Restek at ASMS 2025

Introduction: The quantitative analysis of bile acids in plasma is critical for diagnosing many structural liver diseases. There are two main types of bile acids based upon their functional groups: unconjugated (or free) and conjugated, primarily with glycine- or taurine-based residues. In this study, 17 bile acids were being analyzed by LC-MS/MS using a Raptor C18 column. Through routine validation, a matrix interference was identified to be coeluting with one of the bile acids standards, which resulted in quantitation issues. As the interference could not be resolved on the Raptor C18 column, it was necessary to explore alternative stationary phase chemistries for effectively separating the matrix interference.

Methods: Biphenyl, FluoroPhenyl, and ARC-18 stationary phases were tested on a 100 x 2.1 mm, 2.7 µm column dimension using water with 5 mM ammonium acetate as mobile phase A and 50:50 methanol: acetonitrile as mobile phase B. The column temperature was 50 °C. Data was collected in ESI- mode.

Preliminary Data: The Biphenyl stationary phase was able to partially resolve the matrix interference and showed selectivity for the taurine and glycine conjugated isomers but limited selectivity for the unconjugated isomers. The FluoroPhenyl stationary phase was able to resolve the matrix interference but did not show selectivity for the three isomer sets. The ARC-18 stationary phase was able to resolve the matrix interference and showed selectivity for the three isomer sets. The gradient using the ARC-18 column was optimized to separate all 17 bile acids, including full resolution of the isomer sets and the matrix interference in a 9.5-minute cycle time. Acceptable linearity, accuracy, and precision were demonstrated over an analytical measurement range of 0.5-5 µM for all analytes using the developed method.

Novel Aspect: The developed method overcomes challenges associated with bile acids analysis while also resolving a matrix interference that prevented proper quantitation.

Introduction: The “gold standard” biological matrices for toxicological testing are typically blood and urine, however; collection of these two matrices is invasive. The analysis in oral fluids is an increasingly popular solution due to its ease of collection compared to blood or urine. However, when performing the oral fluid analysis, it can be difficult to remove all of the surfactants present in the collection device’s buffer solution. These surfactants can cause matrix effects and poor column lifetime. Finding a workflow that uses a simple sample preparation paired with accurate and robust quantitation of the analytes is important for laboratories running these tests. In this work, a dilute-and-shoot sample preparation was compared against supported liquid extraction (SLE) and salt-assisted liquid-liquid extraction (SALLE).

Methods: An LC-MS/MS method was developed using a Raptor Biphenyl 50 x 2.1 mm, 2.7 µm analytical column equipped with a Raptor Biphenyl EXP 5 x 2.1 mm, 2.7 µm guard column. A total of 68 analytes were separated using gradient conditions consisting of water (MPA) and methanol (MPB), both containing 0.1% formic acid, for a total cycle time of 10 minutes. Samples were prepared in oral fluid and combined with QuantisalTM buffer. Aliquots from the buffer underwent each of the sample preparation techniques; dilute-and-shoot, SALLE, and SLE. Samples were dried down and reconstituted in 90:10 MPA: MPB and moved to the instrument for analysis.

Results: The biphenyl stationary phase resolved all 68 analytes, including X sets of isobaric analytes, such as methamphetamine and phentermine, isotonitazene and protonitazene, and eutylone and pentylone. All sets of isobars achieved a resolution of 1.5 or greater, providing accurate quantitation of the analytes. Performance metrics, such as recovery, linearity, matrix effects, and accuracy and precision, were evaluated. All analytes passed both intra- and interday accuracy and precision requirements without matrix interferences or effects being observed. Linearity was demonstrated using 1/x weighted linear regression and with an r2 of ≥ 0.991. An evaluation was completed comparing analyte recovery when using dilute-and-shoot to SLE and SALLE approaches. Dilute-and-shoot failed to remove the buffer surfactants compared to SLE and SALLE. Both SALLE and SLE returned increased sensitivity compared to dilute-and-shoot for all analytes (excluding pregabalin, gabapentin, THC, and CBD for the SLE workflow). SALLE yielded higher sensitivity for a broader range of analytes while SLE achieved increased sensitivity for most of the opiates. This work demonstrates an accurate and robust solution for the analysis of these analytes. Additionally, this workflow demonstrates quick and efficient sample cleanup procedures that remove buffer surfactants without the need for SPE or other tedious extraction techniques, leading to faster processing of samples in high through-put laboratories.

Novel Aspect: Comparison of analyte recovery in a newer matrix, oral fluid, when using multiple different sample cleanup techniques.

Abstract: Solid-phase extraction (SPE) is a valuable sample preparation technique for environmental matrices utilized in extracting pesticides, semivolatiles, and emerging contaminants from water samples. Despite its efficacy, SPE encounters challenges in non-potable water matrices where traditional ion-exchange SPE fails to remove certain matrix interferences, leading to the implementation of dispersive carbon protocols. Furthermore, solid particles in the sample can obstruct SPE cartridges, prolonging extraction or needing multiple cartridges to complete the analysis. The recent EPA Method 1633 aims at the analyses of PFAS in aqueous, solids, biosolids, and tissue samples where extract cleanup steps, as well as percent solids, can affect the overall length and performance of the sample preparation. In this work, WAX and Graphitized Carbon Black (GCB) dual-bed SPE cartridges were compared to conventional single-bed SPE formats towards precision, accuracy, and reduction of semi and nonvolatile residues. Furthermore, a novel filter aid was assessed for the mitigation of cartridge clogging and compared to glass wool using an ASTM substitute wastewater matrix. The recoveries for all 40 PFAS analytes in the fortified sample ranged from 80%-120%, and the relative standard deviation was less than 20% for most of the analytes. The advantage of the filter aid was significant as it provided 60% better filtration efficiency when compared to glass wool and over 90% when compared to the unfiltered SPE controls. The experimental SPE format offered a simplified and consistent approach when dealing with dirty matrices while meeting the accuracy and precision of EPA 1633. Details on the HPLC parameters afforded will also be discussed.

Introduction (120): Ultrashort-chain (USC) per- and polyfluoroalkyl substances (PFAS) are small and very polar compounds with carbon chain lengths shorter than C4. Their ubiquitous and high levels of occurrence in environmental aquatic systems are emerging as a significant concern, rivaling the well-established issues associated with long-chain PFAS contamination. Therefore, it is important to analyze both USC and long-chain PFAS together in water samples to comprehensively assess and address the full spectrum of PFAS contamination. In this study, a simple and reliable workflow was developed for the simultaneous analysis of C1 to C14 perfluoroalkyl carboxylic and sulfonic acids, along with other groups of PFAS, in both potable and non-potable waters.

Methods (120): A dilute-and-shoot workflow was evaluated by accuracy and precision analysis of fortified tap water, bottled water, and treated sewage wastewater. Calibration standards were prepared in reverse osmosis water ranging from 1-1000 ng/L. Five and eighteen mass-labeled standard mixtures were added and served as quantitative and extracted internal standards, respectively. Both standard and water samples were diluted two-fold with methanol containing 1% acetic acid for LC-MS/MS analysis. The chromatographic separation was conducted using a polar-embedded reversed-phase LC column with an inert coating on the hardware. Additional potable and non-potable waters collected from various source waters were tested to further demonstrate that the established workflow is suitable for the accurate quantification of targeted PFAS in a wide range of water matrices.

Preliminary Data: (300) The high polarity of ultrashort-chain PFAS poses a challenge to current analytical practices based on reversed-phase liquid chromatography due to insufficient chromatographic retention. This study demonstrated that a polar-embedded reversed-phase column allows for the simultaneous analysis of ultrashort-chain and long-chain PFAS. The targeted analytes included C2 to C14 perfluoroalkyl carboxylic acids, C1 to C13 perfluoroalkyl sulfonic acids, fluorotelomer carboxylic acids and sulfonic acids, perfluorooctane sulfonamides and sulfonamidoacetic acids, and per- and polyfluoroether carboxylic acids and sulfonic acids.

Employing quadratic regression (1/x weighted), all analytes exhibited acceptable linearities with r2 >0.995 and deviations <30%. The linearity ranges varied among analytes, spanning from 1 ppt to 1000 ppt, with some variation observed at the lowest calibration concentration. Accuracy and precision were assessed using fortified water samples at concentrations of 2, 4, 10, 50, and 250 ppt. Three batches of analyses were conducted on different days, totaling nine repetitions at each fortified level. The results showed that all analytes exhibited recovery values within the range of 70-130% across all fortification levels. Satisfactory method precision was demonstrated with %RSD values within 20%. Additionally, the results indicated that all extracted internal standards had recovery values within 30% of the nominal concentration.

The established workflow was applied to measure targeted PFAS in a variety of source waters including five tap waters, three bottled waters, a natural spring water, two well waters, three creek waters, one treated sewage wastewater, one hospital effluent water, one metal finisher effluent water, and one chemical manufacturer effluent water. Consistent with the accuracy and precision analysis, the recoveries of extracted internal standards were within 30% of the nominal concentration across all source waters. This demonstrated that the established method was suitable for accurate measurement of targeted PFAS in a wide range of water matrices.

Novel Aspect (20): A unique method was developed to incorporate ultrashort-chain compounds into comprehensive PFAS analysis in waters.

Introduction: The presence of per- and polyfluoroalkyl substances (PFAS) in drinking water presents a significant concern to human health as these molecules can bioaccumulate and cause a variety of health issues. Coated Blade Spray (CBS) is a sample preparation technology that can be directly interfaced with mass spectrometer (MS) instrumentation for rapid screening and quantitation. In this work, weak anion exchange (WAX) coated blades were used to extract PFAS compounds from water and analyzed by MS with a prototype CBS Ion Source.

Methods: 18 PFAS compounds listed in EPA Method 537.1and internal standards were spiked into LC-MS grade water at concentrations ranging from 10 to 1000 ppt. Extraction from 750 µL samples of spiked water was performed using WAX coated blades for up to 60 minutes. Blades were then rinsed in water and allowed to air dry. Analysis was performed using an Opentrons liquid handling system with a prototype CBS Ion Source coupled to a Thermo TSQ Altis triple quadrupole mass spectrometer at a rate of 30 seconds per sample.

Preliminary Data: Of the 18 compounds analyzed, 14 showed signal-to-noise > 3 at 10 ppt and provided good linearity across a wide concentration range.

Novel Aspect: Coated blades utilizing a WAX coating were used to extract and analyze PFAS compounds.