<p><strong>Ask any analytical chemist which hyphenated technique has become indispensable over the past decade, and the answer will almost certainly be the same: tandem chromatography–mass spectrometry. Whether it is </strong><a target="_blank" rel="noopener noreferrer" href="https://www.organomation.com/preparing-samples-for-lc-ms/ms-analysis"><strong>LC–MS/MS</strong></a><strong> quantifying a novel biologic’s metabolic fate, or </strong><a target="_blank" rel="noopener noreferrer" href="https://www.organomation.com/preparing-samples-for-gc-ms/ms-analysis"><strong>GC–MS/MS</strong></a><strong> chasing per- and polyfluoroalkyl substances (PFAS) down to sub-part-per-trillion levels in municipal water, the combination of chromatographic separation with the specificity of tandem mass detection has become the analytical workhorse of choice across virtually every regulated sector.</strong></p><p>Below we examine the key forces propelling this expansion, from biopharma pipeline dynamics and PFAS regulatory mandates to AI-driven automation and reshoring of pharmaceutical manufacturing—and explore what each trend means for laboratories managing sample preparation and analytical workflows.</p><h3><strong>Biopharma: The Engine Room of LC–MS/MS Demand</strong></h3><h4><strong>GLP-1 Agonists and the Metabolomics Gold Rush</strong></h4><p>The explosive commercial and scientific interest in GLP-1 receptor agonists—<a target="_blank" rel="noopener noreferrer" href="https://www.sepscience.com/advancing-glp-1-peptide-analysis-chromatography-challenges-and-innovations-12163"><strong>semaglutide and related peptide therapeutics </strong></a>for diabetes and obesity—has dramatically expanded the analytical demands placed on tandem MS platforms. Discovery programs require precise quantification of peptide drug candidates and endogenous biomarkers across complex biological matrices. These workflows rely on high-sensitivity LC–MS/MS for pharmacokinetic profiling, receptor occupancy studies, and metabolomics characterization. Metabolomics is, in fact, projected to be the fastest-growing application segment in mass spectrometry, driven in part by its growing role in cancer screening, biomarker discovery, and metabolic disease research.</p><p>I have been spending more time learning about GLP-1s because this is the hot topic that people want to discuss on the Concentrating on <a target="_blank" rel="noopener noreferrer" href="https://open.spotify.com/show/1wZg67rCjoGju4yhqxI3tL?si=n9GLz9zjSCOrVfXPxFPhng&amp;nd=1&amp;dlsi=52868e027aad4d22"><strong>Chromatography podcast</strong></a>. With that said, I was astounded looking at CNBC.com during breakfast this morning to see that <a target="_blank" rel="noopener noreferrer" href="https://www.cnbc.com/2026/03/21/glp-1-diets-restaurants-protein-fiber-weight-loss-drugs.html"><strong>one in eight Americans are currently taking one of these drugs such as Ozempic</strong></a>.</p><h4><strong>Antibody–Drug Conjugates and Gene Therapy: Complexity at Every Step</strong></h4><p>The ascent of antibody–drug conjugates (ADCs) and gene therapies has introduced a new level of analytical complexity. ADCs blend the target specificity of monoclonal antibodies with the cytotoxic potency of small-molecule payloads—and characterizing the linker chemistry, drug-to-antibody ratio (DAR), and conjugate stability demands high-resolution LC–MS/MS at every stage from discovery through QC release testing. Gene therapy vectors require equally rigorous characterization of impurities, aggregates, and empty/full capsid ratios.</p><p>This translates into a direct need for low-evaporation, high-recovery concentration methods that preserve labile protein structures and maintain sample integrity prior to LC–MS/MS injection. <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5392"><strong>Nitrogen evaporation workflows</strong></a> that minimize oxidative stress and maintain sample temperature are increasingly critical in these pipelines.</p><h4><strong>Precision Medicine and Multi-Omics Integration</strong></h4><p>The shift toward precision medicine has accelerated adoption of high-throughput, multi-analyte MS platforms. Proteomics, metabolomics, and lipidomics workflows are converging on a single analytical platform—tandem MS—and generating datasets of a scale and complexity that were unimaginable ten years ago.</p><p>Make no mistake: the triple quadrupole serves an irreplaceable role in quantitative targeted analysis. At the same time, <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=134&amp;manufacturerItems=3"><strong>Orbitrap </strong></a>and <strong>Q–TOF</strong> hybrid platforms are growing rapidly as researchers demand full-scan accurate mass for untargeted discovery.</p><h3><strong>Environmental Mandates: PFAS Testing Drives Adoption</strong></h3><p>Environmental testing is something that every person, chemist or not, can understand the importance of. Mounting mandates from the U.S. Environmental Protection Agency and European regulators regarding PFAS—the “forever chemicals” whose extraordinary environmental persistence is matched only by the analytical challenge of detecting them at trace levels—have created immediate, compliance-driven investment in LC–MS/MS infrastructure.</p><h4><strong>The Regulatory Framework Chemists Need to Know</strong></h4><p>The EPA has codified a suite of LC–MS/MS-based methods as the analytical standard for PFAS in water and solid matrices:</p><ul><li><a target="_blank" rel="noopener noreferrer" href="https://blog.organomation.com/blog/epa-533-drying-down-pfas-samples-after-spe"><strong>EPA Method 533</strong></a><strong>:</strong> Weak anion exchange SPE combined with LC–MS/MS; covers 25 PFAS including short-chain compounds in drinking water.</li><li><a target="_blank" rel="noopener noreferrer" href="https://blog.organomation.com/blog/sample-concentration-epa-method-537-pfas-determination"><strong>EPA Method 537.1</strong></a><strong>: </strong>Reversed-phase SPE followed by LC–MS/MS; covers 18 PFAS including the GenX compound, with method detection limits in the low ng/L range.</li><li><a target="_blank" rel="noopener noreferrer" href="https://www.agilent.com/en/solutions/environmental/water-testing/pfas-in-water/us-epa-method-8327?srsltid=AfmBOooCY1pcavj7B1PH-75d3HO4BGDglNlhmyTyrpDY3zvUksMO1dlg"><strong>EPA Method 8327</strong></a><strong>: </strong>LC–MS/MS for selected PFAS in prepared extracts of aqueous and solid matrices.</li><li><a target="_blank" rel="noopener noreferrer" href="https://blog.organomation.com/blog/preparing-diverse-sample-matrices-for-epa-method-1633"><strong>EPA Draft Method 1633</strong></a><strong>: </strong>The emerging benchmark for PFAS in non-aqueous matrices—aqueous, solid, biosolids, and tissue—quantifying up to 40 PFAS.</li></ul><p>All of these methods designate LC–MS/MS, specifically LC coupled to triple quadrupole or tandem-in-time platforms operating in negative electrospray ionization mode with multiple reaction monitoring (MRM), as the required or recommended analytical technique. PFAS analysis is, at its core, an LC–MS/MS problem.</p><h4><strong>The Sample Preparation Bottleneck</strong></h4><p>Chemists who have worked with PFAS methods understand the particular demands they place on sample preparation. The ubiquity of<a target="_blank" rel="noopener noreferrer" href="https://blog.organomation.com/blog/tackling-pfas-contamination-practical-insights-from-epa-537.1-method-development"><strong> PFAS in laboratory materials</strong></a>—PTFE tubing, some SPE sorbents, standard bench supplies—means that blank contamination control is as analytically demanding as the detection itself. EPA 537.1 requires that sample volumes of approximately 250 mL be extracted and reconstituted in as little as 1 mL, creating a 250:1 concentration factor that can expose even trace contamination in the sample processing chain. The installation of a delay column between the LC pump and autosampler to prevent instrument background interference is now standard practice.</p><p>This is precisely where robust, <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5385"><strong>inert nitrogen evaporation </strong></a>systems earn their keep. PFAS workflows that use nitrogen-assisted evaporation to concentrate extracts prior to reconstitution benefit from materials that avoid PTFE and other fluoropolymers that can leach PFAS background—a consideration Organomation addresses in its concentration system designs.</p><p>The analytical community is also moving beyond targeted PFAS analysis. High-resolution accurate mass (HRAM) spectrometry via Orbitrap technology, paired with non-targeted screening workflows, is gaining traction for the identification of the more than 5,000 currently catalogued PFAS compounds and the novel structures that continue to emerge. LC–MS/MS remains the quantitative gold standard; HRAM provides the discovery layer.</p><h3><strong>Technological Trends Reshaping the LC–MS/MS Workflow</strong></h3><h4><strong>Artificial Intelligence and Automated Data Interpretation</strong></h4><p>The datasets generated by modern tandem MS platforms—particularly in omics applications—are too large and too complex for conventional manual review. AI-driven spectral deconvolution, automated peak integration, and AI-assisted hypothesis generation are now embedded in the software suites accompanying leading instruments. <a target="_blank" rel="noopener noreferrer" href="https://www.agilent.com/about/newsroom/presrel/2022/17feb-gp22004.html?srsltid=AfmBOop3warRIaEojXM2YyMML6RXfMIDzE-Wm0h-SiKkkocBeeET2m7u"><strong>Agilent’s acquisition of Virtual Control’s ACIES </strong></a>software specifically to automate LC–MS and GC–MS data analysis is emblematic of this direction.</p><p>For laboratory managers, AI integration means smaller specialized teams can operate high-throughput multi-analyte workflows—but it also means that the front-end sample preparation must be even more reproducible, since algorithmic interpretation amplifies inconsistencies introduced upstream.</p><h4><strong>Online SPE Coupling and “Chromatography Without Walls”</strong></h4><p>A significant trend is the integration of solid-phase extraction directly into the HPLC flow path—so-called online SPE–HPLC coupling. Systems that combine extraction and chromatographic separation in a single analytical instrument reduce manual transfer steps, minimize the risk of sample loss or contamination, and dramatically increase throughput. For analytes at very low concentrations, online SPE provides the on-column concentration capability that traditional offline preparation achieves with manual evaporation steps.</p><p>That said, offline sample preparation—including <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5392"><strong>nitrogen-assisted evaporation</strong></a> under controlled conditions, <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2407"><strong>liquid–liquid extraction</strong></a>, and protein precipitation—retains significant advantages in flexibility, matrix handling, and the ability to process sample volumes that exceed online system capacity. The two approaches are increasingly complementary rather than competitive in well-designed analytical laboratories.</p><h4><strong>High-Purity Solvents and Reference Materials</strong></h4><p>As detection limits push further into the femtogram range and regulatory methods demand demonstrable blank performance, the quality of analytical consumables—mobile phase solvents, derivatization reagents, reference standards—has moved from background consideration to front-of-mind. Method validation in regulated environments now routinely requires vendor certification of solvent purity at a level that directly interfaces with instrument performance.</p><h3><strong>The Ever-Present Need for Sample Evaporation</strong></h3><p>As LC–MS/MS methods push detection limits lower, the analytical burden shifts increasingly upstream. The quality of the concentration, cleanup, and reconstitution steps preceding injection determines whether the instrument’s theoretical sensitivity is achievable in practice.</p><p><strong>Several specific developments are particularly relevant:</strong></p><ul><li><strong>ADC and gene therapy characterization workflows</strong> require gentle, low-temperature evaporation that preserves protein structure and prevents aggregation prior to LC–MS/MS. Nitrogen evaporation under controlled conditions is the preferred approach.</li><li><strong>PFAS trace analysis</strong> demands evaporation systems constructed from PFAS-inert materials, with rigorous attention to avoiding fluoropolymer surfaces throughout the sample path.</li><li><strong>High-throughput pharmaceutical QC</strong> driven by reshoring demands parallel processing capability—multi-position evaporators that maintain consistent flow and temperature across all positions simultaneously.</li><li><strong>Multi-omics and metabolomics workflows</strong> generate large sample batches that benefit from automated, walk-away evaporation with programmable endpoints to ensure reproducible final volumes across a cohort.</li></ul><p>Tandem chromatography–mass spectrometry is not merely growing; it is becoming structurally embedded in the analytical workflows of every major regulated industry. The convergence of biopharma complexity, PFAS regulatory mandates, AI-enabled data analysis, and pharmaceutical reshoring is creating a multi-year demand cycle that will require laboratories to invest not only in instruments, but in the entire sample preparation ecosystem that makes those instruments perform.</p><p>For analytical chemists, the practical takeaway is this: as mass spectrometry sensitivity continues to advance, the sample preparation step becomes the binding constraint on method performance. Whether you are developing a new PFAS method to meet EPA 1633, characterizing an ADC’s drug-to-antibody ratio, or running a 500-sample metabolomics cohort overnight, the reliability and cleanliness of your concentration and cleanup workflow determines the quality of every data point that follows.&nbsp;</p>

The Continued Rise of Tandem Chromatography–Mass Spectrometry

News from LabRulezLCMS Library - Week 27, 2026

<p><strong>🎤Dr. Christopher Reid (Professor of Chemistry and Biochemistry at Bryant University in Rhode Island)</strong></p><p><strong>In this episode of Concentrating on Chromatography, host Dave Oliva sits down with Chris Reid to explore how chemical biology and LC-MS are being used to tackle one of the most urgent global challenges: antimicrobial resistance.</strong></p><p>As antibiotic resistance continues to rise—and new drug development struggles to keep pace—researchers are looking beyond traditional approaches. Instead of targeting how bacteria build their cell walls, Chris’s work focuses on how they break them down, opening the door to entirely new antimicrobial strategies.</p><h4><strong>What You’ll Learn in This Episode:</strong></h4><ul><li>Why antimicrobial resistance is accelerating globally</li><li>What peptidoglycan is and why it’s essential to bacterial survival</li><li>How targeting cell wall degradation differs from traditional antibiotics</li><li>The strategy behind designing small molecule probes and inhibitors</li><li>How LC-MS is used to analyze peptidoglycan fragments and enzyme activity</li><li>Key analytical challenges: complex mixtures, low-abundance species, and structural similarity</li><li>The importance of sample preparation and reproducibility in LC-MS workflows</li><li>How this research could lead to new antibiotic targets</li></ul><h3><strong>Video Transcription</strong></h3><h4><strong>The Growing Challenge of Antibiotic Resistance</strong></h4><p>According to Reid, antibiotic resistance is increasing across a broad spectrum of bacterial pathogens that were once readily treated with existing therapies. In some cases, clinicians must now rely on only a handful of last-line antibiotics, while resistance is also rising in diseases such as tuberculosis.</p><p>One of the major obstacles is the limited commercial incentive to develop entirely new antibiotics. Because bacterial resistance can emerge relatively quickly after a drug reaches the market, pharmaceutical companies often favor modifying existing antibiotics or combining them with resistance-modifying agents rather than investing in completely new drug classes. As a result, genuinely novel antibacterial targets remain urgently needed.</p><h4><strong>Why the Bacterial Cell Wall Remains an Attractive Target</strong></h4><p>The bacterial cell wall consists primarily of <strong>peptidoglycan</strong>, a highly cross-linked polymer that provides structural integrity, maintains cell shape, and enables bacterial growth and division.</p><p>Many widely used antibiotics—including the β-lactam antibiotics such as penicillins—target enzymes responsible for synthesizing or cross-linking peptidoglycan. These pathways have proven highly successful therapeutic targets because peptidoglycan is unique to bacteria and absent from human cells, minimizing unwanted effects on the host.</p><p>However, most existing antibiotics target only specific stages of peptidoglycan biosynthesis. Reid's research explores another part of this complex pathway: enzymes responsible for degrading the carbohydrate backbone of peptidoglycan during normal bacterial growth and remodeling. These enzymes may represent an underexplored class of antimicrobial targets capable of overcoming existing resistance mechanisms.</p><h4><strong>Targeting the Glycan Backbone</strong></h4><p>A key advantage of focusing on the carbohydrate backbone is its remarkable conservation across both Gram-positive and Gram-negative bacteria.</p><p>Whereas the peptide components of peptidoglycan vary considerably between bacterial species—and frequently acquire mutations associated with antibiotic resistance—the glycan backbone remains highly conserved and tolerates relatively few structural modifications.</p><p>This biological constraint makes enzymes acting on the glycan backbone particularly attractive targets. Reid's group developed a series of <strong>small-molecule probes and inhibitors</strong> designed to recognize enzymes known as <strong>N-acetylglucosaminidases</strong>, which cleave glycosidic bonds within peptidoglycan and generate soluble degradation products.</p><p>Rather than beginning with purified enzymes, the researchers adopted a <strong>top-down phenotypic screening strategy</strong>. Candidate compounds were first evaluated for their ability to inhibit bacterial growth before progressively narrowing the list of potential molecular targets through genetic studies, microscopy, enzyme assays, and biochemical characterization. This iterative workflow ultimately identified compounds capable of inhibiting the desired enzyme class while simultaneously providing valuable chemical probes for studying bacterial cell wall biology.</p><h4><strong>LC-MS as a Central Analytical Tool</strong></h4><p>Liquid chromatography coupled with mass spectrometry plays a central role throughout the project by enabling detailed characterization of structural changes occurring within peptidoglycan after bacterial cells are treated with candidate inhibitors.</p><p>Because intact peptidoglycan is an insoluble polymer, sample preparation begins with isolation of the bacterial cell wall through detergent extraction and differential centrifugation. Additional purification steps remove proteins, nucleic acids, and teichoic acids before enzymatic digestion converts the insoluble material into soluble fragments suitable for chromatographic analysis.</p><p>The resulting digest is analyzed by LC-MS to examine changes in the distribution of peptidoglycan fragments. Chromatographic retention times provide an immediate indication of changes in cross-linking, with larger oligomers such as trimers and tetramers generally eluting later than monomeric fragments. Mass spectrometry then confirms the identity of these fragments by measuring their intact molecular masses, while tandem MS experiments can provide further structural information when required.</p><h4><strong>Sample Preparation Determines Data Quality</strong></h4><p>Reid emphasized that sample preparation represents one of the most critical—and challenging—stages of the entire workflow.</p><p>Because peptidoglycan analysis involves multiple purification, digestion, and reduction steps, even minor procedural differences between samples can introduce analytical artifacts that complicate biological interpretation.</p><p>Maintaining strict consistency throughout sample preparation is therefore essential. Appropriate controls must accompany every experiment, ensuring that observed chromatographic and mass spectrometric differences truly reflect biological responses rather than experimental variability. The complexity of bacterial cell wall composition further increases the analytical challenge, as subtle differences in amino acid substitutions and low-abundance cross-linked species can complicate LC-MS data interpretation.</p><h4><strong>Chemical Biology for Future Antibiotic Discovery</strong></h4><p>Although the identified compounds may eventually serve as starting points for antibiotic development, Reid views them primarily as <strong>chemical biology tools</strong>.</p><p>By selectively inhibiting individual enzymes, these probes allow researchers to investigate previously unexplored aspects of bacterial cell wall remodeling that cannot easily be studied using classical genetic approaches alone.</p><p>The research team is now expanding this strategy by combining their probes with <strong>proximity-labeling proteomics and mass spectrometry</strong>, aiming to map protein interaction networks associated with bacterial cell wall degradation. Such studies may reveal entirely new antibacterial targets and provide deeper insight into the biological processes governing bacterial growth and division.</p><p>Ultimately, the work illustrates how integrating synthetic chemistry, microbiology, molecular biology, and advanced LC-MS can accelerate the search for innovative therapeutic strategies against antimicrobial resistance. As existing antibiotics become progressively less effective, multidisciplinary approaches such as these may play an increasingly important role in identifying the next generation of antibacterial targets.</p><blockquote><p><strong>This text has been automatically transcribed from a video presentation using AI technology. It may contain inaccuracies and is not guaranteed to be 100% correct.</strong></p></blockquote><blockquote><h4><strong>Concentrating on Chromatography Podcast</strong></h4><p>Dive into the frontiers of chromatography, mass spectrometry, and sample preparation with host David Oliva. Each episode features candid conversations with leading researchers, industry innovators, and passionate scientists who are shaping the future of analytical chemistry. From decoding PFAS detection challenges to exploring the latest in AI-assisted liquid chromatography, this show uncovers practical workflows, sustainability breakthroughs, and the real-world impact of separation science. Whether you’re a chromatographer, lab professional, or researcher you'll discover inspiring content!</p><p>You can find <strong>Concentrating on Chromatography Podcast </strong>in podcast apps:</p><ul><li><a target="_blank" rel="noopener noreferrer" href="https://open.spotify.com/show/1wZg67rCjoGju4yhqxI3tL"><strong>Spotify</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podcasts.apple.com/tz/podcast/concentrating-on-chromatography/id1788429185"><strong>Apple podcast</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podscan.fm/podcasts/concentrating-on-chromatography"><strong>podscan</strong></a></li></ul><p>and on <a target="_blank" rel="noopener noreferrer" href="https://www.youtube.com/@ChromatographyTalk/videos"><strong>YouTube channel</strong></a></p></blockquote>

 Fighting Antibiotic Resistance with LC-MS: Targeting Bacterial Cell Walls

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