News from LabRulezLCMS Library - Week 20, 2025

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

1. Agilent Technologies: Scale-Up of Protein Separations from Analytical to Semipreparative HPLC

• Technical note
• Full PDF for download

Method development for preparative HPLC is the same as for analytical HPLC – adjusting conditions for optimal chromatographic performance. That is why it is convenient and cost-effective to modify the analytical column method by scaling up the analytical conditions to achieve the preparative separation needed for the collection and isolation of compounds. This application note demonstrates how easy it is to develop a gradient separation of four proteins on a 4.6 × 250 mm, 5 µm, Agilent ZORBAX 300SB-C8 analytical column, then optimize for sample load on a 9.4 mm inner diameter (id) column of identical length and packing material. Agilent ZORBAX 300SB analytical columns can provide: 

• Quick and easy scale up of protein separations from analytical to preparative HPLC 
• Optimization of separations and reduced costs 
• Minimal time spent on method development

Method development 

Using an analytical column to develop a preparative method not only streamlines the process of preparative method development, but minimizes the amount of solvent and sample required for method optimization. For this protein scale-up exercise, a wide-pore packing, ZORBAX 300SB-C8 was selected, which maximized diffusion of large molecules into the pore structure of the packing, thereby maximizing protein peak separation efficiency and sample loading. The ZORBAX 300SB-C8 analytical and semipreparative columns chosen have ids of 4.6 and 9.4 mm, respectively, and have identical column lengths, making gradient scale-up, in particular, straightforward. 

Using a trifluoroacetic acid/acetonitrile (TFA/ACN) gradient, four proteins were resolved on the selected analytical column, as shown in Figure 1 (A). Gradient conditions were optimized to give a good overall separation. Since the goal was to scale up to a preparative column while achieving maximum resolution between the individual proteins, the actual gradient used was slightly shallower (Figure 1 (B)) with a slight increase in analysis time. In general, gradient conditions accommodate larger injection volumes—up to 100 µL, equivalent to a loading of 1 mg/protein. This separation was performed under these finalized conditions without significant loss in resolution (results are shown in reference 1). Preparative scale-up can now be performed.

Conclusion 

This work demonstrates that with scalable columns such as Agilent ZORBAX analytical, semipreparative, and PrepHT columns, carrying out method development work on less expensive analytical columns can save time and money, especially in solvent costs. The process of optimizing the separation on small matched columns, adjusting mobile phase conditions to achieve the necessary separation factor and plate count, is successful because the column surface chemistry is identical, minimizing problems when implementing the methods on larger scale preparative columns.

2. Metrohm: Oxidation stability of spices and seasonings with the PEG method 

Fast and reliable determination without sample preparation due
to polyethylene glycol as carrier material

• Application note
• Full PDF for download

Herbs, spices, spice blends, flavor enhancers, and other seasonings are integral to modern cuisine. A wide variety of plant parts can be used (e.g., leaves, flowers, bark, seeds, roots, fruits, or sap) which contain flavoring and aromatic compounds as well as essential oils. Thanks to their antioxidant content, spices are also used to preserve foods, beverages, and spice mixtures. This is also known as spices’ antioxidant activity. 

The presence of antioxidants may be natural or added artificially. Rosemary, for example, contains high levels of carnosolic acid and has potent antioxidant, antimicrobial, and anti-inflammatory properties. Furthermore, the oxygen radical absorbance capacity of rosemary helps to scavenge free radicals, lending health benefits and possible protection against heart disease. Rosemary powder or extract is therefore used as a natural favorite antioxidant and is of economic importance in the food industry. 

However, processing spices (especially drying and storage) reduces the total antioxidant content over time and can lead to a loss in quality. It is therefore important to monitor and analyze the antioxidant compounds in spices as a quality parameter. The 892 Professional Rancimat is an analytical system to easily and safely determine the oxidation stability of fresh and dried herbs as well as spices and seasonings with the PEG method according to AOCS Cd 12b-92 and ISO 6886.

CONCLUSION 

Thanks to the PEG method, a reproducible and accurate determination of the oxidation stability of spices and seasonings is possible. Since no sample preparation is required, the direct influence of the complete matrix of the sample is seen—not just the individual components. Using the Rancimat with PEG is therefore a well-suited antioxidant measurement method. 

The results show clear differences between different spices according to their amounts of antioxidants. The induction time for black pepper is nearly twice that of white pepper, while rosemary has the highest induction time of the samples tested in this study. 

With the Rancimat, this quality parameter can easily and simultaneously be determined for eight different samples at a time, increasing quality control laboratory throughput. This is possible due to the eight measuring positions in two heating blocks. The built-in display shows the status of the instrument and each individual measuring position. Start buttons for every measuring position enable the measurement start on the instrument. 

The use of practical disposable reaction vessels and dishwasher-safe accessories reduces cleaning to a minimum. This saves time and money and significantly improves accuracy and reproducibility.

3. Shimadzu: A Study of the Spatial Distribution of Gossypol and Other Terpenoids in Cotton Leaves and Ovules

• Application note
• Full PDF for download

User Benefits

• High spatial resolution(5 µm) of the integrated optical microscopy and MS imaging technique that enables observation of the spatial distribution of endogenous metabolites in tiny areas (e.g., in glands)
• Uniform and fine matrix crystal (sub-microscale in size) coating by matrix sublimation that further supports the high spatial resolution
• Highly-sensitive MS imaging that yields high definition and tissue-specific distribution images of gossypol and hemigossypolone in the glands in cotton ovule despite the MS signal intensity differs by a factor of more than 1000 between the two chemicals 

Terpenoids are a diverse group of metabolites. Plants synthesize different terpenoids in response to different environments, which can attract pollinators and seed dispersers, defend against pathogens and herbivores. Terpenoids are synthesized in specific organs of different cell types. Cotton plants possess lysigenous glands in their leaves, stems, and seeds, which accumulate various non-volatile terpenoids such as gossypol and hemigossypolone. But direct visual evidence islacking. 

iMScope QT mainly consists of a high-resolution optical microscope, an atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) ion source and an quadrupletime-of-flight (Q-TOF) analyzer. We can not only use iMScope QT to observe the morphology of the sample in detail, but also to obtain the distribution and content information of compounds in specific parts of the sample. The spatial distribution of gossypol and other terpenoids (hemigossypol and hemigossypolone) in cotton leaves and cotton ovules was studied using iMScope QT. It was found that gossypol, hemigossypol and hemigossypolone were mainly distributed in the glands, and the content of gossypol in cotton ovule was much higher than that in leaves, which was consistent with literature reports. In addition, the study also revealed that the distribution level of hemigossypolone is high in leaf but low in cotton ovule. The above findings provide a reference for the study of the synthesis and transformation mechanism of gossypol and other terpenoids, and also indicate that iMScope QT is a reliable method for both microscopic observation and MS imaging studies of various compounds.

MS imaging of cotton leaf and cotton ovule

Optical images of cotton leaves and ovules were taken under 5X objective lens and mass spectrum acquisition was performed using iMScope QT. The acquired data were analyzed using the data processing software IMAGEREVEAL MS. Five glands from each leaf and ovule were selected as regions of interest (ROI) for analysis. The signal intensity of gossypol, hemigossypol, and hemigossypolone was shown in Table 3. The spatial distribution images are shown in Figure 3 and Figure 4. 

The results showed that gossypol, hemigossypol, and hemigossypolone are mainly distributed in the glands in cotton leaf and ovule. It was found that Gossypol and Hemigossypol were more abundant in the ovule than in the leaf gland, while Hemigossypolone was much more abundant in the leaf than in the ovule gland (Figure 3 and Figure 4, Table 3). The leaf glands are circular in shape, but their sizes vary somewhat. Glands with larger dimensions contain higher levels of the three compounds n MS imaging of cotton leaf and cotton ovule (e.g., ROI-3 > ROI-5), while glands with similar sizes show relatively comparable amounts of these compounds. The ovule gland reveals irregular shapes and varying sizes, with some glandsshowing cavities, possibly due to the sectioning position. Additionally, we can observe each gland in detail, taking ROI-3 as an example (Figure 3 and Figure 4). The magnified MS image of both glands show that the localization of the three components in the ovule gland is relatively uniform, whereas the localization of Gossypol and Hemigossypol (and Hemigossypolone) in the leaf glands is heterogeneous and higher distribution area is limited to the crescent-shaped region on the left. Such findings can only be obtained by measuring at a high spatial resolution,such as 5 µm.

Conclusions

In this paper, the spatial distribution of gossyphenol, hemigossyphenol and hemigossyphenone in cotton leaves and ovule was analyzed using Shimadzu imaging mass spectrometry microscope iMScope QT. The spatial distribution information of compounds with high spatial resolution of 5 µm was obtained, and the distribution of compounds in tissues could be directly observed at the glandular level through the In situ visual method. It provides clues for the synthesis and transformation mechanism of the terpenoids such as gossyfol. This study provides a reference for the spatial distribution analysis of various endogenous metabolites in plants, and provides a new research method and technical tool for the visual study of endogenous metabolites and the exploration of their physiological functions.

4. Thermo Fisher Scientific: Quantifying more with less: Implementing charged aerosol detection to improve drug safety

• Other document (Case study)
• Full PDF for download

The Pharmacy Department of the University of Wuerzburg has a rich history of collaborating with the pharmaceutical industry to help solve analytical problems, to evaluate new technology, and for modernization of legacy methods to ensure drug safety is improved as the technology and techniques evolve.

Professor Dr. Ulrike Holzgrabe has served as an expert on various committees of the German and European Pharmacopoeia for 25 years, all dealing with the development of high-quality analytics of drugs, with a heavy focus on the gold standard high performance liquid chromatography (HPLC). Especially in the last few years, she has become interested in new column resins, such as HILIC and mixed mode columns. For some time now the group has focused on implementing UHPLC-charged aerosol detection (CAD) as a complimentary detector to UV and mass spectrometry to ensure single methods can measure all the components within a drug product without exception.

Why is charged aerosol detection so powerful? 

For complex separations where multiple analytes in a sample are not compatible with UV and MS detectors, for instance when compounds lack a chromophore or cannot ionize, liquid chromatographers can turn to evaporative aerosol detectors.

In addition to commonly used UV detection, Prof. Holzgrabe’s group initially employed evaporative light scattering detector (ELSD) for APIs and impurities that do not contain a chromophore, or do not readily ionise. However, it was found that ELSD was not specifically suitable for drug purity assessment due to low sensitivity and spike peaks occurring on the tail of the main peak. This led into interest in CAD, which has higher sensitivity than methods based on light scattering or refractive indices, while also offering a higher level of precision.1

“One of our fields of focus is the identification of unknown impurities of a drug substance, many of which lack a chromophore,” according to Adrian Leistner. “We use the CAD to complement UV detection within hyphenated detection techniques, or for preliminary experiments before identifying the compounds by mass spectrometry.” 

Both CAD and ELSD can detect non-volatile and many semivolatile compounds, but how the particles are detected differs between the two technologies. CAD measures particle charge while ELSD measures the ability of the particle to scatter light. Many applications are described in the European and U.S. Pharmacopoeias using ELSD to evaluate the composition of plant extract, however the assessment of a low-amount impurity in the vast majority of drugs is challenging by ELSD due to sigmoidal curve response, which means the content of the impurity can be underestimated. This is not observed with the CAD and thus it makes for a more precise measurement. Laura Backer explains, “We want to make use of CAD technology because we must assume that not all degradation products carry a UV-absorbing structural element. Examples include k-strophanthin ampoules and atropine preparations such as eyedrops and injections.” 

In the evaluation of older CAD systems compared to the Thermo Scientific ™ Vanquish ™ CAD, the data obtained at the University of Wuerzburg shows a superior sensitivity throughout the whole mass range with the Vanquish CAD model, giving better performance for low level impurities.

5. Waters Corporation: Efficient Profiling of Lipid Nanoparticle Formulations Using Waters GTxResolve 2000 Å SEC Column, MaxPeak Premier 3 μm 

• Application note
• Full PDF for download

Benefits 

• Superior resolution for large LNP species as afforded by Waters GTxResolve 2000 Å SEC Column particles and their optimal average pore diameter and finely tuned pore size distribution. 
• Enhanced recovery under low ionic strength conditions as facilitated by novel column and packing material surfaces that are designed to be both hydrophilic and non-ionic. 
• Fast, platform-compatible SEC methodology as made possible by 3 µm particle technology, high efficiency packed beds, and an overall column design that supports orthogonal high-sensitivity UV, MALS, dRI, and fluorescence detection for integrated LNP characterization.

Due to their ability to encapsulate and protect fragile nucleic acid cargo, lipid nanoparticles (LNPs) have emerged as a transformative delivery system for mRNA, sgRNA, and siRNA.1 While the success of mRNA vaccines for COVID-19 underscores the critical role of LNPs in modern therapeutics, it remains essential to continue advancing the ways in which they can be characterized so that new breakthroughs in targeted delivery, stability, and potency can be uncovered.2

LNPs present a unique challenge to analytical scientists due to their diverse formulations, large hydrodynamic sizes, and complex stability profiles. With the increasing prominence of LNP-based therapeutics, particularly for mRNA and gene therapy applications, there is a growing need for reliable and readily accessible analytical techniques capable of resolving LNPs across a wide range of particle hydrodynamic radii while maintaining their sample integrity. Dynamic light scattering (DLS) and field-flow fraction (FFF) are commonly employed for LNP size analysis. DLS provides a rapid assessment of particle size, though it can lack the resolution to accurately quantify polydisperse samples like LNPs. Meanwhile, FFF delivers gentle, size-based separation, and highresolution multi-attribute quantification of LNPs ranging from nanometer to micron radius in a single experiment, without the need for a porous stationary phase.

In contrast, SEC remains a preferred analytical platform for the development and release testing of biologics. It is regularly applied to the analysis of protein therapeutics and is becoming increasingly adopted for the analysis of the megadalton sized drug products in the cell and gene therapy industry. SEC is appealing because it is simple, reproducible, and compatible with UV, fluorescence, and multi angle light scattering (MALS) detection. That said, today’s SEC columns are being pushed to the limits of their capabilities when it comes to the analysis of large LNP species, which has necessitated advancements in both column technology, method considerations and mobile phase selection.3 In particular, low ionic mobile phases that mimic formulation diluent conditions are especially relevant for ex vivo quality control, release testing, and formulation stability assessment, etc. While mechanistic or biophysical studies that explore in vivo behavior may employ physiologically buffered conditions to simulate intracellular state, analytical methods intended to assess product quality and shelf-life stability benefit from using gentle, low-salt mobile phases that preserve native particle structure and avoid salt-induced aggregations. If optimal mobile phase conditions are applied, the particle integrity of the LNP can be preserved while maintaining compatibility with downstream analytical technique.4 This application note explores the performance of Waters GTxResolve 2000 Å SEC Columns for LNP analysis under low ionic strength conditions. Herein, the use of 0.1 x DPBS as a suitable mobile phase for size exclusion chromatography-based analysis while maintaining particle integrity was reported, achieving reproducible recoveries, and providing sizing profiles of LNPs. Further, these observations strongly indicate the possibility of using SEC as an orthogonal method to rapidly probe the LNP characteristics such as polydispersity, stability, and size distributions.

Experimental 

LC Conditions

LC System configuration

• ACQUITY™ Premier System with: 
  • Binary Solvent Manager (BSM) 
  • Flow-Through Needle Sample Manager (SM-FTN)

Detector: ACQUITY Premier eLambda PDA with 5 mm Titanium Flow Cell 

Column(s): 

• Waters GTxResolve 2000 Å SEC Column
• MaxPeak Premier 3 µm 4.6 × 150 mm (SKU: 186010735)
• Agilent Bio SEC-5 2000 Å , 5 µm, 4.6 × 150 mm

Results and Discussion

Waters GTxResolve 2000 Å SEC Column, MaxPeak Premier 3 µm Particles and Column Hardware Design 

New column technologies were required to achieve reproducible profiling and characterization of large delivery vehicles such as LNPs. Figure 2 illustrates the key design features of the Waters GTxResolve 2000 Å SEC Column, optimized for the analysis of large biomolecules and nanoparticle-based therapeutics such as LNPs. One of the primary challenges in SEC analysis of these complex samples is the risk of analyte interaction with exposed metal surfaces in conventional stainless-steel hardware. Positively charged metal oxide layers can attract negatively charged sample components—such as certain ionizable lipids, nucleic acid payloads, and/or the net charge of an LNP—leading to non-specific adsorption, peak tailing, and inaccurate measurements. To overcome this, the Waters GTxResolve 2000 Å SEC Column incorporates MaxPeak High-Performance Surfaces (HPS). This proprietary surface treatment applies a hydrophilic, organic-inorganic hybrid barrier across all internal metal surfaces, dramatically reducing undesired analyte-surface interactions. By preventing metalmediated adsorption, MaxPeak HPS enhances recovery, preserves peak shape, and ensures consistent performance, which is particularly critical under low ionic strength conditions like 0.1 × DPBS where electrostatic interactions are more pronounced. The use of 3 µm particles in SEC allows high-resolution LNP analysis in under 30 minutes using compact 4.6 × 150 mm columns. In contrast, columns packed with larger particles—such as 5, 8, 10, 26, or 27 µm—require longer columns and significantly longer run times, often exceeding 90 minutes for just a single injection. 

In addition to advanced hardware, the Waters GTxResolve 2000 Å SEC Column features a novel 3 µm particle with an extra-large 2000 Å average pore size designed to resolve large, polydisperse analytes. The particle is comprised of high-strength silica, and the surface is chemically modified with a polyethylene oxide (PEO) bonding and bridged ethylene hybrid crosslinks, yielding a uniquely inert and hydrophilic packing material. This combination minimizes hydrophobic and ionic interactions while also improving chemical resilience and enabling high-efficiency separations. The MaxPeak HPS column hardware and wide-pore hydrophilic particle design make the Waters GTxResolve 2000 Å SEC Column a powerful tool for achieving highly reproducible, low-interaction separations of nanoparticle therapeutics, as shown in Figures 3 and 4.

Conclusion

As part of a growing toolbox for the analysis of LNP drug substances, the use of Waters GTxResolve 2000 Å SEC Columns is proposed. Dynamic light scattering, electrophoretic light scattering, and field-flow fractionation can be relied upon as tried and true approaches for rapid screening and multi-attribute quantification, respectively. For orthogonal capabilities and screening studies, GTxResolve SEC Columns can now be considered too. These columns are uniquely engineered to meet the growing demands of LNP characterization, offering superior peak profiles and reproducibility for multiple types of formulations compared to other commercially available options. Its wide pore packing material allows for efficient separation of intact LNPs, while the combination of an inert polyethylene oxide surface chemistry and MaxPeak HPS hardware ensures minimal analyte loss and secondary interactions, even under low ionic strength conditions like 0.1 × DPBS, which has proven to be a viable mobile phase for repeatable LNP studies. Progress on SEC methods for LNPs is promising. While this preliminary study provides an intriguing perspective on things to come, it is future investigations that are bound to show a rich interplay between various analytical approaches and how to apply them to guide development teams toward more potent, more stable lipid nanoparticles. 

As biopharmaceutical pipelines continue to evolve toward larger and more sophisticated delivery platforms, the Waters GTxResolve 2000 Å SEC Column, MaxPeak Premier 3 µm offers a fit-for-purpose SEC solution that balances analytical rigor with practical usability, making it a preferred tool for modern large-molecule separations.