News from LabRulezLCMS Library - Week 08, 2026

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 16th February 2026? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!

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This week we bring you application notes by Agilent Technologies, KNAUER, Shimadzu and Waters Corporation and presentation by MDCW / Clemson University!

1. Agilent Technologies: Development and Optimization for a Comprehensive LC/MS/MS Method for the Detection of 74 PFAS Compounds

• Application note
• Full PDF for download

PFAS represent a large and chemically diverse class of synthetic compounds widely used in industrial processes and consumer products due to their exceptional surfactant properties and thermal stability. These compounds are defined by strong carbon-fluorine bonds, which confer resistance to environmental and biological degradation. Consequently, PFAS are persistent, bioaccumulative, and have been detected globally in water, soil, air, and food.^1-3 

Mounting scientific evidence has linked PFAS exposure to a range of adverse health outcomes, including thyroid problems, immune suppression, and increased cancer risk.^4,5 Among various exposure pathways, dietary intake is considered a primary route for the general population, particularly through consumption of animal-derived foods such as meat, eggs, and dairy products^{1-3, 6-8}, which may accumulate PFAS from contaminated environments. 

To support accurate and sensitive detection of PFAS in complex matrices, regulatory agencies have developed targeted analytical methods. The U.S. Environmental Protection Agency (EPA) Method 1633 includes 40 PFAS compounds⁹ , the U.S. Department of Agriculture Food Safety and Inspection Service (USDA FSIS)¹⁰ has published a method for quantifying 16 PFAS in meat and bovine plasma, and the U.S. Food and Drug Administration (FDA) has published a method for quantifying 30 PFAS in food samples¹¹ using liquid chromatography-tandem mass spectrometry (LC/MS/MS). 

This study presents an optimized LC/MS/MS method capable of detecting 74 PFAS compounds across diverse chemical classes amenable to this technique. Compound transitions and source conditions were developed using the Agilent MassHunter Acquisition Optimizer program. Method development addressed key analytical challenges. These include the instability and cross-contamination risks associated with combining targets into mixed standards, chromatographic optimization to achieve baseline separation of bile acids from PFOS, and the implementation of an injection programming strategy to enhance peak shape and minimize matrix-related background. Although the method was specifically tailored for extracts of animal origin¹², it is broadly applicable to other sample types. IDLs achieved using the 6495D triple quadrupole mass spectrometer are reported, demonstrating the method's sensitivity and robustness.

Experimental

LC/MS/MS conditions 

This study was performed using an Agilent 1290 Infinity III LC system consisting of a 1290 Infinity III high-speed pump (G7120A), a 1290 Infinity III Multisampler (G7167B), and an Agilent 1290 Infinity III Multicolumn Thermostat (G7116A). The LC system was modified using an Agilent InfinityLab PFC‑free HPLC conversion kit (part number 5004‑0006). Injection program and chromatographic separation parameters are detailed in Table 2. 

The LC system was coupled to an Agilent 6495D LC/TQ equipped with an Agilent Jet Stream source. All multiple reaction monitoring transitions are provided in Table 1. Compound-specific parameters for the 6495D MRMs were determined using MassHunter Optimizer. The source conditions for the 6495D are shown in Table 3. To address the wide range of PFAS compounds included in the method, source parameters such as temperature and flow rates were fine‑tuned using MassHunter Source Optimizer. Data acquisition and analysis were carried out with Agilent MassHunter Workstation software.

Conclusion 

This study presents a robust and comprehensive LC/MS/MS method for the detection of 74 PFAS compounds across diverse chemical classes. Through careful optimization of mixed standard preparation, chromatographic separation— particularly for bile acids—and injection programming, the method achieves high sensitivity and reproducibility, with most compounds exhibiting instrument detection limits below 10 pg/mL. Although developed for animal-derived matrices, the method demonstrates broad applicability to other sample types. These findings underscore the importance of addressing compound-specific challenges in PFAS analysis and provide a reliable framework for future monitoring efforts in food safety and environmental research.

2. KNAUER: Fast protein analysis of mucins using an AZURA® SEC System

• Application note
• Full PDF for download

Mucins are glycoproteins, which are characterised by a combination of proteins and carbohydrates¹. They play an important role in biological processes, particularly in the protection of mucous membranes against mechanical, chemical and biological influences². Due to their high content of sugar molecules, mucins have a strong hydrophilic tendency, which helps them to bind water and form a gel-like consistency^3,4,5. They are part of a mixture of water, salts, lipids, cell material and other proteins that form the mucus that covers the surfaces of the mucous membranes of organisms such as the mouth and nose^1,2. Thereby, the mucins are crucial for the physical properties of the mucus, especially its viscosity¹.

An important analytical tool for the characterisation of mucins is SEC¹. This method allows the determination of the molecular weight and molecular weight distribution of mucins, contributing to a better understanding of their biological functions¹. In addition, SEC allows efficient isolation of mucins from biofluids, resulting in purified mucin fractions that can be used for further analysis or medical applications, such as biomarkers in cancer diagnosis and prognosis^6,7. Overall, SEC is a gentle chromatographic technique performed under mild conditions such as room temperature and neutral pH⁸. This ensures that the native conformation and function of the mucins are maintained throughout the separation process, which is crucial for preserving their biological activity and interactions⁸.

CONCLUSION 

The KNAUER AZURA® SEC System in combination with the AppliChrom® VivoSep SEC column offers significant time savings for the analysis of mucins. This solution provides good separation performance and is an innovative addition to the field of protein size exclusion chromatography.

3. MDCW / Clemson University: Two-Dimensional Liquid Chromatography Isolation and Quantitation of IgG and Exosomes from Cell Culture Media

• Presentation
• Full PDF for download

The presentation describes the development of a 2D-LC platform for simultaneous recovery of monoclonal antibodies (IgG) and exosomes from CHO cell culture supernatant. In standard biomanufacturing, IgG is purified using protein A (ProA) affinity chromatography, while the ProA effluent—rich in exosomes—is typically discarded. The authors propose valorizing this waste stream by implementing a second chromatographic dimension to isolate exosomes downstream of IgG purification.

The first dimension employs Protein A affinity chromatography using polypropylene capillary-channeled polymer (C-CP) fibers functionalized with recombinant protein A for selective IgG capture via pH-controlled binding and elution. The second dimension uses Hydrophobic Interaction Chromatography (HIC) on polyester C-CP fibers to isolate exosomes from the ProA flowthrough. A modulation strategy with dual pumps and UV detection enables transfer between dimensions, fraction collection, and quantitation of both IgG and exosomes.

The system demonstrated excellent IgG performance, with linear response (R² = 0.99), quantitative recoveries (RSD <5%), and purity comparable to commercial Agilent columns. SDS-PAGE confirmed product integrity. Exosomes were characterized using nano-flow cytometry (nanoFCM), showing a mean diameter of 83.2 ± 22.6 nm and strong membrane labeling (MemGlow™) and tetraspanin expression (CD9, CD63, CD81). The method achieved >99% reduction of host cell proteins and exosome purity of 5.86 × 10¹⁰ particles per µg protein, meeting NIH guidelines.

Overall, the study demonstrates that 2D-LC combining ProA affinity chromatography and HIC on C-CP fiber columns enables efficient IgG purification and high-purity exosome recovery from the same culture stream. The approach offers low-cost implementation, no IgG carryover or ProA leaching, and potential expansion toward advanced bioprocess monitoring and novel fiber-based separation modalities.

4. Shimadzu: Method Development for Separating Charge Variants of Antibody-Drug Conjugates by Ion- Exchange Chromatography

• Application note
• Full PDF for download

User Benefits

• Shim-pack Bio IEX ion-exchange chromatography column enables analysis of charge variants of antibody-drug conjugates (ADCs). 
• LabSolutions MD can automate the entire workflow for method development, including the generation of an analysis schedule, mobile phase preparation.

Monoclonal antibodies and antibody-drug conjugates (ADCs), like other antibody pharmaceuticals, are produced using animal cells, which results in structural heterogeneity and impurities. Charge variants, which are impurities generated from the heterogeneity of C-terminal lysine, deamidation, oxidation, and other causes, can impact the stability and efficacy of antibody pharmaceuticals. Therefore, it is important to appropriately separate, detect, and monitor charge variant peaks for quality control purposes. 

This article describes using LabSolutions MD, which is dedicated software for supporting method development, to efficiently optimize peak separation between ADC charge variants when using pH gradient ion-exchange chromatography.

In this study, trastuzumab deruxtecan (T-DXd) diluted to a concentration of 5 mg/mL in ultrapure water was used for optimizing separation of charge variant peaks. For screening (analytical conditions in Table 1), the parameters that have a large effect on separation, such as the concentration of the MES, HEPES, and sodium acetate in the mobile phase and the ratio of acetonitrile or methanol in the mobile phase, were considered . Mobile phases adjusted to pH 5.0 and pH 10.0 were prepared by mixing an equimolar aqueous solution of HEPES, MES, and sodium acetate with acetic acid or sodium hydroxide. To determine the condition settings that result in optimal separation, a schedule was prepared with ten MES-HEPESsodium acetate concentration levels (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mmol/L) and six different acetonitrile or methanol ratio levels(5, 10, and 15 % in mobile phase).

LabSolutions MD can quickly and easily generate analysis schedules for specified parameter settings, such as for several types of mobile phases and column oven temperatures (steps (1) to (5) in Fig. 1). In addition, mobile phase blending functionality can automatically prepare mobile phases with different concentrations of the MES-HEPES-sodium acetate, as well as different ratios of acetonitrile or methanol, by simply clicking the mobile phases to use for automated screening (step (1) in Fig. 1). That significantly reduces the amount of work and human errorsinvolved in manual preparation.

Conclusion 

The separation patterns of ADC charge variants differ depending on the concentration of reagents in the mobile phase and the ratio of acetonitrile or methanol in the mobile phase. LabSolutions MD can automate the method development workflow, including the generation of analysis schedules, mobile phase preparation, and data processing thanks to specific functionalities, such as for ranking chromatograms by criteria values.

5. Waters Corporation: HILIC-MS/MS Analysis of Free Inositol Stereoisomers in Foods

• Application note
• Full PDF for download

Benefits 

• A highly specific and reliable HILIC-MS/MS method for the separation and quantification of free inositol stereoisomers in dairy and plant-based milks, grains, and infant formulas
• The ACQUITY BEH™ Amide Column offered efficient separation of inositol stereoisomers and demonstrated ruggedness across different column batches
• The Xevo TQ-S cronos Mass Spectrometer delivered accurate and reliable quantification of free inositol stereoisomers in complex food matrices

Inositols are cyclic carbohydrates with a six-carbon ring structure and six hydroxyl groups (cyclohexane1,2,3,4,5,6-hexol). There are nine stereoisomers: myo-, D-chiro-, L-chiro-, scyllo-, neo-, muco-, allo-, epi-, and cisinositol (Figure 1), most of which occur naturally.^1-5 While the human body can synthesize inositol from glucose, it is an essential nutrient for babies and must be supplied through breast milk, inositol-enriched formula, or milk. For individuals with inositol deficiency caused by certain medical conditions, adequate dietary intake is critical to maintain normal biological functions and help prevent related health issues.⁶ Moreover, evidence suggests that an imbalanced profile of inositol stereoisomers in the body could contribute to certain biological dysfunctions and increase the risk of certain diseases.⁷ An increasing number of food products are now enriched with inositol. Developing an analytical method capable of analyzing inositol stereoisomers in foods is therefore of great interest to many fields, including food manufacturing, nutritional research, and clinical studies. 

Previously, a HILIC-MS method was developed for analyzing inositol stereoisomers in dietary supplements.⁸ In this study, the focus was on improving the original method to make it suitable for more complex food matrices, such as dairy and plant-based milks, grains, and infant formulas. Details of the method development and validation are presented. This study focused exclusively on free inositol isomers; bound inositols were outside the scope of the work.

Experimental

LC-MS/MS system: Arc™ Premier System equipped with a Quaternary Solvent Manager (QSM-R), a Sample Manager (FTN-R), a Column Manager, and coupled with a Xevo TQ-S cronos triple quadrupole Mass Spectrometer

Software: MassLynx™ 4.2 MS Software 

Column: ACQUITY UPLC™ BEH Amide Column (1.7 μm, 2.1 mm x 150 mm, p/n: 186004802, Waters), or ACQUITY Premier BEH Amide VanGuard™ FIT Column (1.7 μm, 2.1 mm x 150 mm, p/n: 186009509, Waters).

Results and Discussion 

Method Improvements for Complex Food Matrices 

Previously, HILIC-MS method was developed for the determination of free inositol stereoisomers in dietary supplements. Sample preparation involved a simple dilute-and-filter process, and detection was performed using selected ion recording (SIR) on an ACQUITY QDa II Mass Detector. When this original HILIC-MS method was applied to more complex food matrices, such as milk, grains, and infant formulas, the initial results were unsatisfactory due to improbably high recovery levels (results not shown). To improve analytical accuracy for these food matrices, a protein precipitation step was incorporated using Carrez reagents into the sample preparation, and a more selective MS detection technique, MRM, using a Xevo TQ-S cronos Mass Spectrometer was adopted. These two improvements resulted in excellent recovery, which will be discussed in detail in the following section.

Conclusion 

We have developed a HILIC-MS/MS method for the separation and quantification of free inositol stereoisomers in dairy and plant-based milks, grains, and infant formulas, employing an ACQUITY BEH Amide Column on an Arc Premier System coupled with a Xevo TQ-S cronos Mass Spectrometer.

• Incorporating protein precipitation into the sample preparation significantly improved matrix cleanup. 
• The ACQUITY BEH Amide Column delivered efficient separation of inositol stereoisomers and effectively resolved them from potential interfering monosaccharides. 
• The Xevo TQ-S cronos Mass Spectrometer further enhanced quality of analysis through highly selective MS/MS detection. 

This HILIC-MS/MS method was validated using both solvent standards and food samples, demonstrating excellent analytical performance in terms of linearity, sensitivity, specificity, accuracy, precision, and ruggedness. The method was successfully applied to commercially available food products and its suitability for routine quantitation of free inositol stereoisomers across a variety of food matrices has been demonstrated.