News from LabRulezLCMS Library - Week 51, 2025

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 15th December 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!

👉 SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES

👉 Need info about different analytical techniques? Peek into LabRulezGCMS or LabRulezICPMS libraries.

This week we bring you application notes by Agilent Technologies, Shimadzu and Waters Corporation and technical note by Thermo Fisher Scientific!

1. Agilent Technologies: Advanced SEC-MALS Analysis of Sodium Hyaluronates

• Application note
• Full PDF for download

Hyaluronic acid is used in a variety of cosmetic and pharmaceutical applications due to its viscoelastic properties. The ability to retain water is the reason for its application in eye drops and gels. In cosmetics, hyaluronic acid is applied to reduce wrinkles. 

As with every macromolecular material, the molar mass and molar mass distribution impact the properties of hyaluronic acids. Thus, it is of high importance to reliably and precisely characterize these molecular features to gain both insight into structure-property relations, as well as in quality control (QC) to assure constant material quality. 

Size-exclusion chromatography (SEC), also known as gel permeation chromatography (GPC), is an essential tool for determination of molar masses and molar mass distributions of polymers and macromolecules. SEC separates the molecules by their hydrodynamic size in solution. By running a series of narrowly distributed polymer standards of known molar mass and assigning the peak elution volumes to the molar masses, a calibration curve is constructed. The calibration curve is then used to assign a molar mass to the eluting fraction of the polymer to be analyzed, allowing determination of the molar mass distribution and molar mass averages of unknown samples^.1,2 

As the hydrodynamic size of macromolecules in a given solvent depends on macromolecule chemical structure, only molar masses relative to the applied standards are obtained. In addition, for very large molecules like hyaluronic acid, the situation may arise that no standards of sufficient size are available to allow assigning a molar mass to the elution volume of the analyte. In these cases, parts of the analyte's chromatogram elute outside the calibrated region, resulting in unreliable molar mass determination. 

The lack of sufficiently large polymer standards can be overcome by using a Multi-Angle Light Scattering (MALS) detector, which enables molar mass determination without the need for column calibration. In addition, information about the size of the eluting molecule (radius of gyration) can be obtained by MALS if the dimension of the molecule is large enough, compared to the wavelength of the laser.^2,3 

Experimental 

Equipment 

Agilent 1260 Infinity II//Infinity III GPC/SEC System: 

• Agilent 1260 Infinity III Isocratic Pump (G7110B) 
• Agilent 1260 Infinity III Vialsampler (G7129A) 
• Agilent 1260 Infinity II GPC/SEC Column Thermostat (G7886A) 
• Agilent 1260 Infinity III Variable Wavelength Detector (G7114A) with Standard Flow Cell (G1314-60186) 
• Agilent 1260 Infinity III Refractive Index Detector (G7162A)
• Agilent 1260 Infinity II Multi-Angle Light Scattering Detector (G7885A)

Results and discussion 

Hyaluronic acids and the corresponding salts are frequently of very high molar mass. Often the pore sizes of SEC columns are not large enough to allow separating such high molar mass materials. In such cases, the very large molecules elute without separation at the column's exclusion limit, resulting in a sharp rising peak onset. In addition, not much variation in peak onset is observed for different samples of high molar mass. Figure 1 shows the RI-traces of three different high molar mass sodium hyaluronate samples. Despite their large size, differences in elution time are clearly observed. Peak onset clearly shifts, indicating that the pore size of the applied SUPREMA column combination is appropriate to separate these high molar mass sodium hyaluronates. Additionally, the chromatogram of a series of pullulan standards having molar masses of MP = 1.45 × 106 , 216,000, 20,700, and 991 g/mol is overlaid. The molar mass of the pullulan with MP = 1.45 × 106 is among the highest molar mass standards commercially available to calibrate SEC columns in aqueous solvent. Despite the high molar mass of the pullulan, it elutes significantly later than the hyaluronates. Thus, even if using one of the highest molar mass standards available for aqueous SEC, large fractions of the hyaluronates will elute outside the calibrated region, questioning the reliability of even relative molar masses provided by SEC.

Conclusion 

Agilent SUPREMA ultrahigh columns provide very large pores which allow for the separation of the sodium salt of high molar mass hyaluronic acids. The reduced risk of exclusion peaks lowers molar mass uncertainties resulting from insufficient separation of the high molar mass fraction of the molar mass distribution. 

The large number of scattering angles in the Agilent 1260 Infinity II Multi‑Angle Light Scattering Detector allows reliable extrapolation to zero angle required for correct molar mass and size determination. 

Using a 1260 Infinity II Multi-Angle Light Scattering Detector in combination with an RI-detector allows molar mass determination without establishing a GPC/SEC calibration curve based on polymer standards. Therefore, reliable molar mass determination of hyaluronic acids can be achieved even if no calibration standards of suitable molar mass or suitable chemical structure are available. This reduces the uncertainties in molar masses resulting from different experimental conditions and calibrants used in different laboratories. The determination of absolute molar masses derived by light scattering between laboratories enables comparability and consistency when providing molar masses e.g. for registration purposes.

2. Shimadzu: Enhancing Method Development Efficiency Using MS Peak Tracking with a Triple Quadrupole Mass Spectrometer

• Application note
• Full PDF for download

User Benefits

• Method development is generally performed using LC detectors such as a photodiode array (PDA) detector. However, incorporating mass information obtained from a mass spectrometer enables more accurate peak tracking.
• By visualizing resolution within the design space, the search for optimal separation conditions in method development can be streamlined without relying on intuition or experience.

In the process of separation optimization during method development, optimal analytical conditions are explored by varying LC parameters such as mobile phase composition, gradient profile, and column oven temperature. However, accurate peak tracking across multiple chromatograms can be challenging because differences in separation conditions may cause variations in retention times or co-elution of compounds. Although peak tracking based on differences in UV spectra obtained with a PDA detector can be applied, accurate identification of impurities and co-eluting peaks is often difficult using UV information alone. In contrast, combining a PDA detector with a massspectrometer can improve tracking accuracy, particularly for low-abundance compounds and impurities. This article describes a case study in which the combination of LabSolutions MD, a dedicated software for supporting method development, and LCMS-8060RX triple quadrupole mass spectrometer enabled efficient exploration of optimal separation conditions through accurate MS-based peak tracking for the simultaneous analysis of six small-molecule pharmaceutical compounds. 

Analytical Conditions

Table 1 summarizes the analytical conditions used for optimizing the simultaneous analysis of six small-molecule pharmaceutical compounds. Gradient elution was performed using 0.1% formic acid in water as the aqueous mobile phase and acetonitrile as the organic mobile phase. The separation of each compound was optimized by varying the gradient conditions. Specifically, gradient times of 4, 5, and 6 minutes (three conditions) were combined with final gradient concentrations of 50%, 60%, and 70% (three conditions), resulting in a total of nine analytical conditions.

LC-MS Conditions

• System: Nexera X3
• System: LCMS-8060RX

MS-Based Peak Tracking with LCMS-8060RX

Fig. 1 shows automatic peak tracking of each compound performed by LabSolutions MD, using chromatograms (partially shown) obtained under different gradient conditions and mass information acquired with LCMS-8060RX. LabSolutions MD enables accurate tracking of impurities and co-eluting compounds based on the pre-registered MRM transitions of each compound (Table 2). For compounds without predefined MRM transitions, peak tracking can also be performed by using mass information from scan measurements (full scan without fragmentation within a specified m/z range), allowing all compounds to be tracked based on mass information. As shown in Fig. 1, although the retention times and separation of compounds varied with changes in gradient time and final gradient concentration, all compounds were accurately tracked. Compound (2) in Fig. 1 is an impurity of quinidine. Because its UV spectrum showed a high similarity (≥0.99) to that of quinidine (Fig. 2), peak tracking using UV spectra alone was considered difficult. However, it was correctly identified using mass information (m/z 327.1; Fig. 3) obtained from scan measurements. In addition, dibucaine (Fig. 1, (6)) and amitriptyline (Fig. 1, (7)) were co-eluted, but accurate tracking was achieved even for these unresolved peaks by using product ion obtained from MRM measurements. The MRM chromatograms of dibucaine (Fig. 1, (6)) and amitriptyline (Fig. 1, (7)) are shown in Fig. 4 for reference. 

Conclusion

The efficient method development using LabSolutions MD and LCMS-8060RX triple quadrupole mass spectrometer is presented. By utilizing the mass information obtained from LCMS-8060RX, precise peak tracking can be achieved even for impurities and coeluted compounds. In addition, visualization of resolution within a design space allows efficient exploration of optimal separation conditions without relying on the intuition and experience of the analyst.

3. Thermo Fisher Scientific: Selective and sensitive measurement of 17 steroids in human serum using a Stellar mass spectrometer

• Technical note
• Full PDF for download

Steroidogenesis is the biological process by which the body synthesizes steroid hormones from cholesterol. It begins in the mitochondria with the conversion of cholesterol into pregnenolone, which serves as the precursor for several classes of steroids, including progestogens, corticosteroids, androgens, and estrogens (Figure 1). Steroidogenesis is vital for numerous physiological functions, including maintaining cell membrane structure, hormone production, cellular signaling, growth and development regulation, reproduction, and immune functions. 1 Disruptions in steroidogenesis can result in endocrine disorders such as congenital adrenal hyperplasia, Cushing's syndrome, and certain forms of hypertension. 1-3 Therefore, accurate clinical research laboratory tests require researchers to prioritize the right analytical testing methods to ensure reliable monitoring of steroid hormones.

Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the gold standard for quantifying steroids in serum due to its ability to measure multiple steroids simultaneously with high sensitivity and selectivity. However, quantifying such a steroid panel in human biofluids is challenged by the structural similarity among different steroids and their low concentrations in biological samples. Researchers are exploring LC-MS systems with sufficient sensitivity, specificity, selectivity, wide dynamic range, and excellent precision and accuracy for reliable concentration measurements. 

In this technical note, we demonstrated the streamlined development of a method for the simultaneous measurement of 17 steroids in human serum using a Thermo Scientific™ Vanquish™ Horizon ultra-high performance liquid chromatography (UHPLC) coupled to the Stellar mass spectrometer (MS) (Figure 2). The Stellar MS is a hybrid quadrupole, dual-pressure linear ion trap MS that offers two types of orthogonal, yet complementary, fragmentation modes with rapid MS2 and MS3 scans: beam-type high-energy collision-induced dissociation (HCD), which is a triple quadrupole (QqQ)-like fragmentation, and resonance-type collision-induced dissociation (CID).4 We utilized TraceFinder software to aid the compound optimization and demonstrate the advantages of utilizing multiple collision activations and MS3 scans performed by the Stellar MS to the selective and sensitive quantification of 17 steroids in serum, highlighting its potential to improve the monitoring of clinical biomarkers and therapeutic drugs.

Experimental

Liquid chromatography – mass spectrometry 

Samples were analyzed on a Vanquish Horizon UHPLC system coupled to the Stellar mass spectrometer in the targeted-MS2 (tMS2 ) and targeted-MS3 (tMS3) scan modes. The mobile phase, analytical column, and LC gradient are specified in Table 1. The Stellar MS was equipped with the Thermo Scientific™ OptaMax™ Plus ion source and heated electrospray ionization (HESI) sprayer. Multiple fragmentation schemes were evaluated and optimized on the Stellar MS. The source and scan properties are listed in Table 2.

Results and discussion 

Compound optimization 

Given the significant variations in the chemical structures of small molecules, it is highly recommended to optimize MS parameters to achieve maximum sensitivity and selectivity for their quantitative analysis in complex matrices using LC-MS/MS methods. The Stellar MS offers several beneficial and differentiating features compared to a conventional QqQ MS commonly employed for targeted quantitative analysis for small molecules, including the rapid acquisition rate with MS2 up to 140 Hz and MS3 up to 40 Hz, fast polarity switching of 5 milliseconds, and the capability of acquiring the full fragmentation spectra of the analyte precursor.4 The resonance-type CID fragmentation and MS3 scan provide alternative, yet complimentary capabilities to the QqQ-like HCD and could improve the detection sensitivity and selectivity. TraceFinder software offers an automated feature to facilitate online optimization of MS parameters in a panel of analytes. Figure 3 illustrates the optimization process using the MS2 normalized collision energy (NCE) for HCD and CID for testosterone. Other MS parameters, such as RF lens and vaporizer temperature, can be similarly optimized using TraceFinder software. The targeted product ions can be determined from literature, MS fragmentation spectra library repositories, such as Thermo Scientific™ mzCloud™ (https://www. mzcloud.org/) spectral library and MassBank of North America (https://mona.fiehnlab.ucdavis.edu/), or a survey MSn scan. 

In this technical note, up to four most intense fragment ions of each steroid and its corresponding IS were selected from the published quantification method using QqQ MS,5 in addition to the online LC-MS injection of the synthetic standards with NCE 30% in both HCD and CID MS2 scans. Our empirical data indicated that NCE 30% offers efficient fragmentations to many small molecules in both HCD and CID MS2 , thus, it was chosen for the survey scan. A series of instrument methods were built for the steroid panel with varying NCE settings: 0 to 90% NCE for HCD (10% interval) and 0 to 50% NCE for CID (5% internal). The same steroid mixture was injected in duplicate for each method, and the average peak areas of the target fragment ions were determined in TraceFinder software from the Group Average. Figure 3 uses testosterone product ion m/z 97 and estradiol product m/z 183 to illustrate the peak area changes with ramping NCE settings. The optimal NCE values and potential MS3 candidates can be selected from the resulting “breakdown curves.” The fast tandem MS scan speed of the Stellar MS allows for the simultaneous optimization of steroids and corresponding IS in both MS2 -HCD and MS2 -CID scan modes without retention time (RT) scheduling.

Conclusion and future work 

Sensitive and selective measurement of steroids in serum using the Stellar MS was demonstrated with a highly multiplexed method. The experimental results show method optimization can be performed using UHPLC separation at extremely low levels to streamline final method development in the presence of matrix. Leveraging the instrument flexibility of the Stellar MS, both beamtype and resonance-type dissociation techniques at the MS2 and MS3 levels were evaluated for the set of analytes across the spiking ranges, resulting in determination of the best parameters for the entire set of steroid hormones. Future work will leverage the information obtained in the current work to extend LOQ performance for the set of steroid hormones. 

Future work will leverage the information obtained in the current work to extend LOQ performance for the set of steroid hormones. We demonstrated that the Stellar MS could perform fast and sensitive tMS2 and tMS3 in one method using HCD and CID with more than seven data points per LC peak at the lower spiking levels with UHPLC peak widths of 6 to 12 seconds. Additional method improvement could be achieved using narrower scheduled RT windows from the 5-minute setting in this work to less than 1 minute or by selecting only one scan event per analyte. Collectively, these steps will increase the injection time and further improve the data quality.

Overall, the Stellar MS showed fast scan speed, alternative fragmentation, novel source design, and software features that enabled high-throughput, sensitive, and selective biomarker quantifications required by clinical research.

4. Waters Corporation: Constant Neutral Loss, Precursor Ion Scanning, Product Ion Scanning in Support of DMPK Studies Using waters_connect for Quantitation Software Solution and Xevo TQ Absolute XR Mass Spectrometer

• Application note
• Full PDF for download

Benefits 

• Rapid, simple screening of biofluids for drug related species in discovery and development DMPK studies Rapid screening of in vitro incubations and in vivo sample for the presence of reactive metabolites such as glutathione
• Confirmation of the identity of potential drug related peaks in metabolite profiling, dose escalation, or species – species comparison studies
• Simplifying troubleshooting in quantitative bioanalysis studies

Drug Metabolism and Pharmacokinetics (DMPK) studies to support drug discovery and development, provide information of the extent of exposure to the drug, route and rate of elimination and the metabolic fate of the molecule. Analytical support for these studies involves the detection and quantification of the dosed drug and resulting metabolites in a variety of biological fluids (blood, plasma, urine, tissue etc.) and in vitro samples. Tandem quadrupole LC-MS/MS is the dominant technique for the quantitative measurement of drug and metabolites concentrations in DMPK studies due to its sensitivity and speed when operated in multiple reaction monitoring (MRM) or single ion monitoring (SIR) mode. Drug metabolite de novo structural elucidation normally accomplished using accurate mass LC-MS/MS and/or NMR. 

During the course of a drug development program multiple studies are performed in cell lines, laboratory animals and humans to identify the effect of dose level on pharmacokinetics, metabolites formation, efficacy, off target pharmacology and toxicity. In these studies it is often necessary to scan for reactive metabolites or confirm and compare the metabolic profile of the drug candidate in multiple studies, e.g., comparing inter species profiles or in a dose escalation study. This metabolite comparison/confirmation can be quickly and efficiently achieved using the neutral loss, precursor ion and product ion scanning functions of a modern tandem quadrupole mass spectrometer such as the Xevo TQ Absolute XR.

Conclusion 

Tracking and confirming drug metabolites in preclinical or human clinical studies is essential to successful drug candidate development. By employing the scanning functions available on the Waters Xevo TQ Absolute XR Mass Spectrometer, precursor ion scanning, constant neutral loss, and product ion scanning, drug related metabolites can be easily detected and their identity confirmed. These acquisition functions are easy to configure in waters_connect for Quantitation software. The rapid acquisition speed of the Xevo TQ Absolute XR Mass Spectrometer allows these information-rich acquisition modes to be performed in the same acquisition method as MRM acquisition without compromising the quality of the quantitative data. Samples derived from the oral administration of methapyrilene to male rats was analyzed using the precursor ion, constant neutral loss, and product ion scanning acquisition modes. The data derived from this analysis allowed for the accurate tracking and confirmation of the major drug metabolites in this study.