News from LabRulezLCMS Library - Week 24, 2025

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 9th June 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 notes by Agilent Technologies, Shimadzu and Thermo Fisher Scientific!

1. Agilent Technologies: Analysis of Oligonucleotides Using Ion-Pairing Alternatives on the Agilent Pro iQ Plus

• Application note
• Full PDF for download

Oligonucleotides are an emerging therapeutic modality that target and modulate gene expression through the silencing or degradation of mRNA. Once an mRNA target is identified, the antisense sequence must be optimized with strategically incorporated chemical modifications to improve pharmacokinetics¹ , affinity, and minimize off-targeting or mismatching.² 

Ion-pair reversed-phase LC/MS is commonly used to confirm the molecular weights of target oligo sequences to ensure proper synthesis. However, using alkylamines as ion-pairing agents often requires dedicated instrumentation. Furthermore, toxic and cost-prohibitive perfluorinated alcohols such as HFIP (hexafluoroisopropanol) are required for optimal chromatographic separation and MS sensitivity. 

In this application, an alternative reversed-phase approach was used for molecular confirmation of oligonucleotides. The method used ammonium bicarbonate instead of ion pairing, while still providing sufficient chromatographic retention and MS sensitivity. Twenty replicate injections of three different antisense oligonucleotides and five replicates of a single siRNA were performed to ensure the applicability and reproducibility of the method.

Experimental 

Instrument configuration 

This experiment was conducted using the following instrument configuration:

• Agilent InfinityLab Pro iQ Plus LC/MS system (G6170A) 
• Agilent Infinity II 1290 bio binary pump (G7120A) 
• Agilent Infinity II 1290 bio multisampler (G7167B) 
• Agilent Infinity II 1290 bio column compartment (G7116B) 
• Agilent Infinity II 1260 diode array detector HS (G7117C) 

Although this analysis used an Infinity II LC configuration, comparable results can be achieved on the Infinity III LC system with no changes to method parameters.

Results and discussion

Established in 1997, an alkylamine ion pair with perfluorinated alcohol as the acidic modifier is the preferred mobile phase for LC/MS analysis of oligonucleotides.³ This is due to its chromatographic performance and electrospray efficiency, especially when compared to mobile phases using acetate as the counter ion. Extensive work has further demonstrated a wide experimental design space when using this powerful ion-pairing system.⁴ 

However, there are consequences when using alkylamine and HFIP for LC/MS methods. First, optimization of buffer components is necessary since oligo modifications and sequence affect electrospray desorption and chromatography.⁵ Second, alkylamine may contaminate ionization sources and LC systems, leading to background peaks if polarity is switched back to positive mode. Consequently, this requires extensive cleaning/passivation of LC components and ion source surfaces. Even then, some labs may dedicate systems to negative mode due to the adsorption of alkylamines onto the LC/MS system. Finally, alkylamine-containing mobile phases have a short shelf life if not kept sealed under argon gas, and thus, mobile phases must be made fresh, sometimes daily, to ensure consistent method performance. 

For labs performing molecular weight confirmation workflows where many oligos are analyzed, optimizing mobile phase conditions may not be feasible. Additionally, LC/MS downtime due to instrument maintenance and daily preparation of buffers may lead to sample backlogs, which for many labs can be a considerable challenge. A more practical approach would be to use a non-ion-pairing, reversed-phase methodology. 

Recent work provides a more practical and cost-effective alternative to ion pairs, using an ammonium bicarbonate (NH₄ HCO₃) buffer and methanol as the strong solvent.⁶ This is an advantageous method for molecular weight confirmation, as ESI sensitivity and chromatographic performance are sufficient, even with minimal optimization of the mobile phase and gradient. Further, this referenced work postulates that carbon dioxide outgassing facilitates droplet formation, while ammonia evaporation contributes to proton adduction, thus allowing for positive mode analysis. This, therefore, eliminates the need to dedicate a system for negative mode analysis.

Conclusion

The method described in this application note provides a practical approach for medium- to high-throughput molecular confirmation of synthetic oligonucleotides. The Agilent Pro iQ Plus demonstrates excellent resolution and sensitivity for a unit mass detector, even at high m/z ranges that exceed the capabilities of many single quadrupole detectors. The method uses a novel ammonium bicarbonate mobile phase, providing sufficient LC/MS performance for labs analyzing many types of oligos, including antisense oligonucleotides and siRNAs. LC and MS results described in this application note are reproducible. Data analysis workflows are simplified using Agilent OpenLab CDS MS spectral deconvolution, which is automated by unattended processing methods that require minimal optimization.

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

• Application note
• Full PDF for download

User Benefits

• A single Shimadzu LCMS-8060RX triple quadrupole mass spectrometer can successfully quantitate both PFAS and cyanotoxins in water with automatic method switching.
• With only a five-minute rinsing time between methods, the system maintains high accuracy and sensitivity over an extended run time, even with multiple injections and method changes.
• This single system approach allows laboratories to respond swiftly to emergencies, like Harmful Algae Blooms, while minimizing disruption to routine PFAS testing and eliminating the need to invest in multiple instruments.

Per- and Polyfluoroalkyl Substances (PFAS) are a group of synthetic chemicals extensively used in consumer products (e.g., food packaging materials and non-stick coatings) and industrial applications such as firefighting foams and polymer/plastics manufacturing. Their remarkable properties, including high stability and resistance to degradation, coupled with widespread usage, have led to their persistent accumulation in the environment. Consequently, regulatory and governmental organizations, including the US Environmental Protection Agency (EPA) and the European Chemicals Agency (ECHA)¹, are working to restrict their presence in the environment. 

In the United States, EPA Method 537.1 is one of the approved methods by EPA for the analysis of PFAS in drinking water. It targets 18 compounds, utilizing solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detection at low ng/L concentrations². EPA Method 533 is the only other EPAapproved method for PFAS analysis in drinking water. It follows a workflow similar to that of EPA Method 537.1 but expands the target list to include 25 PFAS compounds³. 

PFAS is not the only concern—other contaminants in drinking water can also pose significant health risks. Cyanobacteria, also referred to as blue-green algae, are photosynthetic organisms that thrive in both freshwater and marine ecosystems. The frequency and occurrence of Harmful Algae Blooms (HABs) have risen substantially over the years, primarily due to human-induced inputs of phosphorus and nitrogen into aquatic systems, fueling the global increase in harmful algal blooms. These organisms can significantly affect water quality by producing cyanotoxins such as cylindrospermopsin, anatoxin-a and microcystins. Exposure to these cyanotoxins can cause a range of adverse health effects in humans and animals, from mild skin irritations to severe illnesses⁴. 

The US EPA has established Methods 544 and 545 as analytical protocols for detecting cyanotoxins in drinking water and freshwater. EPA Method 544 is designed to detect microcystins and nodularin⁵, while EPA Method 545 targets cylindrospermopsin and anatoxin-a ⁶. Both methods employ liquid chromatography-tandem mass spectrometry (LCMS/MS) for precise analysis.

Ideally, separate instruments should be dedicated to analyzing PFAS and cyanotoxins to minimize the risk of contamination and interference. However, with the growing demand for water testing and rapid turn-around-times during emergencies that may alter routine testing, a single system capable of handling multiple methods offers a more costeffective solution. This application demonstrates the accurate and robust quantification of PFAS and cyanotoxins, performed in accordance with EPA Methods 537.1, 545, and 544, using a single Shimadzu LCMS-8060RX triple quadrupole mass spectrometer with automatic method switching.

Method

System configuration: A Shimadzu LCMS-8060RX triple quadrupole mass spectrometer (Figure 1) was used to quantify the PFAS and cyanotoxins in water. The front-end liquid chromatography (LC) system was configured with the Shimadzu LC-40 series, ensuring seamless automation, method switching, and high efficiency. Key modules include three degassers (one 3-channel and two 5-channel), two LC40 solvent pumps equipped with low-pressure gradient (LPGE) modules, an autosampler, a system controller, a column oven with two column switching valves (one 6-port, 2-position valve and one 7-port, 6-position valve).

PFAS contamination can originate from solvent containers and consumables used in the LC system. To minimize background interference, a delay column is implemented before the autosampler to separate background PFAS from target analytes.

Conclusion 

This study demonstrates the successful quantification of PFAS (EPA 537.1) and cyanotoxins, including microcystins and nodularin (EPA 544) as well as cylindrospermopsin and anatoxin-a (EPA 545), using a single triple quadrupole mass spectrometer with automatic method switching. By leveraging this streamlined approach, we achieved precise and reliable measurements while maintaining high accuracy and sensitivity across extended analytical runs, even with multiple injections and frequent method transitions. 

A key advantage of this approach is the efficient switching between analytical methods with only a five-minute rinse between methods. This ensures complete removal of residual analytes and prevents mobile phase contamination, allowing smooth transitions between PFAS and cyanotoxin analysis without compromising data quality. The rapid automatic rinsing process eliminates the need for extensive manual intervention, reducing instrument downtime and increasing overall laboratory productivity. 

By consolidating multiple analytical methods into a single instrument, this approach minimizes the need for separate systems, thereby reducing both capital investment and maintenance costs. This single system enables laboratories with the capability to respond quickly to emergencies, such as HABs, without major disruption of their routine testing, for PFAS and potentially other organic contaminants. Furthermore, automation significantly enhances operational efficiency by minimizing manual labor requirements, improving reproducibility, and optimizing high-throughput sample analysis. This method provides a cost-effective and reliable solution for laboratories performing environmental monitoring and regulatory compliance testing.

3. Thermo Fisher Scientific: Comprehensive screening of per- and polyfluoroalkyl substances (PFAS) in textiles: Utilizing combustion ion chromatography for total organic fluorine (TOF) analysis

• Application note
• Full PDF for download

Per- and polyfluoroalkyl substances (PFAS) are used globally across various industries and comprise thousands of individual compounds. These amphipathic substances are extensively incorporated into textile-based products, such as clothing, carpets, and other household items to provide waterproofing, oil, dirt, and heat protection, as well as increased durability. PFAS can be released from functional textiles according to an outdoor aging study.¹ The Department of Toxic Substances Control (DTSC) has found that treated textiles and leathers are major sources of PFAS exposure for people and the environment, particularly through inhalation when using these products. PFAS also contribute to widespread environmental contamination, similar to other consumer products like food packaging and cosmetics.²

Traditionally, PFAS testing has been conducted using liquid chromatography coupled with triple quadrupole mass spectrometry (LC-QQQ). While LC-QQQ is a targeted analytical technique, its results are limited to compounds for which standards are available. Consequently, these targeted studies do not necessarily provide a comprehensive measurement of the total PFAS in samples. Recently, laboratories have focused on developing and validating lower-cost alternatives that offer a more comprehensive measure of total PFAS content. This has led to the development of several methods for measuring total fluorine (TF) as a proxy for total PFAS contamination in textiles. Technologies employed in these methods include combustion ion chromatography (CIC)⁵ and particle-induced γ-ray emission spectroscopy (PIGE).6 However, measuring only TF is not a reliable proxy for PFAS, as it includes both organic and inorganic fluorine, the latter of which is not considered PFAS. Using TF as a measure of PFAS may result in overestimating the amount of PFAS in samples. 

California AB 1817 introduces a new methodology for testing PFAS contamination or intentional use of PFAS in fabrics based on the amount of TOF in the sample. This is a significant change from previous approaches, employing a non-specific untargeted method to PFAS detection and quantification. In this study, we developed a method to determine TOF in textiles using CIC. CIC offers excellent sensitivity and versatility, independent of sample thickness, and the capability for direct ion chromatography (IC) analysis to determine inorganic fluorine (IF).

Experimental 

Equipment 

A Thermo Scientific™ Dionex™ Integrion™ HPIC™ System (P/N 22153-60306) including: 

• Eluent generator 
• Pump 
• Degasser 
• Conductivity detector 
• Column oven temperature control 
• Detector-suppressor compartment temperature control 

Nittoseiko Automatic Combustion Unit Model AQF–2100H system* including: 

• Automatic Sample Changer ASC-270LS 
• Horizontal Furnace Model HF-210 
• Gas Absorption Unit GA-211 
• External Solution Selector ES-210 
• *Any combustion oven with equivalent performance will work.

Software 

• Thermo Scientific™ Chromeleon™ Chromatography Data System (CDS) Version 7.3 1 with DDK driver to control the combustion system

Results and discussion

TOF in textile samples by CIC

The TF in a sample is defined as the sum of TIF and TOF. In this study, TOF was determined by subtraction of TIF from TF. After combusting the solid sample, TF was measured using IC. High concentrations of TF (>100 ppm) were observed in five of the six samples tested (Table 2). 

TIF was measured using IC after extracting the ground textile samples with DI water. The contribution of TIF to TF was found to be very small (<1 ppm) as shown in Table 2. Even after subtracting TIF, the TOF remained above 100 ppm for five of the samples. Therefore, the five samples did not meet the regulatory limit of 100 ppm TOF set by the state of California.³

 Figure 5 shows the IC chromatograms of TF and TIF of Sample #1. Fluoride is well separated from other anions that may be present in samples, allowing for accurate determination.

Conclusion 

We developed a sensitive and accurate method using CIC to determine TOF in textiles. The method offers a detection limit of 0.37 ppm, which is significantly lower than the regulatory threshold of 100 ppm, ensuring reliable detection of PFAS. Spiked recovery experiments show the method’s accuracy falls within a range of 85–105%. This TOF method is beneficial for manufacturers who want to ensure compliance with current state regulations. Moreover, the CIC workflow provides a more comprehensive understanding of the total PFAS and fluorinated content in textiles compared to LC-MS targeted approaches, offering greater clarity about the potential PFAS contamination in textiles.