Post-translational modifications (PTMs) are where so much of biology’s complexity lives, but they are also some of the hardest molecular features to analyze with confidence. Phosphorylation, asymmetric dimethylarginine (ADMA), and O-GlcNAcylation (O-GlcNAc) often occur at low abundance, can be highly labile, and frequently require precise site localization to understand their biological meaning. This<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/tn_004377_ov_phosphorylation_ptm_etd_excedion_pro_tn004377_na_en_71f681655f.pdf"> technical note </a>shows how the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1958">Thermo Scientific Orbitrap Excedion Pro Hybrid Mass Spectrometer</a> helps address those challenges by combining fast, sensitive performance with complementary <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/7217">HCD and EThcD fragmentation</a> workflows.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Phosphorylation_ADMA_and_O_Glc_N_Ac_Analysis_on_the_Orbitrap_Excedion_Pro_Mass_Spectrometer_d24a5de98e.jpg" alt="Thermo Fisher Scientific: Phosphorylation, ADMA, and O-GlcNAc Analysis on the Orbitrap Excedion Pro Mass Spectrometer" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Phosphorylation_ADMA_and_O_Glc_N_Ac_Analysis_on_the_Orbitrap_Excedion_Pro_Mass_Spectrometer_d24a5de98e.jpg 245w," sizes="100vw" width="1024" height="512">Using enriched PTM samples and automated data-dependent acquisition, the study demonstrates that the Orbitrap Excedion Pro MS can deliver both depth and confidence across multiple PTM classes. For phosphorylation analysis, the platform identified 5,892 unique phosphosites from IMAC-enriched samples and 2,941 unique phosphotyrosine sites after phosphotyrosine enrichment, highlighting strong performance for both broad and highly targeted signaling studies. Just as importantly, the data show that HCD and EThcD provide complementary value, with each fragmentation method contributing unique site identifications and helping resolve ambiguous assignments.The note also highlights the value of EThcD for more analytically demanding PTMs. In ADMA-enriched samples, EThcD delivered more ADMA peptide-spectrum matches and more unique modified peptides than HCD, supporting improved confidence in site characterization. For O-glycopeptides, the Orbitrap Excedion Pro MS uses an intelligent HCD-product-dependent-EThcD strategy that triggers EThcD only when oxonium ions are detected, improving productivity while preserving the fragmentation detail needed for confident glycosite localization.Overall, this technical note shows how Orbitrap Excedion Pro MS enables researchers to go deeper into PTM biology with greater sensitivity, better site confidence, and smarter acquisition. For scientists studying signaling, regulation, and disease mechanisms, it presents a powerful approach for expanding PTM coverage without compromising analytical rigor.<blockquote><h5><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/tn_004377_ov_phosphorylation_ptm_etd_excedion_pro_tn004377_na_en_71f681655f.pdf">Download the technical note "Advancing phosphorylation, ADMA, and O-GlcNAc PTM analysis with HCD and EThcD on the Orbitrap Excedion Pro mass spectrometer"</a></h5></blockquote>Visit us on&nbsp;<a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/72003132/admin/dashboard/">LinkedIn:</a>&nbsp;#PTM #OrbitrapExcedionPro #glycosylation #phosphorylation

Phosphorylation, ADMA, and O-GlcNAc Analysis on the Orbitrap Excedion Pro Mass Spectrometer

<h3>Refractive Index (RI) Detection in HPLC – Making Invisible Compounds Visible</h3>When HPLC detectors are discussed, UV-Vis absorbance detection is often the first technique that comes to mind. Its popularity is easy to understand: it is sensitive, selective, and suitable for a wide range of compounds. But what happens when the analyte does not absorb ultraviolet or visible light?This is where the <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/en-us/all/products/2429">Refractive Index Detector (RID)</a> becomes particularly useful. Reliable, versatile, and often described as a “universal detector” for HPLC, RI detection can reveal compounds that remain invisible to many optical detectors. For substances such as sugars, polymers, lipids, or alcohols, it is often an important analytical option.Let’s look at how RI detection works, where it performs best, what its main limitations are, and how to achieve stable, reproducible results.<h4>How RI Detection Works</h4>When light passes from one medium into another, its path changes direction, or refracts, depending on the phase velocity and angle of incidence. This phenomenon is described by Snell’s law, with the extent of refraction determined by the refractive index (RI) of the medium (Figure 1).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_1_Refraction_of_light_at_the_interface_between_two_media_with_different_refractive_indices_baa4aad28c.webp" alt="KNAUER: Figure 1: Refraction of light at the interface between two media with different refractive indices, where n₁ &gt; n₂. Since the velocity is lower in the second medium (v₂ &lt; v₁), the angle of refraction α₂ is less than the angle of incidence α₁." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_1_Refraction_of_light_at_the_interface_between_two_media_with_different_refractive_indices_baa4aad28c.webp 238w," sizes="100vw" width="1024" height="670">In HPLC, an RI detector monitors small changes in the refractive index of the mobile phase as compounds elute from the column. Because each substance has its own refractive index, even relatively small changes in the composition of the column effluent can generate a measurable response.Modern RI detectors generally use a differential optical design (Figure 2). Two optical paths are continuously compared: one passes through a reference cell containing pure mobile phase, while the other passes through a sample cell containing the column effluent, i.e. mobile phase plus analyte.A light source, commonly an LED or tungsten lamp, illuminates both cells. Changes in refractive index alter the path of the light, and a photodiode detects the resulting deviation.When only mobile phase is present, the two optical paths are effectively identical and no significant detector response is produced. As an analyte enters the sample cell, however, the refractive index changes. The light beam is slightly deflected, creating an optical imbalance and consequently a change in light intensity.The detector converts this change into an electrical signal, which appears as a chromatographic peak. Because the response is related to analyte concentration, RI detection can also be used for quantitative analysis.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_2_Schematic_of_a_differential_RI_detector_with_dual_flow_cell_6299f55b17.webp" alt="KNAUER: Figure 2: Schematic of a differential RI detector with dual flow cell. The sample cell carries column effluent; the reference cell contains pure mobile phase. A light beam passes through both and forms a slit image on a photodiode. Any analyte-induced change in refractive index alters the beam’s path, shifting the image on the photodiode and generating a signal peak." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_2_Schematic_of_a_differential_RI_detector_with_dual_flow_cell_6299f55b17.webp 245w," sizes="100vw" width="1024" height="524">For reliable measurements, the reference cell must always contain the same mobile phase as the sample cell, but without analyte. It therefore needs to be refreshed regularly with clean mobile phase, commonly using a switching valve and/or T-piece (Figure 3).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_3_Flow_path_in_the_AZURA_RID_2_1_L_during_analysis_left_and_flushing_right_e6bc30acef.webp" alt="KNAUER: Figure 3: Flow path in the AZURA® RID 2.1L during analysis (left) and flushing (right). During analysis, solvent flows from the IN port through the heat exchanger and sample cell, then continues through the T piece and flush valve to the OUT port. During flushing, the valve redirects the flow through the T piece so the solvent passes a second heat exchanger and reference cell before exiting via the OUT port." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_3_Flow_path_in_the_AZURA_RID_2_1_L_during_analysis_left_and_flushing_right_e6bc30acef.webp 245w," sizes="100vw" width="1024" height="410">It is important to remember that refractive index depends on both light wavelength and medium density. The wavelength is fixed by the optical design of the detector, but density—and therefore refractive index—changes with temperature, pressure, and composition.For this reason, RI detection is particularly sensitive to changes in operating conditions. Stable measurements depend heavily on maintaining a tightly controlled analytical environment.<h4>Why Choose RI Detection?</h4>RI detectors become especially valuable when other detector types cannot provide a useful response. They may be less sophisticated than some modern detection technologies, but several characteristics make them indispensable for particular applications.<h5>Broad Applicability</h5>An RID can detect virtually any compound that produces a change in the refractive index of the mobile phase. No UV-absorbing chromophore is required.Because almost every dissolved substance changes refractive index to some degree, RI detection is commonly regarded as a universal detection technique. Typical analytes include sugars, alcohols, lipids, fatty acids, and polymers.<h5>Non-Destructive Detection</h5>RI measurement does not destroy the analyte. This makes it useful in workflows where the separated compound needs to be collected or analyzed further, including preparative chromatography.KNAUER’s <a target="_blank" rel="noopener noreferrer" href="https://store.knauer.net/shop/add38-azura-r-rid-2-1l-highflow-preparative-refractive-index-detector-62927?category=75">AZURA® RID 2.1L for preparative applications</a>, for example, supports flow rates up to 100 mL/min.<h5>Simple and Reliable Operation</h5>RI detectors are suitable for aqueous and many organic mobile phases as long as the solvent composition remains constant.Under stable isocratic conditions, they are relatively straightforward to use, robust, and require comparatively little maintenance.<h5>Direct Detection Without Derivatization</h5>Analytes do not need to be derivatized or labelled before RI detection. If a compound is soluble in the mobile phase and produces a sufficient difference in refractive index, it can be detected directly.This simplifies sample preparation and reduces additional handling steps that could introduce variability or error.These characteristics make RI detection particularly attractive for routine applications such as lactose determination in milk or polymer purity testing.<h5>Limitations of RI Detection</h5>Universal detection comes with several practical compromises. RI detectors require highly stable conditions and can be considerably more sensitive to environmental and system changes than other common HPLC detectors.<h5>High Temperature Sensitivity</h5>Refractive index is strongly influenced by temperature. For aqueous solvents, the change in refractive index with temperature is approximately dn/dT ≈ 10⁻⁴/°C.Even small temperature fluctuations can therefore produce baseline drift that may be larger than the analytical signal itself.Modern RI detectors use thermostated compartments and heat exchangers to stabilize the temperature of the flow cell. This improves accuracy and reproducibility, although the heat exchangers introduce additional system volume that can contribute to peak broadening compared with UV detection.<h5>Limited Compatibility With Gradient Elution</h5>RI detection is essentially restricted to isocratic chromatography.Changing the composition of the mobile phase during a gradient produces a much larger change in refractive index than the analyte itself. The resulting baseline shift can easily overwhelm chromatographic peaks.A stable RI baseline therefore requires the sample and reference cells to contain the same mobile-phase composition throughout the analysis.<h5>Lower Sensitivity</h5>Compared with UV detection, RI detection is generally about one to two orders of magnitude less sensitive.Typical detection limits are in the µg–mg/mL range, making RID less suitable for trace-level analysis or impurity profiling.<h5>Limited Pressure Tolerance</h5>RI flow cells can tolerate only moderate backpressure. When several detectors are connected in series, the RI detector should therefore normally be positioned last.<h5>Longer Equilibration</h5>RID systems require time to reach thermal and optical stability. Warm-up and equilibration can take hours before a sufficiently stable baseline is obtained.<h5>Sensitivity to Baseline Disturbances</h5>Because the detector responds to any change in refractive index, many factors can affect the baseline.Temperature fluctuations, unstable flow or pressure, pump pulsation, air bubbles, inadequate solvent mixing, and impurities in the mobile phase can all lead to noise, drift, or false signals.Clean operation and stable system conditions are therefore essential.<h4>How to Get the Best Performance From an RI Detector</h4>Successful RI detection can largely be summarized in one word: stability.Small variations in temperature, flow, solvent composition, or pressure can quickly become visible in the chromatographic baseline. Careful system preparation and handling can therefore make a major difference.<h5>Maintain a Stable Temperature</h5>Thermal equilibration should be one of the first priorities.Allow the detector, column, and mobile phase to equilibrate fully before beginning analysis, ideally for at least 60 minutes. A column oven can help maintain stable conditions, and the column and detector should be operated at the same temperature whenever possible.Environmental influences such as direct sunlight, air currents, or day-to-night laboratory temperature fluctuations should also be minimized.<h5>Use Isocratic Methods</h5>Keep the mobile-phase composition constant throughout the run.If the separation requires gradient elution, an RI detector is generally not the appropriate choice.<h5>Degas the Mobile Phase and Keep the System Clean</h5>Use well-degassed, particle-free mobile phases and make sure the LC system is properly primed.An in-line degasser can help prevent bubble formation. Tubing, solvent reservoirs, and the entire flow path should also be kept clean because even small bubbles or contaminants can generate spikes, noise, or other baseline disturbances.<h5>Match the Sample Solvent Carefully</h5>Whenever possible, prepare samples directly in the mobile phase or in a solvent with very similar composition.Large differences between the injection solvent and mobile phase can produce strong refractive-index disturbances around the void volume.Filtering samples also helps reduce the risk of blockages and protects the detector flow cell.<h5>Keep Flow and Pressure Stable</h5>Maintain a constant flow rate and avoid abrupt pressure changes, excessive pump pulsation, or leaks.A restriction capillary or back-pressure regulator positioned after the flow cell may help stabilize pressure, although these measures cannot compensate for noise caused by a poorly performing pump.<h5>Maintenance and Practical Considerations</h5>Flush the reference cell regularly with fresh mobile phase. The mobile phase itself should also be replaced periodically to reduce microbial growth or compositional changes caused by evaporation.When changing solvent systems, flush the instrument with mutually miscible solvents and allow sufficient time for the new system to equilibrate fully.Where appropriate, mobile-phase recycling or a low-flow standby mode can help reduce solvent consumption and shorten restart times between analytical sequences.<blockquote>👉 Pro Tip: Keep an eye on the baseline. A stable baseline is one of the best signs that the RID has equilibrated properly. Negative peaks are not necessarily an error—they simply indicate that the analyte has a lower refractive index than the mobile phase.</blockquote><h5>The Bottom Line</h5>With careful control of temperature, mobile-phase composition, pressure, and cleanliness, an RI detector can provide stable baselines and consistent quantitative data.Keep the system clean, maintain constant operating conditions, and allow sufficient equilibration time, and even this demanding HPLC “diva” can deliver highly reliable results.<h4>Typical Applications: Where RI Detection Performs Best</h4>RI detection is particularly useful for substances that do not absorb UV light strongly and therefore cannot be detected effectively using conventional UV/VIS detectors.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_AI_Figure_4_Common_uses_of_RI_detection_in_HPLC_d84d8458dc.webp" alt="KNAUER / AI: Figure 4: Common uses of RI detection in HPLC" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_AI_Figure_4_Common_uses_of_RI_detection_in_HPLC_d84d8458dc.webp 245w," sizes="100vw" width="1024" height="433">Typical analytes include sugars, alcohols, fatty acids, polymers, lipids, and other small organic molecules without strong chromophores.<h5>Some common application areas include:</h5><ul><li>Carbohydrate and sugar analysis, including determination of glucose, fructose, sucrose, or lactose in food and beverages. HPLC-RI has been used for <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/pdf/VFD_0195_sec_for_fingerprint_analysis_of_gummy_candies_ce5349cd95.pdf">product quality control of gummy candies</a>, with further discussion available in this <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/en-us/all/article/7186">related blog post</a>.</li><li>KNAUER’s AZURA® RID 2.1L has also been used for the <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/pdf/VFD_0194_d497a35278.pdf">simultaneous determination of sugars and organic acids in wine</a>, an application explored further in the article <a target="_blank" rel="noopener noreferrer" href="https://www.knauer.net/blog/blog-9/the-hidden-science-in-your-glass-147">“The hidden science in your glass”</a>.</li><li>Polymer and macromolecule characterization in GPC/SEC applications, for example when determining molecular-weight distributions. Examples include <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/pdf/4czsecf32d70a6d2yugpojtljg8rvp4vd4ur_05660bfad3.pdf">optimization of molecular-weight determination with the KNAUER AZURA® GPC/SEC system</a> and <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/pdf/qpwsk0igeqca8b5bqunbjjqnwum09tucb83s_cc4dbbcec9.pdf">analysis of mucin proteins using an AZURA® SEC System</a>.</li><li>Bioprocessing, including monitoring fermentation products such as <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/pdf/VBS_0079_Characterization_of_fructans_in_fermentation_products_with_sec_bc8f294f0e.pdf">fructans</a>.</li><li>Pharmaceutical applications, for example analysis of excipients such as sugars and polyols, as well as identity and purity testing of APIs. Dextranes, which are used as carrier materials in pharmaceuticals and as thickening or moisturizing agents in cosmetics, can be <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/pdf/h2oikq51kfvn6ebxij6hs88pxupstfgdqfyd_3e4f29d933.pdf">characterized by SEC</a>, as their properties depend strongly on molecular-weight distribution.</li><li>Lipid and surfactant analysis for formulation development and quality control.</li><li>Environmental analysis, including determination of selected organic and inorganic impurities in water samples.</li></ul>Overall, RI detection works best for analytes that are soluble, stable in solution, and compatible with straightforward isocratic separation conditions.It is not the preferred solution for trace analysis, gradient chromatography, or workflows requiring direct MS coupling. Nevertheless, it remains a practical and dependable option for compounds that are difficult or impossible to monitor using UV or fluorescence detection.<h4>Final Thoughts: A Detector That Still Deserves Attention</h4>As analytical laboratories adopt increasingly sophisticated detection technologies, refractive index detection can appear relatively simple. Yet for many applications, that simplicity is exactly its strength.For analytes such as sugars, alcohols, polymers, and other compounds without strong UV absorption, RID offers a direct, reliable, and non-destructive method of detection.Its greatest challenges are its sensitivity to temperature and its incompatibility with gradient elution. Under carefully controlled isocratic conditions, however, RI detection remains a dependable workhorse for routine HPLC and GPC/SEC applications.<blockquote>Pro tip: If your analyte seems invisible to conventional optical detection, a change of perspective may be all that is needed. RI detection could make it visible.</blockquote>In the next article in the “Seeing is Believing – Detection Made Easy” series, we will focus on Fluorescence Detection (FLD), a highly selective and sensitive technique for compounds that fluoresce naturally or can be derivatized to do so.

Seeing is Believing – Detection Made Easy Part 4: Refractive Index Detector

News from LabRulezLCMS Library - Week 36, 2026

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A unique company dedicated to serving science helping our customers drive productivity and accelerate results. Through our premier brands – Thermo Scientific, Applied Biosystems, Invitrogen, Fisher Scientific and Unity Lab Services.

Thermo Fisher Scientific

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LabRulez

Based in Berlin, KNAUER is a medium-sized, owner-managed company that has been serving the sciences since 1962. We develop and manufacture scientific instruments of superior quality for liquid chromatography, including: Analytical HPLC/UHPLC, Preparative HPLC, Fast protein liquid chromatography (FPLC), Multi-column chromatography/Simulated moving bed (SMB) chromatography. High pressure dosing, in-line detection systems, and osmometry are additional business areas.

KNAUER

Identification of the substance – what is in your sample? (Bioanalytic)

We are able to identify metabolites and products of chemical reactions, contaminants of biological and chemical substances and preparations, and perform profiling of a series of samples to study the kinetics of transformations.

<h4>Substance Identification - Looking for answers to what's in your sample? - we can help you.</h4>Substance identification based on LC-MS/MS techniques can involve single compounds or mixtures of substances. In our analyses, we rely primarily on the knowledge and skills we have acquired after many years of work. For analysis, we use full laboratory facilities for sample preparation and high-resolution mass spectrometry (<a target="_blank" rel="noopener noreferrer" href="https://labexperts.com.pl/o-nas/">equipment</a>).We use different work algorithms depending on the material we are dealing with and the analytical task to be solved. We use DDA (IDA) or DIA (SWATH) methods or targeted approaches (e.g. MRMhr) if we know what to expect. Thus, we are able to identify metabolites and products of chemical reactions, contaminants of biological and chemical substances and preparations, and perform profiling of a series of samples to study the kinetics of transformations.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_Identification_of_the_substance_what_is_in_your_sample_2_0f425a648b.jpg" alt="Bioanalytic: Identification of the substance – what is in your sample" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_Identification_of_the_substance_what_is_in_your_sample_2_0f425a648b.jpg 245w," sizes="100vw" width="1881" height="1156"><h4>COST OF SERVICE</h4>We determine the cost of the service individually. The final cost is influenced by many factors related to the nature of the sample itself and the client's expectations. Thus, the price depends, among other things, on the complexity of the sample preparation procedure, the scope and number of analyses, etc.The service includes (depending on individual arrangements):<h5>Development of sample preparation procedure</h5><ul><li>Depending on the matrix and test substances.</li></ul><h5>Development of analytical methods</h5><ul><li>Application of high-resolution LC-MS/MS analyses using DDA (IDA) or DIA (SWATH) experiments and software to support the development of targeted screening methods.</li></ul><h5>Analysis of results includes:</h5><ul><li>finding and identifying the substances of interest, interpreting MS/MS spectra and proposing a structural formula.</li><li>Comparative analysis of sample series, e.g. PCA,</li><li>profiling of analytes in sample series</li></ul><h5>Reporting of results</h5><ul><li>Compile results into easy-to-read reports.</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_Identification_of_the_substance_what_is_in_your_sample_3_c1b8adc1d0.jpg" alt="Bioanalytic: Identification of the substance – what is in your sample" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_Identification_of_the_substance_what_is_in_your_sample_3_c1b8adc1d0.jpg 245w," sizes="100vw" width="1919" height="1208">

Characteristics of monoclonal antibodies (Bioanalytic)

At LabExperts, proteomic analyses are performed on a ZenoTOF 7600 high-resolution LC-MS/MS system (SCIEX). For MS/MS analyses, we use Collision induced dissociation (CID) and Electron-Activated Dissociation (EAD) fragmentation.

Antibodies are molecules made up of 4 polypeptide chains: two heavy (H - high-chain) linked to each other by a specific type of chemical bond - a disulfide bridge, and two light (L - light-chain) linked by an identical bond to the heavy chains. Monoclonal antibodies (mABs) are used in various forms - as free molecules, in the form of a drug-antibody combination, and in the form of an immunoconjugate, a drug-antibody-biopolymer combination.Their main use is in highly specific targeted therapy for cancer, cardiovascular, autoimmune and nervous system diseases. They are also used in the treatment of osteoporosis, Alzheimer's disease or transplantation. The range of applications of monoclonal antibodies is systematically expanded. The introduction of a new antibody to the market requires complex product characterization, which involves numerous proteomic analyses, which are performed by LC-MS/MS techniques, among others.<h4>LC-MS/MS</h4>The characterization of monoclonal antibodies involves many aspects, including, but not limited to, a suite of analyses related to confirmation of the structure, chain sequence and degree of modification of the antibody. Post-translational modifications (PTMs) are chemical changes that occur after the translation process. These changes generally result in an increase in the immunogenicity of the drug. For this reason, it is essential to monitor modifications as critical quality attributes (CQAs) during both the production and storage of the drug. To ensure monitoring of CQA points, the primary structure of the protein is characterized based on peptide mapping (peptide mapping) analysis. The protein under study is subjected to proteolytic digestion using enzymes such as trypsin, chymotrypsin, Lys-C, Glu-C. As a result of various proteolytic enzymes, a wide range of peptides is obtained. Ultimately, coverage of the entire sequence of the protein under study is obtained.<figure class="image"><img style="aspect-ratio:1536/947;" src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_3_d94fa1623b.jpg" alt="Bioanalytic: Characteristics of monoclonal antibodies." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_3_d94fa1623b.jpg 245w," sizes="100vw" width="1536" height="947"></figure>At LabExperts, proteomic analyses are performed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=52&amp;search=ZenoTOF+7600">ZenoTOF 7600</a> high-resolution LC-MS/MS system (SCIEX). For MS/MS analyses, we use Collision induced dissociation (CID) and Electron-Activated Dissociation (EAD) fragmentation. This provides us with data for unambiguous identification of protein-building amino acids. We process the results using Sciex OS, Skyline and Spectronaut software.<blockquote>We perform:<ul><li>Whole protein analyses - Intact - to determine the exact mass and confirm the theoretical mass;</li><li>N-glycosylation profile determination (based on MS spectrum reconstruction):<ul><li>Glycosylated form;</li><li>Deglycosylated;</li><li>Glycosylated with reduced disulfide bridges;</li><li>Deglycosylated with reduced disulfide bridges.</li></ul></li><li>Peptide maps:<ul><li>Protein digestion with various proteases so that sequence coverage is close to 100%<ul><li>Analysis of PTMs:</li><li>Location of modifications</li></ul></li><li>[%] of modifications compared to the unmodified sequence</li></ul></li><li>TiPOS testing - (technical proof of similarity) - technical confirmation of the similarity of the protein synthesized by the customer (from several LOTs) with the target protein (provided as a template).</li></ul>In order to maintain the highest possible analytical standards, we perform monoclonal antibody analyses under the regime of an internally developed and implemented laboratory quality system based on GLP principles.See also our other proteomics services: <a target="_blank" rel="noopener noreferrer" href="https://labexperts.com.pl/service/identyfikacja-bialek/">Protein identification</a>, <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1294">Protein quantitative analysis</a></blockquote><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_2_cb93143fe6.jpg" alt="Bioanalytic: Bioanalytic Characteristics of monoclonal antibodies." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_2_cb93143fe6.jpg 245w," sizes="100vw" width="1536" height="908"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_1_0e8ab7cdfa.jpg" alt="Bioanalytic: Bioanalytic Characteristics of monoclonal antibodies." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_1_0e8ab7cdfa.jpg 245w," sizes="100vw" width="1536" height="858">

Determination of nitrosamine content (Bioanalytic)

Our offer includes comprehensive development and validation of methods for determination of nitrosamines in pharmaceutical products. We perform the work according to the criteria and needs of the client based on the EMA note.

Determination of nitrosamines is one of the pharmaceutical industry's top priorities. After the presence of N-nitrosodimethylamine (NDMA) was first reported in an active pharmaceutical ingredient (API), the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) mandated the identification and control of all APIs and finished products in which there is a risk of nitrosamines.The content of nitrosamines in drug products can be controlled primarily by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. More than that, a reliable analysis result in our offer consists of a well-chosen sample preparation procedure, a selective and sensitive analytical method, and the experience of the analyst, who selects the best method parameters at the optimization stage.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Determination_of_nitrosamine_content_Bioanalytic_2_ce4f440837.jpg" alt="Bioanalytic: Determination of nitrosamine content." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Determination_of_nitrosamine_content_Bioanalytic_2_ce4f440837.jpg 234w," sizes="100vw" width="1536" height="1024">We have gained experience working with leading pharmaceutical companies. As part of our ongoing work, we have developed analytical methods for small-molecule nitrosamines of 70-150 g/mol. These compounds are mainly formed during manufacturing processes and depend on the presence and type of excipients (e.g. NDBA - N-nitroso-dibutylamine). We have also developed methods for determining molecules with higher nitrosamine drug substance related impurities (NDSRIs). In our work, we focus on achieving the highest possible sensitivity, selectivity and precision. To this end, we individually select the parameters of the methods depending on the composition of the drug and the expected nitrosamines. Due to the above criteria and the required detection limits, we perform nitrosamine impurity determinations using LC-MS/MS kits based on <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=52&amp;search=QTRAP+5500%2B">QTRAP 5500+</a> and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=52&amp;search=QTRAP+6500%2B">QTRAP 6500+</a> mass spectrometers (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=55">SCIEX</a>).<blockquote>Our offerOur offer includes comprehensive development and validation of methods for determination of nitrosamines in pharmaceutical products. We perform the work according to the criteria and needs of the client based on the EMA note.At the stage of sample preparation, we focus on:<ul><li>obtaining the highest possible recovery of nitrosamines (matrix purification);</li><li>limiting additional synthesis of impurities (selection of appropriate equipment and reagents);</li><li>ensuring the stability of the sample prepared for LC-MS/MS analysis.</li></ul>During the optimization of the LC-MS/MS method, we solve such problems as:<ul><li>selection of an appropriate internal standard;</li><li>separation of the impurity from the API;</li><li>obtaining the expected sensitivity of the analytical method.</li></ul>Quantitative analysis of nitrosamines requires sensitivity, precision and accuracy, which we provide to our customers.</blockquote><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Determination_of_nitrosamine_content_Bioanalytic_1_d629e00e62.jpg" alt="Bioanalytic: Determination of nitrosamine content." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Determination_of_nitrosamine_content_Bioanalytic_1_d629e00e62.jpg 103w," sizes="100vw" width="1130" height="1710">

rHGH fragment or AOD-9604 (LCMS+CHNS) analysis

Combined LC-MS, CHNS and HPLC analysis of rHGH fragment 176–191 and AOD-9604 for identification, amount, peptide purity and contamination screening.

This analysis is designed to identify and determine the amount and purity of recombinant Human Growth Hormone fragment 176–191 and AOD-9604. It combines LC-MS, CHNS and HPLC to provide information about peptide identity, net peptide content, peptide purity and potential contamination with other peptides or degradation products.The test can detect and measure either target compound. It is therefore not necessary to know in advance whether the sample contains rHGH fragment 176–191 or AOD-9604.<h4>rHGH Fragment 176–191</h4>rHGH fragment 176–191, also known as somatropin 176–191, is a peptide corresponding to amino acids 176 to 191 of Human Growth Hormone. It consists of 16 amino acids and represents the C-terminal region of the HGH molecule.Its molecular weight is approximately 1,799 Da. It differs from AOD-9604 in the first amino acid of its sequence.<h4>AOD-9604</h4>AOD-9604 is a synthetically modified peptide derived from Human Growth Hormone fragment 176–191. Like the rHGH fragment, it contains 16 amino acids, but its sequence begins with tyrosine instead of phenylalanine.This structural difference allows the two peptides to be distinguished by LC-MS. The molecular weight of AOD-9604 is approximately 1,815 Da.<h4>Analytical Methods</h4><h5>LC-MS – Identification and Contamination Screening</h5>LC-MS is used to confirm the identity of the peptide in the sample. It also provides screening for detectable contamination with other peptides and peptide degradation products.<h5>CHNS – Net Peptide Content Determination</h5>CHNS elemental analysis measures the carbon, hydrogen, nitrogen and sulphur content of the sample. The measured values can be used to determine Net Peptide Content.Because the sample is combusted during CHNS analysis, the result is not affected by solubility issues that may complicate chromatographic measurement of peptide amount.<h5>HPLC – Peptide Purity Determination</h5>HPLC is used to determine peptide purity and the relative proportion of the main peptide component compared with chromatographically detectable impurities.<h4>What the Analysis Determines</h4><ul><li>Identity of rHGH fragment 176–191 or AOD-9604</li><li>Amount of peptide in the sample</li><li>Net peptide content determined by CHNS</li><li>Peptide purity determined by HPLC</li><li>Presence of other peptides or degradation products detected by LC-MS</li><li>Differentiation between rHGH fragment 176–191 and AOD-9604</li></ul><h4>Analysis Parameters</h4><ul><li>Target compounds: rHGH fragment – somatropin 176–191 and AOD-9604</li><li>CAS number of AOD-9604: 221231-10-3</li><li>Molecular weight of AOD-9604: 1,815 Da</li><li>AOD-9604 sequence: H-Tyr-Leu-Arg-Ile-Val-Gln-Cys(1)-Arg-Ser-Val-Glu-Gly-Ser-Cys(1)-Gly-Phe-OH</li><li>CAS number of rHGH fragment: 66004-57-7</li><li>Molecular weight of rHGH fragment: 1,799 Da</li><li>rHGH fragment sequence: H-Phe-Leu-Arg-Ile-Val-Gln-Cys(1)-Arg-Ser-Val-Glu-Gly-Ser-Cys(1)-Gly-Phe-OH</li><li>Identification method: LC-MS</li><li>Amount determination method: CHNS</li><li>Peptide purity method: HPLC</li><li>Detection limit: less than one twentieth of the usual dosage per vial</li><li>Result format for lyophilizates: mg per vial and peptide purity</li><li>Alternative result format: m/m purity and peptide purity</li><li>Minimum lyophilizate sample: one whole unopened vial containing the lyophilizate</li><li>Minimum raw API powder sample: 30 mg in a sealed glass vial to prevent moisture absorption</li><li>Average turnaround time: 7 business days</li></ul><blockquote><h5>Important Notice</h5>The alternative m/m purity result format is intended exclusively for raw API powders. For lyophilized samples, the standard result format provides the peptide amount in mg per vial together with peptide purity.</blockquote><h4>Additional Analyses</h4><h5>TFA Analysis</h5><ul><li>Detection and measurement of trifluoroacetic acid (TFA) in the sample. Results are reported in mg per vial.</li></ul><h5>Raw Data</h5><ul><li>Delivery of the available raw analytical data from the primary test and all applicable additional analyses.</li></ul><h5>Fentanyl Presence Analysis</h5><h5>Alternative Result Format – m/m Purity</h5><ul><li>Alternative reporting of the result as mass-by-mass purity. This option is intended exclusively for raw API powders.</li></ul><h5>Variance Testing</h5><ul><li>Analysis of multiple sample units to assess variation within a single batch. All submitted samples must originate from the same batch of the same product.</li></ul><h5>pH Measurement</h5><h5><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2522">Endotoxin Analysis</a></h5><ul><li>Measurement of bacterial endotoxins, particularly lipopolysaccharides (LPS), in the sample. This analysis is available only for finished products and should not be ordered for raw powders.</li><li>The standard test provides accurate results up to 10 EU per vial. Values above 10 EU per vial are estimated and require an additional test with a different dilution for precise determination. Sending two vials is therefore recommended.</li><li>Where endotoxin levels above 10 EU per vial are expected, the method can be calibrated for the higher range in advance.</li></ul><h5>Additional Report</h5><ul><li>A separate additional laboratory report issued under a different client name.</li></ul><h5><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2527">Sterility Testing</a></h5><ul><li>TAMC and TYMC testing checks the product for the presence of bacteria, yeasts and moulds. Additional unopened sample units must be submitted for analysis. The average turnaround time for this test is 14 days.</li></ul><h5><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/products/2525">Heavy Metals Analysis</a></h5><ul><li>Detection of arsenic, cadmium, lead and mercury in the sample. Testing for other metals can be arranged individually.</li></ul>

GCMS/LCMS screening for contamination

GCMS/LCMS screening for identification of contaminants in samples, with the analytical method selected according to sample type.

The GCMS/LCMS Screening for Contamination is a standalone laboratory service designed to identify potential contaminants in a sample using either gas chromatography–mass spectrometry (GCMS) or liquid chromatography–mass spectrometry (LCMS).The appropriate analytical method depends on the sample type. GCMS is used primarily for volatile and semi-volatile compounds, while LCMS is suitable for peptide-related contamination.<h4>About the Screening</h4><ul><li>GCMS screening uses the NIST 2014 mass spectral library to identify a broad range of potentially harmful substances in the product. Detection is limited to volatile and semi-volatile compounds and this method is not suitable for peptide samples. &nbsp;</li><li>LCMS screening is intended for peptide products and can detect contamination with other peptides or degraded peptide. Detection is limited to peptides included in the laboratory’s available reference list.</li></ul>Both methods provide identification of detected contaminants only. Quantitative determination of a detected substance can be offered separately.<h4>Key Features</h4><ul><li>Standalone contamination screening by GCMS or LCMS</li><li>Analytical method selected according to sample type</li><li>GCMS screening for volatile and semi-volatile contaminants</li><li>GCMS identification using the NIST 2014 mass spectral library</li><li>LCMS screening for peptide contamination and degraded peptide</li><li>Identification of detected contaminants</li><li>Quantitative determination available separately after identification</li><li>Suitable for tablets, capsules, liquid solutions, raw materials, and vials depending on the selected method</li><li>Average turnaround time of 7 business days</li></ul><h4>Analysis Parameters</h4><ul><li>Testing method: GCMS or LCMS</li><li>Amount of sample required: 1 tablet/capsule, 1 ml solution, 200 mg raw material, or 1 vial</li><li>Average turnaround time: 7 business days</li></ul><blockquote>Important Notice<ul><li>The analytical method is selected according to the type of sample and the expected contaminants.</li><li>GCMS detection is limited to volatile and semi-volatile compounds and cannot be used for peptide samples.</li><li>LCMS detection is limited to peptides included in the laboratory’s available reference list.</li><li>Both GCMS and LCMS screening provide identification only. Quantitative determination of a detected contaminant is not included in the standard analysis and can be offered separately.</li></ul></blockquote><h4>Optional Additional Analyses</h4>The following optional laboratory services are available:<ul><li>Additional Laboratory Report</li></ul>

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