News from LabRulezLCMS Library - Week 36, 2025

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 1st September 2025? 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 note by Agilent Technologies, posters by Shimadzu and Waters Corporation and other document by Thermo Fisher Scientific!

1. Agilent Technologies: PFAS Analysis in Human Plasma Using the Agilent Ultivo Triple Quadrupole LC/MS 

• Application note
• Full PDF for download

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a group of synthetic compounds that have been widely used in various industrial and consumer applications, including cosmetics and personal care products. Due to their widespread use, persistence in the environment, and known bioaccumulation with adverse effects, PFAS are a significant concern for human health. For example, PFAS have been linked to several adverse health effects, including cardiometabolic diseases,¹ reproductive effects,² and increased risk of certain cancers.³ PFAS exposure has been shown to lead to alterations in lipid and energy metabolism, contributing to metabolic dysfunction.^4,5 It is also known that PFAS accumulate in liver and other tissues, contributing to age‑related morbidities.⁶ To increase the understanding the molecular basis of these effects, analysis of PFAS in blood plasma has been combined with lipidomics to identify the lipid biomarkers associated with metabolic risks.⁷ Further, the PFAS concentrations in serum have also been shown to differ by gender^7,8 and ethnicity.⁹ Considering these and other findings, monitoring PFAS levels in plasma is important to understand their health impacts and develop strategies for their mitigation. This application note validates an LC/MS/MS method for targeted quantification of fourteen PFAS in plasma using the Agilent Ultivo Triple Quadrupole (TQ) LC/MS. For the method presented, the Ultivo LC/TQ offers performances on par with mid- to high-end TQ systems in a much smaller footprint. The protein-precipitation-based sample preparation method is fast and effective for plasma samples. Method performance in terms of sensitivity, linearity, carryover, accuracy, and precision are described. Stability of targeted PFAS in quality control (QC) samples and analysis of real human plasma samples are also discussed.

Experimental

Instrumentation and LC/MS/MS method 

Chromatographic conditions were optimized for analyte separation and retention time for each of the PFAS as determined by analyzing the labeled and non-labeled standards. PFAS were separated by reversed-phase LC on an Agilent InfinityLab Poroshell 120 EC‑C18, 2.1 × 50 mm, 2.7 μm (part number 699775-902) and detected in negative electrospray ionization (ESI) mode using an Agilent 1290 Infinity II LC system coupled with the Ultivo LC/TQ. The liquid chromatograph was equipped with a binary pump (part number G7120A), column compartment (part number G7116B), and Agilent 1290 Infinity II Multisampler (part number G7167B). For each experiment, 5 μL of extract was injected. The LC parameters are listed in Table 2. The Ultivo LC/TQ was operated in dynamic multiple reaction monitoring (dMRM) mode. The dMRM parameters and retention times were optimized by analyzing each unlabeled PFAS standard. The MS parameters are listed in Table 3. The Agilent MassHunter Acquisition software dMRM parameters per PFAS are in Table 4. Agilent MassHunter Quantitative Analysis software was used for data processing.

Conclusion 

The quantitative method for the analysis of 14 PFAS in human plasma samples that is presented here is simple and robust, uses relatively small plasma volumes, and relies on fast and effective protein precipitation, facilitating its large-scale application. The method's sensitivity, linearity, carryover, recovery, and precision make it scalable to population-based studies and biomonitoring. The compact, stackable Ultivo LC/TQ and MassHunter software, in combination with columns and consumables from Agilent Technologies, offer a fit-for-purpose, end-to-end solution to enable widespread adoption in both routine monitoring and research. The method has already been used in combination with plasma lipidomics to characterize the relationship of circulating PFAS and lipidomic profiles.7 These types of studies promise to reveal the molecular basis of the adverse effect of PFAS exposure on human health outcomes.

2. Shimadzu / ASMS: One System, Multiple Solutions: Analysis of PFAS & Cyanotoxins in Water Adhering to EPA 537.1, 544, and 545 

• Poster
• Full PDF for download
• PFAS are persistent synthetic chemicals found in consumer and industrial products, while cyanotoxins are harmful substances produced by cyanobacteria in water bodies. 
• EPA Methods 537.1 and 533 are approved by EPA for PFAS analysis in drinking water; Methods 544 and 545 target cyanotoxins like microcystins, nodularin, cylindrospermopsin, and anatoxin-a. 
• A single Shimadzu LCMS-8060RX system with automatic method switching enables efficient, accurate analysis of PFAS and cyanotoxins, reducing equipment needs and turnaround time.

2. Methods

Figure 1 illustrates the LC-MS system’s method-switching capability, enabling seamless transitions between EPA Methods 537.1, 544, and 545 on a single platform. For EPA Method 537.1 (PFAS analysis), the flow path includes the delay column to remove background PFAS. In contrast, for EPA Methods 544 and 545 (cyanotoxin analysis), the delay column is bypassed to prevent unnecessary contaminations, ensuring accurate results. This automation enhances workflow efficiency and analytical flexibility. 

To prevent mobile phase contamination when switching between PFAS and cyanotoxin analysis, a simple five-minute rinse (the flow path indicated by the red lines in Figure 2) with the appropriate mobile phase is shown to be sufficient.

4. Conclusion

This study shows that PFAS and cyanotoxins can be accurately measured using one triple quadrupole mass spectrometer with automatic method switching. A quick five-minute rinse ensures clean transitions between methods, minimizing downtime and manual work. This streamlined setup reduces equipment costs, boosts efficiency, and supports high-throughput environmental testing, including rapid response to events like harmful algal blooms. 

3. Thermo Fisher Scientific: Nestlé develops an Orbitrap Exploris 480 mass spectrometer method for wide-scope analysis of pesticides and natural toxins in foods

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

Nestlé is committed to ensuring the highest standards of food quality and safety through rigorous research and innovation. Located in Lausanne, Switzerland, Nestlé Research plays a pivotal role in developing analytical methods for monitoring chemical contaminants in food products. These robust methodologies are implemented across Nestlé's global laboratory network to screen, identify, and ensure compliance with food safety standards. In response to the challenges posed by climate change, evolving regulations, and shifting consumer preferences, Nestlé Research has developed a QuEChERS-based liquid chromatography high-resolution mass spectrometry (LC-HRMS) workflow. This innovative approach allows for the simultaneous analysis of over 1,100 contaminants, including pesticide residues, mycotoxins, and plant toxins, across various food matrices such as cereals, fruits, and vegetables. The method's sensitivity and low quantification limits ensure compliance with regulatory standards while facilitating the exploration of additional compounds to preemptively address future food safety risks.

Technology selection 

Next, Nestlé Research scientists aimed to identify the most suitable technology to address the challenges associated with broad-scope contaminant analysis. The versatility and high selectivity of LC-HRMS for screening a broad range of compounds offered a solution. Theoretically, in full-scan mode, HRMS instruments can monitor an unlimited number of molecules simultaneously, opening up avenues for continuous extension of the method’s scope. The high-quality, high-resolution accuratemass (HRAM) data provided by the Thermo Scientific™ Orbitrap™ mass analyzer can help differentiate between compounds with similar masses and isobaric interferences from coextracted matrix components, reducing the chance of false positives. Highresolution mass spectrometry (HRMS) systems offer the capability to perform nontargeted or retrospective analyses by leveraging existing data and potentially identify novel contaminants. 

Due to its sensitivity and HRAM data quality, the Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer was evaluated and ultimately selected for workflow development. The developed method uses the full-scan MS/data-dependent MS2 (FS-ddMS2 ) acquisition mode, which combines a full scan with MS2 scans on the targets of interest measured in the full scan. 

Necessary compromises 

Though HRMS technology theoretically offers an unlimited scope of analysis, the true scope of analysis remains limited by the sample preparation method, chromatographic conditions, and MS ionization efficiency. According to Bessaire, “We needed to compromise on sample preparation and extraction, the LC method—the column and the gradient—and MS ionization mode because not all organic molecules can be analyzed using a single ionization mode. The scope of compounds that we dreamt about at the beginning was not the same as what we had at the end.” 

With these compromises in mind, a QuEChERS-based sample preparation followed by dispersive solid-phase extraction (dSPE) cleanup was chosen due to its effectiveness for a wide range of sample types and analyte chemistries. LC method development was carried out on the Thermo Scientific™ Vanquish™ Horizon UPHLC system. A 22 minute LC run using acidic mobile phases on a 15 cm reversed phase (RP) C18 column with a smooth gradient slope was chosen to retain a broad range of chemicals with various polarities, while separating a maximum number of isomers and isobaric compounds within an acceptable run time. The generic sample preparation approach has advantages as Dr. Flavia Nagy (-Badoud), Scientist, Nestlé Research noted, “With the generic multiresidue method, we can reduce the use of solvents and work towards greener chemistry.”

In-house HRAM mass spectral library maximizes compound identification 

To maximize the method’s capability to unequivocally confirm the presence or absence of numerous compounds from a single injection, the group developed an in-house HRAM mass spectral library populated in Thermo Scientific™ mzVault™ software. Creating the library was time-consuming and involved injection and analysis of individual standard solutions for each compound. Dr. Nicolas Christinat, Scientist, Chemical Contaminants, Nestlé Research described the library creation process, “We relied on analytical standards for each molecule. That’s a lot of individual injections and we acquired multiple parameters for each, including different fragments at different collision energies, at different polarities, and for different adducts. All this information was stored in our mzVault spectral library, and we can use that information to build targeted methods that use the most relevant collision energy, most abundant ions, and the best polarity. Bessaire added, “Our internal mass spectral library was key to maximizing the identification capabilities of our method.”

Orbitrap Exploris 480 mass spectrometer essential capabilities 

The Orbitrap Exploris 480 mass spectrometer platform provides capabilities essential to developing methods that provide reliable identification and quantification. 

Sensitivity 

First, the platform offers good sensitivity compared to other HRMS instruments. Aurélien Desmarchelier, Scientist, Nestlé Research explained, “We need state-of-the-art instruments when we develop new methods. We evaluated the Orbitrap Exploris 240 and Orbitrap Exploris 480 mass spectrometers. The Orbitrap Exploris 480 system’s data pointed to a bit more sensitivity and that’s why we selected and are recommending it to the laboratories that will be implementing the method, so we are aligned.” Dr. Nagy (-Badoud) added, “Contaminant analyses are always at low levels—parts per billion normally—so it is very important that we have a sensitive instrument. That’s mostly why in the past HRMS was not considered and contaminant analysis was done by triple quadrupole instruments with targeted approaches. One of the most important criteria was that we reached the required sensitivity.”

4. Waters Corporation / ASMS: Analysis of Amino Acids in Plant-Based Proteins and Pet Foods – Modification of AOAC 2018.06 to Fit for Novel Foods and Ingredients

• Poster
• Full PDF for download

The AOAC International Official Method 2018.06 specifies an analytical procedure for the analysis of amino acids (AA) in infant formula, adult nutrition and dairy products. This procedure is based on the pre-column derivatization with 6-aminoquinoline succinic carbamate (AQC) and reversed-phase ultra-performance liquid chromatography with UV detection.

When applied to pet foods, a limitation was observed in the target AA list. Specifically, hydroxyproline (HyPro), common in pet foods, exhibited poor retention on the column, making it unmeasurable. HyPro is a marker for potential food adulteration using hydrolyzed animal products to artificially boost protein levels in plant-based proteins. It is also an indicator for animal-derived ingredients such as gelatin and collagen, which are critical in halal and kosher food testing.

Therefore, it is necessary to develop an analytical method capable of effectively separating and detecting HyPro alongside other amino acids.

Experimental

LC Conditions

• System: ACQUITY Premier System (BSM) with PDA detector
• Detection: UV (260 nm)
• Tubing: 0.004 mm ID, 10.5 in (Waters p/n 430001784) between column and detector
• Software: Empower CDS
• Columns:
  • ACQUITY UPLC BEH C18, 1.7 μm, 2.1 mm × 150 mm (Waters p/n 186002353)
  • AccQ∙Tag Ultra C18, 1.7 μm, 2.1 mm × 150 mm (Waters p/n 186009954)

Conclusion

• Excellent separation resolution for all amino acids with high repeatability and intermediate reproducibility in RT
• Amino acids in six pet foods and four common plant-based proteins successfully analyzed
• Modified AOAC Method provides a reliable and practical solution for AA analysis in pet foods and plant-based proteins