News from LabRulezLCMS Library - Week 33, 2025

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 11th August 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 posters by Agilent Technologies / ASMS and Thermo Fisher Scientific / ASMS and application notes by Shimadzu and Waters Corporation!

1. Agilent Technologies / ASMS: Fully Automated Sensitive Quantitation of PFAS in Seafood for Regulatory Screening Using Triple Quadrupole LC/MS 

• Poster
• Full PDF for download

Per- and polyfluoroalkyl substances (PFAS) are extensively found in marine life, posing health risks through seafood consumption. Seafood is one of the regulated matrices by the U.S. FDA, EU, EURL POPs, and AOAC. Regulatory and standard-making bodies like the U.S. FDA, EFSA, EURL POPs, and AOAC have set stringent guidelines for PFAS levels in seafood. Detecting trace PFAS level is challenging due to complex sample preparation like QuEChERS extraction followed by SPE cleanup, and evaporation/reconstitution. The manual steps can be labor-intensive and error-prone, affecting accuracy and reliability. This study developed a fully automated workflow for PFAS quantitation in seafood – shrimp.

Experimental

Instrumentation 

An integrated 160 cm PAL3 Series 2 RTC autosampler coupled with a 6495D LC/TQ (Figure 1) was employed. The PAL3 platform performed automated sample preparation, while TQ data acquisition and analysis were conducted in parallel. LC system comprised of two modules; an Agilent 1290 Infinity II high-speed pump and an Agilent 1290 Infinity II multicolumn thermostat. The study utilized the following tools and modules: two PAL park stations with three liquid syringe tools, a dilutor tool, a micro-SPE tool, an LC/MS tool, a vortex mixer, a centrifuge, a dilutor multi, a tray cooler (for 2/10/20 mL vials), tray holders with rack R60 (for 10/20 mL vials), a micro-SPE tray (for 2 mL vials and micro-SPE cartridges), a solvent module, a fast wash module, and an LC injection valve. The details of various consumables used for this study are provided elsewhere.1

Conclusions

• The integrated system allows sample preparation and data analysis to run in parallel, offering a streamlined workflow and improving productivity for routine laboratory operations. 
• The automated workflow significantly reduces manual intervention, minimizing human error and enhancing the precision of the analysis. 
• The integration of automated sample preparation techniques with the highly sensitive 6495D LC/TQ ensures consistent and reproducible results, which are critical for regulatory compliance. 
• The workflow demonstrated excellent analytical performance meeting the stringent regulatory requirements and recommendations for PFAS in seafood matrices set by the U.S. FDA, EU, EURL POPs, and AOAC.

2. Shimadzu: Determination of PFASs in Food Contact Material by LCMS-8050

• Application note
• Full PDF for download

Due to the unique physical and chemical properties of per- and polyfluoroalkyl substances (PFASs), such as reducing surface tension, good stability, hydrophobicity, and hydrophilic, so it is very widely used in the field of food contact materials. It is mainly used for the production of plastic film, PVC food packaging, waterproof, and oil-proof coating on the surface of food packaging (such as waterproof oil paper products) and non-stick cooker coating, etc. Research has shown that PFASs will migrate from food contact materials to food, and then contaminate the food that comes into contact with it, entering the human body. PFASs are very stable, difficult to be metabolized and biodegraded in the body, and exhibit the characteristics of persistence, accumulation, and long-distance migration. It can be accumulated and amplified in the food chain and enriched in the organisms, endangering human health. 

Refer to the sample preparation method in GB 31604.35-2016, determination of both Perfluorooctane Sulfonates (PFOS) and Perfluorooctanoic Acid (PFOA), an analytical method for the determination of 17 perfluorinated compounds in food contact materials was built.

Analysis Conditions

System: Nexera XS 

• Column : Shim-pack GIST C18-AQ HP (2.1 mm I.D.×100 mm, 1.9 μm)
• Temperature : 30 ˚C 
• Injection volume : 5 µL 
• Mobile phases : A-5 mM ammonium acetate in Water B-ACN 
• Flow rate : 0.3 mL/min 
• Elution mode : Gradient elution 
• Time program (%B) : 15% (0 min) → 15% (2.5 min) →98% (10-12 min) → 15% (12.1-15 min) 

System: LCMS-8050 (ESI Negative) 

• Nebulizing gas : 3 L/min 
• Drying gas : 10 L/min 
• Heating gas : 10 L/min 
• DL temp. : 250 ˚C 
• Heat block temp. : 400 ˚C 
• Interface temp. : 300 ˚C

Conclusion 

Utilizing the LCMS-8050 system for quantitative analysis of PFASs in Food Contact Material revealed that the method can accurately determine PFASs levels within a broad concentration range of 0.05 to 5 ng/mL. This approach exhibits several notable advantages, including excellent repeatability, stability, and reliability.

3. Thermo Fisher Scientific / ASMS: Leveraging advanced mass spectrometry technology with Orbitrap Astral Zoom MS for in depth immunopeptidome profiling 

• Poster
• Full PDF for download

Immunopeptidomics is the study of the peptides presented by MHC molecules on the surface of cells. These MHC peptides have major implications for many areas of research, including immunotherapy and personalized medicine. Mass spectrometry allows for direct immunopeptidomics analysis, enabling simultaneous identification and quantification of thousands of MHC peptides in a single run. The new Orbitrap Astral Zoom MS has enabled new levels of sensitivity and selectivity to provide deeper insights into the immunopeptidome.

Materials and methods

LC-MS/MS method 

Peptides were separated on a Vanquish Neo UHPLC System using Aurora Ultimate column (25 cmx 75µm). Total run time was 72 min. Thermo Scientific EASY-Spray Ion Source was used coupled to the Thermo Scientific FAIMS Pro Duo interface. Peptides were analyzed by the Orbitrap Astral Zoom MS.

Data Processing 

The data analysis was performed using PEAKS Studio 12 with DeepNovo Peptidome workflow for database search and de novo peptides identification. Spectra were searched against UniProt human database (20,607 sequences) with no-enzyme option. The sequence motif and binding properties of 9-mer peptides were analyzed using MHCMotifDecon 1.2 and NetMHCpan 4.0.

Conclusions 

• Orbitrap Astral Zoom MS equipped with FAIMS Pro Duo interface and coupled to Vanquish Neo UHPLC system provides high sensitivity and the dynamic range necessary for deeper coverage of immunopeptidomic samples
• The low input application mode on Orbitrap Astral Zoom MS offers the sensitivity necessary to analyze low levels of material equivalent to samples ectracted from tissue biopsy samples
• Loading capacity is not limited by MS allowing analyses from ultra low (1e5) to high (1e8) loads of sample 
• FAIMS enables the analysis of singly and multiply charged ions enhancing the immunopeptidome coverage 
• AGC modulates injection times for each peptide, producing high quality spectra across a wide range of loading levels. This spectral quality is crucial for downstream analysis and confident identification and quantitation of neoantigens

4. Waters Corporation: Optimization of Detector Parameters to Improve Sensitivity using the Alliance™ iS HPLC System with PDA Detector

• Application note
• Full PDF for download

Benefits 

• The Alliance iS HPLC System with PDA Detector allows the user to easily modify PDA settings to obtain optimal performance, including signal-to-noise
• The Alliance iS HPLC System with PDA Detector has a range of parameters, including variable slit width and wide resolution range for method optimization
• Optimization of the PDA settings produced a S/N ratio increase of 7x

Default PDA instrument method settings are frequently used for running HPLC methods. When running a method for the first time, these default settings are a good starting point and often produce suitable chromatography. However, detector parameters may need to be modified to obtain optimal separations. In this study, the USP method for organic impurities in ibuprofen tablets was used to show how the Alliance iS HPLC System with PDA Detector parameters may be optimized to improve sensitivity. The USP S/N ratio was used to define sensitivity. The method is a reversed-phase isocratic method with UV detection (254 nm). The method’s system suitability criteria includes a S/N ratio of not less than (NLT) 10 for a 5-ppm solution of ibuprofen. 

The Alliance iS HPLC System with PDA Detector settings that were evaluated and optimized for their impact on sensitivity included data rate, filter time constant, slit width, resolution, and absorbance compensation. 

• Data rate: The data rate defines the rate that the detector collects data, measured in hertz (Hz). The data rate should be set to yield reproducible peak area and retention times. Data rates that are too low will result in poorly defined peaks; high data rates may result in increased noise. Generally, the data rate is set to collect 25-50 points across the narrowest peak in the chromatogram. The data rate of the Alliance iS HPLC Systém with PDA Detector has settable values of 1–160 Hz. The default data rate is 10 Hz. 
• Filter time constant: The filter time constant is a noise filter that filters out high frequency noise. Faster time constants produce narrower peaks and remove less baseline noise. Slower time constants result in broader peaks and decreased baseline noise. The Alliance iS HPLC System with PDA Detector has the following filter time constant options: no filter, slow, normal, fast, and custom. The default selection is normal.
• Slit width: Slit width is an added feature available in the Alliance iS HPLC System with PDA Detector. The slit width determines the amount of light that reaches the photodiode array sensor which impacts resolution. Smaller slit widths result in improved resolution. Larger slit widths produce less noise and increased sensitivity, but at the cost of lower resolution. The Alliance iS HPLC System with PDA Detector has a variable slit, with available slit width sizes of 35 µm, 50 µm, 100 µm, and 150 µm. The detector’s default slit width is 50 µm.
• Resolution: The resolution setting determines bandwidth, which is the number of diode responses that are averaged when calculating the absorbance at a specified wavelength. Larger values result in less noise and a better S/N ratio, but they also produce decreased spectral resolution and can impact linearity. Resolution on the Alliance iS HPLC System with PDA Detector is settable over a range of 1–20 nm. The default value is 4 nm. 
• Absorbance compensation: The absorbance compensation feature of the Alliance iS HPLC System with PDA Detector provides a way to reduce non-wavelength dependent noise. This is achieved by collecting absorbance data over a user-specified wavelength range where there is little or no absorption and calculating the average absorbance. This average absorbance is then subtracted from the absorbance value. The Alliance iS HPLC System with PDA Detector default setting for absorbance compensation is ‘off’.

Using the USP method, each of these detector settings were individually evaluated and optimized. A method was created using the optimized settings and the results were compared to those obtained using default values.

Experimental

LC Conditions 

• LC system: Alliance iS HPLC System with PDA Detector 
• Detection: PDA Detector with 10 mm flow cell
• Column: XBridge™ BEH™ C18, 250 x 4.6 mm; 5 µm (p/n: 186003117)

Data Management 

• Chromatography data system: Empower™ Chromatography Data System (CDS)

Conclusion

While default PDA instrument method settings may be suitable for routine HPLC analysis, they may need to be adjusted to produce optimal separations. This study demonstrated how the Alliance iS HPLC System with PDA Detector allows the user to easily modify PDA settings to obtain optimal separations. Using the USP method for organic impurities in ibuprofen tablets, the data rate, filter time constant, slit width, resolution, and absorbance compensation settings were optimized. The optimized settings produced a 7-fold increase in sensitivity over that obtained using default values.