News from LabRulezLCMS Library - Week 05, 2026

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 26th January 2026? 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, Thermo Fisher Scientific and Waters Corporation!

1. Agilent Technologies: Quantitative Analysis of Nitrosamine Impurities in Synthetic Oligonucleotides

Using the Agilent 6495D triple quadrupole LC/MS system

• Application note
• Full PDF for download

The discovery of N-nitrosodimethylamine (NDMA) in certain drug products in 2018 prompted global regulatory agencies, including the FDA and EMA, to mandate nitrosamine risk assessments across all pharmaceutical classes. Nitrosamines are genotoxic impurities of significant concern due to their high carcinogenic potential, requiring stringent control to ng/day levels. While chemically synthesized oligonucleotides—such as ASOs, small interfering RNAs (siRNAs), and aptamers—contain primary aromatic amines in their nucleobases, these groups typically act as scavengers rather than forming stable nitrosamines. Consequently, the intrinsic risk of nitrosamine formation from oligonucleotide APIs is considered low.¹ 

However, potential contamination from raw materials, reagents, or process conditions necessitates a robust risk assessment and control strategy to ensure patient safety and regulatory compliance. FDA² , EMA³ , and ICH⁴ require nitrosamine risk assessment and control for all drug products, including synthetic oligonucleotides. Manufacturers must document risk evaluation, confirmatory testing, and mitigation strategies. In this application note, a quantitative analysis of eight nitrosamine compounds was carried out on the Agilent 6495D triple quadrupole LC/MS (LC/TQ) system coupled with the Agilent 1290 Infinity II LC system and Agilent atmospheric pressure chemical ionization (APCI) source. The results demonstrated ppt-level detection of the nitrosamine targets in an ASO sample.

Experimental

Instrumentation 

For separation, the Agilent 1290 Infinity II bio LC system was used, including: 

• Agilent 1290 Infinity II bio high-speed pump (G7132A) 
• Agilent 1290 Infinity II bio multisampler (G7137A) with Agilent Infinity II sample cooler 
• Agilent 1290 Infinity II multicolumn thermostat (G7116B) equipped with Agilent InfinityLab bio-inert Quick Connect heat exchanger 

Samples were analyzed on the Agilent 6495D LC/TQ equipped with the Agilent APCI source.

Software 

The following software was used in this study: 

• Agilent MassHunter acquisition software (LC/TQ), version 12.2 
• Agilent MassHunter Quantative Analysis software, version 12.1

Results and discussion 

The calibration curve of the eight nitrosamine compounds was established with concentrations ranging from 0.05 to 25 ng/mL in the ASO matrix. Excellent chromatographic separation and peak shapes were achieved for all analytes, as shown in Figure 1. The NPIP peak baseline was impacted by matrix interference due to coelution with the ASO sample. To reduce matrix effects and improve method performance, a divert valve can be programmed to direct the matrix peak to waste at its elution time window. 

Excellent calibration linearity with R2 > 0.99 (Figure 2) was achieved for all the eight analytes within 0.05 to 25 ng/mL concentration range, except NPIP (0.25 to 25 ng/mL) and NDBA (0.1 to 25 ng/mL). Accuracy was consistently within the range of 90 to 120% at all tested levels (Table 4). High analytical sensitivity was achieved, enabling quantification of all the targeted analytes down to ppt level. Table 4 summarizes limits of detection (LODs) and limit of quantification (LOQs) for each nitrosamine. LOD values were determined using software-driven automated based on eight LQC samples. The LOQ values corresponded to the lowest calibration level.

Conclusion 

This application note underscores the critical importance of mutagenic impurity analysis in oligonucleotide‑based therapeutics, which is in alignment with regulatory requirements. 

A sensitive and robust LC/MS/MS method was successfully developed on Agilent 6495D LC/TQ system. The method can quantify nitrosamine impurities in ASO sample down to ppt levels. The method demonstrated excellent performance across eight nitrosamines, with strong calibration linearity (R² > 0.99), high accuracy (80 to 120%), and outstanding retention time and area precision (%RSD < 0.17 and < 3.71, respectively). LOD and LOQ were below 0.10 and 0.25 ng/mL, respectively, confirming high sensitivity in complex matrices. Recovery and reproducibility results further validated the method’s robustness. Overall, the findings confirm the method’s reliability and suitability for routine nitrosamine analysis in oligonucleotide therapeutics, supporting regulatory compliance.

2. Shimadzu: Simultaneous Analysis of Food Allergens, Including Nuts and Fruits, Using a Triple Quadrupole Mass Spectrometer

• Application note
• Full PDF for download

User Benefits

• Simultaneous detection of allergens from seven specified ingredients and ten ingredients equivalent to the specified ones is possible, and calibration curves can be prepared from 1 ppm.
• The LC-MS/MS method enables detection of all target ingredients with a single sample preparation.

Food allergies are caused by excessive immune responses to specific food proteins (allergens) and can occasionally result in severe symptoms. To prevent health risks associated with food allergies, food labeling is strictly regulated in many countries. In Japan, based on the severity and frequency of past health incidents, labeling is mandatory for seven specified allergens and recommended for twenty allergens equivalent to the specified onesin prepackaged processed foods. 

In recent years, LC-MS/MS has attracted increasing attention for allergen analysis due to its various advantages. This application news presents an example of the simultaneous analysis of seven specified food allergens and ten allergens equivalent to specified ingredients in processed foods using LC-MS/MS. Specific peptides and corresponding MRM transitions for four types of nuts and four types of fruits were newly selected and developed using Skyline software. In addition, examples of calibration curve preparation using extraction reagents and standard materials optimized for food allergen analysis are presented.

Various Analytical Methods for Allergens

The quantity and type of allergens responsible for allergic symptoms vary among individuals; therefore, it is essential to accurately determine the presence or absence of allergenic ingredients in food on an ingredient-by-ingredient basis. In Japan, the enzyme-linked immunosorbent assay (ELISA) has been designated as a quantitative testing method, whereas the polymerase chain reaction (PCR) and Western blotting have been designated as qualitative testing methods. The characteristics of each analytical method are summarized in Table 1.

ELISA enables quantitative determination of allergens; however, it carries a risk of false-positive results due to cross-reactivity with homologous proteins. When it is difficult to distinguish true positives from false positives based on ELISA results and manufacturing records, PCR or Western blotting is employed as a complementary technique. Moreover, ELISA cannot analyze multiple ingredients simultaneously, requiring a dedicated kit for each ingredient along with individual sample preparation and measurement procedures.

PCR detects target DNA sequences specific to each ingredient through amplification reactions. This provides highly specific discrimination even among closely related species, such as wheat and barley. In contrast, PCR is not suitable for distinguishing foods that share identical genetic sequences, such as eggs and chicken meat. In such cases, Western blotting is used instead, targeting proteins that are present in the same species but not designated as allergens. Because Western blotting detects proteins via antigen–antibody reactions following electrophoresis, it allows evaluation of molecular weight information in addition to protein detection. Although both PCR and Western blotting exhibit high specificity, quantitative determination of allergenic proteins remains difficult using these methods. 

LC-MS/MS, which has recently attracted increasing attention, involves extracting allergenic proteins from food, enzymatically digesting them into peptide fragments, and subsequently analyzing these peptides. The amino acid sequences of the detected peptides enable identification of the corresponding allergens. This approach allows simultaneous analysis of multiple ingredients and provides high specificity because it targets amino acid sequences. Quantification has also become feasible through the use of allergen protein standards. Nevertheless, challenges remain, including the difficulty of selecting appropriate target peptides and MRM transitions to ensure accurate analysis, as well as the lack of an established protein extraction method optimized for LC-MS/MS. This application news introduces sample preparation procedures and analytical methods developed to addressthese challenges.

Development of MRM Transitions

Although several studies have reported the analysis of eight specified allergens using LC-MS/MS, this application describes the development of new analytical methods for walnut, which was newly added to the list of specified ingredients in Japan, as well as for three nuts and five fruits categorized as nonmandatory food allergens (allergens recommended for labeling under Japanese regulation).

Food-derived allergen extracts from walnut, almond, cashew nut, macadamia nut, banana, kiwifruit, orange, peach, and apple were used as standard materials. Each standard was digested into peptide fragments using S-Trap (ProtiFi) and trypsin. For MS/MS analysis used in qualitative evaluation, a system consisting of an ultrafast liquid chromatograph Nexera X3 UHPLC coupled with a quadrupole time-of-flight mass spectrometer LCMS-9030 was employed, and data were acquired in DDA mode. Data analysis was performed using Skyline, with MS Amanda applied as the peptide identification algorithm for high-accuracy matching. Amino acid sequence information for the proteins derived from each ingredient was obtained from the Allergen Nomenclature Database (www.allergen.org) and UniProt. Candidate MRM transitions obtained were optimized using a triple quadrupole mass spectrometer, and transitions were selected based on sensitivity, specificity, and ionization stability. The workflow isshown in Fig. 2, and the identified peptides and their corresponding source proteins are summarized in Table 2. 

When MRM transition candidates are generated solely using Skyline, it is known that several transitions that can actually be ionized are not included among the proposed candidates. By exploring transitions from MS/MS analysis data as performed in this method, such omissions can be effectively reduced.

Allergen Analysis in Processed Foods

To verify the quantitative capability of the analytical method for food allergen analysis, calibration curves were prepared and evaluated. Allergen-free processed food (chicken rice) was spiked with standard allergen materials at concentrations of 1, 5, and 10 mg/kg. After sample preparation (n = 2), LC-MS/MS analysis was performed, and the background and linearity of the calibration curves were assessed. 

As standard materials, protein fractions extracted from 17 food ingredients were used, consisting of eight specified allergens (milk, egg, wheat, buckwheat, shrimp, crab, walnut, and peanut) and nine allergens categorized as equivalent to the specified ingredients under Japanese labeling regulation (soybean, almond, cashew nut, macadamia nut, banana, kiwifruit, orange, peach, and apple). 

Quantitative analysis was conducted using a triple quadrupole mass spectrometer LCMS-8060RX coupled with a Nexera X3 UHPLC system (Fig. 4). A Shim-pack GIST-HP C18 column was employed, and the analysis was performed under a 25-minute method including column washing and equilibration. The analytical conditions are summarized in Table 5. 

In all food samples, good calibration curves were obtained within the range of 1 to 10 mg/kg. Fig. 5 shows the MS chromatograms and calibration curves of nuts and fruits, comparing samples without spike and those spiked at 10 mg/kg. 

No peaks derived from raw ingredients were detected in the non-spiked samples, whereas in the spiked samples, not only the quantifier ion but also multiple qualifier ions were successfully detected.

Conclusion 

New MRM transitions for LC-MS/MS analysis were developed for the specified allergen walnut and eight additional ingredients equivalent to specified ones (almond, cashew nut, macadamia nut, banana, kiwifruit, orange, peach, and apple). Using a triple quadrupole mass spectrometer, spiking experiments were conducted with standard materials for a total of 17 food allergens in processed foods, confirming that good calibration curves could be obtained. The specificity of each peptide was also evaluated, and analyses without false-positive detections were achieved for all ingredients except orange. These results demonstrate that the developed LC-MS/MS method enables simultaneous analysis of 17 food allergens, including those equivalent to the specified ones, in processed foods.

3. Thermo Fisher Scientific: The benefit of binary pump stroke synchronization for more reliable peak identification based on improved retention time precision

• Technical note
• Full PDF for download

Benefits 

• Repeatability: Stable retention times in peptide mapping ensure that the same peptide elutes at exactly the same time in repeated runs. This precision is essential for comparing results across different experiments and for validating the consistency of the method. 
• Accurate peak identification: Consistent retention times help in accurately identifying peaks corresponding to specific peptides. When retention times are stable, it is easier to assign peaks to known peptides based on their expected elution times. 
• Cost savings: Traditionally, LC-MS is the standard method utilizing top-tier UHPLC-MS instrumentation. The Thermo Scientific™ Vanquish™ Flex Binary UHPLC System configured with a variable wavelength detector (VWD) complemented with the binary pump stroke synchronization (HPG Sync) feature provides a biocompatible, cost-effective, robust, UHPLC instrument to standardize the peptide mapping application.

Retention time stability in liquid chromatography (LC) is a critical factor that significantly impacts the robustness of an analytical application. Consistent retention times ensure reproducibility, which is essential for the reliable identification and quantification of analytes. When the same compound elutes at the same time in repeated analyses, it will reduce the risk of misidentification and incorrect quantification. This reproducibility is vital for method validation, as it demonstrates that the method can produce consistent results over time, thereby ensuring the accuracy and reliability of the analytical data.

Moreover, stable retention times are crucial for quality control processes, as they help ensure that products meet required specifications. Any fluctuation in retention time can indicate potential issues with the method or the system, necessitating re-analysis, troubleshooting, or, at the very least, manual peak assignment changes. 

Retention time stability in peptide mapping is crucial for ensuring the accuracy and reproducibility of the analysis. Peptide mapping is a key technique used in the characterization of proteins where peptides generated from enzymatic digestion are separated and identified. Stable retention times allow for consistent identification of peptides across different runs, which is essential for comparing results and detecting any modifications or variations in the protein structure. Variability in retention times can lead to difficulties in matching peptides to their corresponding proteins, potentially compromising the integrity of the analysis. As highlighted by Gilar et al. in their study on peptide mapping, maintaining stable retention times is fundamental for reliable peptide identification and quantification, which is critical for applications in proteomics and biopharmaceutical development.¹

Results and discussion 

The peptide mapping application using tryptic digested Cytochrome C was based on the LC-MS method2 with a variable wavelength detector in place of the MS. A binary pump consists of two pump blocks/drives: A and B. At 250 µL/min and an initial solvent B composition of 1%, pump B responsible for solvent B is delivering 2.5 µL/min. At 2.5 µL/min, pump block B is moving relatively slowly and requires time to come into position for the synchronization. Conversely, the waiting time between injections is reduced when the B block delivers more than 2.5 µL/min. The purpose of the HPG Sync is to time the injection with the exact same piston position for pump block B, which in turn delivers the exact same conditions for pump block B for every injection. This, as a result, increases the retention time stability for all components, especially over a long shallow gradient. 

The implementation of the HPG synchronization (Figure 2) begins in Chromeleon CDS with the instrument configuration where the “Support HPG Stroke Synchronization” is activated to enable the synchronization feature (Figure 2A). Next, the instrument method is created with the “Enable Stroke Synchronization” so that the specific instrument method utilizes the HPG Sync feature (Figure 2B). Finally, the mode in which the HPG stroke synchronization takes place is specified (Figure 2C). The option is scheduling the “prepare inject” command to prepare the injection (needle wash, sample draw, etc). The waiting for the correct piston position is then an extra.

Conclusion 

The positive effects of the HPG Sync implementation on the retention time precision is expressed in a significant decrease in the standard deviation of the retention time of every component during the eluting gradient. The impact of the HPG Sync ON for this example of peptide mapping was shown to improve the application robustness in terms of: 

• Retention time precision: The average retention time standard deviation with HPG Sync OFF is ±0.023 minutes, and the average retention time standard deviation with HPG Sync ON is ±0.007 minutes for all components over the gradient, highlighting an average improvement of retention time repeatability by a factor of more than three times. With a selected customer’s criterion of 0.03 minutes, both modes provide passing results; the HPG Sync ON increases confidence on peak identification. 
• Accurate peak identification: Cytochrome C digest was used as a proxy sample representing a monoclonal antibody digest. The peptide mapping application presents, in general, a challenging gradient method for most HPLC systems, resulting in fluctuating peak retention times, potentially leading to inaccurate peak identification. The impact of the HPG Sync feature on the retention time reproducibility makes for simpler and more accurate component identification, increasing the compatibility for automated workflow integration. 
• Cost savings: Elimination of the MS dependency after the component characterization has taken place can substantially reduce the investment for the routine quality control workflow for peptide mapping. Retention time stability due to exactly controlled cam shaft position at the time of injection increases the method robustness and component mapping and could, therefore, not require the additional MS peak characterization.

4. Waters Corporation: Deep Characterization of Aspartic Acid Isomerism Using High-Resolution Cyclic™ Ion Mobility and Electron-Capture Dissociation

• Application note
• Full PDF for download

Benefits 

• Achieve complete separation – High-resolution cyclic ion mobility fully resolves Asp and isoAsp isomers that co-elute during conventional LC analysis.
• Accelerate workflows – Rapidly distinguish isomeric peptides using multipass Cyclic IMS, reducing reliance on extended LC gradients.
• Lower development costs – Confidently identify Asp and isoAsp variants with ECD fragmentation, minimizing the need for synthesizing reference peptides.

Isomeric amino acid residues have an effect on protein function, sometimes significant enough to contribute to diseases such as Alzheimer’s disease.1 In the realm of biopharmaceutical drug products, these isomerized protein species can have undesired effects on safety or efficacy and therefore must be investigated and characterized. The introduction of structural isomers and stereoisomers can occur during cell culture, downstream processing, or while in storage.^1,2 Asp, specifically, can be present in a protein in four forms: L-Asp, D-Asp, L-isoAsp, or DisoAsp. The most common form is L-Asp, which can isomerize or racemize into the other three forms through a succinimide intermediate (Figure 1).1 This modification is particularly challenging to characterize as it is a relatively small spatial change that is isomeric and therefore cannot be distinguished by typical characterization methods such as mass spectrometry. Various methods for the investigation of Asp and isoAsp are being employed in the industry today. The most common is peptide mapping, in which the protein is enzymatically digested and the resulting peptides are separated chromatographically in order to quantify isomer species by either UV or ion peak areas.³

Cyclic ion mobility mass spectrometry is highly powerful in the separation of isomeric species, including small molecules,⁴ natural products,⁵ lipids,⁶ peptides,_⁷ and oligonucleotides.⁸ By separating on the basis of molecular shape in a high-resolution ion mobility device, Cyclic IMS provides a means to distinguish ions with identical elemental formulae but distinct arrangements of atoms in space. 

Collision-induced dissociation is by far the most widely used fragmentation technique in tandem MS studies, providing high efficiency fragmentation with broad applicability in the structural elucidation of both small and large molecules. However, some types of isomerism are out of reach of CID, which leads to the need for alternative techniques. ECD is an emerging tool capable of identifying peptide isomers directly from tandem MS spectra, depending on the type of isomerism.^9,10 It is well established that ECD can distinguish some isomeric amino acids in leucine- and isoleucine-containing peptides, as well as Asp- and isoAsp-containing peptides, by virtue of characteristic product ions. In the latter case, for an isomerization at position n in a peptide of length m, characteristic ions c(n-1)+57 Da and z(m-n-1)-57 Da can be observed. For further information on comprehensive biotherapeutic characterization using ECD, there is an existing Waters application note.¹¹ 

In this application brief, the depth of characterization achieved using the SELECT SERIES Cyclic IMS Mass Spectrometer equipped with the Waters ECD cell option is demonstrated. The power of high-resolution cyclic ion mobility enables full separation of the four isomer mix of a mAb tryptic peptide with L-Asp, L-isoAsp, D-Asp and D-isoAsp forms, where only partial chromatographic separation is achieved. Furthermore, ECD fragmentation clearly distinguishes the Asp isomers from their isoAsp counterparts, providing unprecedented clarity in the analysis.

Conclusion

High-resolution cyclic IMS combined with ECD fragmentation delivers a powerful solution for resolving and identifying aspartic acid isomers in complex biotherapeutic samples. Unlike conventional LC-MS peptide mapping, which provides only partial separation, multipass cyclic IMS achieves complete resolution of L-Asp, DAsp, L-isoAsp, and D-isoAsp forms, even at low abundance. The integration of ECD further enhances specificity by generating diagnostic fragment ions that distinguish Asp from isoAsp residues. This approach reduces reliance on synthetic standards, accelerates analysis, and improves confidence in structural characterization. Overall, the workflow represents a significant advancement for monitoring critical quality attributes in monoclonal antibodies and other protein therapeutics.