News from LabRulezLCMS Library - Week 11, 2026

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 9th March 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, KNAUER, Shimadzu and Waters Corporation and technical note by Thermo Fisher Scientific!

1. Agilent Technologies: Drug Metabolite Identification with a Streamlined Software Workflow

Combining Agilent Revident Q-TOF LC/MS and MassHunter Explorer 2.0

• Application note
• Full PDF for download

Understanding how drugs are metabolized is essential for predicting their efficacy, safety, and potential interactions in vivo. Drug metabolite identification provides critical insights into pharmacokinetics and pharmacodynamics, yet remains a complex task, partly due to the absence of biotransformation products in public spectral libraries. This limitation is especially pronounced during early drug discovery, where novel compounds and their metabolites lack reference spectra, complicating efforts to characterize low-abundance or structurally diverse transformation products. 

To overcome these challenges, we developed an integrated workflow that combines high-resolution mass spectrometry with advanced data analysis and structure prediction tools (Figure 1). The approach uses the Agilent Revident Q-TOF LC/MS system for sensitive and accurate mass detection, Agilent MassHunter Explorer 2.0 for chemometric feature extraction and statistical filtering, and SIRIUS for molecular fingerprint-based structure elucidation. A custom structure database generated using BioTransformer further enhances the workflow's ability to identify metabolites not present in conventional libraries.

Verapamil was selected as a model compound due to its well‑documented metabolic profile, making it a reliable benchmark for evaluating the workflow's performance. As a widely used cardiovascular drug, its known biotransformation products support validation of untargeted metabolite identification strategies. In this study, human liver microsomes were used to simulate in vitro metabolism, and differential analysis was applied to isolate statistically significant features associated with drug treatment. The workflow successfully identified known verapamil metabolites, demonstrating its utility for untargeted metabolite discovery and its adaptability for future applications involving new chemical entities

Experimental

Instrumentation 

An Agilent Infinity III LC system was used, consisting of: 

• Agilent 1290 Infinity III high-speed pump (product number G7120A) 
• Agilent 1260 Infinity III hybrid multisampler with thermostat (product number G7167C) 
• Agilent 1290 Infinity III multicolumn thermostat (product number G7116B) 

The setup also included the Agilent Revident Q-TOF LC/MS system equipped with an Agilent Dual Jet Stream technology source (product number G6575A).

Identification of verapamil metabolites 

As verapamil is a well-characterized drug, its parent compound and several metabolites are likely present in established MS/MS libraries. MassHunter Explorer supports spectral library matching, and to demonstrate this capability, a library search was performed against the Agilent Applied Markets PCDL, which contains numerous known drug compounds and metabolites (Figure 7A). Verapamil and a few of its known metabolites, norverapamil, D-617, and D-620, were confidently identified using MS1 scoring and MS/MS reference library matching. Furthermore, these metabolites were included among the 88 significant compounds, reinforcing the effectiveness of the statistical filtering strategy for prioritizing compounds of interest. 

However, in typical drug discovery pipelines, resulting novel drugs and their metabolites would not be found in public spectral libraries, necessitating an alternative identification approach. For this purpose, the 88 compounds and their extracted MS and MS/MS spectra were exported to SIRIUS software through the single-click option in MassHunter Explorer (Figure 7B). The extracted compound corresponding to verapamil was additionally exported and added to the same project within SIRIUS.

Conclusion 

This application note demonstrates a streamlined workflow for drug metabolite identification, integrating the Agilent Revident Q-TOF LC/MS system with Agilent MassHunter Explorer 2.0 and SIRIUS software. Using verapamil as a model compound, the workflow successfully identified both known and novel biotransformation products through a combination of chemometric filtering, MS/MS acquisition, and molecular fingerprint-based structure elucidation. 

Key innovations include the use of the Agilent 1260 Infinity III hybrid multisampler with Feed Injection, which enabled direct injection of minimally prepared microsomal supernatants while preserving chromatographic integrity and peak shape— critical for accurate feature extraction and quantification. The differential analysis strategy in MassHunter Explorer 2.0 effectively prioritized drug-related features from complex datasets, while the coordination with SIRIUS software and its integration of a custom BioTransformer-generated database allowed confident identification of metabolites not present in traditional spectral libraries. 

Although verapamil is a well-characterized drug, the workflow was designed with flexibility to accommodate novel compounds, making it particularly valuable in early-stage drug discovery where reference spectra are unavailable. The approach is additionally applicable to other xenobiotics and other types of transformations, offering a powerful solution for metabolite identification in broad research and development settings.

2. KNAUER: Optimizing molecular weight determination: A comprehensive guide to SEC/GPC

• Application note
• Full PDF for download

Gel Permeation Chromatography (GPC), Size Exclusion Chromatography (SEC) and less colloquial Gel Filtration Chromatography (GFC) are names used to describe the same liquid chromatographic technique required to determine important properties of macromolecules, like polymers, biopolymers, polysaccharides, or proteins. For example, the molecular weight distribution and some structural properties of polymers and proteins can be determined in a single injection using standard equipment for liquid chromatography, providing critical insight into their functionality and performance. 

The separation process (Fig. 1) behind each of these terms, is the same, it is based on hydrodynamic volume, not molecular weight, but scientists from different backgrounds talk about the technology in different ways. The term GPC is mostly used by chemists and scientists working with synthetic polymers or plastics. Many of these materials are only soluble in organic solvents, so the term GPC is often associated with separations in organic solvents. The term SEC, and less commonly GFC, is mostly used by biologists or scientists who need to purify or separate biological molecules such as proteins or oligonucelotides. This usually requires an aqueous mobile phase, so the term SEC or GFC is frequently used for separations in aqueous solvents.

RESULTS

The coupling of columns in GPC/SEC is a common method to increase the resolution or extend the separation range. The choice of columns to be coupled is therefore crucial. If columns with identical pore sizes are combined, the resolution increases. In contrast, combining columns with different pore sizes expands the separation range. A direct comparison of the two coupling methods is summarized in Tab. 2. In this application, two columns with same base material but different pore sizes of 150 Å and 350 Å were coupled together with increasing pore size, in order to increase the separation range to 100 - 1 000 000 Da (Tab. 1).

Before combining columns with different pore sizes, make sure that your columns do overlap in their separation ranges to avoid gaps in your resolution range. An example of an inefficient combination would be the use of a SuperOH-P-100 column (separation range: 100 - 2 500 Da) together with a SuperOH-P-400 column (separation range: 10 000 - 5 000 000 Da), which would result in not covering the range between 2 500 - 10 000 Da. At the same time, it should be noted that a significant loss of resolution can be caused in the overlapping separation range. This is because molecules may be retained differently in these areas due to different retention mechanisms of the columns, resulting in broader peaks in the chromatogram and incorrect molar masses. Consequently, the choice of columns when combining is critical. Regarding the order in which the columns should be combined, it can be said that the order has no impact on retention times or molecular weight distribution calculations^3,4. Columns can be coupled with either increasing or decreasing pore size, as long as the sequence remains consistent^3,4.

CONCLUSION 

Using the KNAUER AZURA® GPC/SEC system it is possible to create calibrations with strong organic as well as aqueous solvents. Furthermore, the system can be customised or upgraded to meet specific requirements. For example, an additional detector can be integrated. 

To ensure good reproducibility of test results, it was essential to meet the minimum requirements for peak broadening, as indicated by the number of theoretical plates. Therefore, all used columns were successfully tested based on official DIN EN ISO regulations. For calibration with pullulan standards in water, a combination of two columns with different pore sizes was employed to increase the separation range, while for calibration with PS standards in THF, a column with a larger separation range was used. The calibrations were evaluated using samples of known molar mass, specifically sugar for the pullulan calibration and PS standards for the PS calibration. The deviations were found to be less than 4% for the PS calibration and less than 3% for the pullulan calibration. Consequently, the accuracy of the calibration can be confirmed within these ranges. In summary, users can significantly improve the reproducibility and reliability of their analytical results by following common guidelines and considering key factors.

3. Shimadzu: Achieving Solvent Reduction and Lower Running Costs with Nexera X4

• Application note
• Full PDF for download

In recent years, there has been growing interest in sustainability within analytical laboratories. In liquid chromatography, the large volumes of organic solvents used as mobile phases present challenges from both environmental and cost perspectives. One approach to reducing solvent consumption is to downscale analyses by using small-diameter columns. Such solvent-saving strategies not only contribute substantially to lowering running costs, but also reduce the amount of waste solvent, enabling more environmentally conscious laboratory operations. However, analytical downscaling requires effective suppression of band broadening within flow channels and highly accurate low-volume injections. Nexera X4 (Fig. 1) delivers exceptional instrument performance that meets these demands and provides stable analytical results even when small-diameter columns are used. This article presents a case study in which analysis was downscaled and solvent consumption was significantly reduced by combining Nexera X4 with a 1-mm-ID column (Shim-pack NovaCore C18-HB).

Conclusion

This article introduced an approach for reducing solvent consumption by using small-diameter columns. Reducing solvent usage not only lowers running costs but also contributes to reducing environmental impact by decreasing the amount of waste solvent. Although suppressing dispersion within the flow path is essential when scaling down an analysis, Nexera X4 maximizes the performance of small-diameter columns through itslow-dispersion design.

4. Thermo Fisher Scientific: Beyond the box: A ready-to-run workflow for quantitating 80 drugs of abuse in whole blood with the TSQ Certis Triple Quadrupole MS

• Technical note
• Full PDF for download

Application benefits 

• A quantitative workflow for 80 drugs of abuse in whole blood featuring polarity switching and a 4.5-minute analysis equaling 320 samples per day on the TSQ Certis mass spectrometer
• Proven instrument robustness for high-throughput toxicology workflows in complicated matrices
• Streamlined sample preparation with efficient protein precipitation and filtration using INTip™ Filtration (DPX Technologies)

The accurate and reliable quantitation of drugs of abuse in whole blood is critical for forensic toxicology and clinical research investigations. Analytical laboratories face ongoing challenges in meeting increasing demands for high-throughput, sensitive, and reproducible testing while maintaining compliance with regulatory standards. Advances in mass spectrometry technology have significantly enhanced analytical capabilities, enabling broader compound coverage and improved confidence in results.

This technical note presents an end-to-end workflow for the quantitation of 80 commonly encountered drugs of abuse in whole blood using the TSQ Certis triple quadrupole mass spectrometer. The workflow integrates efficient sample preparation, robust chromatographic separation, and optimized mass spectrometric detection to deliver exceptional sensitivity, selectivity, and quantitative accuracy. Leveraging the latest innovations in instrument design of >900 SRM/second acquisition speed and less than 5 ms polarity switching, this method provides a powerful solution, enabling analysis of 320 samples/day for laboratories seeking to streamline operations and ensure consistent, high-quality data in complex biological and forensic matrices.

Experimental

Liquid chromatography 

Drug analytes were separated with a Thermo Scientific™ Accucore™ Biphenyl Column (2.1 × 50 mm, 2.6 µm, Cat. No. 17826-052130) connected to a Vanquish Flex UHPLC system. Mobile phases consisted of 0.1% formic acid and 2 mM ammonium formate in water for mobile phase A and 0.1% formic acid and 2 mM ammonium formate in MeOH mobile phase B.

Mass spectrometry 

Data was acquired on the TSQ Certis mass spectrometer using SRM mode for quantitative and confirming transitions for each target compound and a quantitative transition for the internal standards. Table 2 highlights the source parameters and values. Q1 resolution was 0.7 FWHM, and Q3 resolution was 1.2 FWHM. The SRM list contained the retention times, precursor and product ions m/z, optimized RF lens voltages and collision energies, and retention times (Appendix 1).

Data analysis 

Data was acquired and processed with Thermo Scientific™ TraceFinder™ Software, version 5.2. In cases where no existing method parameter set is available, the Method Forge tool in TraceFinder software can be used to automatically build a complete target list directly from an instrument raw file. In this workflow, a raw file from one of the calibrators was loaded into the Method Forge tool, which extracted the precursor ions, product ions, retention times, and associated transition parameters defined in the instrument method (Figure 4). 

The automated extraction tool created a fully populated master method without the need for manually entering compound information, and the resulting information was then saved into the TraceFinder software compound database, allowing the target list and associated transition parameters to be reused for future analyses. This newly created master method was subsequently applied to process all calibration and study samples.

Conclusion 

The developed workflow enables rapid, accurate, and reproducible quantitation of 80 drugs of abuse in whole blood using the TSQ Certis triple quadrupole mass spectrometer. With low limits of quantitation down to 0.05 ng/mL, excellent linearity, and stable performance across more than 450 injections, the method demonstrates strong sensitivity and robustness. Additionally, TraceFinder software further enhances this workflow by streamlining and automating data processing method creation and data analysis with a comprehensive set of tools that are applicable to a broad range of toxicology workflows. By combining efficient sample preparation, fast chromatographic separation, and reliable SRM detection, this workflow offers a practical and high-performance solution for forensic and clinical toxicology laboratories seeking confident, high-throughput results.

5. Waters Corporation: A Combined Method for Anionic, Cationic, and Zwitterionic PFAS using a Direct Injection UPLC™-MS/MS Method for Environmental Water Samples

• Application note
• Full PDF for download

Benefits 

Expanding the scope of PFAS analysis in a single injection to include anionic, cationic, and zwitterionic forms.

• A sensitive, direct injection method for the analysis of PFAS in environmental water samples in accordance with the regulatory requirements for environmental water testing in the EU and U.K.
• High sample throughput with reduced downtime due to the extreme robustness of the Xevo TQ Absolute XR Mass Spectrometer.
• Quantitative method for 58 native and 23 ILIS PFAS with fast polarity switching demonstrating that the analytical scope can be readily extended to include emerging PFAS with different chemical properties.
• Time and cost savings due to the efficiency gains in all aspects of method development, data processing, and review using waters_connect™ for Quantitation Software.

This application note describes an increase to scope of the direct injection MRM methodology previously reported 1 to include representative cationic and zwitterionic PFAS suitable for the analysis of surface, ground, treated and wastewater samples. To achieve the sub ng/L level of detection in a variety of water types, an ACQUITY™ I-Class PLUS System was coupled to the Xevo TQ Absolute XR Mass Spectrometer with UniSpray Ionization Source. Due to the chemical properties of the cationic/zwitterionic PFAS, positive polarity ionization mode was selected as the most sensitive mode for these compounds. The rapid polarity switching capability of the Xevo TQ Absolute XR Mass Spectrometer was utilized to develop a quantitative MRM method for 58 native PFAS and 23 isotopically labeled internal standards.

Experimental

• LC system: ACQUITY I-Class PLUS System with BSM and FTN. Sample Manager fitted with Waters PFAS Kit and Atlantis™ Premier BEH™ C18 AX 5 µm, 2.1 x 50 mm Isolator Column (p/n: 186009407)
• MS system: Xevo TQ Absolute XR Mass Spectrometer
• Software: waters_connect for Quantitation Software

Results and Discussion

Detection of PFAS in an Unknown Wastewater Sample 

A sample of previously uncharacterized ‘trade effluent’ wastewater was analyzed in duplicate following the developed procedure. The trade effluent sample was quantified against solvent standards covering the concentration range 0.24 to 240 ng/L. A total of 16 PFAS were identified (passing the confirmatory requirements) in the wastewater exceeding the Environmental Quality Standard (EQS) reporting level of 0.65 ng/L.16 The PFAS with concentrations exceeding 20 ng/L are shown in Figure 9. The highest concentration detected was L-PFHxS at 172 ng/L, with L-PFOS at 134 ng/L and L-PFOA at 119 ng/L. 

Figures 10 and 11 show the ‘Unknown’ workflow views in MSQuan. The ‘By Analyte’ view comparing the trade effluent sample (replicate 1) to a solvent standard at 120 ng/L showing retention time alignment and closely matching ion ratio for is shown in Figure 10 for PFOA. Figure 11 shows ‘By Injection’ view visualizing the concentrations of the zwitterionic PFAS, 6:2 FTAB detected in the samples (unknowns and QCs) across the batch. 

The data demonstrates the potential of high concentrations of PFAS in U.K. wastewater with identifications of anionic, cationic, zwitterionic forms and degradation products. The concentrations of PFAS identified ranged from 0.975 (PFDA) to 172 ng/L (L-PFHxS). These findings reinforce the requirements to expand the scope of PFAS in targeted analytical methods to monitor the occurrence of contamination in environmental water samples. Due to the complexity of such samples and the advantages of direct injection the choice of MS system is critical in terms of adequate sensitivity, linear dynamic range and robustness to withstand 1000’s of injections of complex extract with minimal maintenance.

Conclusion 

Historically, most of the research into PFAS contamination, ecotoxicity and remediation strategies has focused on the anionic forms of PFAS. More recently, increasing reports of the occurrence of cationic and zwitterionic forms, frequently originating from AFFF impact sites is driving the need to expand the scope of targeted MRM methods for quantitative analysis of PFAS in environmental water samples. Due to the chemical properties of the cationic and zwitterionic PFAS, they are found to ionize more favorably in positive polarity mode in contrast to the anionic forms where negative polarity mode predominates. UniSpray Ionization Source has previously been shown to offer unique benefits in terms of enhanced sensitivity for PFAS ionization of the labile species.¹ 

Expanding the analytical scope to include anionic, cationic and zwitterionic forms demands the MS system to be capable of fast polarity switching speed to deliver dwell times necessary to achieve (at least) the minimum number of data points per peak for reproducible quantitative performance. Direct injection methods have become increasingly popular as they allow laboratories to maximize throughput efficiency, however, they can put strain on the analytical system in terms of maintaining optimum performance in the presence of often complex extracts injected at large volumes (> 20 µL) with minimal downtime.