News from LabRulezLCMS Library - Week 41, 2024

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 7th October 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 applications by Shimadzu, Thermo Fisher Scientific, Agilent Technologies, and presentation by James Little Mass Spec Interpretation Services!

1. Agilent Technologies: Simplified Analysis Workflow – Determination of Vitamins B1, B2, B3, B5, and B6 in Infant Formula

• Application

Using the Agilent 1260 Infinity II Prime LC system with diode array detection

Abstract

This study developed and validated an efficient workflow for analyzing vitamins B1, B2, B3, B5, and B6 in infant formula. The workflow employed a straightforward sample preparation protocol, using trichloroacetic acid (TCA) for protein precipitation. For enhanced detection sensitivity, the analysis was performed using the Agilent 1260 Infinity II Prime LC system, equipped with a 60 mm Agilent InfinityLab Max-Light cartridge cell in the diode array detector (DAD). The developed methods demonstrated robustness and reliability in quantifying B vitamins in infant formula, ensuring accurate nutritional labeling and compliance with regulatory standards.

Introduction 

B vitamins are essential for numerous bodily functions, such as supporting aerobic metabolism of glucose for energy production and maintaining immune function. Deficiencies in B vitamins can adversely affect human health. Since the human body cannot produce these vitamins in sufficient quantities, it is necessary to obtain them from dietary sources of animal origin or through supplementation.¹ Foods such as infant formula are allowed to be fortified with vitamins to meet infants' nutritional requirements, subject to strict regulatory control. Table 1 lists the forms of B vitamins that are commonly used in food fortification. Robust analysis techniques are imperative for accurately quantifying these essential compounds. The quantification of vitamin B7, B9, and B12 in infant formula has been detailed in previously published application notes.^2,3

Acidic hydrolysis and enzymatic digestion are generally used to release protein-bound and phosphorylated B vitamins. However, in infant formula, the vitamin content is declared based on the amount added during the manufacturing. This means that the extraction protocol and the vitamin forms to be determined can be simplified and minimized. Traditionally, microbiological assays were employed to measure the B vitamin concentrations due to their high sensitivity. With technological enhancements, HPLC with ultraviolet (UV) detection has emerged as a more rapid and reliable quantification tool.⁵

In this study, a unified sample preparation workflow using TCA as protein precipitation agent was developed for: (1) the simultaneous analysis of vitamins B1, B2, B3, and B6, and (2) the analysis of vitamin B5 in infant formula. Each uses distinct HPLC analytical conditions. The structure of vitamins B1, B2, B3, B5, and B6 are shown in Figure 1. The 1260 Infinity II Prime LC, with a high-pressure limit of 800 bar, is ideally suited for coupling with a small particle size column, ensuring exceptional resolution and sensitivity. Additionally, the use of a 60 mm Max-Light cartridge cell in the DAD further enhances the detection of B vitamins, particularly, vitamin B5, which lacks chromophore.

Conclusion

This study successfully developed and validated an efficient workflow for quantifying B vitamins in infant formula. The workflow uses a simple and straightforward sample preparation protocol to precipitate proteins in the formula. Despite its simplicity, the study demonstrated excellent linearity, repeatability, and recovery, providing reliable measurements of vitamins B1, B2, B3, B5, and B6. The use of the Agilent 1260 Infinity II Prime LC system, paired with a 60 mm Agilent InfinityLab Max-Light cartridge cell in the DAD, significantly enhanced detection sensitivity.

2. James Little - Mass Spec Interpretation Services: LCMS Unknown Identification with NIST Search Using MSMS Libraries

• Presentation

Table of content

• Overview of Process
• Some Typical User Options and Setting for NIST MSMS 2023 Search Version 3
• Library Search Menu in NIST MSMS Search
• Other Settings in NIST MSMS Search
• Other Settings in NIST MSMS Search
• Sorting of the Search Results
• Important!! Save Your Settings for Future Sessions!
• Libraries Searched

3. Thermo Fisher Scientific: Development and implementation of a comprehensive fecal metabolites LC-MS library for dietary intervention studies using the Thermo Scientific Stellar mass spectrometer

• application

Goal

The primary goal of this application note is to demonstrate the capabilities of the Thermo Scientific™ Stellar™ mass spectrometer in developing and implementing a comprehensive fecal metabolites LC-MS library for high-throughput quantitation analysis. This study aims to:

1. Showcase expedited method development: Illustrate the intuitive and efficient method development process facilitated by the rapid scanning and high selectivity features of the Stellar mass spectrometer.
2. Assess analytical performance: Evaluate the sensitivity, accuracy, and selectivity of the Stellar mass spectrometer in quantifying fecal metabolites from mice subjected to various dietary interventions, ensuring minimal data loss even with low sample loads.
3. Demonstrate high-throughput capabilities: Highlight the instrument's ability to perform high-throughput analyses without compromising data quality, employing three distinct LC columns and various gradient methods.
4. Validate compound detection: Validate the robustness and reliability of the Stellar mass spectrometer in detecting and quantifying a wide range of metabolites, including challenging co-eluting isomers, using advanced MSⁿ fragmentation techniques.

Keywords: Stellar mass spectrometer, targeted quantitative analysis, LC-MS library, fecal metabolites, high-throughput analysis, method sensitivity, compound coverage, quantitation accuracy, selectivity.

Introduction

The Thermo Scientific Stellar mass spectrometer (Figure 1) revolutionizes biomarker verification with its remarkable sensitivity and compound coverage, surpassing existing technologies tenfold and fivefold, respectively. By integrating the robust quantitative capabilities of triple quadrupole technology with the hyper-fast full scan MS n acquisition capabilities of the dual-pressure linear ion trap technology, the Stellar mass spectrometer broadens its analytical scope to encompass a wider range of compounds. Enhanced single-ion detection facilitates precise quantitation even with minimal sample loads, minimizing the risk of data loss. This advanced approach distinguishes itself  from traditional methods by significantly improving sensitivity and accuracy, thereby ensuring more reliable analytical results. Cutting-edge software tools, in addition, simplify the development, implementation, and data acquisition of complex targeted quantitative methods, eliminating the need for time-consuming replicate injections. These advancements establish the Stellar mass spectrometer as an indispensable tool for transitioning putative biomarker candidates from discovery to validation in translational metabolomics and lipidomics research.

This application note presents the development of a fecal metabolites LC-MS library on the Stellar mass spectrometer, using three distinct LC columns for expanding metabolite coverage. The library was then implemented to quantify selected fecal metabolites from mice subjected to different dietary interventions. The experiments here aimed to assess the ability of the Stellar mass spectrometer to conduct high-throughput quantitative analysis without sacrificing data quality, such as method sensitivity, accuracy, and selectivity. In addition, the work presented here describes an intuitive method development procedure facilitated by the fast-scanning mass spectrometry, where the user can obtain information on retention time, optimal fragmentation collision type and energy level, and optimal ion transmission conditions using multiple experiments [e.g., MS1 full scan with polarity switching, and MS n with Higher Energy Collisional Dissociation (HCD) and Collision Induced Dissociation
(CID) fragmentation methods] within a single-injection method.

Conclusion

The Stellar mass spectrometer’s exceptional capabilities in biomarker verification has been demonstrated through this application note. By combining the robust quantitative power of triple quadrupole technology with the rapid full-scan MSⁿ acquisition of dual-pressure linear ion trap technology, the Stellar mass spectrometer has shown a remarkable increase in both sensitivity and compound coverage. This study illustrated the effective development and implementation of a fecal metabolites LC-MS library using three distinct LC columns, showcasing the instrument's ability to conduct high-throughput quantitation analysis without compromising data quality.

Key findings include the Stellar mass spectrometer’s performance capabilities delivering rapid scanning, high sensitivity across a broad dynamic range, and efficient fragmentation using both HCD and CID techniques. These features facilitated intuitive method development and enabled the precise quantitation of numerous fecal metabolites from mice subjected to different dietary interventions. The experiments underscored the instrument's high selectivity and robustness, with minimal data loss and consistent signal responses, demonstrating its utility in translational
metabolomics and lipidomics research.

4. Shimadzu: AI-Driven Automated Column Screening and Gradient Optimization for LC Method Development

• application

User Benefits

• The AI algorithm of LabSolutions MD can automatically optimize gradient conditions to greatly reduce labor of LC method
  development.
• Gradient conditions are automatically optimized for multiple columns by column switching valve.
• Automatic optimization of gradient conditions can be applied not only to new method development, but also to existing method to efficiently improve resolution.

Introduction

In the typical LC method development, the process begins with “preparation” which includes mobile phase preparation, column installation, and creation of analysis schedules, then the analysis is started. After that, the acquired data is analyzed and “preparation” for the subsequent analysis is carried out, followed by starting the next analysis again. The method development progresses by repeating these processes, but in addition to the significant time required to repeatedly create analysis schedules, expertise in chromatography is necessary to explore optimal conditions based on data analysis. In other words, typical method development requires “human intervention”. Therefore, eliminating human involvement and automating such method development processes would be desirable to improve labor efficiency. This article introduces an automatic exploration of a combination of the column and gradient conditions that meet the criteria of resolution by automatic optimization of gradient conditions on multiple columns consecutively.

 Conclusion

Automatic optimization of gradient conditions using AI algorithm of LabSolutions MD was applied to a model sample (mixture of six compounds of small molecule) with three columns. As a result, the column and gradient conditions that met the resolution criteria were successfully explored. In method development, human intervention, such as analysis batch creation and data analysis, is required to optimize gradient conditions. However, LabSolutions MD can provide significant labor savings in this area. For more information on LabSolutions MD, please refer to the Technical Report “Efficient Method Development Based on Analytical Quality by Design with LabSolutions MD Software (C190-E284)”.