<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 22th July 2024? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p>
<blockquote>
<h4>👉 <a href="https://lcms.labrulez.com/library"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></a></h4>
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<p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p>
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<p>This week we bring to you poster presentations from <a href="https://lcms.labrulez.com/events/568"><strong>ASMS 2024</strong></a> by <strong>Agilent Technologies</strong>, <strong>Shimadzu</strong>, <strong>Thermo Fisher Scientific</strong>, and <strong>Waters Corporation</strong>!</p>
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<p>💡 <strong>Almost 1000 ASMS posters and presentations <a href="https://lcms.labrulez.com/library?mainauthorItems=284">available in the LabRulezLCMS library</a></strong> (unique search and filtering)</p>
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<h4><strong>1. Waters Corporation: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/2023_asms_king_liver_178c54e990.pdf">HIGH THROUGHPUT PLASMA PROFILING OF HUMAN LIVER DISEASE SAMPLES USING RAPID CHROMATOGRAPHY AND A MULTI- REFLECTING TIME-OF-FLIGHT MASS SPECTROMETER</a></strong></h4>
<ul>
<li>poster - ASMS 2024</li>
</ul>
<p><strong>Introduction:</strong> Metabolomics is a valuable research strategy in the determination  FIG. 5  FIG. 1  FIG. 7  FIG. 3  of disease progression, disease stratification, the identification of  biological markers for early diagnosis and for the development of  new therapeutics. Acute-on-chronic liver failure (ACLF) is a  condition characterized by systemic inflammation, multiple organ  failure, and high short-term mortality1. ACLF develops from the  acute decompensation of cirrhosis and currently there is no  biomarker identified for the early diagnosis of this disease.  Therefore, fully understanding the role metabolites and lipids play  in the progression of ACLF and its underlining mechanisms could  greatly help determine new diagnostic markers driving  development of new therapeutics. Investigating biomarkers for  large scale studies requires robust, high-throughput analytical  methods.</p>
<p>Rapid microbore metabolic profiling (RAMMP) methods and  conventional high-resolution mass spectrometry have previously  been shown to considerably reduce analysis time2-3. Here we  compare these methods with a vacuum jacketed column  technology (VJC)4-5 and ultra high-resolution mass spectrometry6  for the analysis of plasma from liver disease patients by reversed-  phase (RP) for lipid and hydrophilic interaction chromatography  (HILIC) for small molecules.</p>
<p><strong>Conclusion:</strong></p>
<ul>
<li>Conventional LC-MS methods used for large cohort studies suffer from batch response variation.  The rapid chromatographic methods reduce this impact and demonstrated reproducible response  across the entire batch of 168 injections per polarity (Fig. 2).</li>
<li>The SELECT SERIES MRT mass spectrometer generated highly accurate mass measurements  (&lt;1 ppm), improving annotation confidence for both lipids and small molecules, including essential  amino acids and carnitines from the HILIC analysis (Fig. 3).</li>
<li>PCA of the RAMMP data showed good analytical reproducibility from the clustered QC samples and  group separation with some overlap between the different disease groups (Fig. 4).</li>
<li>Group comparisons were performed by OPLS-DA where statistically significant features were  highlighted for database annotation (Fig. 5).</li>
<li>A number of lipids classes, particularly lysophosphatidylcholines (LPC), phosphatidylcholines (PC)  and sphingomyelins (SM), showed dysregulation in the diseased samples when compared to healthy  controls and were annotated through Lipostar2 with mass accuracies between 0.1-0.3 ppm (Fig. 6).</li>
<li>For the HILIC small molecule analysis, differences in amino acid response were detected in  comparisons between diseased and healthy controls.</li>
<li>The SELECT SERIES MRT MS achieved mass resolution of 170-200K FWHM at 30 Hz compatible with  RAMMP (a) and VJC (b) methodologies for example lipids, PC36:1 (0.18 ppm) and SM42:3, O2 (0.16  ppm), with the VJC providing better peak shape and resolution of adjacent peaks (Fig. 7)</li>
<li>Comparison of the RAMMP and VJC negative lipid analysis showed very similar distribution of  disease and healthy control groups demonstrating minimal impact on the overall statistics (Fig. 8).</li>
<li>Both RAMMP and VJC methodologies combined with the SELECT SERIES MRT provided highly  accurate reproducible metabolic profiling for a biomarker study, with the VJC providing improved  peak fidelity complemented by the fast scanning high resolution capabilities of the SELECT SERIES  MRT.</li>
<li>This work demonstrated an accurate, reproducible and high throughput metabolomic method for the  rapid screening of a large number of biological samples for biomarker discovery.</li>
</ul>
<h4><strong>2. Thermo Fisher Scientific: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/po_143_characterization_structure_adenopeptin_lc_hram_ms_po143_asms2024_en_7ae893f36c.pdf">In-depth characterization and structure elucidation of non-ribosomal peptides related to adenopeptin using LC-HRAM-MSⁿ</a></strong></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
<h5><strong>Abstract</strong></h5>
<p><strong>Purpose:</strong> Characterization of adenopeptin and related peptaibol analogs in a crude fermentation broth extract using LC-HRAM-MSn to identify adenopeptin congeners and elucidate their structures. Methods: Adenopeptin and its analogs were separated using reversed phase chromatography and analyzed using multi-stage fragmentation on a Thermo Scientific Orbitrap IQ-X Tribrid mass spectrometer. </p>
<p><strong>Results:</strong> Several analogs of adenopeptin differing by one or more methyl groups could be detected and the site of their modification could be narrowed to specific amino acid residues based on their fragmentation patterns.</p>
<h5><strong>Conclusions</strong></h5>
<p>The LC/MS analysis of the crude adenopeptin extract revealed the presence of eight analogs above 0.5% relative MS-abundance, mainly resulting from ”demethylations”, i.e., substitution of Aib for Iva residues. Their modification sites could be determined based on shifts in the fragmentation pattern relative to adenopeptin. Additionally, a potentially crucial substitution of pipecolic acid with proline could be detected and verified based on MS3 spectral evidence for compound 4.</p>
<h4><strong>3. Thermo Fisher Scientific: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/po_146_evaluation_automated_low_flow_lc_ms_setup_po146_asms2024_en_1d935eb142.pdf">Evaluation of an Automated “Quick” Optimization Routine to Streamline Low-Flow LC-MS Setup</a></strong></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
<h5><strong>Abstract</strong></h5>
<p><strong>Purpose:</strong> When setting up low flow LC-MS experiments, a user regularly positions an  emitter at an arbitrary fixed distance from the inlet based on visual alignment. A  prototype ion source featuring an emitter mounted on a 3D motorized stage was used  to evaluate a “quick” optimization strategy used to define the active position for  subsequent LC-MS measurements.</p>
<p><strong>Methods:</strong> The basis of the quick optimization routine was established using m/z- and  position-dependent intensity distributions. The routine was evaluated against two  methods to manually set the emitter position.</p>
<p><strong>Results:</strong> All optimization strategies yielded a statistically equivalent number of protein  and peptide identifications. However, the quick optimization routine yielded superior  peak area reproducibility that translated into significantly better emitter-to-emitter  consistency.</p>
<h4><strong>4. Shimadzu: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/ASMS_poster_PESI_v_DMS_170524_fed75d0c1e.pdf">DPiMS-MS (PESI) combined with vacuum differential mobility spectrometry for rapid forensic analysis.</a></strong></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
<p><strong>Introduction</strong></p>
<ul>
<li>Ambient ionisation (AI) mass spectrometry techniques facilitate rapid turn-around of samples, often in a walk-up format with limited sample preparation, with obvious benefits for forensic applications.</li>
<li>The absence of a chromatographic separation enables sub-minute data acquisition. However, this comes with the cost of greater chance of problems with chemical noise and/or interference between isomeric/isobaric compounds.</li>
<li>One way to remove chemical noise and to separate interfering compounds is to add an ion mobility separation to the method.</li>
<li>Here we implement a novel low pressure differential mobility on a DPiMS-8060 (PESI) system and demonstrate performance for steroids and small molecule drugs in saliva.</li>
</ul>
<p><strong>Conclusion</strong></p>
<ul>
<li>These results demonstrate the potential of vDMS to enhance AIMS by removing chemical noise and separating isobaric species.</li>
<li>DPiMS-vDMS-8060 (PESI) retains the high acquisition speed of PESI while adding an additional dimension of separation.</li>
<li>The additional selectivity may lower the LOQ and reduce false positives.</li>
<li>vDMS-MS methods can be applied in a wide range of complex matrices (e.g. urine, plasma).</li>
</ul>
<h4><strong>5. Shimadzu: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/MP_045_Targeted_quantitative_screening_pesticides_using_high_resolution_DIA_spectral_library_matching_238a221a67.pdf">Targeted quantitative screening pesticides in food matrices using high resolution DIA spectral library matching.</a></strong></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
<p><strong>Overview</strong></p>
<ul>
<li>A single, generic untargeted data independent acquisition (DIA) method was developed for the analysis of extended panels of pesticides in food matrices.</li>
<li>The high-resolution LC-MS/MS method was designed for multi-residue screening and quantitation of pesticides to meet the European Union SANTE 11312/2021 v2 guidelines capable of working with extended target pesticide panels.</li>
<li>The MS and DIA-MS/MS method was applied to quantify a routine panel of pesticides but also included an additional target panel of fungicides for qualitative screening.</li>
</ul>
<p><strong>Conclusions</strong></p>
<ul>
<li>Using a single generic LC-MS and DIA-MS/MS method targeted quantitation using ion ratio confirmation (to meet the EU SANTE guidelines) and extended search capabilities with library identification were supported.</li>
<li>The method results in the quantitation of &gt;250 pesticides at or below the default MRL concentration (10 µg/kg).</li>
</ul>
<h4><strong>6. Shimadzu: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/MP_045_Targeted_quantitative_screening_pesticides_using_high_resolution_DIA_spectral_library_matching_238a221a67.pdf">Identification of positional isomers of linoleic acid containing phospholipids involved in pancreatic ductal adenocarcinoma.</a></strong></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
<p><strong>Overview</strong></p>
<ul>
<li>To enhance lipid identification, Oxygen Attachment Dissociation (OAD) MS/MS has been applied to provide double bond specific dissociation, enabling C=C position assignments.</li>
<li>High resolution OAD MS/MS was applied in positive and negative ion mode to provide further confidence in lipid identification in a pancreatic adenocarcinoma biomarker study</li>
</ul>
<p><strong>Conclusions</strong></p>
<ul>
<li>OAD-MS/MS is a high specificity fragmentation technique to enable C=C positional assignments in lipids. Analysis is easy to perform alongside CID-MS/MS and spectra are easy to interpret.</li>
<li>OAD-MS/MS has been applied to structurally characterize lipids found to be potential markers of PDAC in a previous study using high resolution metabolomics analysis by LC-CID-MS/MS and DPiMSᵗᵐ.</li>
<li>The use of this new technology aims to enhance pancreatic cancer research by providing more comprehensive identification needed to understand the biological roles of the lipids identified as potential markers of PDAC.</li>
</ul>
<h4><strong>7. Agilent Technologies: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/po_2dlc_6545xt_monitoring_oxidation_mabs_asms_2024_wp030_en_agilent_77f2bb18d5.pdf">Monitoring oxidation in recombinant monoclonal antibodies at subunit level through two-dimensional liquid chromatography coupled with mass spectrometry</a></strong></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
<p><strong>Introduction:</strong> Oxidation, induced by reactive oxygen species, is a prevalent chemical modification in monoclonal antibodies (mAbs), affecting amino acid residues such as cysteine (C), methionine (M), tryptophan (W), tyrosine (Y), lysine (K), and others (Gupta et al., 2022). Each residue&#39;s oxidation uniquely influences the protein&#39;s structure and function. Methionine is highly prone to oxidation in mAbs, typically resulting in the formation of a methionine sulfoxide product with increased polarity. These methionine residues are found in both the antigen-binding (Fab) and effector activity (Fc) domains of a mAb, with those situated on the surface being particularly susceptible to oxidation (Hageman et al., 2019). The increasing popularity of two-dimensional liquid chromatography (2D-LC) has led to a significant rise in the integration of two complementary chromatographic techniques, coupled with mass spectrometry (MS), aiming to provide comprehensive insights into mAb post-translational modifications (PTMs) (Sarin et al., 2022). The proposed 2D WCX-RP-MS workflow enables the analysis of mAbs at the subunit level, providing an automated analytical platform for characterizing and identifying oxidized regions within both the Fc and Fab domains.</p>
<p><strong>Conclusions:</strong> The oxidized peaks not resolved by 1D methods are successfully resolved by the D2 RPC column with additional HC-LC information. It should be highlighted that the proposed method completely uses MS compatible buffers and is a faster method to identify mAb modification and their correlation in different mAb fragments compared to the tedious bottomup approach. A general mAb bottom-up analysis takes up to 24-48 hours with the risk of sample loss, degradation, and induced modifications. In the present study, sample preparation took only 4-10 hours (depending on oxidation incubation), and sample analysis took only 3 hours. Hence, 7-13 hours is enough for the subunit analysis with a low risk of sample loss and degradation. The present study focuses on oxidation as the chemical modification of interest, but the workflow can be extended to other critical charge modifications easily resolved by the WCX column.</p>
<h4><strong>8. Agilent Technologies: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/po_2dlc_6545xt_monitoring_oxidation_mabs_asms_2024_wp030_en_agilent_77f2bb18d5.pdf">Flash Characterization of mAbs Using a Combination of Reagents via Automated On-Line Microdroplet Reaction</a></strong></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
<p><strong>Introduction:</strong> Recently, an unmodified Agilent Jet Stream ESI source (AJS) was used to demonstrate “Flash Characterization” of mAbs by microdroplet facilitated reductions or enzyme digestions with good reproducibility and high yield 1. These results motivated us to evaluate treatments that combined enzymatic digestion, reduction, and/or deglycosylation of mAb in a single microdroplet experiment. New optimization parameters for the Agilent Injection Program on Agilent 1290 Infinity II LC were presented. Microdroplet experiments, including GlycINATOR ® deglycosylation, the combination of IdeS cleavage and DTT reduction and IdeS cleavage and GlycINATOR ® deglycosylation were examined for reproducibility, intact mAb remaining after treatments and simplified subunit characterization. GlycINATOR is an endoglycosidase that cleaves N-linked glycans and leaves the core GlcNAc residue with or without a fucose on the asparagine. Additional antibodies of Herceptin and sigmamab are also studied with the combination treatment in the microdroplet reaction.</p>
<p><strong>Conclusions:</strong> </p>
<ul>
<li>Flash Characterization of mAbs with an automatic injector, combination treatments, and microdroplet reaction in the AJS source demonstrates simplification, reproducibility, and cost reduction of protein characterization. </li>
<li>Combination treatments are viable with microdroplet reactions to accelerate the reduction, cleavage, and deglycosylation of native IgG1 significantly.</li>
</ul>


News from LabRulezLCMS Library - Week 30, 2024

<p><strong>In the research article recently published in ACS </strong><i><strong>Analytical Chemistry</strong></i><strong> journal the researchers led by Lucie Nováková from the Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, present a novel analytical approach for mixtures of natural products.</strong></p><p>This study addresses the complexity of plant extract analysis by developing a holistic two-injection method that integrates <strong>ultrahigh-performance </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?instrumentationItems=38"><strong>supercritical fluid chromatography</strong></a> (<strong>UHPSFC</strong>) with <strong>multimodal ionization tandem mass spectrometry</strong> (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?instrumentationItems=32"><strong>MS/MS</strong></a>). This method, which does not require manual intervention, significantly improves the efficiency and accuracy of analyzing various small molecules, such as terpenes, flavonoids, and phenolic acids, in plant extracts. The authors applied this method to Eucalyptus sp. extracts, demonstrating its potential as a robust tool for comprehensive plant metabolite analysis.</p><blockquote><h4><strong>The original article</strong></h4></blockquote><blockquote><h5><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c03599"><strong>Ultrahigh-Performance Supercritical Fluid Chromatography–Multimodal Ionization–Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts</strong></a></h5><p>Kateřina Plachká, Veronika Pilařová, Štefan Kosturko, Jan Škop, Frantisek Svec, and Lucie Nováková</p><p><i>Analytical Chemistry</i> <strong>2024</strong> <i>96</i> (7), 2840-2848</p><p>DOI: 10.1021/acs.analchem.3c03599</p><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><h5>Selected sections from the article follow.</h5><h3><strong>Abstract</strong></h3><p>Complex analysis of plant extracts usually requires a combination of several analytical approaches. Therefore, in this study, we developed a holistic two-injection approach for plant extract analysis, which is carried out within one instrument without the need for any manual intervention during the analysis. <strong>Ultrahigh-performance supercritical fluid chromatography</strong> (<strong>UHPSFC</strong>) was employed for the analysis of 17 volatile terpenes on a <strong>porous graphitic carbon column</strong> within 7.5 min, followed by analysis on <strong>short diol column</strong> where flavonoids, phenolic acids, and terpenoic acids were analyzed within 15.5 min. A <strong>multimodal ionization</strong> source <strong>combining </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=electrospray"><strong>electrospray</strong></a> and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=atmospheric+pressure+chemical+ionization"><strong>atmospheric pressure chemical ionization</strong></a> (<strong>ESCi</strong>) was selected for mass spectrometry detection as a simultaneous ionization of both lipophilic and polar compounds was required. The quantitative aspects of the final <strong>UHPSFC-ESI/ESCi-MS/MS two-injection approach</strong> were determined, and it was applied to the <strong>analysis of Eucalyptus sp. extracts</strong> prepared by <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=SFE"><strong>supercritical fluid extraction</strong></a>. Current methods reported in the literature typically require a labor-intensive combination of liquid and gas chromatography for the complex analysis of plant extracts. We present for the first time a new UHPSFC approach requiring only a single instrument that provides an alternative approach to the analysis of complex plant extracts.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Analytical_Chemistry_2024_96_7_2840_2848_cover_image_2a6c4c6d6b.jpg" alt="Analytical Chemistry 2024 96 (7), 2840-2848: graphical abstract."></p><h4><strong>Subjects</strong></h4><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=chromatography"><strong>Chromatography</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=Flavonoids"><strong>Flavonoids</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=Hydrocarbons"><strong>Hydrocarbons</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=Ionization"><strong>Ionization</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=Solvents"><strong>Solvents</strong></a></li></ul><h3><strong>Experimental Section</strong></h3><h4><strong>Chromatographic Conditions</strong></h4><p>All experiments were carried out using the supercritical fluid chromatography system <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?instrumentationItems=38&amp;mainauthorItems=5&amp;search=Acquity+UPC2"><strong>Acquity UPC2</strong></a> (Waters, Milford, MA, USA) equipped with a binary pump, an autosampler, a column thermostat, a back pressure regulator (BPR), and a PDA detector. The system was coupled to a triple quadrupole mass spectrometer <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?instrumentationItems=38&amp;mainauthorItems=5&amp;search=Xevo+TQ-XS"><strong>Xevo TQ-XS</strong></a> (Waters) via a commercially available SFC-MS dedicated pre-BPR splitter device with an additional isocratic or binary pump for makeup solvent delivery (Waters).</p><p>Several stationary phases were tested during optimization including Viridis BEH (hybrid silica), Viridis BEH 2-ethylpyridine (2-EP), Torus 2-picolylamine (2-PIC), Viridis HSS C18 SB (C18), Torus 1-amino anthracene (1-AA), all in 3.0 × 100 mm, 1.7 μm, Torus Diol (diol) (3.0 × 50 mm and 100 mm, 1.7 μm), BEH HILIC (2.1 × 100 mm, 1.7 μm), CORTECS HILIC (silica) (3.0 × 100 mm, 1.6 μm), all from Waters, YMC Carotenoid C30 (4.6 × 100 mm, 5 μm) from YMC (Dinslaken, Germany, and porous graphitic carbon (PGC) columns Hypercarb (3.0 × 50 mm and 100 mm, 3.0 μm, Thermo Fisher Scientific Inc., Waltham, MA, USA) and Supel Carbon LC (3.0 × 150 mm, 2.7 μm, Merck KGaA, Darmstadt, Germany). Analyses were carried out using methanolic organic modifiers with or without an additive that included 10 mmol/L ammonia, 5% water, and 10 mmol/L formic acid. Final chromatographic conditions were as follows: Method 1: 150 mm Supel Carbon LC column at 60 °C, BPR pressure at 3300 psi (22.75 MPa), flow rate 1.5 mL/min, MeOH as organic modifier in gradient elution: 0% for 1.5 min, 0–40% in 1.5–4.0 min, 40–41% in 4.0–6.0 min, followed by 1.5 min equilibration at starting conditions. Method 2: 50 mm Torus Diol column at 20 °C and 5% water in MeOH as organic modifier with specific gradient conditions listed in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c03599#tbl1"><strong>Table 1</strong></a>. The partial loop with needle overfill injection mode was used to inject 2 and 10 μL samples for methods 1 and 2, respectively. The autosampler was cooled to 5 °C to reduce the evaporation of volatile compounds.</p><h4><strong>MS Conditions</strong></h4><p>The four ionization sources, ESI, APCI, multimodal ESCi, and US, all from Waters were tested. MassLynx Software 4.1 was used for MS control, data acquisition, and processing. Optimization of the ionization sources conditions was carried out using a design of experiment (DoE) approach, followed by the optimization of the makeup solvent composition and flow rate. The makeup solvents tested included pure MeOH and MeOH with various additives, such as water, ammonia, formic acid, and their combinations in different concentrations. The makeup solvent flow rate was tested in the range of 0.0–0.6 mL/min. Selected reaction monitoring (SRM) transitions were determined for each analyte (Supporting Information S2), and their selectivity was verified. MODDE Software 13.0.1 was used to design the experiments for the ion source optimization and subsequent data evaluation. The parameters tested and their respective ranges for each ionization source are listed in Table 2. The DoE were selected as a compromise between the number of experiments required and the power of the study. Three replicates were always included to test the repeatability of the model. The data evaluation included its logarithmic transformation when necessary and the exclusion of outliers to achieve the highest possible linearity (R2), model validity, predictability (Q2), and reproducibility. The limits for the applicable model were set according to the MODDE software: R2 showing model fit &gt;0.5, Q2 estimating future prediction precision &gt;0.1 for significant model and &gt;0.5 for a good model, reproducibility &gt;0.5. Model validity tests diverse model problems and should be &gt;0.25. However, model validity can be lost for very good models with Q2 &gt; 0.9 due to high sensitivity in the test or extremely good replicates (&gt;0.9). Therefore, model validity was always carefully checked manually. The critical factors (marked with “!“ in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c03599#tbl2"><strong>Table 2</strong></a>) were identified as having a significant effect on the model, i.e., contributing more than 50% to the final response. Therefore, these factors were optimized separately, and a second DoE was run.</p><h3><strong>Results and Discussion</strong></h3><p>The aim of this study was to develop a holistic method for the simultaneous analysis of compounds typically found in plant extracts, namely, flavonoids, terpenoic and phenolic acids, and terpenes. These compounds include molecules with a wide range of physicochemical properties, from small lipophilic terpenes to polar flavonoids, as listed in Table S1.</p><p>First, a stationary phase had to be selected that would allow the retention of all of the target compounds. We expected that the retention and elution of hydrophilic flavonoids, and especially their glycosylated forms, i.e., rutin, hirsutrin, and hesperidin, would be the main challenge of the UHPSFC-MS method due to the low polarity of the CO₂-based mobile phase. On the other hand, less polar terpenes should be easily retained and eluted. A systematic column screening was carried out on a set of selected stationary phases, including hybrid silica, 2-EP, C18, 2-PIC, and diol with preferred H-bonding and π–π interactions. A generic gradient from 0 to 45% of organic modifier, i.e., MeOH, 10 mmol/L ammonia in MeOH, 10 mmol/L formic acid in MeOH, and 2% water in MeOH, was tested on all five columns. As expected, the addition of water to the organic modifier had a beneficial effect on the elution of polar flavonoids. However, it was necessary to increase the percentage of organic modifiers up to 58% to enable the elution of all target analytes. Unfortunately, these stationary phases were not able to retain most of the lipophilic terpenes, especially those without hydroxy groups. That was surprising as more lipophilic compounds are commonly analyzed by SFC. However, cymene, limonene, pinene, caryophyllene, and citronellal eluted in the dead volume even when using pure CO₂. Volatiles containing hydroxy groups were retained on C18, diol, silica, and 2-EP stationary phases but still coeluted in 1 peak near to the dead volume. Therefore, the retention and separation of terpenes became the main challenge. Accordingly, the 1-AA column with dominant π–π interactions was tested, where the higher retention of nonpolar compounds was expected. This hypothesis was confirmed. However, flavonoids were retained to such an extent that they could not be eluted within a reasonable analysis time under any conditions tested.</p><p>Thus, a holistic approach to the analysis of a plant extract consisting of two UHPSFC methods carried out on two columns and the same system without any manual intervention was developed and tested as a proof of concept. Indeed, most commercially available SFC instruments use a column manager that allows two or more columns to be connected simultaneously. Thus, it is still possible to analyze a complex sample in just two injections, taking advantage of the two stationary phases with different selectivity.</p><h4><strong>Application of UHPSFC-ESI/ESCi-MS/MS Method on Eucalyptus Extracts</strong></h4><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Analytical_Chemistry_2024_96_7_2840_2848_Figure_4_Comparison_of_the_percentage_abundance_of_targeted_analytes_i_e_A_terpenes_B_terpenoic_acids_C_16211a715a.jpg" alt="Analytical Chemistry 2024 96 (7), 2840-2848: Figure 4. Comparison of the percentage abundance of targeted analytes, i.e., (A) terpenes, (B) terpenoic acids, (C).jpg"></p><h3><strong>Conclusions</strong></h3><p>UHPSFC-MS/MS is an interesting alternative to LC and GC methods, permitting the analysis of a wide range of compounds with a single instrument. Unfortunately, a single holistic method for the simultaneous analysis of terpenes and polar flavonoids remains unattainable due to two main challenges. No tested stationary phase enabled the retention and elution of both groups of analytes. However, new stationary phases are being introduced to the market every year, which is promising. Second, a universal ionization source combining GC and LC approaches is required for the simultaneous analysis of small lipophilic terpenes and polar glycosylated flavonoids.</p><p>Despite these challenges, we were able to develop a two-injection approach for complex plant analysis as a proof-of-concept based on the ability of the UHPSFC to cover supercritical, subcritical, enhanced-liquid, and liquid conditions within one instrument. Here, volatile terpenes are measured using a PGC stationary phase within supercritical to subcritical conditions achieved by adding up to 40% of MeOH to CO₂. More polar phenolic and terpenoic acids and flavonoids are then analyzed on short diol column with gradient from 0 to 90% MeOH with addition of 5% water.</p><p>The APCI source proved to be beneficial in the case of lipophilic volatiles when compared to ESI, but it was not possible to detect flavonoids using APCI. Conversely, US resulted in higher sensitivity for flavonoids, but similar to ESI, it was not possible to detect terpenes without hydroxy groups. The finally selected multimodal ESCi source was able to detect all targeted volatiles although the sensitivity was significantly lower than in the case of APCI. However, it was possible to switch from ESCi to ESI without any need for manual intervention, which was crucial for the applicability of the developed approach.</p><p>Finally, the quantitative aspects of the two developed UHPSFC-ESI/ESCi-MS/MS methods were determined, and the approach was successfully applied for the analysis of SFE extract from Eucalyptus sp. The novel approach can be beneficial for analysis of complex samples containing analytes with a wide range of physicochemical properties.</p>

Ultrahigh-Performance Supercritical Fluid Chromatography–Multimodal Ionization–Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts

<p><strong>In the research article published in ACS <em>Analytical Chemistry</em> journal the researchers from the University of Geneva, Switzerland, and Waters Corporation took a deep dive into understanding the retention mechanism of oligonucleotides (ONs), evaluation of the applicability of the linear solvent strength (LSS) retention model, and exploration of the potential of ultra-short columns.</strong></p>
<p>Ion-pairing reversed-phase liquid chromatography (<a href="https://lcms.labrulez.com/webinars?search=IP-RP"><strong>IP-RPLC</strong></a>) serves as a key method for characterizing oligonucleotides and their impurities. This study delved into understanding the retention mechanisms of oligonucleotides, validating the linear solvent strength (LSS) retention model for molecules ranging from 3 to 30 kDa, and confirmed the model&#39;s ability to predict retention times accurately. It introduced the use of ultra-short 5 mm columns which facilitated rapid separations of oligonucleotide mixtures in just 30 seconds without compromising separation efficiency. Despite oligonucleotides displaying &quot;on-off&quot; elution patterns similar to smaller proteins, the effectiveness of these shorter columns suggests no further gains in selectivity or efficiency from using longer columns. This finding sets the stage for future research on more complex oligonucleotide formulations using this accelerated method.</p>
<blockquote>
<h4><strong>The original article</strong></h4>
</blockquote>
<blockquote>
<h5><a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c01934"><strong>High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns</strong></a></h5>
<p>Honorine Lardeux, Szabolcs Fekete, Matthew Lauber, Valentina D’Atri, and Davy Guillarme</p>
<p><em>Analytical Chemistry</em> <strong>2023</strong> <em>95</em> (27), 10448-10456</p>
<p>DOI: 10.1021/acs.analchem.3c01934</p>
<h6>licensed under <a href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6>
</blockquote>
<h5>Selected sections from the article follow.</h5>
<h3><strong>Abstract</strong></h3>
<p>Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the reference separation technique for characterizing oligonucleotides (ONs) and their related impurities. The aim of this study was to better understand the retention mechanism of ONs, evaluate the applicability of the linear solvent strength (LSS) retention model, and explore the potential of ultra-short columns having a length of only 5 mm for the separation of model ONs. First, the validity of the LSS model was evaluated for ONs having sizes comprised between 3 and 30 kDa, and the accuracy of retention time predictions was assessed. It was found that ONs in IP-RPLC conditions follow an “on–off” elution behavior, despite a molecular weight lower than that of proteins. For most linear gradient separation conditions, a column length between 5 and 35 mm was found to be appropriate. Ultra-short columns of only 5 mm were therefore explored to speed up separations by considering the impact of the instrumentation on the efficiency. Interestingly, the impacts of injection volume and post-column connection tubing on peak capacity were found to be negligible. Finally, it was demonstrated that longer columns would not improve selectivity or separation efficiency, but baseline separation of three model ONs mixtures was enabled in as little as 30 s on the 5 mm column. This proof-of-concept work paves the way for future investigations using more complex therapeutic ONs and their related impurities.</p>
<p>...</p>
<h3><strong>Experimental Section</strong></h3>
<h4><strong>Chemicals and Samples</strong></h4>
<p>Water was obtained from a Milli-Q water purification system from Millipore (Bedford, MA). LC-MS grade methanol (MeOH) was purchased from Thermo Fisher Scientific (Reinach, Switzerland). Oligonucleotides were purchased from Eurogentec (Seraing, Switzerland) and Integrated DNA Technologies (IDT, Leuven, Belgium). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, ≥99%), triethylamine (TEA, ≥99.5%), and RNase-free water were purchased from Sigma-Aldrich (Buchs, Switzerland).</p>
<h4><strong>Sample and Mobile Phase Preparation</strong></h4>
<p>100 μM oligonucleotide aliquots were initially prepared by reconstituting lyophilized material in the appropriate volume of RNase-free water and stored at −20 °C.</p>
<p>Equimolar oligonucleotide mixtures were prepared by mixing aliquots and diluting the products to 5 μM in RNase-free water prior to the IP-RPLC analysis (if not stated otherwise). Sample mixtures used to illustrate the retention of oligonucleotides were (first part of the study) the following: dT10–40 (a mixture of seven poly-deoxythymidylic (dT) acids of 10-, 15-, 20-, 25-, 30-, 35-, and 40-mer) and dT40–100 (a mixture of four poly-dT acids of 40-, 60-, 80-, and 100-mer). For the second part of the study, dT40–100, 4 dT (a mixture of four poly-dT acids of 10-, 20-, 30-, and 40-mer), and 4 PS (a mixture of four 20-mer poly-dT acids with 0, 6, 9, and 19 phosphorothioate modifications that replace the phosphodiester linkages) were used.</p>
<p>Mobile phase A was 14 mM TEA in water, containing 100, 160, or 400 mM HFIP, pH 8.2, 8.1, or 7.8, respectively. Mobile phase B was a mixture of 50:50 mobile phase A and methanol.</p>
<h4><strong>Chromatographic System, Columns, and Software</strong></h4>
<p>All chromatographic separations were performed on a Waters <a href="https://lcms.labrulez.com/library?mainauthorItems=5&amp;manufacturerItems=5&amp;search=ACQUITY+UPLC+I-Class"><strong>ACQUITY UPLC I-Class</strong></a> System (Milford, MA) equipped with a binary solvent delivery pump, an autosampler with a flow-through-needle (FTN), and a UV detector with a 0.5 μL UV flow cell. The overall extra-column volume was measured as 5.6 μL from the injection seat of the autosampler to the detector cell, while the offset time was equal to 0.87 s. These values were found after plotting system residence time vs reciprocal flow rate (1/F) for various experiments conducted at several flow rates in the absence of a column (zero-dead volume union). Waters <a href="https://lcms.labrulez.com/library?from=2020&amp;mainauthorItems=5&amp;manufacturerItems=5&amp;to=2024&amp;search=ACQUITY+UPLC+BEH"><strong>ACQUITY UPLC BEH</strong></a> C18 1.7 μm Column, 5 mm × 2.1 mm, 130 Å <a href="https://lcms.labrulez.com/library?from=2020&amp;mainauthorItems=5&amp;manufacturerItems=5&amp;to=2024&amp;search=VanGuard+Pre-column"><strong>VanGuard Pre-column</strong></a> was used in this study. Commercial 50 mm and 150 mm × 2.1 mm columns (Waters ACQUITY Premier Oligonucleotide BEH C18 1.7 μm, 130 Å Column) were also used. Data acquisition and instrument control were performed by <a href="https://lcms.labrulez.com/library?mainauthorItems=5&amp;manufacturerItems=5&amp;search=Empower"><strong>Empower</strong></a> 3 Software (Waters). The freely available Excel Spreadsheet developed by Guillarme et al. was used to derive LSS parameters and predict retention times. (23,24)</p>
<h4><strong>Apparatus and Methodology</strong></h4>
<p>Sample volumes of 1 μL were injected using linear gradients (for all separations). The column temperature was set at 60 °C unless stated otherwise. Various semi-empirical models are used in LC to describe the relationship between experimentally observed retention times and mobile phase composition or gradient conditions. In this study, the following model was considered...</p>
<h3><strong>Results and Discussion</strong></h3>
<h4><strong>Retention Properties of Oligonucleotides</strong></h4>
<p>Several studies have reported the elution behavior of ONs in IP-RPLC in the presence of various ion-pairing reagents and using different stationary phases. (25−27) Efforts were mainly focused on (1) improving the chromatographic resolution (selectivity and peak capacity) of ONs or (2) improving the LC-MS sensitivity. (25) Relative retention indices and retention time calibration of homo-oligonucleotides in IP-RPLC have been published, (28) but absolute dimensionless measures of retention (like the S parameter of the LSS model) have not been reported yet, at least to the best of our knowledge. In this study, we focus our attention especially on the sensitivity of solute retention to mobile phase composition (S parameter), as this determines the required (effective) column length. We also discuss the log k0 parameter that corresponds to the extrapolated value of k for φ = 0. Here, the change in mobile phase composition was approximated by the change of organic modifier composition (methanol), while neglecting the effect of the small change in additive concentration. (29) It has also been reported that IP-RPLC separations of ONs benefit from shallow gradients, as they elute in relatively sharp peaks, despite the shallow gradient applied. (25) This may indicate that the retention of the solute is very sensitive to the eluent composition (on–off-like behavior). In practice, 50–150 mm long columns are often used for the IP-RPLC analysis of ONs, which is not necessary if the on–off behavior is valid.</p>
<p>...</p>
<h4><strong>Illustration of the On–Off Mechanism of Oligonucleotides</strong></h4>
<p>In general, IP-RPLC acts as a mixed-mode chromatographic mode combining hydrophobic and electrostatic (ionic) retention mechanisms. However, if the ion-pairing reagent concentration is sufficiently high, elution is mainly driven by hydrophobic interactions occurring between the stationary phase ligands and the hydrophobic part (i.e., alkyl chain) of the ion-pairing reagent molecules.</p>
<p><a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c01934#fig2"><strong>Figure 2</strong></a>A shows plots of the log retention factor as a function of mobile phase composition (φ) for ATP, dT20, dT100, and a monoclonal antibody (mAb) as a reference. The figure was constructed on the basis of data obtained with the 100 mM HFIP mobile phase, resulting in the highest S values. It clearly illustrates that dT20 already shows an on–off behavior (very steep curve, S = 60). For the dT100, we observed a 2.5 times higher S value (S = 264) compared with an intact mAb (S typically ranges between 100 and 150). The higher S values observed with ONs are probably due to the differences in molecular shape and specific surface available for interaction with the stationary phase (helical straight shape versus Y-shape).</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Anal_Chem_2023_95_27_10448_10456_Figure_2_Logarithmic_solute_retention_factor_d0e09e3597.jpg" alt="Analytical Chemistry 2023, 95 (27), 10448-10456 - Figure 2. Logarithmic solute retention factor (A) and relative migration velocity (u/u0) (B) as a function of the volume fraction of the organic mobile phase φ for ATP (black, MW = 0.5 kDa; S = 13), dT20 (green, MW = 6 kDa; S = 60), dT100 (red, MW = 30 kDa; S = 264), and an intact mAb (blue, MW = 150 kDa, S = ∼116)."/></p>
<p><a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c01934#fig2"><strong>Figure 2</strong></a>B shows the relative migration velocity as a function of mobile phase composition. Such a plot illustrates the transition between fully adsorbed (“on”) and fully released (“off”) states. The value of S = 264 (and the log k0 = 45.2) means that up to a mobile phase composition of φ = 0.164, the migration velocity of the dT100 is practically zero (bound at the column inlet, its velocity is less than 1% of the interstitial mobile phase velocity), whereas from φ = 0.179, it moves at a velocity greater than 99% of the mobile phase velocity, indicating that the solute is fully desorbed from the column. The transition range between the adsorbed and desorbed states therefore corresponds to a Δφ range of only 1.5% (0.179–0.164). The transition range for the dT20 is somewhat wider, namely, 6.5% of Δφ. This transition typically occurs between 3 and 4% Δφ for an intact mAb in RPLC mode. For the small ATP molecule, we observed a transition range of 31.4% Δφ (from φ = 0.116–0.430).</p>
<p><a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c01934#fig3"><strong>Figure 3</strong></a> shows a plot of the column length (Leff) required to effectively retain dT20 and dT100 (at least at ke = 0.5, which is a common practice for small molecule separations) as a function of gradient time and flow rate.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Anal_Chem_2023_95_27_10448_10456_Figure_3_Color_coded_surface_plot_of_the_column_length_Leff_required_to_retain_d_T20_and_d_T100_with_ke_0_5_7271adf747.jpg" alt="Analytical Chemistry 2023, 95 (27), 10448-10456 - Figure 3. Color-coded surface plot of the column length (Leff) required to retain dT20 and dT100 with ke = 0.5, as a function of gradient time and flow rate (dc = 2.1 mm ID, ε = 0.68, and Δφ = 0.2)."/></p>
<p>...</p>
<h3><strong>Conclusions</strong></h3>
<p>The goal of this proof-of-concept work was to evaluate if ultra-short columns can be used in IP-RPLC for the analysis of model oligonucleotides in gradient elution mode.</p>
<p>First, the validity of the LSS model was evaluated, and the accuracy of retention time predictions was assessed. It was found that ONs in IP-RPLC conditions follow the on–off elution behavior and that for most linear gradient separation conditions an effective column length between 5 and 35 mm was sufficient to obtain the best selectivity and separation efficiency. Therefore, the applicability of ultra-short columns was explored to speed up ON separations. In this regard, the extra-column band broadening induced by the instrumentation was carefully evaluated. Interestingly, the impact of injection volume and post-column connection tubing on peak capacity were found to be negligible. Next, the impact of column length on apparent efficiency was also monitored. First, column length was varied (150, 50, and 5 mm), while flow rate and gradient time were kept constant. Then, various gradient times and flow rates were tested on the three column lengths. In all cases, the on–off retention mechanism of ONs was confirmed. In addition, 5 mm column proved to be an advantageous choice to (i) increase the overall resolution achievable in gradient mode and (ii) perform ultra-fast separations with reasonable kinetic performance.</p>
<p>Optimized ultra-fast separations were therefore developed on the 5 mm column by using a flow rate of 1 mL/min and a gradient time of 1 min. Three model oligonucleotide mixtures including small, large, and PS-modified ONs were analyzed, and a remarkable resolution was especially obtained for the separation of the small ONs mixture. The separations were subsequently pushed to their limits at a higher flow rate (1.75 mL/min) and temperature (90 °C against the previous 60 °C), enabling baseline separation of all of the mixtures in as little as 30 s.</p>
<p>For the first time, a comprehensive study on the demonstration of the applicability of the LSS model to model oligonucleotides’ IP-RPLC separations in gradient elution mode was performed. The understanding of the ONs retention behavior enabled the evaluation of ultra-short columns and the selection of a 5 mm column to boost the ONs baseline separations in less than 1 min. These findings represent the first proof of concept for achieving ONs high-throughput analysis and pave the way for further investigation using more complex therapeutic oligonucleotides.</p>
<p>Interestingly, there are two possible reasons for using ultra-short columns for ONs: (i) achieving ultra-fast separation (1 min or less), while maintaining a reasonable efficiency, provided that the flow rate is sufficiently high and (ii) increasing the overall resolution achievable in gradient mode (corresponding to Pmin) within a reasonable analysis time (few minutes). The second aspect has not yet been explored but will be investigated in an upcoming work.</p>


High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns

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