Gas Chromatography Meets Non-Targeted Screening

We take a look at the past, present, and future of applying gas chromatography–mass spectrometry (GC–MS) techniques to non-targeted screening (NTS) in various disciplines, assessing both the opportunities and the challenges.

The application of gas chromatography (GC) to non-targeted screening (NTS) has a short (but already fine) history. For instance, a recent search for the term gas chromatography–mass spectrometry (GC–MS) in article titles, abstracts, and keywords in the literature database Scopus, linked with keywords such as untargeted screening, non-targeted screening, or untargeted analysis, led to 53, 80, and 395 scientific reference hits, respectively. Initial research studies on this topic were published in the late 1990s; however, most publications have been released in the last decade.

In practical laboratory environments, many scientists feel that GC–MS has already been quite well implemented for NTS applications. This assessment mostly reflects a misunderstanding of real NTS. A typical GC–electron impact (EI)–MS run, in which the ionization is performed by “hard” EI, followed by a National Institute of Standards and technology (NIST) database search, is really more of a suspects screening approach (for “known unknowns”) than a classical NTS.

This year, however, GC–MS vendors and several users came together for the first time to present and discuss their experience with “real” NTS by GC–MS, and to explore the options for such studies. This workshop, which took place online in February 2021, was consequently called “GC meets NTS.” In this meeting, participants discussed, clarified, and accentuated the power and excellent usability of GC–MS in NTS, and decided to form a nonaffiliated expert group. A 20-minute video that summarizes the two days of presentations and platform discussions is available online at https://afin-ts.de/gc-meets-nts/.

Topics like analytical instrumentation and data analysis strategies in NTS were the focus of that workshop, and they also represent the tasks that have to be addressed for GC–MS to have a successful future in NTS. Some of these challenges are discussed in this article.

Instrumental Needs for NTS with GC–MS

Comprehensive NTS requires a broad analytical view of (mainly) organic molecules—a smart combination of analytical techniques in sample preparation, extraction, and injection, as well as GC separation coupled to highly accurate and high-resolution MS (HRMS) systems. One key aspect of GC–MS and NTS is the use of soft ionization (SI) sources in addition to the classical EI interfaces. Soft ionization sources, such as chemical ionization (CI), mainly form molecular ions and fewer “in-source” fragments (as observed in EI). Recent developments of various soft ionization sources open the door to perform GC–MS analysis within a new context. Several options for coupling analytical instruments and performing NTS workflows on instrumental level are shown in Figure 1. In addition to low pressure ionization techniques, such as CI, nowadays many atmospheric pressure ionization sources can be used in GC–MS. Examples are atmospheric-pressure chemical ionization (APCI), atmospheric-pressure photoionization (APPI), atmospheric-pressure laser ionization (APLI), and dielectric barrier discharge ionization (DBDI). Finally, a combination of GC–EI-MS and GC–SI-MS might be the perfect solution for enabling comprehensive analysis in NTS. The value of such a combination is underscored by a rumor that the first GC–CI & EI-time of flight (TOF)-MS system may soon be available on the market.

FIGURE 1: Schematic showing multiple options for coupling analytical instruments, such as gas chromatography and mass spectrometry, for performing non-targeted screening (NTS).

To gain the most comprehensive perspective on samples in NTS, it is important to detect as many molecules as possible. Therefore, sample preparation, extraction, and injection are very important not only in terms of their efficiency, repeatability, and robustness, but also in ensuring that all techniques used allow the recovery of as many molecules as possible. As a result, one has to take sample preparation into account and to document the processes (such as whether chemical derivatization or other such techniques were used) in a detailed and meaningful way. Various effective sample preparation–injection systems are available for GC, but all affect samples differently, and because some are highly orthogonal, they can and should be used thoughtfully. The documentation of all applied steps of the analytical procedure is key to ensuring the comparability of NTS results. Sample preparation and injection strategies should not complicate the data analysis that will be performed later; rather, they can contribute to better understanding of the analytical results.

GC–MS allows the use of multiple dimensions (in separation and detection), and more dimensions might come in the future. Independent of the analytical strategy applied, GC, GCxGC, and GCxGC–MS—including GCxGC–MS using soft ionization, using ion-mobility MS, and using ion-mobility tandem MS, all have the high repeatability, robustness, and quality that are of critical importance in GC analyses. Laboratories conducting routine analysis tend to focus more on stability and reproducibility, whereas research laboratories favor the flexibility. Both reproducibility and flexibility can be handled in NTS today, and for the long term requirements, it should be possible to combine both aspects effectively. Just as in many other aspects to our lives, a good approach is to keep things as simple as possible and combine only as much components as is truly needed.

Applying NTS Strategies Across Analytical Techniques

NTS is used in different analytical fields, and developments in one analytical technique can often be applied to others. Some NTS developments and solutions are specific to modern GC–MS/MS systems. However, some strategies and workflows from liquid chromatography–MS/MS (LC–MS/MS) analysis can and should be included wherever possible in GC NTS strategies, and vice versa. Of course, LC–MS also can provide solutions that complement those of GC–MS. A Scopus database search related to NTS, like the one described previously, but with substituting “LC–MS” for “GC–MS” led to 57, 111, and 796 scientific references, respectively (most of them from the last 20 years). Of course, GC and LC are highly orthogonal techniques with very different application areas because of the different classes of compounds they can separate. However, in NTS we aim to assess samples in a highly comprehensive way. As a consequence, the combination of LC-SI-MS, GC-SI-MS, and GC-EI-MS will bring enormous benefits.

Data Analysis for NTS

Taking data evaluation strategies from one discipline and applying them to another will strengthen our analytical concepts. Of course, there are some specialties for data evaluation in one technique or another, but in the end, they all rely on the same basic steps. The idea is always to use all available data and information about a sample and thus find an answer to the analytical question. This approach will be helpful for identifying molecules as well as for specifying statistical solutions. Characteristic workflow schemes are presented in Figure 2.

FIGURE 2: Schematics showing examples of characteristic workflows.

The range of NTS applications of GC–MS spans qualitative needs (such as authenticity and identification) and quantitative specifications (such as pesticides in foods). For such qualitative and quantitative applications of complex molecular information, an automated and digitalized data handling process are needed. Careful documentation and harmonization of parameters such as polarity, boiling point, empirical formula, molecular fragments, and signal intensities, are needed, along with correct statistical handling of these data.

Analytical Fields in Which GC–MS Is Being Applied to NTS

Currently, the majority of applications of NTS are focused on identifying unknown organic molecules, but improved implementation of robust NTS strategies opens the door to other (statistically based) solutions, potentially yielding more detailed molecular analysis. This opens up the possibility of answering questions that have not yet been asked. GC NTS techniques may find their way into applications and fields such as biological screening, metabolomics, biomarker screening, brand piracy (to ensure authenticity of origin as well as for safety concerns about impurities in counterfeit products), environmental analysis (air and water), food fraud (in products like olive oil, vanilla, herbs, and additives), food safety and stability (for fingerprinting, batch tests, and aroma stability), leachables and extractables (for pharmaceuticals or consumer products), and monitoring of production processes (like chemical synthesis).

What Will Come Next in “GC Goes NTS”?

At this point, all of us in the analytical science community have to keep in mind that NTS is a complex analytical strategy, and that we should all aim for the highest standards in robustness, comparability of results, and validity. Bringing together highly complex analytics, advanced data evaluation, and powerful statistical methods in NTS is a huge challenge. Next, each laboratory has to decide if data handling solutions should be centralized or decentralized, on site or in the cloud, and whether vendor-driven or open-source solutions should be used. Finally, data handling must be professional and sustainable; and care must be taken to determine whether real retrospective analysis of NTS data can be done in the future, or whether claims of retrospective analysis capabilities will just become a publicity ploy.

Let’s come together to take advantage of the current momentum for NTS using GC–MS to create something global and sustainable. If you want to participate, don’t hesitate to join the group! You can also present your projects and thoughts at the International Conference on Non-Target Screening (ICNTS 21), which will be held October 4–7, 2021, in Erding, Germany, and online (https:// afin-ts.de/icnts-21).

Thomas Letzel is an analytical chemist with more than 20 years of experience in analytical screening using liquid and gas chromatography with mass spectrometric detection. Formerly, he was the head of the Analytical Research Group at the Technical University of Munich (TUM), in Germany. Now he is a lecturer at TUM and the founder and Executive Director of AFIN-TS GmbH. He is an author or co-author of more than 150 journal papers, book contributions, and conference proceedings, and of four books. Direct correspondence to: t.letzel@afin-ts.de

Stefan Bieber was formerly a researcher and the Chair of Urban Water Systems Engineering at TUM. He received his PhD in 2017 following studies on the use of polarity-extended chromatographic separation techniques and water management strategies. Bieber is currently the Executive Director of AFIN-TS GmbH, where he conducts research and provides analytical support for companies in non-targeted screening. Direct correspondence to: s.bieber@afin-ts.de