Non-targeted analysis (NTA) is designed to find what researchers do not yet know to look for. That objective creates a fundamental sample-preparation challenge: laboratories must recover chemicals spanning a wide range of physicochemical properties while limiting matrix interference, instrument fouling, and background contamination.
At RTI International’s Center for Analytical Sciences, Research Scientist Dr. Imari Walker-Franklin uses liquid and gas chromatography coupled with high-resolution accurate mass spectrometry (LC-HRMS and GC-HRMS) to investigate environmental contaminants. Her work illustrates why NTA sample preparation cannot be treated as a conventional targeted method with a longer analyte list.
“When setting up non-targeted analysis for unknown or emerging contaminants across diverse matrices, the goal is not to optimize for a single compound,” Dr. Walker-Franklin explains. “Instead, the focus shifts toward maintaining broad-spectrum coverage without fouling sensitive high-resolution instruments or introducing laboratory contamination.”
Designing Extractions for Broad Chemical Coverage
Unknown contaminants can span wide ranges in molecular weight, polarity, ionizability, volatility, and partitioning behavior, including differences reflected by octanol–air (Koa) and octanol–water (Kow) partition coefficients. An extraction optimized for one chemical class can exclude others before they reach the mass spectrometer.
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“Designing extraction workflows for non-target analysis inevitably involves some compromise,” Dr. Walker-Franklin says. “To avoid unintentionally excluding entire compound classes, I build the workflow around broad chemical coverage rather than optimizing for a single analyte group. We use a diverse quality-control mixture of representative emerging contaminants to evaluate recoveries across chemical classes and matrix types.”
These recovery patterns reveal where compound losses occur, guiding adjustments to extraction solvents, cleanup intensity, or matrix handling.
“When needed, I use complementary extraction approaches so that no single workflow becomes overly selective,” she adds. “When a project prioritizes a specific compound class, such as PFAS, I review established methods for those analytes and adapt relevant elements to improve coverage while maintaining the broader goals of the non-target workflow.”
Protecting HRMS Performance Without Losing Trace Analytes
Complex environmental extracts can cause ion suppression, contaminate the source, and compromise chromatographic performance. However, aggressive cleanup can also remove the trace-level unknown analytes researchers are trying to identify.
“My main approach is to balance matrix cleanup with preservation of trace-level analytes,” says Dr. Walker-Franklin. “I typically use targeted cleanup steps, such as solid-phase extraction and post-extraction filtration, to reduce particulate matter and remove the most problematic matrix components without making the workflow overly selective. Guard columns are also useful for preserving analytical column performance.”
“I incorporate procedural blanks and solvent blanks throughout the sequence to monitor carryover and background contamination, often placing blanks after every 10 samples or after samples expected to have higher matrix load,” she explains. “In addition, I run an LC reference mixture before and after sample batches to assess instrument response, mass accuracy, and retention time stability.”
These checks help determine whether additional cleaning, source maintenance, or method optimization is needed without automatically subjecting samples to more aggressive cleanup.
The need to balance sample preparation with analytical coverage also extends to Walker-Franklin’s work with microplastics.
Balancing Digestion With Microplastic Integrity
Isolating microplastics from complex samples presents a distinct analytical trade-off: digestion steps that remove organic material can affect synthetic polymers or deplete chemicals associated with their surfaces.
For water samples, Dr. Walker-Franklin’s lab filters the liquid to isolate solid particles for micro-Fourier transform infrared spectroscopy (µFTIR) or pyrolysis gas chromatography–mass spectrometry (Py-GC-MS), while processing the filtrate separately via SPE to investigate dissolved chemical constituents, including polymer additives.
Biological matrices such as oysters present a more difficult compromise because conditions that effectively remove organic material may not preserve all associated chemical information.
“For more complex biological matrices, we generally prioritize polymer isolation over additive analysis because digestion conditions that efficiently remove organic material can also alter or deplete sorbed chemicals,” Dr. Walker-Franklin explains.
Her laboratory uses controlled digestion approaches such as potassium hydroxide treatment combined with wet peroxide oxidation, keeping temperatures below 60 °C to minimize potential effects on polymer integrity. Representative polymer controls undergo the same digestion
process to evaluate recovery and determine whether preparation affects polymer types differently.
The analytical method can provide additional information about polymer-associated chemicals.
“Double-shot Py-GC-MS can be useful because the first shot can provide information on polymer-associated additives, while the second shot supports polymer identification and quantitation,” Dr. Walker-Franklin notes. “Overall, I view the most reliable approach as matrix-specific, temperature-controlled, and supported by recovery checks that help preserve both particle integrity and chemical interpretability.”
Separating Sample Signals From Laboratory Contamination
Microplastics create a difficult QA/QC problem because potential contaminants exist throughout modern laboratory environments. QA/QC controls must therefore help distinguish sample-derived particles from laboratory background.
“Strict contamination control is essential,” says Dr. Walker-Franklin. “I generally follow QA/QC practices commonly recommended in the microplastics field, including the routine use of sample-preparation blanks, analytical blanks, and spiked quality-control samples to assess background contamination, recovery, and method performance.”
She also minimizes plastic use during sample collection, extraction, and analysis where possible. Glassware is preferred when appropriate, baked before use, and handled to limit airborne or contact contamination.
“Sample preparation is also conducted in a dedicated microplastics workspace to reduce external sources of contamination,” she explains. “We use sticky mats at the room entrance to limit dust transfer from shoes, wear cotton laboratory coats, and work to minimize any plastic materials and supplies utilized, including reduced-plastic writing tools, whenever practical. Reagents, solvents, and rinse solutions are filtered prior to use.”
These controls allow background contamination to be evaluated alongside the samples rather than treated as an unexpected interference after analysis.
Using Automation to Reduce Sample-Preparation Delays
Processing large sets of environmental water samples within short holding times can create a sample-preparation bottleneck. Delays matter in NTA because researchers do not know in advance which compounds may be vulnerable to losses or degradation.
“For non-targeted analysis of environmental waters, one of the biggest sample-preparation bottlenecks was the reliance on manual solid-phase extraction, particularly when large sample sets had to be processed within short holding times,” Dr. Walker-Franklin recalls. “Because the
target analytes are unknown, minimizing delays is important to reduce the potential for losses, degradation, or changes in the chemical profile before extraction.”
Her team has recently moved toward automated SPE, including acquiring the system and optimizing the sample setup.
“Automation helps improve throughput, consistency, and timing across sample batches while reducing the hands-on burden associated with manual extraction,” she says.
For NTA, that consistency has significance beyond laboratory efficiency. Reducing delays and variation during sample preparation can help limit changes to the chemical profile before instrumental analysis begins.
Building an NTA Workflow From the Ground Up
For chromatographers transitioning from targeted quantitative methods to non-targeted workflows, Dr. Walker-Franklin cautions against treating NTA as an expanded targeted method.
“My main advice would be to start by building a strong foundation in established best practices rather than trying to adapt targeted workflows directly to non-target analysis,” she says.
She points to resources from the Best Practices for Non-Targeted Analysis (BP4NTA) organization, along with introductory short courses and conference workshops, as useful starting points. These resources can help chromatographers consider the full workflow, including study design, sample preparation, data processing, quality control, and interpretation.
“Overall, the transition is much easier when it is approached as a structured workflow with clear QA/QC expectations rather than simply an expanded version of targeted method development,” Dr. Walker-Franklin concludes.
