News from LabRulezLCMS Library - Week 49, 2024

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 2nd December 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 you applications by Thermo Fisher Scientific, Shimadzu, Waters Corporation, Agilent Technologies, and Metrohm!

1. Metrohm: Potassium assay in potassium citrate and citric acid oral solution

• Application

Method validation according to the U.S. Pharmacopeia (USP)

Potassium citrate and citric acid oral solutions are systemic alkalizers beneficial for health conditions where long-term maintenance of alkaline urine is desirable, and administration of sodium salts is contraindicated [1,2]. To comply with the strict quality standards for pharmaceutical products, validated methods such as those from the United States Pharmacopeia – National Formulary (USP-NF) are mandatory for manufacturers and laboratories. As part of the USP modernization initiative, the potassium monograph was updated, replacing the previous identification procedure of titration with ion chromatographic (IC) analysis [3]. For quality control, the USP specifies ion chromatography using a cation-exchange column with L76 column material and non-suppressed conductivity detection to quantify the potassium content [3].

The present IC method uses the Metrosep C 6 - 150/4.0 column (L76) to separate potassium from other potentially present ions. This method has been validated according to USP General Chapters <621> Chromatography [4] and <1225> Validation of Compendial Procedures [5]. All acceptance criteria for
the potassium assay of the USP monograph «Potassium Citrate and Citric Acid Oral Solution» are fulfilled [3].

CONCLUSION

As per USP «Potassium Citrate and Citric Acid Oral Solution» [3], the assay for potassium is carried out with an IC using a Metrosep C 6 separation column
(packing material L76). All validation results fulfilled the requirements of the monograph and followed the guidelines of the USP General Chapters <621> [4] and <1225> [5]. The presented IC method is suitable to quantify potassium in potassium citrate and citric acid oral solutions.

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

• Application

Abstract

This application note details a methodology for toxicological drug screening in complex biological matrices. The method was developed on the Agilent Revident liquid chromatography/quadrupole time-of-flight mass spectrometer (LC/Q-TOF MS) with the Agilent ChemVista spectral library manager and Agilent MassHunter Quantitative Analysis software, version 12.1. The LC Screener tool, which is embedded in MassHunter Quantitative Analysis software, was used to quickly review results of a data-independent acquisition (DIA) method for a wide range of target analytes over a typical large concentration range. This application note describes a complete screening workflow including sample preparation, suspect screening, and data analysis results for the screening of toxicological drugs in relevant biological matrices.

Introduction

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.

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.

3. Waters Corporation: Monitoring Impurities Using a High Throughput Focused Gradient With the Alliance™ iS Bio System 

• Application

Abstract

The Alliance iS Bio HPLC System provides an efficient HPLC platform for routine monitoring of quality indicating attributes in protein drug substances. Innovative products such as Waters IonHance™ difluoroacetic acid, when used in conjunction with the Titanium diffusion bonded mixer, and the Integrated sample metering pump, provides improved UV-detection through reduced baseline noise, increased sensitivity, and reliable detection for improved peak integration. This unique configuration increases confidence in monitoring low-abundant impurities by retention time matching in UV-based assays, making it particularly well-suited for quality control environments. In this study, a shortened focused gradient targeting an oxidized peptide and its native species was developed via mass confirmation and transferred to the Alliance iS Bio HPLC System which provides high throughput and efficient monitoring.

Benefits

• The Titanium diffusion bonded mixer reduces baseline noise for increased confidence in detecting low-abundant impurities via retention time matching
• The sample metering pump and large volume needle loop of the Alliance iS Bio HPLC System is capable of precise injections up to 100 μL, bypassing the need for external loops
• The Alliance iS Bio HPLC System consistently monitors low-abundant impurities over extended analysis periods

Introduction

Monoclonal antibody (mAb) therapies have a distinct advantage in their capability to target specific cellular epitopes and have proven performance in disease diagnosis and treatment of infections and cancer. As a biologic, the complexity in the structure of mAb-based drug substances requires robust methods in the analysis and monitoring of impurities to ensure safety and compliance standards are met. Peptide mapping is frequently used as part of upstream development activity to identify undesired modifications to amino acid residue side chains such as deamidation, isomerization, N-terminal cyclization, and oxidation. Characteristically, these assays typically require lengthy analyses to increase the resolution of chromatographically resolved impurities and predominantly utilize mass spectrometry for analysis and identification. As part of the drug development process, these characterization methods are often transferred downstream to manufacturing labs for further optimization and routine monitoring of quality indicating attributes.

In manufacturing activities, these methods may be translated into a UV-based assay and optimized as a targeted assay with reduced run time to increase assay efficiency and robustness as part of routine monitoring. The purpose of this study is to demonstrate instrument and method considerations that can expedite development and transfer of targeted RPLC-UV assay from a RPLC-MS method for easier deployment in a QC lab environment. For this study, an enzymatically treated standard of NISTmAb RM 8671 was analyzed with the Alliance iS Bio HPLC System under Empower™ 3 CDS control as a representative sample and LC platform deployed in QC labs. A methionine containing peptide fragment was monitored for its oxidation degradation to
evaluate assay reproducibility and robustness.

Conclusion

Consideration of ion-pairing agents and various mixer designs as part of the method development process can be critical when monitoring low-abundant species. In this study, DFA emerged as a suitable mobile phase ion pairing agent which improved peak shape in UV-detection while also supporting upstream MS-based workflows, facilitating easier method transfer between labs. The Titanium diffusion bonded mixer, available as an optional mixer for the Alliance iS Bio HPLC System, provides reliable baseline performance and increases the confidence in detecting low-abundant impurities via retention time matching. Additionally, the sample metering pump of the Alliance iS Bio HPLC System in conjunction with the large volume needle loop, allows for precise injections up to 100 μL thus eliminating the need for extension loops and enabling direct method scaling, which saves both time and resources. In conclusion, the Alliance iS Bio HPLC System proves to be an ideal HPLC platform for QC labs that can deliver reliable and consistent results for UV-based monitoring assays. It efficiently monitors low-abundant impurities over extended analysis times, thereby reducing errors and operation costs in manufacturing environments.

4. Shimadzu: Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Drinking Water in Accordance with EPA Method 537.1

• Application

User Benefits

• Based on EPA Method 537.1, 18 PFAS in drinking water can be accurately analyzed at concentrations equivalent to EPA’s Final MCLs.
• PFAS analysis can be done immediately by using the LC/MS/MS Method Package for PFAS in Drinking Water, minimizing the effort required to set up an analytical system.

Introduction

Per- and polyfluoroalkyl substances (PFAS) are organofluorine compounds that are used in a wide range of consumer products and other applications due to their water repellency, heat resistance, chemical resistance, and other beneficial properties. However, their extremely high chemical stability prevents them from breaking down easily. Consequently, due to their persistence in the environment and possible toxicity to biological organisms, some PFAS have been designated as being subject to the Stockholm Convention on Persistent Organic Pollutants (POPs Convention), which restricts their manufacture and use. In recent years, there have been efforts to strengthen PFAS restrictions and determine their actual levels, resulting in a need to standardize the methods used to analyze them.

In April 2024, the U.S. Environmental Protection Agency (EPA) announced the following final maximum contaminant levels (Final MCLs) for certain PFAS in drinking water,1) 4 ng/L PFOA, 4 ng/L PFOS, 10 ng/L PFHxS, 10 ng/L PFNA, and 10 ng/L HFPO-DA. This article describes the results from using the LC/MS/MS Method Package for PFAS in Drinking Water to simultaneously analyze 18 PFAS target compounds in drinking water in accordance with EPA Method 537.1, 2) which was published by the EPA in 2018.

Conclusion

By using an LCMS-8060RX liquid chromatograph mass spectrometer and the LC/MS/MS Method Package for PFAS in Drinking Water, 18 PFAS in drinking water were measured in accordance with EPA Method 537.1.

The above set up was able to measure concentration levels that were one-tenth those published by the EPA as Final MCLs (4 ng/L PFOA, 4 ng/L PFOS, 10 ng/L PFHxS, 10 ng/L PFNA, and 10 ng/L HFPO-DA).

Based on spike-and-recovery test results using ultrapure water, good recovery rates and repeatability results were obtained when spiking the ultrapure water with the equivalent of 1/4 the Final MCLs (1 ng/L in sample water). Similarly, good recovery rates and repeatability results were obtained when spiking drinking water samples with the Final MCLs (4 ng/L in sample water). These results confirm that PFAS compounds can be simultaneously analyzed in accordance with EPA Method 537.1 with good accuracy.

In addition, the LC/MS/MS Method Package for PFAS in Drinking Water makes it easy to set up an analytical system, and it enables accurate analysis.

5. Thermo Fisher Scientific: Method transfer case study for instrument and LC column migration of the purification and analysis workflow for synthetic oligonucleotides

• Application

Application benefits

• A method transfer of semi-preparative purification and QC from a non-Thermo Fisher Scientific liquid chromatographic system to the Thermo Scientific™ Vanquish™ LC platform
• Purification and quality assurance columns to facilitate a Thermo Fisher workflow solution
• A simple method to purify oligonucleotides via the Vanquish Analytical Purification LC system and perform quality control using UHPLC high-resolution mass spectrometry (HRMS)

Goal

Demonstrate the complete method transfer of oligonucleotide purification and qualification from a non-Thermo Fisher LC and column workflow to a Thermo Fisher solution using high-performance liquid chromatography (HPLC) for purification and HPLC-HRMS for quality control, all managed under Thermo Scientific™ Chromeleon™ Chromatography Data Solution CDS.

Introduction

Nucleic acids play a vital role in the existence of life. In recent years, there has been a growing interest in oligonucleotides within the fields of biochemical research, diagnostics, and pharmaceuticals.¹ As a result, significant efforts have been made to optimize and automate their synthesis. However, the process of oligonucleotide synthesis involves multiple reactions, leading to the accumulation of impurities, such as truncated nucleotide sequences, partial deprotection, and fluorophore/quencher degradation. Therefore, it is crucial to purify the desired oligonucleotides effectively and with high purity for downstream applications, such as quantitative polymerase chain reactions (qPCR), integrated human identification (HID) solutions for forensics, and gene transfer agents (GTA).

Since the 1970s, various chromatographic methods have been utilized for the analysis and purification of synthetic oligonucleotides.² In recent years, significant advancements have been achieved in terms of instrument performance and stationary phase.³ Reversed-phase high-performance liquid chromatography (RP-HPLC) is the widely employed technique for high-resolution separation of nucleic acids.⁴ However, the purity specification for oligonucleotides is on the rise. For example, the chromatographic analysis of active pharmaceutical ingredients (API) is required to ensure the detection of contaminants at concentration levels down to trace amounts relative to the drug.⁵ For other applications, oligonucleotide purity needs to exceed 90%. The increasing demand for oligonucleotides as therapeutic agents necessitates the development of a HPLC purification scheme that satisfies a high purity specification.

When developing methods to separate and purify oligonucleotides, it is essential to consider their unique characteristics. These include the length of the oligo, specific sequence, fluorophore/quencher combination, and ability to form secondary structures. Other factors that influence an oligonucleotide’s interaction with the stationary phase, and therefore retention time, include buffer (pH and salt concentration), reversed-phase column, and LC system
selection.²

Additionally, column selection, LC hardware/software, and automation can also affect purity. As a result, the ability to seamlessly transfer oligonucleotide purification methods from one vendor instrumentation and columns to another while meeting or exceeding the expected quality criteria is a valuable tool.

This work demonstrates the effective method transfer of the semi-preparative RP-HPLC purification of two different dual-labeled 15mer oligonucleotides from a source HPLC instrument and column to an alternate vendor column and HPLC. By-products of the DNA synthesis are resolved, and the target oligonucleotides are successfully isolated and collected. LC-HRMS and HPLC analyses were used to confirm oligonucleotide purity, resulting in passing QC standards for the yield and purity, and suggesting a successful method transfer for the preparation of the high-purity oligonucleotides for downstream application (Figure 1).

Conclusions

• A simple method transfer was successfully shown for the purification of the dual-labeled oligonucleotide samples from a non-Thermo Fisher Scientific LC purification system to the Vanquish Analytical Purification LC system.
• The Vanquish system purification yields quantitation/QC data comparable to the Agilent system purification for the ABY/JUN-MGB probes (Figure 5).
• The Hypersil GOLD semi-preparative column yields similar quantification and purification performance to that of the non-Thermo Fisher Scientific semi-preparative column when used to isolate the short-chain dual-labeled oligonucleotide samples. This has the added benefit that the fraction volume is reduced making for less solvent to be evaporated from the purified products.
• For QC, the Hypersil GOLD analytical column successfully passed the criteria demonstrating to be a suitable substitute of the non-Thermo Fisher Scientific analytical column.