News from LabRulezLCMS Library - Week 16, 2026

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This week we bring you application notes by Agilent Technologies, KNAUER, Shimadzu and Waters Corporation and technical note by Thermo Fisher Scientific!

1. Agilent Technologies: Oligonucleotide Analysis Using the Agilent InfinityLab Pro iQ and Altura Oligo HPH-C18 Column

• Application note
• Full PDF for download

The pharmaceutical industry is witnessing a rapid surge in oligonucleotide-based therapeutics, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs). These nucleic acid therapies represent a paradigm shift in drug development, offering the ability to directly modulate genetic targets that were previously considered "undruggable" by conventional small molecules or monoclonal antibodies. This makes them particularly innovative for treating rare genetic disorders and chronic diseases.¹ 

In the manufacturing and quality control (QC) of oligonucleotides, purity is a critical parameter for ensuring drug safety and efficacy. While HPLC-UV has traditionally been the method of choice, the increasing complexity of therapeutic structures is exposing the limitations of relying solely on UV detection. Specifically, identifying and separating n-1 mer impurities—which share nearly identical physicochemical properties with the main component—is exceptionally challenging. Consequently, mass spectrometry (MS) has become essential for comprehensive purity analysis, impurity profiling, and molecular weight confirmation.² 

Due to their negatively charged phosphate backbone, oligonucleotides are highly polar and exhibit poor retention on standard reversed-phase columns. This necessitates the use of ion-pairing agents such as triethylamine (TEA), dibutylamine (DBA), or hexylamine (HA).³ While higher concentrations of these alkylamines generally improve peak resolution, they often lead to significant sensitivity suppression and instrument contamination in LC/MS analysis. Therefore, researchers are frequently forced to find a compromise between chromatographic resolution and MS sensitivity. 

In this application note, we present an optimized oligonucleotide analysis using the Altura Oligo HPH-C18 column, the Pro iQ mass detector, and the 1260 Infinity III Prime bio LC system. The Agilent AdvanceBio Oligonucleotide column is already well-regarded for its superior resolution, particularly in separating n-1 mer species under UV-optimized conditions. The Altura Oligo HPH-C18 column utilizes the same high-performance packing material as the AdvanceBio line but features ultra-inert column hardware. This technology minimizes secondary interactions with the metal surfaces of the hardware, allowing for improved resolution even when ion-pairing reagent concentrations are reduced for MS compatibility. 

Experimental 

Instrumentation 

The following instrumentation was used in this study: 

• Agilent 1260 Infinity III bio flexible pump (product number G7131C) 
• Agilent 1290 Infinity III bio multisampler (product number G7137C) with sample thermostat 
• Agilent 1290 Infinity III multicolumn thermostat (product number G7116B) with Quick Connect bio heat exchanger standard flow (product number G7116-60071) 
• Agilent 1260 Infinity III diode array detector (product number G7117C) with Max-Light cartridge cell LSS, 10 mm (product number G7117-60020) 
• InfinityLab Pro iQ (G6160B)

Conclusion 

This application demonstrates that high-resolution separation and molecular weight verification of oligonucleotide impurities are achievable even at a reduced ion-pair concentration of 15 mM HA, leveraging the combined power of the Agilent InfinityLab Pro iQ and Agilent OpenLab CDS deconvolution. While mitigating column hardware-induced peak broadening typically necessitates high concentrations of ion-pair reagents, the implementation of ultra-inert coating technology significantly enhances chromatographic resolution under MS-friendly conditions. This advancement offers several critical advantages: it ensures more accurate sample purity assessments, reduces operational costs through lower reagent consumption, minimizes chemical noise, and streamlines LC/MS method development. Furthermore, the rapid scan speed of the InfinityLab Pro iQ preserves the integrity of the improved peak resolution, enabling the precise characterization of larger species such as the 39 nt oligonucleotide impurity. This capability is particularly significant, as such high-molecular-weight impurities have historically been challenging to resolve and analyze under standard LC/MS conditions.

2. KNAUER: SEC for fingerprint analysis of gummy candies

• Application note
• Full PDF for download

Gummy candies are one of the most widely consumed confectionery products globally, with consumption reaching over 3.2 million tons just in 2023 [1]. Their popularity is attributed not only to their diverse flavour profiles, but also to their characteristic texture, which is determined by the used gelling agent [2] [3]. Typical formulations contain high levels of sugars, organic acids, colourings and flavourings, combined with a gelling agent. Traditionally, gelatin of animal origin has been used, but plant-based alternatives such as pectin are becoming increasingly popular [3]. Gelatin imparts the characteristic chewiness and elasticity, whereas pectin produces a noticeably firmer structure. In addition, the gel strength of these gelling agents depends on factors such as their concentration and the composition of other ingredients [2] [3]. Consequently, even minor variations in the manufacturing process can significantly affect texture, elasticity, transparency, and taste [2] [4]. For this reason, reliable and reproducible monitoring of these ingredients is essential for the food industry. In this context, SEC represents a powerful analytical approach for the characterization of gummy candies. SEC separates molecules according to their hydrodynamic size/radius, generating a highly reproducible chromatographic profile, so called “fingerprint” for each formulation of gummy candy. This fingerprint enables rapid quality control by detecting production errors and verifying batch consistency. It can clearly differentiate between manufacturers, as well as between products from the same manufacturer. Furthermore, it allows monitoring of time-dependent changes during storage, providing insights into product shelf life and stability. SEC is also well suited for the characterization of polymers and biopolymers, including the gelling agents found in gummy candies, such as gelatin an animal-derived protein from collagen and pectin, a plant-derived polysaccharide. By coupling SEC with special detectors and applying appropriate calibration, the method can be extended to provide quantitative information on the molecular weight and molecular weight distribution of gelling agents such as gelatin [2] [4]. This is relevant not only for confectionery analysis but also for the pharmaceutical industry, for example in the quality assessment of hard and soft gelatin capsules, coatings, and other drug-delivery systems [4].

Results

Column selection 

All measurements were performed either with a AppliChrom® Multipore SuperOH-P column (column A) or with a combination of a AppliChrom® SuperOH-P 350 column coupled to a AppliChrom® SuperOH-P 150 column (column set B). Both approaches cover the same separation range of 100 to 1 000 000 Da, but coupling the columns provides twice the separation length. Consequently, an improved resolution was expected, and the results confirmed this assumption. Fig. 7 shows the chromatograms of porcine and bovine gelatin measured on both column sets with a DAD at 230 nm. Only the DAD trace was shown, since the presence of maltodextrin from sample 5 (Tab. 1) in the RID signal would interfere with the interpretation of the gelatin peaks. Furthermore, the DAD provides a more selective response to gelatin due to its UV activity, resulting in a clearer and more reliable evaluation. As expected, column set B provided better separation. Subsequently, sample measurements taken using column set B produced a more detailed fingerprint.

CONCLUSION 

It was demonstrated that SEC can be used as a reliable and versatile chromatographic technique for product quality and process control for various gummy candies. This study involved investigating sample preparation procedures and testing different columns. Characteristic chromatographic fingerprints were thereby generated, enabling the identification of production deviations, verification of batch consistency, differentiation between products and manufacturers, and monitoring of storagerelated changes using UV and RI detection. Additionally, the method enables the semi-quantitative estimation of low-molecular-weight components, such as sugars. Moreover, this approach can be expanded and adapted for use with other suitable products.

3. Shimadzu: Detection of PFAS in Aqueous Samples by Matrix Assisted Laser Desorption Ionisation Time-of-Flight (MALDI-TOF) Mass Spectrometry

• Application note
• Full PDF for download

User Benefits

• Obtain results indicative of PFAS in aqueous samples within minutes for further study.
• Minimal sample preparation required prior to acquisition.
• Method can be applied to numerous sample types following additional preparation.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants which accumulate in water, soil and living organisms. PFAS negatively impact human health with links to cancer, immune system depression and hormone disruption, amongst others, already being established. Worldwide, concern regarding the exposure of biological organisms to environmental PFAS continuesto grow. PFAS contamination in water is a particular focus for both regulations and research. Widespread water contamination has been reported, and removal continues to be difficult and expensive. We developed a simple, cost-effective method for the detection of PFAS in aqueous samples using an entry-level benchtop linear MALDI-TOF mass spectrometer (MALDI-8030).

Q-TOF LCMS Analysis 

To confirm the presence of PFAS in the water samples, solid phase extraction (SPE) was used to prepare the samples. Chromabond WAX SPE columns (Avantor, UK) were conditioned with methanol and UHQ water. 50 mL of sample was loaded and washed with methanol:UHQ water, 5:95 (v/v). The samples were eluted in 2 mL methanol and concentrated by SpeedVac (Thermo Fisher, UK). Finally, the samples were reconstituted in 1 mL methanol:water, 80:20 (v/v). The SPE extracts were analysed on an LCMS-9050 Q-TOF mass spectrometer (Shimadzu Corporation). Detection of the PFAS seen in the MALDI spectra was achieved by data-independent acquisition (DIA) analysis. The obtained DIA data for the water samples were processed using LabSolutions Insight Explore for compound identification. Using the MS/MS library search, all potential PFAS contaminants flagged by MALDI analysis were confirmed and matched to reference spectra in the PFAS library. Library similarity scores (SI) for each compound by sample are shown in Table 2 below. Representative MS/MS spectra obtained are shown in Fig. 8 alongside library reference spectra.

Conclusions

We have shown MALDI-TOF mass spectrometry applied to the detection of PFAS in aqueous samples in this proof of principle analysis. The samples can be analysed using an entry-level MALDI-8030 linear benchtop MALDI-TOF mass spectrometer. These instruments are robust and compact in size, so could be installed with minimal resources into a wide variety of laboratories. 

Spot imaging at 30 µm spacing during method development highlights the differences in homogeneity of PFOS and PFOA spots suggesting that multiple internal standards would be necessary to pursue quantitative analysis. 

Analysis of bottled and pond water samples were chosen to be representative of a significant proportion of samples in environmental research and monitoring laboratories. We were able to identify peaks corresponding to low levels of PFAS in some of these samples.

4. Thermo Fisher Scientific: Advancing phosphorylation, ADMA, and O-GlcNAc PTM analysis with HCD and EThcD on the Orbitrap Excedion Pro mass spectrometer

• Technical note
• Full PDF for download

There is increasing recognition of the critical role that protein posttranslational modifications (PTMs) play in regulating protein structure, function, stability, and interactions. PTMs are essential for modulating a wide range of cellular processes, including signal transduction, cell growth and differentiation, and responses to environmental stimuli. Phosphorylation, for example, is a central regulator of cellular signaling networks, where the addition of a phosphate group can alter protein function, structure, localization, and stability. Other PTMs, such as asymmetric dimethylarginine (ADMA), can significantly influence protein activity and have been implicated in endothelial dysfunction as well as various cardiovascular and renal diseases. O-linked glycosylation also plays an important role in modulating protein–protein interactions, trafficking, and cellular recognition processes, but its structural diversity and lability make it one of the most analytically challenging PTMs to study. Collectively, such modifications contribute to the complexity of cellular regulation, and aberrant PTM patterns have been linked to numerous diseases, highlighting the importance of accurately identifying and characterizing these modifications in complex biological systems. 

However, the detection and analysis of PTMs present significant analytical challenges. Many PTMs occur at low stoichiometry, are transient in nature, or are distributed across multiple potential modification sites, often within specific subcellular locations. These characteristics demand highly sensitive and precise analytical techniques capable of distinguishing modified from unmodified peptide species and accurately localizing modification sites. 

Mass spectrometry (MS) has emerged as the gold standard for comprehensive proteome and PTM analysis due to its high sensitivity, mass accuracy, and ability to detect a wide dynamic range of peptide abundances. Among the various MS fragmentation techniques, electron transfer dissociation (ETD) has proven particularly powerful for PTM analysis, as it preserves labile modifications and minimizes neutral losses during peptide fragmentation, resulting in confident site localization. Furthermore, the addition of supplemental collisional energy to ETD—known as EThcD—enables secondary fragmentation of ETD product ions, thereby increasing sequence coverage and enhancing localization confidence of the modified residues. 

In this study, we demonstrate the improved detection and characterization of PTM-enriched samples using HCD and EThcD fragmentation on the Orbitrap Excedion Pro mass spectrometer. The results highlight the benefits of employing multiple fragmentation methods to enhance spectral quality, expand PTM site coverage, and enable deeper insights into complex PTM landscapes across phosphorylation, ADMA, and O-glycosylation.

Experimental

Instrumentation 

• Thermo Scientific™ Vanquish™ Neo UHPLC System (Part No. VN-S10-A-01)
• Orbitrap Excedion Pro hybrid mass spectrometer (Part No. BRE725572) 
• Thermo Scientific™ EASY-Spray™ Source (Part No. ES081) 

Data analysis software 

• Thermo Scientific™ Proteome Discoverer™ Software, version 3.2 
• Protein Metrics™ Byonic™ Software, version 5.2.5 
• MSFragger via FragPipe, version 22.0 
• O-Pair via MetaMorpheous, version 1.1.4

Conclusion 

• Expand PTM coverage across multiple modification classes: the Orbitrap Excedion Pro mass spectrometer identified 5,892 unique phosphorylation sites from IMAC enrichment and 2,941 phosphotyrosine sites, showcasing deep coverage of both abundant and low-level PTMs. 
• Confidently localize challenging sites using EThcD fragmentation, which uniquely detected 15% of phosphotyrosine sites not observed with HCD and enabled clear site discrimination where HCD alone produced ambiguous assignments (e.g., ARHGEF5 peptide). 
• Increase sequence and site confidence in ADMA analysis with EThcD identifying more unique ADMA-modified peptides and more PSMs than HCD despite HCD producing a higher total peptide count—resulting in ~300 confidently assigned ADMA sites. 
• Improve O-glycopeptide characterization with HCD-pdEThcD, achieving confident localization of modifications on highly complex peptide, such as 2 localized O-glycosylation sites across a peptide containing 12 potential sites (HCFC1) and accurate site assignments for multi-glycosylated peptides where stepped-HCD mislocalized modifications (PCLO example). 
• Enhance productivity and duty cycle through intelligent triggering—EThcD scans are acquired only for precursors with oxonium-ion signatures, increasing dynamic range and avoiding unnecessary fragmentation overhead. 
• Deliver high-quality spectra across PTM chemistries and charge states with both HCD and EThcD producing complementary identifications—40% of phosphotyrosine sites were unique to HCD and 15% unique to EThcD, demonstrating the power of combined fragmentation strategies.

The Orbitrap Excedion Pro mass spectrometer demonstrates exceptional capability for comprehensive PTM analysis through the integration of flexible fragmentation options. The combination of HCD and EThcD enables complementary peptide fragmentation, resulting in broader sequence coverage and more confident PTM site localization. EThcD fragmentation, in particular, provided enhanced detection and characterization of labile PTMs such as phosphorylation, asymmetric dimethylation, and O-linked glycosylation, improving localization accuracy and sequence information compared to traditional methods. Thousands of unique modification sites were identified across multiple enrichment strategies, illustrating the system’s sensitivity and performance across diverse PTM classes. 

The combination of speed, sensitivity, and versatility supports confident PTM identification in complex biological samples. Overall, the Orbitrap Excedion Pro mass spectrometer empowers researchers to achieve deeper proteome coverage and unlock new biological insights into cellular regulation, signaling, and disease mechanisms through advanced PTM analysis.

5. Waters Corporation: HPLC Analysis of Phosphorylated Peptides on the Alliance™ iS Bio System with MaxPeak™ High Performance Surface (HPS) Technology - A System Designed for Bioseparations

• Application note
• Full PDF for download

Benefits 

• The Alliance iS Bio HPLC System with MaxPeak HPS technology reduces NSA of phosphorylated peptides
• The Alliance iS Bio HPLC System is suitable for HPLC peptide mapping applications, delivering precise gradients with quaternary delivery over extended periods

HPLC analysis of peptides is often performed in regulated laboratories to monitor CQAs as part of process control strategies to ensure drug quality. However, HPLC peptide mapping analysis can be challenging given the method conditions, which typically require small changes in the gradient over an extended period to provide adequate separation. Additionally, one type of peptide, phosphorylated peptides, can exhibit NSA with metal surfaces via Lewis acid-base interactions. Phosphorylated peptides are produced through post transitional modifications. Under common reversed-phase HPLC conditions, the phosphorylated moiety can result in NSA with metal surfaces, including stainless steel. While these unwanted interactions can occur with any biomolecule that contains an electron-rich functional group, the phosphate group has a strong negative charge and as such molecules containing them tend to exhibit strong NSA.² 

NSA can have a significant impact on chromatographic results, including but not limited to peak tailing, poor area precision, and decreased recovery. While there are a variety of mitigation techniques available, they are often time-consuming and may need to be repeated frequently, leading to inconsistent results. 

A more modern approach to address NSA is to provide instrumentation that is not prone to this interaction. Bioinert instrumentation has been developed to specifically address these issues. One example of this is MaxPeak HPS technology, which provides a protective barrier over the metal oxide layer minimizing interactions between the metal surfaces and metal-sensitive compounds such as phosphopeptides. The Alliance iS Bio HPLC System uses this technology, along with bio-compatible materials, in a single system solution, to reduce NSA of sensitive biomolecules, such as phosphopeptides. In addition, the design of the Alliance iS Bio HPLC quaternary pump provides precise gradient delivery under HPLC peptide mapping conditions.

Experimental

• LC system: Alliance iS Bio HPLC System, Bio-inert HPLC System X, Legacy Stainless-Steel HPLC System 
• Column: XSelect™ Premier Peptide CSH™ C18 Column, 130A, 2.5 µm, 4.6 x 100 mm (p/n: 186009908)
• Chromatography software: Empower™ 3.8.0.1 Chromatography Data Software

Conclusion 

HPLC analysis of phosphorylated peptides on the Alliance iS Bio HPLC System demonstrated high level of performance. For this challenging method with both a long shallow gradient and analytes prone to NSA, the Alliance iS Bio HPLC System met all method criteria. In addition, chromatographic analysis demonstrated improved repeatability as compared to legacy stainless-steel system. The Alliance iS Bio HPLC quaternary pump delivered highly repeatable retention times for all peaks, compared to another modern bio-inert solution and a legacy stainless-steel system. Due to key factors such as a robust flow path and MaxPeak HPS technology, the system yields the best performance for biomolecules prone to NSA. Continued improvements of instrumentation plays a key role in advancement of biomolecule analysis, and the mitigation of NSA demonstrated in this study shows one example of how this is possible.