News from LabRulezLCMS Library - Week 12, 2025

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 17thMarch 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 Agilent Technologies, Knauer, Shimadzu and Waters Corporation and technical notes by ALS Europe and Thermo Fisher Scientific!

1. Agilent Technologies: Peptide Mapping of Trastuzumab Tryptic Digests on an Agilent 6545XT AdvanceBio LC/Q-TOF

• Application note
• Full PDF for download

There are several LC/MS techniques that can report on protein sequence and posttranslational modifications (PTMs) present for monoclonal antibodies (mAbs). For example, intact protein mass spectrometry will use MS1 information and deconvolution tools to report on the mass correlation to the original and potentially modified sequence of the mAb, as well as the relative abundances of glycosylation present in the sequence. Since PTMs can play a role in the safety, efficacy, and binding of a therapeutic to its target¹ , the ability to digest an intact mAb into smaller peptides and identify the location of unknown PTMs is an important technique in the biotherapeutic process. Peptide mapping uses a combination of MS1 and MS2 information to confidently assess the primary sequence of an mAb, as well as identify the location of PTMs and sequence variants.² A 6545XT AdvanceBio LC/Q-TOF was used in combination with Agilent MassHunter BioConfirm software, version 12.1 to characterize trastuzumab for sequence confirmation, glycosylation heterogeneity, and unknown PTMs.

Experimental

LC/MS analysis 

LC/MS analysis was performed on an Agilent 1290 Infinity II Bio LC system coupled to a 6545XT AdvanceBio LC/Q‑TOF system (Figure 1). To prepare the 6545XT AdvanceBio LC/Q-TOF system for acquisition, a TOF transmission tune was performed with the Fragile Ions checkbox selected. An Agilent AdvanceBio Peptide Mapping column (2.1 × 150 mm, 2.7 µm) was used for chromatographic separation. LC and MS parameters used are listed in Tables 1 and 2. 

Data processing 

Raw data from LC/MS/MS analysis was processed using MassHunter BioConfirm software, version 12.1. Trastuzumab, Trypsin, and LysC sequences were entered into BioConfirm biopharmaceutical software using the Sequence Manager tool. For trastuzumab, cysteines involved in disulfide bonds were modified directly in the sequence to incorporate iodoacetamide alkylation as a fixed modification. The peptide mapping search includes several filters to restrict the appearance of low-quality MS/MS mass spectra that can artificially inflate the sequence coverage calculation. For example, mass matching used thresholds of 5 ppm for MS1 and 20 ppm for MS². In addition, a minimum BioScore of 5 for the sequence coverage calculation was set to restrict the inclusion of false positive peptide identifications. The BioScore was calculated as the –log¹⁰ of the spectral E-value score, which is a metric that evaluates the statistical significance of individual peptide-spectrum matches relative to a decoy database. Practically speaking, an E-value of 0.00001 (corresponding to a BioScore of 5) means that there is a very low chance of finding a match by random chance within the searched database. More details on the calculation of the spectral E-value score can be found in reference 3.3 The processing parameters used for the peptide mapping search are listed in Table 3.

Conclusion 

Peptide mapping is a routinely used LC/MS/MS technique, employed in the biopharmaceutical industry to confirm mAb sequence and identify PTMs. This application note presents a workflow solution that generates a sequence coverage of 98% for trastuzumab when digested with Trypsin and LysC at a protein-to-enzyme ratio of 25:1. The peptide mapping coverage map was collected with a fragmentor voltage of 95 V, since higher fragmentor voltages (170 and 125 V) resulted in the appearance of peptide fragments that were not reflective of the digestion protocol outlined here. Furthermore, the extended dynamic range capabilities of the Agilent 6545XT AdvanceBio LC/Q-TOF are also highlighted, where two peptides that have MS1 signals in the ~ 1e⁴ range show near-complete fragment ladders. The native and deamidated forms of the FTISADTSKNTAYLQMNSLR peptide show unique fragment ions that unambiguously identify the site of deamidation. Finally, the TKPREEQYNSTYRVVSVLTVLHQDWLNGK with G0F glycopeptide was identified from the MS/MS spectrum, despite the dominant fragmentation pathway for the glycopeptide generating the glycan fragment at m/z 204.0865. The combination of quality MS/MS spectra and depth of coverage for low-abundant peptides highlight the capabilities of the 6545XT AdvanceBio LC/Q-TOF for peptide mapping applications.

2. ALS Europe: Environmental Challenges of Pharmaceuticals: Persistence and Accumulation in Ecosystems

• Technical note
• Full PDF for download

Pharmaceuticals as "New" Pesticides...

Pharmaceuticals represent a broad and chemically diverse group of substances that is continuously expanding and is characterised by a wide range of clinical effects and extensive use, often leading to overuse, in both human and veterinary medicine. From an environmental perspective, they are classified as organic micropollutants due to their presence in water at low concentrations (ranging from ng/L to μg/L). Both the public and experts are concerned not only about their negative impacts on natural ecosystems but also about their presence in drinking water and the related effects on human health. Their contribution to the emergence of antimicrobial resistance (AMR) is also significant, as it greatly complicates the treatment of infectious diseases and represents a major challenge for current and future medicine.

Drugs can enter the environment through various pathways. One of the primary sources of contamination is wastewater from households and health and social care facilities, which contains human urine and faeces, as well as unused or expired pharmaceuticals. Another significant source is wastewater from the pharmaceutical industry. Consequently, wastewater treatment plants (WWTPs) themselves become sources of environmental contamination, as they are unable to effectively remove pharmaceuticals, allowing them to be transported further into the environment [1].

This occurs through treated wastewater discharged into surface waters and sewage sludge used as fertiliser. Livestock production also contributes significantly, as animals excrete pharmaceuticals in their urine and faeces, leading to contamination via aquaculture, grazing, and manure application on agricultural land. Pharmaceuticals enter the environment in both unchanged and metabolised forms; the latter results from the body's conversion of substances into more mobile polar forms.

European Legislation

EU legislation governing the quality of various types of water is gradually incorporating pharmaceuticals into its relevant documents at both European and regional levels. Regarding municipal wastewater, a proposal for a revised directive on wastewater treatment has been introduced, and the legislative process is nearly complete. Member States will now be required to ensure both the monitoring of antimicrobial resistance (for agglomerations with populations of 100,000 or more) and the removal of the widest possible range of micropollutants, particularly pharmaceuticals (by the end of 2045 for all wastewater treatment plants with a load of 150,000 or more). To determine whether the required minimum removal rate of 80% has been met, 12 parameters will need to be monitored, almost all of which are pharmaceuticals. The list of the relevant compounds is summarised in Table 1.

Pharmaceuticals at WWTPs

ALS laboratory research projects have examined the rate of pharmaceuticals removal through conventional mechanical-biological treatment of wastewater as conducted at standard municipal WWTP. The results are shown in the graphs in Figure 3. Figure 3 (a) presents the results for the analytes most frequently detected in wastewater and at the highest concentrations, their removal rate is as high as 80-90%. Figure 3 (b) illustrates the pharmaceuticals proposed for monitoring under the mentioned EU regulations. It is evident that the removal rate for these analytes is lower, averaging only about 20%, highlighting the importance of their monitoring.

ALS Laboratories have developed and accredited a multiresidue methods for the determination of more than 100 different pharmaceuticals and their metabolites in various types of water. All of our analytical methods use the LC/MS/MS technique for analytes determination, which provides high sensitivity, selectivity and precision of measurement and allows the determination of target compounds at the very low limits required for residue analysis.

3. Knauer: Determining molecular weights for P(D,L)LA in ethyl acetate – an internal validation

• Application Note
• Full PDF for download

Plastics are one of the most widely used most versatile materials in the 21st century. However, the majority of the world’s annual production of approximately 370 million tons of plastics is neither biodegradable nor produced from renewable raw materials.(1) In contrast, poly lactic acid (PLA) is one of the most promising plastics with sustainable properties. The physical properties of plastics are mostly determined by their molar mass distribution, with the parameters Mn, Mw and the PD being the most important. GPC is the method of choice for the determination of these values. When no absolute method is available, the two most common calibrations are conventional and universal calibrations. For a conventional calibration, calibration standards of the polymer to be analyzed, or at the least a polymer of high chemical similarity, are required.

For a universal calibration, first published by Benoit(2), a universal calibration curve is created via the Mark-Houwink relationship, so that a wide variety of polymers can be analyzed, for example with a narrow polystyrene (PS) calibration. In 1998, S. Mori published the results of a multistage round-robin test in which PS standards were analysed by GPC using conventional calibration (CC) with PS under various framework conditions.(3) The relative interlaboratory standard deviation (%RSD) for Mn of more than 20% was reduced by setting strict framework conditions for the analysis, such as injection volume, sample concentration, and sample size.

RESULTS AND DISCUSSION 

Initially, a calibration was made using PMMA, since polystyrene (PS) proved unsuitable in EtOAc due to interactions with the stationary phase (VTN0021). The two largest molar masses were excluded due to the peak shape, resulting in an 10-point universal calibration with a 5th degree fit function. The overlay of calibration function and the chromatograms is shown in Fig. 1. For universal calibration with PMMA in EtOAc, the following Mark-Houwink parameters were used: K= 21.1·105 dL/g, α=0.64.(5) For PLA K=15.8·105 dl/g and α=0.78 were used.(6)

CONCLUSION 

These experiments have shown that the determination of the Mn and Mw of low molecular weight P(D,L)LA is easily possible using a KNAUER HPLC system combined with a gel permeation chromatography (GPC)-column. The intralaboratory validation proves that the developed method is robust within the limits shown here with respect to changes in temperature, concentration, flow rate and injection volume. Furthermore, the repeatability of the molar mass determination as well as the intermediate precision of the sample preparation was verified. It was shown that the CC fails for this column/solvent/ polymer combination, whereas the UC provides good results. The conventional calibration did not fulfil the specification because the different hydrodynamic radii of PLA and PMMA in EtOAc were not considered. The flow rate has a very strong influence on the molar mass determination and must be as constant as possible. Slight fluctuations affect the retention volume. In order to take fluctuations into account, a flow marker, in this case BHT, was used and is recommend as mandatory for all GPC/SEC applications.

4. Shimadzu: Determination of Aminoglycoside Drugs Residual in Bee Products by LC-MS/MS

• Application Note
• Full PDF for download

User Benefits:

• Heptafluorobutyric acid is added to the injection vial to enhance the retention of aminoglycosides
• There is no need to use ion-pairing reagents and highly concentrated salt solutions in the mobile phase, which might inhibit the mass spectrometry signal

Aminoglycosides (AGs) are composed of glycoglycans and aminocyclic alcohols combined with glycoside bonds. Figure 1 shows the structure of streptomycin as an example of an aminoglycoside. Their main role isto hinderthe protein synthesis of bacteria, so the permeability of bacterial cell walls changes, which exerts antibacterial effects. In recent years, it has been reported that AGs have significant ototoxicity, nephrotoxicity, and vestibular function damage, which can lead to shock and even death in severe cases. GB 31650-2019 “Maximum Residue Limits of Veterinary Drugs in Food” stipulates residue limits of gentamicin, kanamycin, spectinomycin, streptomycin, dihydrostreptomycin, and neomycin B in different matrices.

In this paper, a method for the detection of aminoglycoside residuesin honey was established. The extract was divided into two equal parts and purified by MCX and WCX SPE cartridges, respectively. This method covers AGs commonly used in the livestock and poultry industry. Ion-pairing reagents and highconcentration salt solutions are not needed in the mobile phase, and the results are accurate and reliable, and can effectively detect the residues of aminoglycosidesin bee products.

Sample Preparation

Purification

The MCX SPE column (200 mg/6 mL) and WCX SPE column (150 mg/3 mL) were activated with 5 mL of methanol and 5 mL of water, respectively. The prepared solution was divided into two aliquots, one passed through the MCX SPE cartridge, then rinsed with 7.5 mL of water and 7.5 mL of methanol, and eluted with 5 mL of ammonia methanol solution for the analysis of neomycin, kanamycin, apramycin, spectinomycin, hygromycin and tobramycin. The other solution passed through WCX SPE cartridge after adjusting the pH to 7.5 with sodium hydroxide solution, then washed with 7.5 mL of water, and eluted with 5 mL of methanol acetate for the analysis of streptomycin, dihydrostreptomycin and gentamicin. The two parts of the eluate were dried at 40℃ with nitrogen atmosphere, dissolved with 2 mL of 0.3% acetic acid waterHFBA (99:1), filtered through a 0.22 μm membrane, and placed in a plastic vial for LC-MS/MS analysis (system Nexera LC-40 X3).

Conclusion

A method for the detection of aminoglycoside residues in honey samples was established. This method only adds ion-pairing reagents to the vials, and analytes were well retained on the C8 column. Aminoglycoside drugs have good linearity in the concentration range of 5 ng/mL to 500 ng/mL, with a correlation coefficient R2>0.996. The recoveries of the samplesspiked at 25 and 50 μg/kg ranged from 68.5 to 93.4%. This method is sensitive, accurate, and can be used for the determination of aminoglycoside drug residuesin bee products.

4. Thermo Fisher Scientific: Comparison of InVial and AboveVial mode in fraction collection on a Vanquish Analytical Purification LC system

• Technical Note
• Full PDF for download

The Thermo Scientific™ Vanquish™ Analytical Purification LC system provides highresolution sample separation followed by precise sample purification, which is facilitated by the integrated Thermo Scientific™ Vanquish™ Fraction Collector (FC). The Vanquish FC comes with a 3-axis motion control, which enables the two collection modes—AboveVial and InVial—for flexible multi-application purposes (Figure 1).1 

AboveVial mode provides low cross-contamination of fractions due to the non-wetted needle and the fast time of transferring to an adjacent vial (<0.75 s). The adjustable needle height enables the use of a wide variety of sample containers from 384 or 96 well plates to racks holding 2–10 mL vials. InVial mode enables sample collection from the bottom of the container (just 3 mm from the bottom) to avoid sample splashing during droplet formation. Notably, the collection needle can puncture sealed caps like the Thermo Scientific™ Vanquish™ Autosampler, thereby reducing the risk of sample evaporation 

In this technical note, five compounds were employed as standards, and gradient elution was used to evaluate the two collection modes. To be noted are risks associated with InVial collection and AboveVial collection. When collecting InVial, the Automatic (default) setting positions the needle 3 mm above the bottom of the vial. Therefore, the risk is that the fill level in the collecting vessel reaches the collection needle and this could result in the contamination to the outside of the collection needle. This can be avoided by adjusting the InVial needle height below the top level of the collection vessel but above the expected fill level of the liquid inside the collection vessel. When collecting AboveVial, the Automatic (default) setting positions the collection needle 2 mm above the specified vial or plate height. This is a safe setting for the needle when it moves along the collection path above the collection vessel. The risk associated with this collection mode is that various well plate or vial manufacturers have different well plate or vial heights and this could result in a collision of the collection needle with the side of the collection vessel. To be safe, test the needle height before beginning the fraction collection as described in the technical note: Principles of fraction collection using the Vanquish HPLC and UHPLC systems1 .

Experimental

Instrument configuration

The Vanquish Analytical Purification LC system included a pump, autosampler, column compartment, diode array detector (DAD), and fraction collector, all controlled by the Thermo Scientific™ Chromeleon™ Chromatography Data System 7.3.1 and newer. Refer to Table 1 for configuration details.

Conclusions 

The Thermo Scientific Vanquish Fraction Collector controlled by Chromeleon CDS enables excellent fractionation, providing purer samples for further research. The two collection modes of AboveVial and InVial can be adopted for numerous applications with low cross contamination. While the AboveVial collection mode enables continuous fractionation with the fastest transition from vessel to vessel, the InVial mode offers the advantage of the exact positioning height of the needle within the vial to minimize the risk of any splashing even at higher flow rates. Moreover, combining the cap piercing capability and adjustable needle height, the collection function of filling from bottom effectively reduces volatile gradient solvent evaporation during fraction collection.

6. Waters Corporation: Optimizing IP-RP CRISPR sgRNA Purification Passes With High Efficiency Oligonucleotide Certified BEH 300 Å C18 5 µm Preparative Sorbent

• Application Note
• Full PDF for download

Benefits:

• Batch tested and selected XBridge™ Oligonucelotide BEH C18 5 µm 300 Å sorbent particles for predictable, ow secondary interaction adsorptive interactions 
• Widepore organosilica sorbent columns with optimized diffusion kinetics for CRISPR sgRNA, chemical rugged for many repeat runs, and mechanically stability confered by specialized optimal bed density packing procedures
• Increases in accessible surface area per column length as a result of reduced particle size and pore size optimization

The application of CRISPR for gene editing and therapeutic interventions demands a high level of precision. The specificity of the sgRNA-Cas complex in targeting the intended sequence is crucial, as off-target effects can lead to unintended genetic modifications, which may compromise the safety and efficacy of the approach. Thus, the purity of sgRNA used in CRISPR experiments is of paramount importance. Impurities present in the sgRNA preparation can affect the binding efficiency and accuracy of the sgRNA-Cas complex, increasing the likelihood of off-target DNA cleavage events. Therefore, optimizing the purification process to remove impurities and ensure the highest possible sgRNA purity is vital for minimizing these risks. The purification of sgRNA typically employs chromatographic techniques that exploit the physicochemical properties of nucleic acids.3 On a laboratory scale, ion-pairing reverse-phase (IPRP) liquid chromatography is a commonly used method, particularly effective for separating oligonucleotides like sgRNA based on their hydrophobic interactions with the stationary phase. IPRP is often chosen due to its ability to discriminate between full-length products and various truncated or chemically modified versions of sgRNA, which may arise during synthesis. sgRNA sequences are frequently prepared by solid-phase phosphoramidite chemical synthesis so that important synthetic modifications such as 2'-O-methylation or phosphorothioate linkages can be readily incorporated for enhanced stability and functionality. The crude sgRNA mixture, which contains both the desired full-length product and various synthesis by-products or impurities, is subjected to IPRP chromatography. Using specific buffer systems and optimized gradients, impurities can be separated based on retention time differences, allowing for the isolation of high-purity sgRNA fractions. 

Traditionally, large particle size columns containing 8 to 20 µm diameter sorbents have been applied. However, to enhance separation efficiency and loading capacity of sgRNA, column characteristics such as pore size and particle size should be carefully considered.

In this work, we therefore investigate the use of a new oligonucleotide batch tested and selected widepore BEH C18 sorbent. Herein, BEH C18 Columns with smaller particle sizes (e.g., 5 µm) and optimized pore dimensions (e.g., 300 Å) are applied to achieve new single pass, purity levels that are required for high-precision CRISPR applications.

Experimental

Purification IP-RPLC

LC system: Waters™ LC Prep 150 System, comprised of a 2545 Quaternary Solvent Manager, 2489 UV/Visible, (UV/Vis) Detector™, and WFC III. Fraction collection was triggered by peak detection.

Analytical IP-RPLC-UV/MS

LC system: Waters ACQUITY™ UPLC H-Class Plus System, comprised of BioQuaternary Solvent Manager, Bio Sample Manager FTN-H, and ACQUITY™ UPLC and ACQUITY Premier Tunable UV Detectors

Results and Discusion

Assessing an XBridge Oligonucleotide BEH C18 OBD 300 Å 5 µm Column The 5 µm Oligonucleotide BEH 300 Å C18 Column showed a marked improvement over its 10 µm counterpart in terms of purity and the separation of impurities. Initially, the sgRNA purification on the 10 µm column resulted in a purity level of ~35% (Best Fraction), with significant front impurity peaks (~16%) still present. Subsequent purification attempts failed to yield substantial improvements in separating these impurities, highlighting the limitations of the 10 µm sorbent in handling closely eluting species. In contrast, the XBridge Premier Oligonucleotide BEH C18 300 Å 5 µm Column delivered superior performance across all purification rounds. The increased surface area provided by the smaller particle size of the 5 µm sorbent significantly enhanced the binding efficacy, resulting in improved retention and better separation of impurities. This column achieved purity levels of approximately 41–45% in repeated purifications, with a consistent reduction of front impurity peaks to around 11%, demonstrating its efficiency in removing early-eluting contaminants. The enhanced binding and retention characteristics of the 5 µm sorbent column also can be translated into a higher loading capacity. The ability of the sorbent to retain and effectively separate higher concentrations of the sample contributed to more efficient purification cycles. This is especially beneficial for handling complex biomolecules like sgRNAs, where higher loading often necessitates greater separation capabilities due to the increased potential for co-eluting impurities.

Conclusion 

These results demonstrate that the XBridge Oligonucleotide BEH C18 300 Å 5 µm OBD Column provides significant advantages over larger particle size options, particularly in enhancing impurity separation and increasing loading capacity. These benefits underscore its utility in the high-resolution purification of sgRNAs and similar oligonucleotide products, emphasizing its role as a preferred choice in high-performance liquid chromatography (HPLC) methods. Future investigations will focus on addressing the late-eluting impurities through synthesis optimization and exploring alternative gradients and purification schemes that combine weak and strong ion pairing passes as well as anion exchange coupled with IP-RP purification.