News from LabRulezLCMS Library - Week 33, 2024

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 12th August 2024? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!

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This week we bring to you posters, applications, and other documents from Agilent Technologies, Shimadzu, Thermo Fisher Scientific, and Waters Corporation!

1. Agilent Technologies: Identification of Amino Acid Isomers Using Electron Capture Dissociation in the Agilent 6545XT AdvanceBio LC/Q-TOF System

• Application

Abstract: Accurately determining the amino acid sequence is crucial for understanding a protein's structure and function. However, distinguishing between leucine (Leu) and isoleucine (Ile) poses a challenge, because they are positional isomers and cannot be differentiated using traditional collision-based fragmentation techniques. Electron‑based fragmentation offers a promising solution by producing side chain fragments that can be used to distinguish between Leu/Ile and other isobaric residues such as aspartate (Asp) and isoaspartate (isoAsp). This application note demonstrates the identification of isobaric residues in peptides and intact proteins using the Agilent 6545XT AdvanceBio LC/Q-TOF system equipped with an ExD cell for electron capture dissociation. Agilent ExDViewer software enables intuitive analysis of side chain fragmentation in Q-TOF datasets, enhancing the ability to discern between isobaric amino acids.

Conclusion: This application note describes the analysis of isobaric amino acids using an Agilent 6545XT AdvanceBio Q-TOF LC/MS system equipped with an Agilent ExD cell and the Agilent ExDViewer software tool for analysis. Electron capture dissociation is a powerful fragmentation technique that provides complementary information to CID fragmentation. These methods can be applied to investigate peptide backbone and side chain fragmentation for a range of molecules, reducing ambiguity in protein sequence analysis. The sensitivity of the Q-TOF to detect ECD fragments combined with ExDViewer for fragment analysis provides an effective solution for targeted amino acid isobar identification.

2. Waters Corporation: MODERNIZATION OF A LEGACY NORMAL PHASE METHOD ON A MODERN HPLC SYSTEM

• Poster / HPLC 2024

INTRODUCTION

In regulated laboratories, particularly those within the pharmaceutical industry, High-Performance Liquid Chromatography (HPLC) systems play a crucial role in routine analysis. However, some challenges arise due to legacy methods that were developed during the early days of HPLC technology. These methods have not kept pace with recent advancements and modernization in the field and do not fully leverage the latest technological improvements. Advancements in column chemistry and system robustness allow for better separation and detection of analytes.

Modern HPLC systems are more robust, reliable, and capable of handling challenging analyses and can accommodate various chromatographic modes, including Normal Phase chromatography. While legacy methods persist, regulatory agencies such as the United States Pharmacopeia (USP) provide guidance on how to adapt and adjust these methods. This helps bridge the gap between legacy methods and modern HPLC systems.

In this study, we examine the modernization of a legacy normal phase method, USP Propofol Assay, for use with a HPLC system. The original method specified an older L3 column in a non-standardized column configuration (4.6 mm x 200 mm). Given these column specifications, we compared a suitable column to coupling two columns to match the specified column dimensions. We also explored the use of a column that was scaled in accordance with USP Chapter 6211 that meets the L/dp requirement.

CONCLUSION

• Legacy columns, like the L3 used for column 1, has an older packing technology and is unable to provide a suitable chromatography.
• The use of a newer packing as in BEH Columns helps provide a successful analysis
• Coupling two columns, albeit successful, may prove to be a much more expensive option and may be introducing more opportunities for issues to arise.
• The third column derived from the use of the columns calculator may be the more suitable route since it adheres to the USP guidance as well as not being more expensive and time consuming as the coupling of two columns.

3. Waters Corporation: THE UTILITY OF CYCLIC ION MOBILITY TO IMPROVE SELECTIVITY AND ANALYSIS EFFICIENCY OF ENVIRONMENTAL PFAS CONTAMINATION AND EXPOSURE

• Poster / ASMS 2024

INTRODUCTION

Polyfluorinated alkyl substances (PFAS) exposure is a potential contributor to increased cancer occurrence in the human population. The bio-accumulative nature of PFAS enables monitoring of levels in human biofluids to help gain understanding into exposure levels and pathways. However PFAS isomeric compounds, can be challenging to efficiently separate using liquid chromatography.

Cyclic ion mobility (cIM) provides an added dimension of separation and collision cross section (CCS) values can provide a a complimentary identification descriptor. CCS values provide an additional identification point across multiple application areas. At low concentrations where fragment information may be absent, CCS and arrival time distribution (ATD) provide an additional identification point alongside retention time (tr).

To highlight confidence of the utility of CCS values, independent of ion mobility (IM) technology, when performing PFAS non-targeted screening assays, we have used an internally developed PFAS library to cross correlate PFAS CCS reproducibility. The SELECT SERIESTM CyclicTM IMS mass spectrometer (see Figure 1) has been used to perform an inter-site/intra-site comparison and compared to published drift tube (DT) ion mobility PFAS CCS values.1

Using LC-cIM-MS, branched and linear PFAS isomers have been fully resolved. Detection of individual PFAS isomers provides opportunity to correlate cancer risk with exposure to specific PFAS isomers. Human serum samples were analysed, and data generated for PFAS isomers and isobaric biomarker isomer composition.

LC-cIM-MS (cIM resolution (R)65-145) analyses were performed using a SELECT SERIES Cyclic IMS mass spectrometer. PFAS standards and human serum samples were analysed, using a 22 min reversed phase separation gradient. Using the combined peak capacity of LC-cIM, enabled resolution of linear and branched isomers, whilst simultaneously achieving an increase in analysis efficiency of 75 %. The enhanced resolution has the potential to facilitate a time efficient correlation between PFAS isomeric structure, concentration, and exposure. Linear and branched PFOS isomers were IM resolved in anonymized human serum extracts, using a 5.5-minute LC gradient. Importantly chromatographic separation was achieved for PFOS and bile acid biomarkers. Using ion mobility resolution (145), we report IM enhanced specificity and resolution for biomarker isomers Taurodeoxycholic, Taurochenodeoxycholic and Tauroursodeoxycholic acid.

A focus on LC-cIM-MS as a potential strategy to generate time efficient, additional toxicological correlation specificity with individual PFAS isomeric species and resultant elevation of isobaric biomarker isomers is presented. Identification of perfluoroalkyl carboxylic acids (PFCAs), cIM conformeric profiles and corresponding CCS identification finger prints provides newfound specificity.

4. Shimadzu: Qualitative Analysis of Drug Metabolites Using LCMS-9050

• Application

User Benefits

• The LCMS-9050 quadrupole time-of-flight (Q-TOF) mass spectrometer and LabSolutions Insight Explore™provide qualitative analysis of drug metabolites.
• Analyze and Target Screening function in LabSolutions Insight Explore enables peak extraction of candidate drug metabolites

Introduction: After a drug is ingested into the body, it is metabolized by various reactions, including oxidation, reduction, hydrolysis, and conjugation. The prediction of drug metabolites is important because the metabolites may differ from the original drug in terms of efficacy and toxicity.

This Application News introduces an example of qualitative analysis of drug metabolites using the LCMS-9050 quadrupole time-of-flight (Q-TOF) mass spectrometer. Lenvatinib, a known anticancer drug, was administered orally to rats, and the metabolites in the liver were analyzed. Drug metabolite peaks were extracted using the Analyze function in LabSolutions Insight Explore. The Target Screening function narrowed down the candidate peaks based on a list of drug metabolites, and the MS and MS/MS spectra were analyzed in detail.

Conclusion: Several metabolites derived from lenvatinib were detected by the LCMS-9050 quadrupole time-of-flight mass spectrometer and LabSolutions Insight Explore. By preparing a list of drug metabolites, it was possible to easily extract candidate metabolite peaks. This workflow can also be applied to the metabolite analysis of drugs otherthan lenvatinib.

5. Agilent Technologies: Quality Control of Lithium-Ion Battery Electrolytes and Solvents by UV-Vis Spectroscopy

• Application

Color measurements and safe chemical handling using an Agilent Cary 3500 Flexible UV-Vis with Cary Sipper pump

Abstract: Ensuring the quality of electrolytes used in lithium-ion batteries (LIBs) is crucial for maintaining the safety, performance, and longevity of these energy storage devices. LIB electrolytes consist of lithium salts dissolved in organic solvents. Any discoloration (yellowness) of these near-clear solutions can indicate contamination or degradation. Industry methods such as ASTM D5386-16 Standard Test Method for Color of Liquids Using Tristimulus Colorimetry can be applied to assess the quality of electrolytes by conducting instrumental color measurements using UV-Vis spectroscopy. Given the low absorbance of colorless-to-near-colorless liquid samples like LIB electrolytes and solvents, a highly accurate and sensitive UV-Vis spectrophotometer is essential.

This study demonstrates an effective quality assessment method for LIB electrolytes and solvents using the Agilent Cary 3500 Flexible UV-Vis spectrophotometer fitted with the optional Agilent Cary Sipper flow cell pump. The method is suitable for production quality control of hazardous samples, ensuring safe sample handling, high throughput, and high quality results for production-critical chemicals.

Conclusion: The Agilent Cary 3500 Flexible UV-Vis spectrophotometer provided accurate and precise color measurements for colorless to near-colorless LIB electrolytes and solvents in accordance with the ASTM D5386 standard method. Advantages of the Cary 3500 Flexible method and findings based on the data set include:

• Hazardous samples were introduced directly into a 10 mm path length flow cell using the Cary Sipper pump, ensuring user safety and high sample throughput without compromising data quality.
• The superior sensitivity (high signal-to-noise ratio) of the Cary 3500 combined with Agilent Cary WinUV Color application software enabled the reliable monitoring and control of electrolyte properties by analyzing light absorption in the visible spectrum.
• Higher Pt-Co color (APHA) values of the used LIB electrolytes compared to fresh samples suggested a deterioration in quality due to the color changes.
• Color measurements could be used to improve the quality control of LIB electrolytes and solvents by identifying discoloration—an indication of contamination or degradation that could affect battery performance.
• This effective method could be implemented in both manufacturing QC settings and R&D laboratories.

QC color measurements using the Cary 3500 Flexible UV-Vis would ensure that only high-quality electrolytes are used in the production of LIBs, enhancing their safety, efficiency, and durability.

6. Thermo Fisher Scientific: A Helpful Guide To Modernizing Your Old HPLC Methods

• Guide

Modernizing older or compendial methods can help increase your lab’s productivity and lower your cost and carbon footprint. This guide gives you an overview of what method parameters to consider changing, as defined by USP 621.

7. Thermo Fisher Scientific: Transitioning biomarkers from discovery to validation at unprecedented scale with the Stellar mass spectrometer

• Technical note

Expanded target capacity, higher sensitivity with greater specificity with unrivaled quantitative productivity for translational research

Keywords: Stellar mass spectrometer, biomarker verification, targeted quantitative solutions, protein, metabolite, lipid, dynamic retention time adjustment, automated method creation, fast polarity switching, translational research.

Introduction: The Thermo Scientific™ Stellar™ mass spectrometer establishes a new paradigm to drive biomarker verification by achieving 10X the sensitivity for 5X more compounds at scale compared to existing technologies. By synergistically combining the robust quantitative performance of triple quadrupole technology with the sensitive, rapid full scan MSn acquisition of dual-pressure linear ion trap technology, the Stellar mass spectrometer extends the unprecedented analytical capabilities to a wider range of compounds. Single-ion detection capabilities expand robust targeted quantitation for single cell or low protein load studies minimizing the potential for missing data. Novel software tools streamline highly multiplexed targeted quantitative method creation, implementation, and data acquisition, bypassing lengthy and costly replicate injections associated with existing technology. The new experimental capabilities of the Stellar mass spectrometer make it ideally suited for transitioning putative biomarker candidates from discovery to validation for translational proteomics, metabolomics, and lipidomics research.