Restek at HPLC 2025

Per- and polyfluoroalkyl substances (PFAS) are everywhere – in the environment, food, and even the human body. Although they are prevalent in all areas chemists are interested in, their analysis remains a challenge. Different matrices require tailored sample preparation strategies, and the getting sensitivity and selectivity for a given analyte panel requires the choice of the correct analytical column.

In this talk, we explore how modern LC-MS/MS methods can be optimized to reliably quantify PFAS in complex samples; which columns provide the best separation performance, and which sample preparation techniques maximize recovery whilst minimizing matrix effects. We will highlight best practices, common pitfalls, and innovative approaches for robust and precise PFAS analysis – from environmental samples to clinical research.

LC method optimization, in general, is labor intensive. It requires unique skill sets to understand column chemistry, analyte chemistry, and method goals to optimize methods. With expert chromatographers at work, Restek scientists have mastered the skills of method development and have created a seamless platform that can transform your method development and optimization experience along with column recommendations. In this learning exercise, we will demonstrate the benefits of Restek's Pro EZLC platform.

Per- and Polyfluoroalkyl substances(PFAS) have been a trending global issue for a number of years now. As more research is being carried out on these compounds, their breakdown paths, and their bioavailability, additional methods are required to quantify at lower concentrations, and to look for novel chemistries. As this search has expanded, shorter chain, and particularly ultrashort chain (USC) PFAS with carbon backbone lengths shorter than C4 have become areas of focus. In the most recent EU Commission Notice for Drinking Water (C/2024/4910) the fact TFA was not included in their definition of "PFAS Total" is highlighted as potential cause for concern, and both TFA and USC PFAS more generally are highlighted as areas for more specific monitoring.

In this poster we examine two separate approaches to examining USC PFAS compounds. The first is a comprehensive look at a panel of PFAS compounds (C1-C14) with a dilute and shoot method using an inert coated, polar embedded alkyl LC column. The second is a more targeted screen via direct injection, focussed upon USC PFAS utilising a novel HILIC/ion exchange LC column. These methods were then confirmed utilising a variety of fortified potable and non-potable water samples (industrial effluents, along with bottled, tap and spring waters). Both workflows demonstrate accuracy, sensitivity and linear calibration ranges of ppt levels, within industrially acceptable recovery values (70-130%).

Cannabis consumption (and commercialisation) has been on the rise globally, with numerous countries decriminalising or legalising medical or personal consumption across the last decade. With this has come an increased interest in the various forms of the psychoactive compound tetrahydrocannabinol(THC) which are present in varying quantities within the cannabis plant. Even with decriminalisation or legalisation of cannabis, the vast majority of workplaces require staff to carry out their duties unimpaired for legal purposes, and it remains a criminal offence to perform tasks like operating heavy machinery under the influence of substances. These circumstances have combined to require more robust blood testing programmes focussed not only on the historically prevalent Δ-9-THC, but increasingly Δ-8-THC along with each of their hydroxylated and carboxylated metabolites (11-OH-Δ8/9-THC and Δ8/9-THC-COOH).

The separation of Δ-8- and Δ-9-THC along with their metabolites pose a particular chromatographic challenge, with three pairs of isomers differentiated positionally by one double bond, making quantification of these compounds particularly challenging. In this paper we explore the impact C18, Biphenyl and Fluorophenyl (PFP) HPLC column phases have upon separation of these isomers and their metabolites. With a suitable candidate phase selected, a  method was then developed for optimal separation for these analytes in whole blood samples. This method demonstrated suitable response across a biologically relevant calibration range, and with suitable separation to minimise interference from other structurally similar or commonly encountered cannabinoids.

As LC-MS/MS continues to grow as the dominant separation technique within a host of industries, more laboratories are trying to run broad panels of analytes from single injections, requiring robust chromatography capable of separating analytes, isobars, and matrix elements suitably to afford suitable detector sensitivity for qualification and quantitation. Column selection is the single most impactful factor when it comes to tackling these issues, and yet the most common method development approach globally remains scanning gradients using a C18-type column.

In this poster we leverage a virtual method development tool to compare the selectivity of a Biphenyl column against several C18-type phases for a panel of pesticide standards. Initial runs were performed to verify modelled retention times against real-world LC-MS/MS data utilising a Biphenyl phase, RT differences were compared, with results demonstrating no greater than 10.8s across a 14 minute programmed gradient. Various C18-type columns were then similarly modelled, with the Biphenyl column demonstrating enhanced retention and selectivity for molecules containing phenyl rings, and increased retention for compounds which eluted early into the run (which may typically overlap with matrix elements otherwise).