News from LabRulezLCMS Library - Week 26, 2025

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This week we bring you application notes by Agilent Technologies, Shimadzu and Waters Corporation and technical note by Thermo Fisher Scientific!

1. Agilent Technologies: Assessing the Purity of an Antisense Oligonucleotide Sample by LC/MS

• Application note
• Full PDF for download

Antisense oligonucleotides (ASOs) are an established therapeutic nucleic acid modality, manufactured by solid‑phase synthesis. Because of their complexity, a common way to assess the purity of ASOs is liquid chromatography/mass spectrometry (LC/MS). Although high-resolution MS might be appropriate for workflows such as characterization and sequence confirmation, unit mass detection provides a more robust and practical method for routine testing labs.

Single quadrupole LC/MS has previously been shown to effectively support purity, assay, and impurity profiling for routine testing of single-stranded therapeutic oligos, such as ASOs.¹ This method relies on MS full scan to quantitate closely related impurities that elute under the main peak. The LC/MS conditions ensure that analytes are predominantly in the 4– charge state when entering the gas phase. For example, an oligo that is ~ 8 kDa would have a predominant m/z value of ~ 2,000. This would either be at the upper limit or possibly exceed the scan range for some unit mass detectors. Furthermore, this method has a limit of quantitation (LOQ) of ≥ 0.2%, requiring a sensitive mass detector with efficient high mass transmission. 

In this application note, the Agilent InfinityLab Pro iQ Plus LC/MS system is used to determine the purity of an ASO. The full scan spectrum from this unit mass detector enables the identification and quantification of low-level impurities at method specifications. This study establishes proof of concept for the use of this system in the routine testing of oligonucleotides for QC lot release.

Experimental

Instrument configuration

This experiment was conducted using the following instrument configuration:

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

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

Software 

Data acquisition was performed in Agilent OpenLab CDS, version 2.8, using the LC/MS parameters shown in Tables 1 and 2. Data analysis was performed in Oligo Analysis Accelerator (OAA) for OpenLab CDS, version 1.0.

Results and discussion 

This method demands exceptional sensitivity, precision, and a broad mass range, making the Agilent InfinityLab Pro iQ Plus an ideal solution for the task. To determine the purity of an ASO, a full scan spectrum is extracted from the main peak, which tends to be broad (2 to 3 minutes) due to the shallow gradient program. Any m/z values in the average spectrum that exceed an established threshold can then be used to generate an extracted ion chromatogram (EIC). The limit of quantification (LOQ) is established at 0.2%, and because many impurities are near isobaric, any spectral overlap may interfere with the data. Thus, a sensitive and selective method is required for this analytical workflow. 

The Pro iQ Plus has a mass range of m/z 2 to 3,000. This method typically requires a full scan of m/z ± 150 around the 4– charge state of the full-length product (FLP). For example, an oligo with a molecular weight of 8,000 Da would require a scan range from m/z 1,750 to 2,150. Additionally, sufficient ion transmission at higher m/z values is important, as EICs are used for the relative quantitation of each impurity. Figure 1 shows the ion transmission stability for mass axis assignment and peak width at m/z 2,234, monitored over 24 hours. These data demonstrate the robustness and stability of large molecule transmission on the Pro iQ Plus. 

Prior to selecting the ions that exceed the threshold for EIC integration, the method requires a comparison of the sample under different MS conditions. Figure 2 shows the overlay of so-called "standard" and "harsh" spectra from the ASO-1 sample. This overlay of spectra on a relative scale is used to determine if any ions exceeding the threshold are adducts. Ions that exceed the threshold under standard conditions, but not under harsh conditions, are regarded as adducts. This is because the higher temperature conditions used for harsh conditions minimize adduct formation during electrospray desorption of the ASO. The spectra obtained under both conditions demonstrate excellent selectivity and sensitivity for the ions in the 4– charge state.

Conclusion 

This method, as cited in previous literature1 , has become the gold standard for LC/MS-based purity, assay, and impurity profiling of therapeutic, single-stranded oligonucleotides. The method requires a selective and sensitive detector capable of acquiring full scan spectra from the main peak to ensure accurate quantitation of impurities. Additionally, depending on sample, the method may require scans above m/z 2,000, which is a limiting factor for many unit mass detectors. Due to its excellent ion transmission at high mass ranges, the Agilent InfinityLab Pro iQ Plus can meet these method requirements for both sensitivity and selectivity, thereby allowing QC analysis of oligonucleotide impurities.

2. Shimadzu: Simultaneous Analysis of CHO Cell Culture Supernatant Components Using a Single Quadrupole Mass Spectrometer

• Application note
• Full PDF for download

User Benefits

• A single quadrupole LC-MS system can be used to simultaneously analyze amino acids, organic acids, vitamins, and other substances in a culture supernatant.
• Changes in the components in a culture supernatant can be easily visualized as a function of time by analyzing culture supernatant samples acquired over time with the Multi-omics Analysis Package.

When using cells to produce beneficial substances or manufacture antibody drugs, the pH level of the culture medium, dissolved gases, carbon sources, nitrogen sources, and other factors are monitored to optimize or manage the culturing process. Cell cultures can contain a variety of substances, including not only glucose and glutamine but also vitamins, nucleic acid-related compounds, and metabolites secreted from cells. Therefore, by comprehensively analyzing all the components in a culture medium, useful information about biological processes can be obtained. 

This Application News article describes using a single quadrupole LC-MS system to monitor time-course changes in the components in a supernatant from culturing CHO-K1 cells, which are commonly used for antibody production, by simultaneously analyzing all the components. Because single quadrupole LC-MS systems have a simple configuration process, it is easy even for first-time users to simultaneously analyze components in culture supernatants.

Instruments and Analysis Conditions 

Analysis was performed with a Nexera series HPLC system that was combined with an LCMS-2050 LC-MS system (Fig. 1). Although compact, the LCMS-2050 single quadrupole mass spectrometer offers easy operability and outstanding performance. Equipped with a DUIS heated ion source, which provides the benefits of both ESI and APCI, it can analyze masses ranging from m/z 2 to 2000. That makes it well suited for the simultaneous analysis of components in culture supernatants.

Conclusion 

As described in Application News No. C106A, if simultaneous analysis of all components is necessary, including trace components secreted from cells, a triple quadrupole LC-MS system is suitable. However, a single quadrupole LC-MS is more than adequate for analyzing the main components in culture media, such as glucose, glutamine, and lactic acid. Widespread use of the single quadrupole LC-MS for simultaneous analysis of the components in culture supernatants can be expected to lead to continuous advancements in cell-related R&D work.

3. Thermo Fisher Scientific: Configuring the Thermo Scientific Cindion C-IC system for a 2-in-1 operation:

Seamless switching between combustion-IC and standalone IC with an AS-AP autosampler

• Technical note
• Full PDF for download

In Thermo Scientific Application Note AN003644, a method was developed to measure total organic fluorine (TOF) in food contact materials (FCM) using combustion-ion chromatography (C-IC).¹ In this method, total inorganic fluorine (TIF) was measured by directly injecting water-extracted samples through four external injection channels. This approach, while effective, required large sample volumes and manual sample changes after every four analyses.

The Thermo Scientific™ Cindion™ Combustion Ion Chromatography System integrates the Thermo Scientific™ Dionex™ Inuvion™ Ion Chromatography System, featuring reagentfree ion chromatography (RFIC), with the Thermo Scientific™ Cindion™ Combustion/ Absorption Module. This integration provides versatile 2-in-1 operation capability, enabling seamless switching between combustion-IC and standalone IC with a Thermo Scientific™ Dionex™ AS-AP Autosampler. With this system, TIF analysis can now be fully automated, eliminating manual sample changes and minimizing sample volume usage, as described in Thermo Scientific Application Proof Note AP003822.² 

Moreover, when the Cindion combustion/absorption module is not in use, the standalone IC with the Dionex AS-AP autosampler can be employed for other IC applications, thereby maximizing the utilization of the IC system. This technical note provides step-bystep instructions for configuring the Cindion C-IC system for 2-in-1 operation.

Procedure

1. Install the auxiliary valve

The Dionex Inuvion IC system should already have a 6-port injection valve installed. Install an additional 6-port auxiliary valve next to the injection valve. Connect the auxiliary valve as shown in Figure 1. The auxiliary valve has two positions. 

2. Add the Dionex AS-AP autosampler to the instrument configuration 

In the instrument configuration, follow the setup instructions for both the Dionex Inuvion IC system and Cindion C-IC system as described in the user manuals or Thermo Scientific Technical Note TN003733.3 With the auxiliary valve installed on the Dionex Inuvion IC system, the Dionex Inuvion IC system instrument configuration should display the HP_valve as checked, as shown in Figure 2, and the IC panel should display the HP valve with A and B positions, as shown in Figure 3. Connect the Dionex AS-AP autosampler cable to the Dionex Inuvion IC system and add it to the instrument configuration (Figure 4a). The default device name “Sampler“ has been used for the Cindion C-IC system. Therefore, change the device name to a different name such as “SamplerASAP,” as shown in Figure 4b. During the configuration check, you may encounter the warning: “More than one inject device installed for instrument” (Figure 4c). This warning can be ignored.

3. Create separate sequences for combustion-IC and standalone IC 

Since the sample position information differs for combustion-IC and standalone IC sequences, it is essential to create separate sequences for each type of application. Figures 5a, 5b, and 5c illustrate the three steps for creating a sequence in combustionIC mode. In the sequence creation wizard (Figure 5a), ensure that “Cindion.Absorber” is selected as the sampler. In sequence preview (Figure 5b), set the sample start position to SolidTray: #, where # is the first tray position containing a sample.

4. Create separate instrument methods for combustion-IC and standalone IC

Combustion-IC method: 

• Create the instrument method using the wizard (Figure 8a) 
• Manually insert the injection valve load position and HP valve_B command at the beginning of the instrument method script (Figure 8b). Here are the detailed step-bystep instructions

Standalone IC method:

• Create the instrument method using the wizard (Figure 9a)
• Manually insert the HP valve_A command at the beginning of the instrument method script (Figure 9b)
• Remove any Cindion C-IC system-related commands from the script (Figure 9c)

Conclusion 

The integration of the Dionex Inuvion IC system with the Cindion combustion/absorption module enables a flexible and efficient 2-in-1 operation, seamlessly switching between C-IC and standalone IC with the Dionex AS-AP autosampler. This configuration simplifies the total inorganic fluorine (TIF) analysis process, eliminating the need for manual sample changes while minimizing sample volume usage. The added flexibility in operation not only enhances the system’s utility for TIF analysis but also allows for other ion chromatography applications when the combustion/absorption module is not in use. By following the provided step-by-step instructions for configuring the system, users can ensure a smooth transition between applications, thus maximizing both efficiency and productivity.

4. Waters Corporation: Evaluation of System Robustness of the Alliance™ iS Bio System Using Peptide Mapping Analysis

• Application note
• Full PDF for download

Benefits

• Alliance iS Bio HPLC System produces reproducible results over extended periods of testing for a difficult gradient method.
• Biocompatible design maximizes performance for biomolecules. 

Peptide mapping analysis is a critical component to biologics development in the pharmaceutical industry for assays such as impurity monitoring. The complexity of protein digest samples also makes them ideal for LC system performance evaluation.¹ This is due to the demanding method conditions required to achieve separation of the many digestion products. These samples, along with many other biological samples, often have high levels of interactivity with the metal components in LC systems. These non-specific adsorption interactions are problematic in chromatography because they may result in peak broadening, large precision differences, and long passivation times.² 

The Alliance iS Bio HPLC System is a modern solution to the challenges presented by these samples. The system contains an inert flow path to minimize interaction between sample and system. Components designed to resist corrosion include a titanium mixer and MP35N tubing. These allow for the best performance on applications that employ high salt concentrations and harsh pH conditions, commonly found in bio methods. Components designed to reduce non-specific adsorption include a PEEK needle and MaxPeak HPS tubing. The 680 µL mixer and reduced default stroke volume of 66 µL minimize noise and provide a flatter and more consistent baseline, particularly when using applications that use mobile phase additives such as trifluoroacetic acid.³ These instrument characteristics are ideal in a QC environment where it is critical to achieve repeatable results and minimize errors.⁴ 

The robustness of a system is most challenged with demanding method conditions such as small changes in gradient composition per unit time, particularly for low pressure pumps. Low pressure quaternary pumps contain a gradient proportioning valve with four independent channels which send small packets of solvent along the flow path. With a long shallow gradient, each channel can be open for only fractions of a second for packet delivery, and this must be done repeatedly over the course of long run times and multiple injections. In contrast, binary pumps contain only two solvent lines, but undergo high pressure mixing in the mixer which can yield more consistent performance. Therefore, a quaternary system must exhibit high performance to be suitable for these applications. In this study, a peptide mapping method with a long, shallow gradient (0.5% B/min) is used and injections of an enolase digest standard are completed repeatedly for 30 days. The standard used serves as a representative complex sample commonly found in biopharmaceutical applications. This extended timeframe allows for observations about the stability of the HPS technology and the ability of the gradient composition delivery to remain consistent.

Experimental

LC Conditions

• LC system: Alliance iS Bio HPLC System
• Column: XSelect™ CSH™ C18, 130Å 2.5 µm, 4,6 x 150 mm
  (p/n: 186006729) 

Data Management

• Chromatography software: Empower™ 3.8.0.1

Conclusion 

Pharmaceutical testing, including peptide mapping analysis, requires demanding method conditions to achieve separation such as long, shallow gradients. The Alliance iS Bio HPLC System displayed robust performance over an extended 30 day period of study for these challenging conditions. Results included retention time standard deviation within sample sets of not more than 0.018 minutes, overall retention time %RSD not more than 0.77%, critical pair resolution above baseline resolution for the entire study, and area %RSD within sample set not more than 0.92%. These results demonstrate a system that is fully capable of handling difficult applications while maintaining a high level of performance.