News from LabRulezLCMS Library - Week 16, 2025

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 14th April 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, Knauer, Shimadzu, Thermo Fisher Scientific and Waters Corporation!

1. Agilent Technologies: Toxicological Drug Screening Using the LC Screener Tool with High‑Resolution LC/Q-TOF 

• Application note
• Full PDF for download

Screening biological matrices for the presence of toxicological drugs by DIA offers the benefits of full screening. This screening method is not possible with targeted acquisition since it lacks the ability to retroactively analyze for the continuous emergence of new compounds of interest. Using DIA with applied collision energies furthers the level of information gathered allowing for the ability to differentiate coeluting isomers. The All Ions acquisition technique provides the analyst with full and unfettered access to the analytes of interest along with fragmentation information for identification confidence. 

High-resolution LC/Q-TOF facilitates identification even further with an extended dynamic range, stable accurate mass, and isotopic fidelity. The DIA workflow in complex matrices with the new Revident LC/Q-TOF features key performance elements including a new detector, better mass accuracies, and increased dynamic range compared to previous generations of similar instruments. In addition, the use of ChemVista with LC/Q-TOF spectral libraries and databases can be combined with the LC Screener tool for routine drug analysis testing.

Experimental

Equipment 

Sample separation was performed using the 1290 Infinity II LC system, consisting of the following modules: 

• Agilent 1290 Infinity II high-speed pump 
• Agilent 1290 Infinity II multisampler with thermostat 
• Agilent 1290 Infinity II multicolumn thermostat 

The LC system was coupled to the Agilent Revident LC/Q-TOF mass spectrometer equipped with the Dual AJS ESI source. Agilent MassHunter Workstation software, version 12.0, was used for data acquisition. Agilent MassHunter Quantitative Analysis software, version 12.1, was used for the LC Screener tool and Agilent ChemVista library manager software, version 1.0, was used to curate a forensic toxicology library.

Results and discussion

Confident identifications: mass accuracy, stable area counts, and coeluting fragment ions 

Independent of the matrix, compounds were measured with high mass accuracies, on average within ± 1 ppm and percent relative standard deviations (%RSDs) under 20% for good chromatographic precision. All compounds showed good linearity over all the acquired concentration levels, 1 to 100 ng/mL.

Typical screening parameters were set to mass accuracies of  3, and mass match score. Using the screening parameters, MassHunter Quantitative Analysis 12.1 and the LC Screener tool assign a designation of detected, questionable, or not detected based on alignment that is easy to review. The mass accuracy measurement of all individual analytes is shown in Figure 2, displaying the lowest analyzed concentration (1 ng/mL) on the left to the greatest analyzed concentration (100 ng/mL) on the right, including over 1,900 data points. 

This designation of detected or undetected status by the LC Screener tool is accompanied by clear chromatograms displaying coeluting fragment ions, as shown in Figure 3. The fragment ions are imported from available spectra in curated libraries with retention times developed from neat standards and are available in the Agilent LC/Q-TOF Applied Markets library in ChemVista.

Conclusion 

Using the latest software releases from Agilent including MassHunter Acquisition software, ChemVista library manager, and MassHunter Quantitative Analysis software combined with the Agilent Revident LC/Q-TOF delivers excellence in mass accuracy. With these tools, untargeted screening of drugs in multiple matrices can be done quickly and confidently. This application illustrates high mass accuracy and isotopic fidelity for the toxicology analytes in this study. These results are illustrated across a high concentration range together with low chromatographic %RSDs provided from the Revident LC/Q-TOF. The use of a curated custom forensic toxicology library in ChemVista and use of LC Screener tool provided by MassHunter Quantitative Analysis software achieves a simple and effective screening workflow for forensic and toxicological compounds in complex matrices.

2. KNAUER: Targeted LC-MS/MS detection of lipid impurities in lipid nanoparticles

• Application note
• Full PDF for download

Lipid nanoparticles (LNPs) are a novel drug delivery system which helped combating the COVID-19 pandemics with novel vaccines. The nanoparticles are formed by rapid mixing of an organic phase containing the lipids and an aqueous phase with oligonucleotides. During this process, the active pharmaceutical ingredient (API) is encapsulated in a self-assembled lipid envelope. Currently, the potential of LNPs for several other vaccines and therapeutic applications is investigated. 

With the success of this new drug type comes the responsibility of quality control. Besides other criteria, lipid content and identity must be assessed [1]. In our application note VPH0078, a high-throughput HPLCELSD (evaporative light scattering detection) method for analyzing the lipid content is suggested. Here, we demonstrate the development of an HPLC-MS/MS method for the detection of lipid impurities with KNAUER AZURA® HPLC and the Sciex Triple Quad™ 5500+ mass spectrometer system. 

A suitable separation method for lipids and possible impurities was derived from the KNAUER application note VPH0078 [2]. The MS was optimized and calibrated using standards of the lipids used in the BioNTech-Pfizer vaccine against COVID-19. A list of the most important impurities of this LNP formulation was compiled from literature research. Some of the impurities were already detected in lipid raw materials, including one impurity which can reportedly affect mRNA stability. The newly developed LC-MS/MS method was applied for a stability study on empty and loaded LNPs formulated with the KNAUER Impingement Jets Mixer (IJM) NanoScaler.

RESULTS 

HPLC-MS/MS method development: lipids 

Method development was started by optimizing MS-MS transitions of the parent lipids for multi reaction monitoring (MRM). Lipid solutions were diluted in mobile phase B (Tab. 7) and directly injected into the MS. After selecting the molecule peak from a Q1 scan for the following steps, the masses of two product ions were selected and the single reaction monitoring (SRM) parameters were optimized. 

For the chromatographic separation, an LC method using a gradient with a mix of methanol and acetonitrile as eluent was adapted from the application note VPH0078 [2]. For lower noise and improved separation, the concentration of the modifier ammonium acetate was reduced to 5 mM. The method was transferred to the use of a binary pump by pre-mixing of the solvents of mobile phase B. 

The source parameters were optimized using the LC system with column and a mix standard of the lipids. After optimization, the lipids of the vaccine BNT162b2 were separated and detected within their retention time windows (Fig. 2). A calibration of the lipids was performed to determine the linear response range for each lipid and to enable quantification (Tab. 2).

CONCLUSION 

With the workflow presented here, a MS/MS method for the quantification of lipids and detection of lipid impurities was developed. The method provides the flexibility to monitor targets that are available as a standard or present as impurities in raw materials, as well as targets that are initially not available. At the same time, lipids used for LNP formulations can be identified and quantified. The method was successfully applied to detect impurities and monitor the stability of LNPs formulated with the KNAUER IJM NanoScaler.

3. Shimadzu: Determination of Counter Ions of Synthetic Peptides Using Ion Chromatograph

• Application note
• Full PDF for download

User Benefits

• The purity of synthetic peptides can be confirmed using ion chromatograph.
• Trifluoroacetic acid and chloride ions can be determined simultaneously.
• The system can be selected according to the purpose, such as a suppressor type when used in conjunction with high-sensitivity analysis such as the impurity measurement of inorganic anions in pharmaceuticals, or a non suppressor type with a simple equipment configuration when focusing on cost performance.

The middle molecule drug is attracting attention as a next generation drug discovery modality. The middle molecule drugs have a smaller molecular weight than biopharmaceuticals, and there are oligonucleotide therapeutics using oligonucleotides and peptide therapeutics with peptide skeletons. Among them, peptide therapeutics have the advantage that they can be produced at low cost, easily taken into cells due to their small molecular weight, and can suppress degradation when taken into the body by adopting a specific conformation. 

Since trifluoroacetic acid (TFA) is used to extract the synthesized peptides from the stationary phase, the recovered peptides are converted into peptide TFA salts in which TFA is ionically bonded. The weight of the lyophilized peptide includes the weight of this TFA, which greatly affects the actual peptide content. In addition, TFA may affect bioavailability, and it is necessary to replace it with a salt such as hydrochloride. Therefore, it is essential to quantify the counterion to confirm the purity ofsynthetic peptides. 

In this paper, we report an example of counter ion analysis using an ion chromatograph (IC) using crude purified linear and cyclic synthetic peptides.

Suppressor-type Ion Chromatograph

The suppressor-type ion chromatograph is equipped with a suppressor to improve sensitivity by reducing the background conductivity contained in the eluent, and is compatible with high-sensitivity analysis of the order of μg/L. Fig.2 is a flow chart of a suppressor ion chromatograph (Shimadzu HIC-ESP). The analytical conditions are shown in Table1, and the chromatogram of 10 mg/L trifluoroacetic acid (TFA) and 5 mg/L chloride (Cl) ion mixed standard solution is shown in Fig. 3.

Analysis of Synthetic Peptides 

TFA and Cl ion were determined using PTH (Bachem AG code: H4835.001) and Somatostatin (Bachem AG code: H-1490.005) as real samples. The calibration point was set at 3~4 points over a concentration range of approximately 20 times, centering on the quantitative value. Peptides that were replaced to Cl ion were quantified using HPLC, and the quantified values were used to calculate counter ion concentrations. 

Analysis of TFA 

PTH and somatostatin were each dissolved in ultrapure water to determine TFA. The chromatogram of PTH is shown in Fig. 6, the chromatogram of somatostatin is shown in Fig. 7, and the quantitative results of TFA are shown in Table 3. We confirmed that the ratio between the actual quantitative value and the theoretical TFA value calculated from the structure of each peptide was 0.9~1.2, which was almost the same. Similar results were obtained for both the non-suppressor and suppressor systems.

Analysis of Cl ion 

PTH and somatostatin were each dissolved in ultrapure water, hydrochloric acid was added and freeze-dried. They were dissolved in ultrapure water and analyzed for Cl ion. The chromatogram of PTH Cl salt is shown in Fig. 8, the chromatogram of Somatostatin Cl salt is shown in Fig. 9, and the determination result of Cl ion is shown in Table 4. It was confirmed that the ratio of the quantitative value to the theoretical value of Cl ion was approximately equal to 1.1 for both peptides. Similar results were obtained for both the nonsuppressor and suppressorsystems.

Conclusion

In order to confirm the purity of synthetic peptides, counter ions were quantified using IC. It was confirmed that both the suppressor-type and the non-suppressor-type ICs can obtain quantitative values similar to theoretical values calculated from peptide structures. Therefore, when performing this analysis, it is possible to select a system according to the purpose, such as the suppressor-type system when combined with highsensitivity analysis such as the impurity measurement of inorganic anions in pharmaceuticals, or the non-suppressortype system with a simple equipment configuration when focusing on cost performance. From these results, we confirmed that multiple counter ions can be quantified stably without depending on differences in the amino acid sequence or chain length of peptides. This method is a useful method for confirming the purity of synthetic peptides, because the amount of synthesized peptides can be accurately calculated by determining the quantitative value of counter ions.

4. Thermo Fisher Scientific: Comprehensive PFAS screening in pharmaceutical packaging and medical devices by LC-HRAM-MS

• Application note
• Full PDF for download

Application benefits

• Combined targeted quantitation and non-targeted screening for PFAS compounds from one injection is achieved.
• One LC-MS method provides both PFAS-specific and general extractables screening.
• Targeted analysis of a list of PFAS compounds yields unequivocal identification and quantification down to sub-ppb levels.
• Non-targeted analysis reveals additional PFAS contaminants in the sample extracts that could be quantified using surrogate standards.
• Use of the PFAS analysis kit and delay column minimizes background interference and increases confidence in the analytical results.
• Use of Thermo Scientific™ Chromeleon™ CDS provides a 21 CFR Part 11 complianceready solution for data acquisition and quantitative analysis in extractables screening.

Per- and polyfluoroalkyl substances (PFAS) are known for their persistence in the environment and in the human body, leading to potential health issues. Regulatory agencies like the FDA and EPA have set stringent guidelines and limits for PFAS. For example, on April 10, 2024, the EPA announced the final National Primary Drinking Water Regulation (NPDWR) requiring monitoring for six PFAS in the nation’s public water supplies.1 The EPA expects that over many years the final rule will prevent PFAS exposure in drinking water for approximately 100 million people, prevent thousands of deaths, and reduce tens of thousands of serious PFAS-attributable illnesses. However, there is still no regulatory guidance on PFAS levels present in pharmaceutical products and medical devices, which could compromise product safety and efficacy for drug products. As such, medical device and pharmaceutical companies should be proactive by staying up to date with current and future regulations and develop risk mitigation strategies to avoid costly product recall or delays in approvals. To that end, having the ability to detect and quantitate PFAS in various pharmaceutically relevant test materials is essential. 

Here, we report an LC-MS based analytical strategy to test for PFAS that could be extracted from manufacturing components and containers as part of extractables screening. To demonstrate its utility, it was applied to extracts of two fluorine-containing polymer components in collaboration with the E&L group at SGS Health Science, Fairfield, NJ.

Experimental

Instrumentation

The LC-MS analysis was performed using a Thermo Scientific™ Vanquish™ Horizon UHPLC system coupled to an Orbitrap Exploris 120 high-resolution mass spectrometer (P/N BRE725531) equipped with the Thermo Scientific™ OptaMax™ NG source housing and using the heated electrospray ionization (HESI) probe. 

Software 

The Thermo Scientific™ Chromeleon™ Chromatography Data System (CDS) 7.3.2 was used for data acquisition and quantitative analysis of the LC-MS data. For qualitative MS data processing and differential analysis, data were imported into Thermo Scientific™ Compound Discoverer™ 3.3 SP3 software for spectral deconvolution and compound identification using the workflow template “PFAS Unknown ID w Database Search and Molecular Networks” with modifications to also include positive mode data and search against the Epoxidized Soybean Oil Library5 and a custom E&L-specific library in the mzVault node, as well as a mass list generated from the PFAS standard mixture including retention times.

Results and discussion

Concurrent screening for non-fluorinated extractables 

As described above, the data acquisition in this work was carried out using a polarity-switching method, which enabled the simultaneous detection and identification of other extractables originating from the tubing and bottle, respectively, in either ionization mode. Especially the more non-polar isopropanol extract was found to contain various plasticizers at appreciable levels, including trioctyl trimellitate (TOTM) and several epoxidized triglycerides - common constituents of epoxidized soybean oil (ESBO)—that could be identified with high confidence based on matching to the custom spectral library generated from such compounds in a separate application note (and included with the Compound Discoverer 3.3 SP3 software).5 

Lastly, an additional benefit of the delay column was found for the detection of extractables in the sample that are frequently present in LC-MS systems or solvents, leading to large background interference, such as aliphatic acids (e.g., palmitic acid, stearic acid, or oleic acid) and surfactants (e.g., dodecylbenzene sulfonic acid). As shown in Figure 8, the system peak was shifted to later retention times with the delay column (positioned ahead of the autosampler in the flow path). This enabled the interferencereduced detection of the compounds originating from the sample, which might otherwise be filtered out in the data processing due to the peak area in the sample not significantly differing from that in the extraction blank, caused by the introduction from the system instead of being an actual extractable compound.

Conclusion 

In this work, we have developed a comprehensive solution for the targeted and non-targeted screening for PFAS as part of the E&L analysis of pharmaceutical packaging and processing material components using the Vanquish Horizon UHPLC system coupled to the Orbitrap Exploris 120 mass spectrometer and the combination of Chromeleon CDS 7.3.2 and Compound Discoverer 3.3 software.

• The LC-MS analysis with a polarity switching Full Scan-ddMS2 method allowed the simultaneous identification of known and unknown suspected PFAS, as well as unknown extractables, with high confidence due to the excellent sensitivity and mass accuracy of the Orbitrap detector. 
• The screening and targeted quantitation of 17 common PFAS could be carried out with the Full Scan data with high sensitivity (LOQs ranging from 0.1 to 1 ppb) and minimal background interference from the analytical system with the use of the PFAS analysis kit. 
• The result of the analysis of fluorinated test materials for pharmaceutical applications demonstrated the ability to detect and identify PFAS at low ppb to sub-ppb levels, including five suspected PFAS found in the non-targeted analysis. 
• The use of the delay column also benefits the analysis of extractables that are frequent contaminants of LC-MS systems by separating the system peak from the sample peak. 

The presented approach should have broad applicability to the screening for PFAS compounds in E&L, as well as other pharmaceutical testing and beyond.

5. Waters Corporation: Characterization and Sequencing of Duplex siRNA Using the BioAccord™ LC-MS System and Vion IMS QTof Mass Spectrometer in Combination With the waters_connect™ CONFIRM Sequence App 

• Application note
• Full PDF for download

Benefits

• Develop quality control methods with automated compliance-ready LC-MS workflows that are accessible to non-MS expert
• Assess duplex purity, duplex identity, and duplex sequence from a single analysis
• Facilitate fast and automated interpretation of complex tandem mass spectra (MS/MS) and data-independent MSE (no specific precursor ion selection) siRNA Duplex mass spectra using the CONFIRM Sequence App within the compliance ready waters_connect informatics platform 

Here, we introduce a routine LC-MS methodology employing ion-pairing reversed phase chromatography (IPRP) coupled with data-independent (no precursor selection) MSE fragmentation for the detection and sequencing of siRNA duplexes. This method leverages automated data processing coupled with a highly accessible BioAccord LC-HRMS System, operating under waters_connect informatics, to support complianceready data acquisition, processing, and reporting.

The BioAccord LC-MS System (Figure 1) was introduced in 2019 as a compact, robust platform for routine biopharmaceutical analysis that is accessible to non-MS experts. The fully integrated BioAccord LC-MS System used here is comprised of an ACQUITY™ UPLC™ Premier I-Class PLUS System, a Tunable Ultraviolet (TUV) Optical Detector, and an ESI-TOF ACQUITY RDa™ Mass Detector. The sequencing results were then compared against targeted LC-MSMS analysis using the Vion IMS-QTof MS instrument. 

Unlike the regular (unmodified) oligonucleotides, siRNA molecules can be challenging to characterize by LC-MS due to their complexity and stability. By controlling and adjusting column temperature we can induce duplex conformer dissociation into Sense and Antisense strands increasing MS response and decreasing spectral complexity. This allows for improved sequence coverage, strand identification, and purity to be evaluated from a single injection. Alternately, we can decrease column temperature to preserve duplex conformation on column, allowing identification, and purity to be measured at full duplex level. The combination of these two approaches within a single analysis were evaluated for their ability to provide insights into the quality attributes of the duplex siRNA molecule studied.

Results and Discussion 

With the goal to streamline and efficiently evaluate duplex purity and identity, identify impurities, and confirm the correct sequences of both sense and antisense strands, we attempted to apply LC-MS/MS sequencing to denatured and nondenatured duplex siRNA oligonucleotides in a single analytical analysis. BioAccord LC-MS data were acquired in full scan MSE data-independent fragmentation mode and was acquired and processed on the waters_connect informatics platform. Automated spectral deconvolution was performed in the waters_connect INTACT Mass App using the combined ESI-MS spectra under the TIC peak to obtain an intact oligonucleotide mass measurement for strand identification, purity assessment, and modification localization. Oligonucleotide sequence analysis was then conducted using the waters_connect CONFIRM Sequence App. Finally, Vion IMS QTof data were collected in a targeted MS/MS mode to provide comparison against the BioAccord data-independent acquisition (DIA) sequence results.

Experiments conducted using LC column temperatures of 25 °C (non-duplex denaturing) and 60 °C (denaturing) using the BioAccord System or Vion IMS QTof mass detection demonstrate how these instruments can assess various parameters of double stranded oligonucleotides in a single analytical run. 

At a column temperature of 25 °C, the duplex siRNA molecule is preserved on column (see Figure 2). Individual samples of the sense and antisense strands are chromatographically resolved from each other and the duplex molecule. While the moderate temperature maintains the duplex molecule’s intact structure throughout the LC analysis, the use of ion pairing reagents and low pH result in the denaturing of the complex upon electrospray ionization. This enables duplex spectra to be reviewed and characterized as individual strands (Figure 3A). 

The data were processed through the automated waters_connect INTACT Mass App which enables scientists to analyze both shorter and longer oligonucleotides that supports processing capabilities such as TIC and UV peak detection, BayesSpray charge deconvolution, modification, and product-related impurity assignments and purity calculations for streamlined nucleic acid analysis without requiring significant user interventions. (Figures 3B and 3C).

The INTACT Mass application results (Figure 3C) facilitated the assignment of deconvoluted species co-eluting under the duplex chromatographic peak at 5.13 minutes. Due to this co-elution, the LC-UV purity is reported as 90% for all species. The INTACT Mass App also calculated the MS purity by considering all assigned co-eluting species as the product of LC and MS relative responses (LCxMS). Low ppm (<10 ppm) mass errors were observed for all deconvoluted identifications for both major and minor intensity species.

Conclusion 

Denaturing (higher column temperature) and non-denaturing (lower column temperature) LC-MS methods have been developed that can assess the single strand purity, single strand identity, duplex purity and duplex identity of a duplex siRNA molecule using TOF or a QTof MS detection. 

These methods made use of the waters_connect INTACT Mass and CONFIRM sequence Apps provide data analysis automation for intact mass and fragmentation based sequencing analysis. 

Complete sequencing was obtained for both siRNA strands on the BioAccord LC-MS System using a dataindependent MS fragmentation approach, that benefited from the chromatographic separation obtained in the denaturing LC separation. 

Complete sequencing was obtained for both siRNA strands on the Vion IMS-QTof system by targeted MS/MS that was more tolerant of oligonucleotide strand co-elution in the non-denaturing LC separation. 

The workflows explored in this application note show the complementary nature of the use of non-denaturing and denaturing duplex siRNA analysis, and the ability to make use of more complex QTof instrumentation in a development environment, and the more fit-for-purpose BioAccord LC-MS System for routine analysis in development or for validatable assays in regulated manufacturing and quality organizations