News from LabRulezLCMS Library - Week 48, 2025

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This week we bring you application notes by Agilent Technologies, Shimadzu and Waters Corporation and poster by Thermo Fisher Scientific / HPLC!

1. Agilent Technologies: Determination of 40 PFAS in Tilapia Tissue Following EPA 1633 Method Guidance

Using Agilent Captiva EMR PFAS Food II passthrough cleanup and LC/MS/MS detection

• Application note
• Full PDF for download

Determination of PFAS residues in tissue, especially in fish, has been an important avenue for monitoring and regulating PFAS residues in the environment. In 2021, the EPA published method 1633 for the quantitative analysis of PFAS in aqueous, solid, biosolid, and tissue samples using LC/MS/MS1 for 40 PFAS targets. The acidic groups contained in PFAS compounds enable them to be ionized easily and efficiently under negative mode, providing advantages in method sensitivity and selectivity. Method quantitation based on the use of stable labeled internal standards (ISTD) allows easy and reliable quantitation using neat calibration standard curves. Two sets of internal standards were used: extracted internal standards (EIS) and nonextracted internal standards (NIS). The EIS compounds included 24 isotopically labeled PFAS compounds and were used for PFAS target quantitation. EIS spiking solution was spiked directly into the sample matrix before extraction so EIS standards could track PFAS targets through the sample preparation procedure. NIS compounds included seven different isotopic PFAS compounds and were used for EIS recovery calculation. NIS solution was spiked into the final sample extract, thus only tracking the matrix effect.

QuEChERS extraction has been reported for PFAS analysis in food sample preparation.5,6 Compared to the alkaline MeOH digestion and extraction procedure used in EPA method 1633, QuEChERS extraction reduces the extraction time from about 20 hours to approximately 1 hour, uses approximately 80% less organic solvent, and delivers high extraction efficiency. Captiva EMR PFAS Food II cartridges were developed and optimized specifically for PFAS analysis in animal‑origin food matrices. The crude extract after QuEChERS extraction is mixed with 10% water, followed by passthrough cleanup on the Captiva EMR PFAS Food II cartridge. The PFAS targets flow through the cartridge, while the unwanted matrix co-extractives are retained on the cartridge based on the mixed-mode interactions with the sorbents. The method demonstrated a validated analysis of PFAS in animal‑origin food matrices.7,8 The objectives of this study were to apply this method to the analysis of 40 PFAS in fish tissue and validate it following EPA 1633 method guidance to meet the acceptance criteria. Considering the different requirements on method limits of quantitation (LOQs) for environmental tissue analysis, the previous sample preparation method used for food analysis7 was modified accordingly, saving time by eliminating the drying step. An Agilent 6495D Triple Quadrupole LC/MS was used for LC/MS/MS detection and quantitation, and instrumental conditions were adjusted to accommodate differences in the samples.

Experimental

Equipment and material 

The study was performed using an Agilent 1290 Infinity II LC system consisting of an Agilent 1290 Infinity II binary pump (G4220A), an Agilent 1290 Infinity II high-performance autosampler (G4226A), and an Agilent 1290 Infinity II thermostatted column compartment (G1316C). The LC system was coupled to an Agilent triple quadrupole LC/MS system (G6495D) equipped with an Agilent Jet Stream iFunnel electrospray ion source (ESI). Agilent MassHunter workstation software was used for data acquisition and analysis.

Results and discussion 

LC/TQ instrument method 

The MS detection method used here was adopted directly from previous studies7,8 but with additional targets and ISTD compounds from the Agilent PFAS MRM database. More modifications were applied on the LC method side. The LC method still used the same LC column as the previous studies, but with different mobile phase B, gradient, and injection programs. The modified LC method provided better chromatographic distributions on native targets, EIS, and NIS compounds within the acquisition window. It also improved the chromatographic separation for some targets with their isomers, and provided baseline separation for PFOS and cholic acid interferences. Figure 2 shows the chromatogram of all the targets, EIS, and NIS peaks with partial identification (A), and PFOS isomer and cholic acid interferences (B), demonstrating improved peak distribution over the retention time window and baseline separation for critical targets and possible matrix interferences. 

Sample preparation procedure 

The method using QuEChERS extraction followed with EMR mixed‑mode passthrough cleanup on Captiva EMR PFAS Food II cartridges greatly simplified the entire sample preparation procedure. The ACN solvent extraction followed with salt partition improved extraction efficiency and thus reduced the extraction time. Comparing the long extraction process with alkaline MeOH (~ 18 hours) used in the traditional EPA 1633 method, the QuEChERS method significantly reduced the extraction process time down to approximately 1 hour for the same number of samples, without compromise in extraction efficiency. The salt partition step played a critical role for more polar PFAS target extraction, and also helped with cleaning the matrix polar co-extractives, as the polar matrix co‑extractives were retained in the aqueous phase after the salt partition.

Conclusion 

A simplified, rapid, and reliable method using QuEChERS extraction followed by Agilent Captiva EMR PFAS Food II passthrough cleanup was developed and validated for 40 PFAS targets in fish tissue (tilapia) by LC/MS/MS under EPA 1633 guidelines. When comparing to traditional EPA 1633 SPE-based sample preparation methods, the newly developed method saved > 80% time, reduced solvent and chemical consumption by ~ 80%, and uses fewer consumables. The method was validated following the EPA 1633 guideline, providing acceptable EIS and NIS recoveries and native PFAS acceptable quantitation accuracy and precision. The MDL and LOQ levels for all targets using this method are lower than those reported by the EPA 1633 method. The method described here demonstrates an alternative solution with improved performance, efficiency, and cost compared to traditional EPA 1633 method sample preparation for PFAS analysis in tissue samples.

2. Shimadzu: Analysis of PFAS in Wastewater Based on ISO21675 Using Triple Quadrupole LC/MS/MS

• Application note
• Full PDF for download

User Benefits

• Based on ISO 21675, 30 PFAS can be analyzed in various water samples, including short-chain PFAS with 4 carbon atoms and long-chain PFAS with 16 and 18 carbon atoms.
• Accurate measurements are possible even in low concentration spiking recovery tests using ultrapure water and wastewater

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a large group of over 10,000 chemical compounds that are mainly composed of a carbon chain and fluorine atoms. The characteristics of PFAS depend on the carbon chain length and attached functional groups, though excellent water and oilrepelling properties and heat and chemical stability of some PFAS have led to them being used in a wide range of consumer products and industrial applications. However, their exceptional stability also makes them resistant to degradation. Concerns over the persistence of PFAS in the environment and their toxicity to organisms have led to stricter regulations globally and the development of various analytical methods to determine PFAS levels. ISO 216751) is an international standard for determining PFAS in water samples that was drafted by the National Institute of Advanced Industrial Science and Technology (AIST) and then developed by the International Organization for Standardization (ISO). The U.S. Environmental Protection Agency (EPA) Method 1633 targets PFAS with 4 to 14 carbons in water samples, while ISO 21675 includes additional long-chain PFAS with 16 and 18 carbons, which are not covered by EPA Method 1633. ISO 21675 enables the simultaneous analysis of 30 short- to long-chain PFAS (Table 1) using liquid chromatography-tandem mass spectrometry (LC/MS/MS). This article describes the results of PFAS analysis in industrial wastewater conducted using the LCMS-8060RX (Fig. 1) based on ISO 21675.

Spike Recovery Tests Using Industrial Wastewater 

A matrix blank test was conducted using industrial wastewater, and a spike recovery test was performed by spiking industrial wastewater with either a “Low” concentration (1 ng/L in wastewater, 0.1 ng/mL in solution) or a “High” concentration (10 ng/L in wastewater, 1 ng/mL in solution). Samples were prepared for analysis (Fig. 2), and the results of the spike recovery test using industrial wastewater are shown in Table 5. For the “Low” concentration samples, percentage recoveries ranged from 59 to 170 %. Recovery rates were not sufficient for compounds detected in the matrix blank at concentrations exceeding the spike level, suggesting interference from the blank. In contrast, the “High” concentration samples showed generally good recoveries, ranging from 81 to 116 %.

Conclusion 

PFAS analysis in wastewater was conducted using the LCMS8060RX, in accordance with ISO 21675. Analysis of calibration standards showed good peak shapes, good repeatability at the limit of quantification (0.01 ng/mL or 0.05 ng/mL in solution), and good calibration curve linearity for all the compounds. 

The spike recovery test in ultrapure water also showed good percentage recoveries from samples spiked at 1 ng/L and 10 ng/L in water. In contrast, the spike recovery test using industrial wastewater showed insufficient percentage recoveries at lower concentrations (1 ng/L in water) when the unspiked industrial wastewater contained relatively high concentrations of PFAS. However, good percentage recoveries were obtained at the higher concentration (10 ng/L in water).

3. Thermo Fisher Scientific / HPLC: Achieving consistent SEC performance through the use of 3 μm, 550 Å monodisperse media in novel bioinert column hardware

• Poster
• Full PDF for download

Size exclusion liquid chromatography has been widely used for detecting and quantifying aggregates due to its ability to differentiate molecules based on size. SEC effectively separates aggregates from their native state by exploiting the ability of analytes to access the pores of the stationary phase, with larger molecules being more pore-restricted and eluting earlier than smaller ones. 

A 3 µm monodisperse media with narrow pore size distribution centered at 550 Å was specifically engineered for the high-resolution analysis and precise measurements of adeno-associated virus (AAV) monomeric capsids from high molecular weight species (HMWS) and was recently introduced to the market [1]. This media features monodispersed silica particles covalently modified with a proprietary diol hydrophilic layer, which minimizes secondary interactions and works perfectly together with the hydrophilic-coating of the column hardware. These properties reduces secondary interactions and ensures the optimal performance for all analytes from the first injection. 

The consistent size of the monodisperse particles maintained from batch-to-batch synthesis allows for precise control over column packing, resulting in reliable lot-to-lot performance. The narrow 550 Å pore size distribution, ensures a wide separation range, providing accurate, and reproducible analysis. This enables exceptional and robust separation of various macromolecules, not only AAVs. 

This study investigated the physical parameters of the 3 µm, 550 Å monodisperse media and shows it‘s excellent batch-to-batch consistency. The media packed in the novel bioinert column hardware provided superior recovery of analytes compared to a conventional one. Additionally, the optimal testing conditions were determined by performing a Van Deemter curve study and were then used for protein and other macromolecule analysis to create a calibration curve based on both molecular weight and hydrodynamic radius. 

This study underscores the capabilities of the 3 µm, 550 Å, monodisperse media packed in coated hardware to provide robust and accurate characterization of various protein aggregates, making it an invaluable tool for researchers in the field of large molecule separation.

Results 

Monodisperse vs. Polydisperse Silica Particles 

The SurePac Bio 550 SEC MDi 3 µm column monodispersed silica particles are covalently modified with a proprietary diol hydrophilic layer. This proprietary process brings an extremely low level of non-desired interaction sites. Compared to traditional polydisperse particles (Figure 1B), the consistent size distribution of the monodisperse particles (Figure 1A) not only facilitates precise control over media synthesis and column packing, but also significantly improves column-to-column and lot-to-lot reproducibility.

Conclusions 

The SurePac columns utilize the use of monodisperse particles and bioinert hardware. This technology shows both batch-to-batch and column-to-column consistency. Novel bioinert hardware provides stable results from 1st injection. 

• Bioinert hardware allows for nearly complete recovery of 100% from first injection, significantly outperforming conventional hardware. 
• The monodisperse particle technology provides consistent results from batch-to-batch, even when media with proximity to limits of specification are taken into account. 
• The calibration curves of SurePac columns were introduced for PEG/PEO and proteins samples and show good linear dependency (R2>0.9). The extensive study of proteins and PEG/PEO indicates a separation range of 4–30 nm in hydrodynamic radius.

4. Waters Corporation: Ultra-Sensitive Quantification of Tirzepatide in Human Plasma Using Xevo TQ Absolute Mass Spectrometry with waters_connect Quantitation Software

• Application note
• Full PDF for download

Benefits 

• Advanced quantification platform: Xevo TQ Absolute Mass Spectrometer enables ultra-sensitive, robust, and selective quantification of tirzepatide for clinical and research applications.
• Efficient sample preparation via SPE: SPE ensures consistent recovery, enhanced analyte enrichment, and superior matrix cleanup compared to protein precipitation or liquid-liquid extraction (LLE).
• Minimized matrix effects: SPE effectively removes plasma interferences, reducing matrix effects and improving accuracy at sub-nanogram concentrations.
• High sensitivity and dynamic range: Achieve an LLOQ of 0.250 ng/mL with excellent signal-to-noise and broad dynamic range for pharmacokinetic applications.
• Optimized for peptide analysis: The method supports accurate quantification of low-abundance peptide drugs like tirzepatide in complex biological matrices.

Tirzepatide is a novel, long-acting dual incretin receptor agonist that targets both glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors. As a synthetic peptide, it demonstrates potent antihyperglycemic and weight-reducing effects, making it a promising therapeutic candidate for the treatment of type 2 diabetes mellitus (T2DM) and obesity. 

Structurally designed for enhanced receptor affinity and metabolic stability, tirzepatide combines the efficacy of dual receptor activation with a favourable pharmacokinetic (PK) profile, allowing for once-weekly subcutaneous administration. Preclinical and clinical studies have consistently demonstrated tirzepatide’s superior effects on glycemic control, insulin sensitivity, and body weight reduction compared to traditional GLP-1 receptor agonists.

Tirzepatide exhibits a complex pharmacokinetic profile, characterized by a long half-life of approximately five days, supporting its weekly dosing regimen. Plasma concentrations vary considerably based on dosage and formulation, with peak levels reaching several hundred ng/mL and declining gradually over time. Therefore, the development of a highly sensitive and reliable bioanalytical assay is critical to support PK and pharmacodynamic (PD) studies. Such an assay must be capable of accurately quantifying tirzepatide across a wide concentration range from ultra-trace levels during the elimination phase to high concentrations following peak exposure. This is essential for dose optimization, therapeutic monitoring, and throughout the drug development lifecycle.

Experimental

• LC system: ACQUITY H-Class PLUS UPLC System with FTN Sample Manager 
• Vials: QuanRecovery™, MaxPeak™ 12 x 32 mm PP 300 µl Screw Cap Vials (p/n: 186009186)
• Column: ACQUITY Premier Peptide BEH C18, 300 Å, 1.7 µm, 2.1 x 100 mm) Column
• MS system: Xevo TQ Absolute Mass Spectrometer

Data Management 

• MS software: waters_connect for Quantitation Software

Results and Discussion 

In this study, a robust and optimized sample preparation protocol was established in conjunction with a UPLC–MS/MS analytical method for the accurate quantification of Tirzepatide in human plasma. Solid-phase extraction (SPE) was employed using a Waters WCX 1 cc (30 mg) mixed-mode cartridge, which combines weak cation exchange and hydrophobic interactions, offers enhanced selectivity for peptide-based analytes and enables efficient removal of matrix components, thereby improving assay robustness and sensitivity. The 30 µm particle size provided a larger surface area, contributing to improved extraction efficiency and method reproducibility. This comprehensive and reliable extraction approach, utilizing Waters mixed-mode solid-phase extraction (SPE) cartridges, provided a strong foundation for sensitive and reproducible chromatographic and mass spectrometric analysis. recovery. The use of the Waters WCX 1 cc (30 mg) mixed-mode cartridge provided enhanced selectivity for peptide-based analytes, enabling efficient removal of matrix components with minimal interference observed in blank plasma. The method demonstrated a consistent recovery of approximately 55%. 

Instrument tuning played a pivotal role in ensuring the sensitive and accurate detection of Tirzepatide ions during mass spectrometric analysis. Optimal tuning parameters were essential to maximize the instrument’s response for the target analyte, thereby enabling reliable identification and quantification. Tirzepatide, like many peptide-based molecules, demonstrates multiple charging behaviour under electrospray ionization (ESI) conditions due to its structural composition. This behaviour is typical for large biomolecules, which contain several basic amino acid residues such as lysine and arginine that readily accept protons during ionization, as a result, the multiple charge state offer different precursor ions to investigate in order to obtain the most sensitive and selective MRM method. Tirzepatide forms multiple protonated species, as shown in Figure 4.

Conclusion 

A robust and reliable UPLC–MS/MS method was successfully developed and validated for the accurate quantification of tirzepatide in human plasma. The sample preparation strategy, employing a Waters WCX 1 cc (30 mg) mixed-mode reversed-phase and ion-exchange sorbent, enabled efficient extraction with high reproducibility. The Xevo TQ Absolute Tandem Quadrupole Mass Spectrometer operating in ESI positive mode ensured highly sensitive and precise quantification, with optimized tuning parameters contributing to analytical performance. 

Chromatographic separation on the ACQUITY Premier Peptide BEH C18 Column provided excellent retention, selectivity, and resolution, suitable for complex peptide analysis. Method validation results confirmed outstanding linearity, accuracy, and precision across the concentration range of 0.125–55.0 ng/mL, with all QC levels meeting regulatory acceptance criteria.

This validated method offers a powerful analytical tool for pharmacokinetic, clinical, and therapeutic monitoring of tirzepatide, supporting its continued development in diabetes research and contributing to advancements in bioanalytical methodologies for peptide therapeutics.