News from LabRulezLCMS Library - Week 38, 2025

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This week we bring you application notes by Agilent Technologies, Shimadzu and Waters Corporation and poster by Thermo Fisher Scientific / HPLC Symposium!

1. Agilent Technologies: A Direct Aqueous Injection Method for Contaminants in Drinking and Nonpotable Water

Analysis of acrylamide, haloacetic acids, and β-estradiol in water with an Agilent 6495D LC/TQ

• Application note
• Full PDF for download

Recent regulations, such as EU 2020/218, have included the addition of new emerging contaminants in drinking water and raw waters, which supply water-treatment works. Some of these new contaminants to be analyzed include acrylamide, HAAs, and β-estradiol. However, acrylamide and HAAs can be difficult to analyze due to their high polarity and high interferences from hard water samples. Additionally, β-estradiol requires a very low level of detection (1 ng/L) compared to the other compounds (acrylamide 0.1 µg/L and HAAs 60 µg/L). The analysis uses a 1290 Infinity II LC coupled to a 6495D LC/TQ for analysis of water samples.

Experimental

Instrumentation 

Chromatographic separation was performed using an Agilent InfinityLab Poroshell 120 Aq-C18, 3.0 × 150 mm, 2.7 µm (part number 693675-742) installed on the 1290 Infinity II LC system. The individual modules of the 1290 Infinity II LC system included:

• Agilent 1290 Infinity II high‑speed pump (G7120A) 
• Agilent 1290 Infinity II autosampler (G7167B) 
• Agilent 1290 Infinity II multicolumn thermostat (G7116B) 

The 6495 LC/TQ with an Agilent Jet Stream (AJS) electrospray ion source was operated in dynamic multiple reaction monitoring (dMRM) mode. This mode allows more MRM transitions if future development is required for additional compounds. The LC/TQ autotune was performed in both unit and wide modes. All data acquisition and processing were performed using Agilent MassHunter software (version 12).

Conclusion 

This application note describes a highly sensitive and reproducible method for the fast and reliable quantitation of the listed compounds in water by direct injection. The dMRM method was created and optimized using Agilent MassHunter software and allows the addition of more MRM transitions if future development is required for additional compounds. An Agilent 1290 Infinity II LC coupled to an Agilent 6495D LC/TQ was used for the analysis. The 12-minute LC gradient method using an Agilent Aq-C18 column offered good chromatographic separation and RT distribution of all targets. The LC/TQ data acquisition was in dMRM mode with fast polarity switching for the most efficient use of instrument cycle time. The method performance was verified based on requirements for calibration curve linearity, instrument LOD, recovery, and precision. The results demonstrate the ability of the quantitative analytical method for emerging contaminants in water by direct injection.

2. Shimadzu: Short-Chain Oligonucleotide Analysis Using Supercritical Fluid Chromatography

• Application note
• Full PDF for download

Oligonucleotide therapeutics are drugs in which oligonucleotide is the active ingredient. Oligonucleotide therapeutics are manufactured through chemical synthesis, but impurities are produced during the process, so measurement and characterization of the impurities are required. Currently, reversed-phase ion-pair chromatography is often used for this analysis, but it is difficult to separate some impurities that are structurally similar to the target product. Therefore, a separation method with selectivity different from existing methods is required. 

SFC is an analytical method that employs carbon dioxide as the mobile phase at temperatures and pressures above the respective critical points. Compared to liquids, carbon dioxide is reported to have high resolution and special selectivity as a component of SFC mobile phase because of its low viscosity and high diffusivity. 

On the other hand, since the mobile phase containing carbon dioxide has low polarity, SFC was considered unsuitable for the analysis of highly polar compounds. However, in recent years, the composition of the modifier mixed with carbon dioxide and the choice of stationary phase have made it possible to analyze highly polar compounds,such as peptides and nucleotides2). 

In this study, we evaluated the applicability of SFC to oligonucleotide analysis. Single-strand oligonucleotides of around 20-mer are often used as oligonucleotide therapeutics, but due to the extremely high polarity of the molecules and the difficulty of analysis, we first used 4-mer oligonucleotides for evaluation and investigated their basic retention behaviors.

Experimental

• System: LCMS-9030

Conclusions 

To evaluate the applicability of SFC for oligonucleotide analysis, we developed a separation method using 4-mer oligonucleotides with varying PS contents as model compounds and investigated their basic retention characteristics. First, we identified conditions that allowed oligonucleotides to elute, finding that a methanol-water mixture containing 50 mmol/L ammonium formate enabled elution and peak detection. Using these conditions, we analyzed T4 with varying PS contents on 12 columns, finding good separation with the Diol II column. To apply this method to more polar sequences, we re-evaluated modifier conditions and found that adding aminoethanol improved peak shape. Applying the optimized method to 14 oligonucleotide sequences showed that sequences with two or fewer G or C bases were suitable for this method. Additionally, higher-polarity sequences showed stronger retention, while oligonucleotides with varying PS contents showed weaker retention with higher polarity, indicating unique retention behavior for PS modifications. The high selectivity for sequences with different PS contents suggests that SFC can be applied to separating impurities with a PS linkage replaced by a PO linkage that can be included in PS-modified oligonucleotides in the synthesis process. 

In the future study, the applicability of SFC to longer oligonucleotidesthan 4-mer will be evaluated.

3. Thermo Fisher Scientific / HPLC Symposium: Sensitive LC-MS method for the quantitative analysis of semaglutide and liraglutide in human plasma

• Poster
• Full PDF for download

GLP-1 analogs have emerged as pivotal therapeutic agents in the management of type 2 diabetes and obesity. These analogs mimic the physiological actions of endogenous GLP-1, including enhancing glucose-dependent insulin secretion, suppressing glucagon release, delaying gastric emptying, and promoting satiety. Given their significant clinical utilities, accurate detection and quantification of GLP-1 analogs in human plasma are essential for pharmacokinetic studies, therapeutic monitoring, and ensuring patient safety. This study aims to develop a sensitive LC-MS method for the detection and quantitation of semaglutide and liraglutide in human plasma.

Materials and methods

Mass Spectrometry

 See Table 2 for detailed MS source and scan settings for the analysis GLP-1 analogs on TSQ Altis Plus MS. 

Data Analysis 

Thermo Scientific Enterprise compliance ready Chromeleon CDS 7.3.2 software was used for all instrument control, data acquisition, processing, and reporting.

Conclusions 

We demonstrated a comprehensive LCMS solution for highly sensitive quantitation of GLP-1 analogs in human plasma using the Vanquish UHPLC coupled to TSQ Altis Plus MS. The solution enables: 

• Robust quantitation of semaglutide and liraglutide with a LOQ of 0.1 ng/mL 
• Excellent precision and accuracy across 3 orders of LDR for the analysis of GLP-1 analogs 
• Minimal carryover observied with post-injection signals remaining below 10% of the LOQ for semaglutide and below 14% for liraglutide following the highest concentration injection (100 ng/mL)

4. Waters Corporation: Method Migration of a Reversed Phase TFAAcetonitrile Gradient Method From a Binary to a Quaternary System: Impact of Mixing

• Application note
• Full PDF for download

Solvent mixing is critical to obtaining optimal gradient LC separations. The most commonly available pump mixing designs for reversed phase gradient HPLC separations are high pressure (typically binary) and low pressure (quaternary) systems. High pressure systems use independent pumps to deliver different solvents. Mixing occurs at high pressures with solvent composition controlled by the flow rates of the pump heads. In low pressure systems, solvent composition is controlled by a gradient proportioning valve. Each solvent is delivered in packets (based on the gradient specified in the method) which are mixed as they go through the pump head. High pressure systems have reduced dwell volumes and generally have improved compositional accuracy (leading to better retention time precision) over low pressure systems. Both high- and low-pressure systems are subject to mobile phase composition fluctuations. Mixers of various volumes and designs are utilized to help minimize these composition fluctuations by reducing baseline noise and oscillations in mobile phase composition. 

The use of certain mobile phase modifiers is known to impact chromatographic baselines. Trifluoroacetic acid (TFA) is a commonly used ion-pairing reagent used in combination with acetonitrile for many gradient reversed phase applications. TFA absorbs strongly at wavelengths below 250 nm. Additionally, TFA is slightly retained on reversed phase columns which results in fluctuations in TFA concentration as the acetonitrile gradient passes through the column. In combination, these factors result in significant baseline disturbances (ripples) when TFAacetonitrile gradients are used at low wavelengths. These baseline ripples may make peak integration difficult and can impact the sensitivity along with peak area precision and retention time precision of the method. 

Most modern HPLC systems typically include a standard mixer in the pump design. Additionally, different mixers may be available to improve mixing performance for specific applications. The effect that mixers have on chromatographic performance was demonstrated during the migration of a reversed phase TFA-acetonitrile gradient method from a binary system to the quaternary Alliance iS HPLC System.

Experimental

• LC systems: Alliance iS HPLC System Comparable Binary HPLC system (System X) 
• Detection: UV Detector with 10 mm HPLC flow cell
• Column: XBridge™ C18 Column, 5 µm 4.6 x 250 mm (p/n: 186003117)

Data Management 

• Chromatography data system: Empower™ 3.8.0.1

Conclusion 

The differences in mixing performance between binary and quaternary HPLC Systems may impact method migration. Mixing performance is particularly important when migrating gradient TFA-acetonitrile methods at low wavelengths, as these methods are known to produce significant baseline noise (ripple). In this study, effect of mixers on chromatographic performance was demonstrated through the migration of the USP Tryptophan Organic Impurities method from a binary system to the quaternary Alliance iS HPLC System. The results show that the characteristic baseline ripple associated with TFA-Acetonitrile gradients at low wavelengths is impacted by the choice of mixer. The Alliance iS HPLC System with the 690 µL Ti diffusion bonded mixer displayed a significant reduction of baseline noise when compared against both the binary and quaternary systems equipped with their respective standard mixers. This resulted in easier peak integration, improved peak area and retention time reproducibility, and increased method sensitivity.