News from LabRulezLCMS Library - Week 18, 2025

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 28th April 2025? 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 Shimadzu, Thermo Fisher Scientific and Waters Corporation and presentation by Agilent Technologies!

1. Agilent Technologies: Optimize Your Polymer Analysis by Selecting the Ideal GPC/SEC Column

• Presentation
• Full PDF for download

GPC/SEC Separation Mechanism

• A GPC/SEC column is packed with porous beads of controlled porosity and particle size
• Sample is prepared as a dilute solution in the eluent and injected into the system
• Large molecules are not able to permeate all pores and have a shorter residence time in the column
• Small molecules permeate deep into the porous matrix and have a long residence time in the column
• Sample molecules are separated according to molecular size, eluting largest first, smallest last

Conventional GPC/SEC Workflow

• Calibrate the GPC/SEC column with a set of narrow polymer standards
• Plot retention time (RT) versus peak log molecular weight (logM)
• Calibration is used to generate the molecular weight (averages and distribution) of unknowns on the same system/column set
• Molecular weights are relative to the standards used

Considerations for GPC/SEC Column Selection

Key questions to ask  

• What polymer are you analysing?
• Which solvent (or solvents) is your polymer soluble in?
• What is the expected molecular weight range of your polymer?
• What is the requirement for your analysis or what would you like to improve about
• your existing GPC/SEC separation?
  • Resolution is important
  • Reproducibility of sample chromatography and results
  • Speed of analysis or sample throughput is something to improve on

Further considerations

• Know the properties of the sample
• Be familiar with the properties of the columns being considered
• It is important to balance polarities for the sample, solvent, and column packing

Common Column Chemistries for GPC/SEC

Polymer chemistries particles with their rigid pore structure

These have a higher pore volume. You might see differences in mechanical stability between vendor packings. Due to the polarity of the stationary phase, observed interactions are reduced.

Common polymer packing types:

• Polymethacrylate packings
• Polyester copolymers
• DVB, divinylbenzene
• PS-DVB, polystyrene divinylbenzene

Silica chemistries

Typically have a lower pore volume versus polymeric but are mechanically stronger. They exhibit enthalpic properties due to presence of silanols.

Common silica packing types:

• Diol 
• Surface-modified hydroxyl
• Surface-modified polymeric

2. Shimadzu: HPLC Analyses of Nucleotides in Powdered Infant Formula and Liquid Infant Formula

• Application note
• Full PDF for download

User Benefits

• Five nucleotides in powdered infant formula and liquid infant formula can be simultaneously analyzed by HPLC.
• Nucleotides can be determined precisely owing to the optimization of pretreatment procedure and analytical method to reduce the influence of foreign substances with reference to ISO and AOAC official methods.

Powdered infant formula and liquid infant formula are essential for providing optimal nutrition to infants. They are manufactured to make their composition close to those of breast milk.

Nucleotides in breast milk have not been fully characterized in comparison with other key nutrients such as proteins and fats. However, important physiological functions of nucleotides have been revealed in recent years. 1),2) For example, nucleotides have been reported to improve intestinal development, digestion/absorption, and allergy-prevention of an infant. Furthermore, nucleotides play an important role in the development of lipid metabolism and brain function and are now included in infant formulae as a semi-essential nutrient. 

In this application news, analyses of five nucleotides in powdered infant formula and liquid infant formula using the Nexera XR high performance liquid chromatograph is introduced. The pretreatment method and HPLC analytical conditions are based on ISO 20638:2015(E) 3) published by the International Organization for Standardization (ISO) and the AOAC Official Method 2011.20 4) published by AOAC INTERNATIONAL, a North American organization that standardizes food testing methods and validates analytical methods.

Analysis of a mixed standard solution

Table 1 shows the analytical conditions. Shim-pack GIST C18- AQ, which shows excellent retentions for highly polar compounds, was used as the analytical column. Fig. 1 shows chromatograms of a mixed standard solution of five nucleotides (IMP, AMP, GMP, CMP, and UMP). TMP was used as the internal standard (ISTD). A PDA detector was used and detection wavelengths were 250 nm (IMP), 260 nm (AMP , GMP, TMP), and 270 nm (CMP, UMP).

Calibration curves

The concentrations of the four mixed standard solutions are shown in Table 2. Each reagent used in the standard solutions was the sodium salt or sodium salt hydrate instead of the free acid of the nucleotide, which is difficult to obtain. Absorbance at λmax of each nucleotide in the mixed standard solutions was measured with UV-visible spectrophotometer (UV-1900i) according to ISO and AOAC methods to determine the purity. Concentrations were corrected by multiplying the purity of each nucleotide in standard solution in Table 2.

Analysis of powdered infant formula and liquid infant formula

Fig. 2 shows the pretreatment method for infant formula and liquid infant formula. Two commercially available powdered infant formulae and one liquid infant formula were pretreated according to Fig. 2 and subjected to HPLC analyses. Fig. 3-Fig. 5 show the chromatograms of each sample at 250, 260, and 270 nm. In the pretreatment, the samples were purified using a solid-phase extraction cartridge with a strong anion exchange group in order to reduce the influence of interfering components.

Conclusion

Five nucleotides in infant formula and liquid infant formula were analyzed using Nexera XR high-performance liquid chromatograph. Shim-pack GIST C18-AQ column provided excellent performances both in terms of "pressure fluctuations" and "retention time repeatability" even employing the gradient profile in which 100% aqueous mobile phase delivery interval was included. The influence of interfering components in the sample was suppressed using a solid-phase extraction cartridge.

3. Thermo Fisher Scientific: Instrument and LC column migration for the purification and analysis of synthetic oligonucleotides

• Application note
• Full PDF for download

Application benefits 

• A workflow migration of semi-preparative reversed-phase chromatographic purification and subsequent HPLC-UV purity analysis from a non-Thermo Fisher Scientific liquid chromatography system to the Thermo Scientific™ Vanquish™ LC platform  
• Preparative and analytical column comparative study to facilitate a Thermo Fisher Scientific workflow solution for purification and purity characterization methods 
• A simple method to purify oligonucleotides via the Thermo Scientific™ Vanquish™ Analytical Purification LC System and to perform quality control using UHPLC hyphenated to HRAM-MS

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, in some cases, sub-ppb 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 the wetted parts of the HPLC system.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 successful method transfer of the semi-preparative RP-HPLC purification of two different duallabeled 15mer oligonucleotides from an Agilent™ 1260 Infinity™ II Preparative-Scale LC Purification system and third-party columns to the Thermo Scientific™ Vanquish™ Analytical LC Purification system and Thermo Scientific™ columns. By-products of the DNA synthesis are resolved, and the target oligonucleotides are successfully isolated and collected. HPLC-UV was used to assess the oligonucleotide purity, UV spectroscopy to determine yield, and high-resolution mass spectrometry for identity confirmation. The alignment of yield, purity, and identity obtained from the two systems demonstrates a successful workflow migration (Figure 1).

Experimental

Instrumentation

LC-UV QC

Thermo Scientific™ Vanquish™ Flex Binary UHPLC System consisting of:

• System Base Vanquish Horizon/Flex (P/N VF-S01-A-02)
• Vanquish Binary Pump F (P/N VF-P10-A-01)
• Vanquish Split Sampler FT (P/ VF-A10-A-02)
• Vanquish Column Compartment H (P/N VH-C10-A-03) with Active Pre-Column Heater (P/N 6732.0110)
• Vanquish Diode Array Detector FG (P/N VF-D11-A-01 with standard biocompatible flow cell, 11 µL, PEEK, 10 mm (P/N 6083.0540)

Results and discussion

Step 1 - Sample preparation 

The dye-oligonucleotide combination generates a molecule which is quite hydrophobic. Therefore, adding the aqueous Solvent A to the crude dual-labeled oligonucleotide did not initially result in a homogeneous solution. It was only after the organic Solvent B was pipetted that the sample became a homogeneous solution. 

Step 2 – Purification 

Initially, both 200 nmol crude samples were purified using both the Daisogel Prep and the Hypersil GOLD prep columns. The ABY-MGB crude was purified, and the differing retention and selectivity of the column performances can be observed in Figure 2. Although the target oligonucleotide elutes at an earlier time, the Hypersil GOLD prep column resolves neighboring peaks with higher resolution than the Daisogel column. This difference in elution time is likely due to the differences in pore size and carbon load between the Hypersil and Daisogel columns. The Daisogel column has a shoulder peak at 10.9 minutes, which makes for a more challenging purification versus the Hypersil GOLD prep column.

Figure 3 presents the chromatograms obtained for the JUN-MGB crude sample analyzed on both the Daisogel and Hypersil GOLD semi-preparative columns. This sample purification proved more challenging than that of the ABY-MGB sample due to neighboring peaks prior to the target JUNMGB oligo and a co-eluting shoulder after the target peak. By focusing the collection window on the UV saturation region, one can increase the purity of the JUN-MGB oligo where the later eluting impurity shoulder is sharper using the Hypersil GOLD semi-preparative column. Furthermore, the peak volume for the Hypersil GOLD column provides a smaller collection volume compared to that of the Daisogel column (Table 8). 

Step 3 - LC-UV QC 

Prior to evaporation of the solvent, the LC-UV was used to control the quality and determine if the Hypersil GOLD analytical column could be used as a substitute for the Waters analytical column. A successful transfer of the analytical method would be represented by comparable purity of the purified fractions. The crude sample chosen was that of the ABY-MGB purified on the Hypersil GOLD prep column shown in Figure 4. As shown in Table 9, the Hypersil GOLD analytical column can perform equivalently to the Waters analytical column in the objective of isolating a target oligonucleotide with similarly high purity and yield.

Conclusions 

• A simple method transfer is successfully shown for the purification of the dual-labeled oligonucleotide samples from a non-Thermo Fisher Scientific LC purification system, specifically an Agilent 1260 Infinity II Preparative-Scale LC Purification system, to the Vanquish Analytical Purification LC system. 
• The Vanquish system purification yields quantitation/QC data comparable to the non-Thermo Fisher Scientific HPLC system system purification for the ABY/JUN-MGB samples (Figure 5). 
• The Hypersil GOLD semi-preparative column yields similar quantification and purification performance to that of the Daisogel SP-100 ODS-P 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 Waters ACQUITY UPLC BEH C18 analytical column

4. Waters Corporation: Improving Chromatographic Resolution of the JECFA Method for the Analysis of Steviol Glycosides

• Application note
• Full PDF for download

Benefits

• LC-UV separation of steviol glycosides was optimized following an enhanced approach to method optimization as recommended in the ICH Q14 Analytical Procedure Development guideline 
• Higher resolution of critical pairs has been achieved, which is beneficial to the analysis of complex Stevia extracts

Steviol Glycosides (SG) are constituents of the leaves of the plant Stevia rebaudiana Bertoni (stevia) and have a sweet taste that is 100 to 300 times sweeter than sucrose. They are often used as non-caloric sweeteners in foods and beverages. More than 40 SG have been identified.¹ The most abundant are rebaudioside A (Reb A) and stevioside (SV). However, some minor SG are becoming more readily available and are in demand due to their higher sweetness intensity and less bitter aftertaste.^1–2 

Liquid Chromatography (LC) is the main technique used in SG analysis and reversed-phase (RP) C18 columns are the most frequently used columns in the LC analysis of SG.^3–6 The Food and Agriculture Organization of the United Nations, and the World Health Organization (FAO/WHO) Joint Expert Committee on Food Additives (JECFA) has published a series of monographs on SG since 2006. The latest monograph, published in the FAO/WHO JECFA Monographs 26 (2021), recommended two methods for the determination of the major and the minor SG by LC-UV and LC-UV-MS, respectively.¹ In both methods, the same C18 column and the same gradient elution was used to separate the major SG (13 compounds) and the minor SG (17 compounds and their isomers) within a total run time of 35 minutes. The main difference between these two methods was that a mass spectrometer (MS) was used for the minor SG analysis, while an ultraviolet/visible (UV/Vis) detector was used for the major SG analysis. With these methods, the chromatographic resolution has been improved over the previously published JECFA method, however, it is still not adequate. A resolution of about 1.0 was estimated for the critical pair (Reb A/SV) in these methods, however, more efficient separation of SG is highly desired. 

The objective of this study was to improve the chromatographic resolution of the RPLC of SG, without extending the run time. To achieve this goal, we screened five C18 columns, optimized the elution conditions, and evaluated the analytical method performance. Elements of the enhanced approach to analytical procedure development in the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline Q14 (Final Version, 1 November 2023)7 were adopted in the method development. Commercial Stevia Extracts were analyzed by the developed method.

Experimental 

Method Conditions

• System: Arc Premier System (BSM) with a 2998 PDA Detector
• Detection: UV (210 nm) and PDA (200–400 nm)
• Software: Empower™ 3 CDS
• Column: XSelect™ Premier HSS T3 VanGuard™ FIT Column, 130 Å, 2.5 µm, 4.6 mm x 150 mm (p/n: 186009863)

Results and Discussion

Method Development

We screened five C18 columns using the same gradient elution program that has been recommended by FAO/WHO JECFA Monograph 26. The screened columns are listed in Table 4. All columns showed comparable separations, however, the XSelect Premier HSS T3 Column showed the best resolution for the critical pair (Reb A/SV). Further optimization of the separation conditions for better resolution was carried out on the XSelect Premier HSS T3 Column. The conditions that have been investigated include column temperatures (30 °C–45 °C), flow rates (0.7–1.0 mL/min), and gradient elution. These conditions were investigated using a uni-variate approach. A resolution of 1.5 for the critical pair (Reb A/SV) was achieved in this initial optimization.

The gradient elution parameters, such as elution times and the mobile phase composition, were strongly interdependent on each other, therefore, a multi-variate approach was adopted in fine-tuning the gradient elution conditions for the optimal separation resolution. A multi-variate approach is recommended by the ICH Q14 guideline.7 A resolution of 1.7 for the critical pair was achieved by fine-tuning the elution conditions. Figure 2. shows the acceptable performance region for resolutions of at least 1.70 for the four most challenging pairs. Robustness testing around the optimal LC conditions was also conducted following the ICH Q14 guideline. Figure 3. shows the robustness testing results. A resolution minimum of 1.50 for the critical pair (Reb A/SV) and 1.70 for other challenging SG pairs (Reb O/Reb D, Reb E/Reb O, and SV/Iso Reb A) were obtained. Table 5. shows the range of deviation used in the robustness testing.

Conclusion 

We optimized a JECFA gradient LC-UV method for the analysis of steviol glycosides on an Arc Premier System coupled with a 2998 PDA Detector and an XSelect Premier HSS T3 Column. Chromatographic resolution of SG has been significantly improved. Resolution of 1.5 and higher for the critical pair (Reb A/SV) was reliably achieved on the XSelect Premier HSS T3 Columns (2.5 µm, 4.6 mm x 150 mm), and resolution of 2.0 (for the same critical pair) was achieved on an ACQUITY UPLC HSS T3 Column (1.8 µm, 3 mm x 150 mm). Excellent analytical performance in linearity, sensitivity, accuracy, precision, and robustness has also been demonstrated. This developed method could be a useful alternative method for the analysis of steviol glycosides.