-MS data can be searched against such databases, spectral differences are often observed because of the different instrument types. This means even if a compound is in a database, it can be more challenging to match it to the high-resolution spectral data. Therefore, working with the nominal mass data is often preferable for most compounds in a typical extractables study. Other factors that reduce the likelihood of widespread adoption of accurate mass GC-MS include increased study costs to cover up-front, operational and maintenance costs associated with this advanced technology.
The future of SVOC profiling for E&L could include a combination of both low- and high-resolution analyses. Compounds above the AET in the low-resolution data would be identified, semi-quantitated, and reported using initial database searches, leveraging the high-resolution data on an as-needed basis to support more challenging identifications. With this approach, it would be possible to manage the need for more expensive instruments by collecting high resolution data and analyzing representative samples from a study when additional information is warranted.
Consider the example of a tentatively identified compound of special concern, such as a carcinogenic nitrosamine. For the unknown spectrum below, N-Nitroso-diisopropylamine was the top NIST database hit and showed good retention index agreement with the reported value. With the nominal mass spectrum for this low-level compound, it is not possible to rule out the identity being the nitrosamine.
High-resolution analysis using GC-QTOF-MS resulted in the spectrum below for the unknown. N-Nitroso-diisopropylamine as a potential identification was ruled out based on the 130.0862 m/z ion corresponding to C6H12NO2+ rather than the molecular formula of N-Nitroso-diisopropylamine (C6H14N2O). An alternate identification was proposed based on the analysis of the entire spectrum and utilizing information about the sample.
The example above demonstrates the power of high-resolution GC-MS data to aid in the identification of SVOCs. Rather than requiring costly investigations to evaluate compounds of concern, the approach of running samples by high-resolution GC-QTOF-MS alongside low-resolution GC-MS can greatly reduce the likelihood of time-consuming investigations. Additional benefits include more complete and accurate identifications and a decreased reliance on databases.