News from LabRulezLCMS Library - Week 31, 2024

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 29th July 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 to you applications, brochures and specifications from Agilent Technologies, Shimadzu, Metrohm, Thermo Fisher Scientific, and Waters Corporation!

💡 Almost 1000 ASMS posters and presentations available in the LabRulezLCMS library (unique search and filtering)

1. Metrohm: Determination of total iodine in thyroid tablets with polarography

• Application Note

Polarography in pharmaceutical analysis: indirect determination of iodine after dry ashing in a muffle furnace

Thyroid hormones are made by the thyroid gland and play an important role in regulating metabolism and growth. Iodine acts as a building block and the specific number of iodine atoms determines the type of the hormone: four for thyroxine (T4) and three for triiodothyronine (T3). The number of iodine atoms is critical for the functionality of thyroid hormones. Levothyroxine and liothyronine (synthetic forms of thyroid hormones T4 and T3) are essential components of thyroid tablets. T4 is less active and needs to convert to the more active T3 to be fully effective. Accurate iodine determination in thyroid tablets is a crucial quality control measure, ensuring the effectiveness and safety of thyroid treatments. A robust method is introduced for indirect determination of total iodine content in thyroid tablets as iodate, according to United States Pharmacopeia (USP) guidelines using the 884 Professional VA and the Multi-Mode Electrode pro.

2. Shimadzu: Efficient Preparative Purification Workflow of Synthetic Peptide Using Analytical/Preparative Switching LC-MS System

• Application Note

User Benefits

• A multi-step seamless preparative purification workflow of synthetic peptides can be performed within a single LC-MS system setup.
• LabSolutions MD can provide easy investigation for optimal separation conditions for target compounds.
• Target compounds can be purified from crude samples with high purity and high recovery based on the excellent identification capability of LCMS-2050.

Introduction: In the 2000s, biopharmaceuticals such as antibody drugs have emerged but there are many challenges to overcome because of their manufacturing process using genetic technology. Consequently, middle molecule drugs are paid attention, peptide therapeutics, one of middle molecule drugs which have advantages of being manufactured at low cost, easily taken into cells because of theirsmall molecular weight, and prevented from degradation by adopting a specific three-dimensional structure when they are taken into human body. Since such peptides are produced by chemical synthesis like small molecule drugs, it is essential to purify, fractionate, and confirm the purity of the final synthesized product. In this article, we present a case study of multi-step seamless preparative purification workflow (optimizing separation conditions, scaling-up, fractionation, and confirming (purity/recovery) of a peptide using preparative purification liquid chromatograph Nexera Prep (Fig. 1) based on Application News 01-00650-EN and 01-00651-EN.

Conclusion: The seamless preparative purification workflow can be executed using analytical/preparative switching LC-MS system. In addition, LabSolutions MD can automatically create an analytical batch schedule in which various HPLC parametersettings are examined, resulting in efficient optimization ofseparation conditions. Furthermore, LCMS-2050 enables highly selective fractionation of target compounds based on MS trigger. The analytical/preparative switching LC-MS used in this article has both analytical and preparative flow paths, enabling an efficient preparative purification workflow in the preparative purification of peptide including itssynthesis confirmation process.

1. Seamless Purification Workflow from Analytical to Preparative in Single LC-MS System, 01-00650-EN
2. High Purity Preparative Purification Enabled by UV/MS Trigger on LC-MS System,01-00651-EN.

3. Shimadzu: Rewriting the Book on Supercritical Fluid Chromatography

• Technical Note

Fluids become supercritical when they are highly compressed and take on the properties of both a liquid and a gas. Supercritical carbon dioxide (CO₂) can be used as the mobile phase in supercritical fluid chromatography (SFC). Until recently, this technique had been overlooked in favor of high-performance liquid chromatography (HPLC) and gas chromatography (GC). However, new developments in the field have demonstrated that SFC can be a highly cost-effective, accurate and sensitive solution capable of outperforming HPLC and GC. In light of these new technologies, it’s time to reshape the field of chromatography with SFC.

4. Agilent Technologies: Advanced SEC-MALS Analysis of Monoclonal Antibodies with the Agilent 1260 Infinity II Multi-Angle Light Scattering Detector

• Application Note

Abstract: Size exclusion chromatography (SEC) in combination with multiangle light scattering (MALS) detection enables accurate and reliable separation and molecular weight (MW) determination of monomer and aggregate protein species. The Agilent 1260 Infinity II Multi-Angle Light Scattering detector coupled to the Agilent 1290 Infinity II Bio LC System offers the possibility to measure the intensity of the scattered light at multiple angles to gain more precise absolute MW for the analysis of biopharmaceuticals. The 1290 Infinity II Bio LC is specially designed for conditions used in biochromatography with a completely iron-free flow path and a high tolerance to salt-based buffers such as those used in SEC.

Conclusion: The possibility of using a MALS detector with 20 angles enables highly precise calculations of absolute molar mass. The MALS detector shows higher sensitivity than UV detection especially for higher protein aggregates, adding valuable information to a conventional SEC setup. The smaller angles allow the determination of higher molecular mass aggregates with high sensitivity and precision. The excellent resolution of the Agilent AdvanceBio SEC 300 Å column enables clear separation of monomer, dimer, higher aggregates, and mAb fragments, leading to unique MW determinations for the separated peaks. The combination of the Agilent 1290 Infinity II Bio LC System with the Agilent 1260 Infinity II Multi-Angle Light Scattering detector provides a complete biocompatible flow path to ensure trusted results for challenging solvent conditions or iron-reactive samples.

5. Agilent Technologies: Evaluation of Agilent RoHS-Compliant LC/MS Ion Injectors

• Technical Note

Introduction: Recently, governments across the globe have started to reduce the use of hazardous materials in electronic components. Specifically, the Restriction of Hazardous Substances in Electrical and Electronic Equipment (RoHS) Directive in the European Union has been driving this effort. The Agilent resistive ion injector has been updated to a leaded glass–free, RoHS-compliant-without-exemption part for use in Agilent LC/MS quadrupole instruments that do not have an ion funnel. The ion injector acts to pull desolvated ions from the desolvation chamber into the MS (Figure 1). In doing so, the ions are culminated into a single, narrow beam, allowing the MS to separate the ions according to their mass-to-charge ratio (m/z). The ion injector is resistive, which allows for fast polarity mode switching, and has metal contacts at either end for electrical connection to the MS. In this technical overview, the performance of the Agilent RoHS ion injectors was compared to that of their respective legacy ion injector models (Table 1).

Conclusion: In this technical overview, the Agilent RoHS-compliant ion injectors were compared to their respective Agilent legacy ion injectors. The studies presented in this technical overview demonstrate equivalent functionality between the legacy and RoHS ion injector models. Specifically, tune performance was shown to be equivalent between RoHS and legacy ion injectors, and lifetime performance under acidic and basic conditions was also shown to be comparable between RoHS and legacy ion injections, including at high mass on the Agilent 6545XT AdvanceBio LC/Q-TOF. Moreover, IDLs and S/N were within specifications on multiple of the tested RoHS ion injectors. Furthermore, the RoHS ion injectors held up to 10,000 injections of bovine urine and maintained tune criteria throughout, demonstrating excellent robustness. Finally, no differences in performance were observed on two different MS platforms when running target pharmaceuticals, both in neat samples and diluted human urine. In conclusion, the RoHS Agilent FS and Agilent Ultivo and MSD iQ ion injectors are suitable drop-in replacements for the legacy Agilent FS and Agilent Ultivo ion injectors.

5. Waters Corporation: PEGS: SIMPLE AND AUTOMATABLE SAMPLE PREPARATION FOR TRYPTIC PEPTIDE MAPPING WITH THE PEPTIDEWORKS KIT

• Poster, PEGS 2024

NTRODUCTION

Reliable day-to-day sample preparation is a critical factor in generating an effective peptide mapping method. Sample preparation for peptide mapping can result in method-induced peptide modifications, over- or under-digestion of the protein, and autolysis of the proteolytic enzyme, each of which complicate data analysis and interpretation.

Waters’ PeptideWorkTryptic Protein Digestion Kits deliver fast and reliable sample preparation for routine peptide mapping of therapeutic proteins. The sample preparation kit is centered around RapiZymTrypsin, Waters’ homogeneously methylated, recombinant trypsin. RapiZyme Trypsin enables speed, digestion fidelity, and low levels of trypsin- derived peptides through its autolysis resistance, purity, and high activity. RapiZyme Trypsin can be used at high concentrations to achieve faster digestions without requiring high temperatures or sacrificing digestion completeness.

CONCLUSION

• Comprehensive kit and protocol for preparation of tryptic protein digests
• Automatable preparation of 24 samples in under 2.5 hours
• Reproducible sample prep without sacrificing digestion completion or generating high levels of method-induced peptide modifications
• 93% reduction in missed cleavages and 55% reduction in non-specific cleavages compared to a leading immobilized trypsin digest kit
• Suitable for peptide mapping of ADCs

6. Thermo Fisher Scientific: Determination of selenite and selenate in environmental waters by ion chromatography-mass spectrometry (IC-MS)

• Application note

Keywords: Single quadrupole mass spectrometer, Dionex Integrion HPIC, Dionex IonPac AS11-HC column, ISQ-EC, ADRS 600 suppressor

Goal: To develop a method to determine selenite and selenate in spiked environmental water by coupling ion chromatography (IC) with single quadrupole mass spectrometry.

Conclusion: An IC-MS method that enables the determination of selenium species in simulated contaminated environmental waters was developed. The reagent-free ion chromatography system provides excellent reproducibility, yielding exceptional quantification accuracy and consistently reliable results. Recoveries for selenite and selenate in the three water samples ranged between 90–110%, with a method LOD of 4 and 2 µg/L for selenite and selenate, respectively. This method is helpful for highly selective screening of selenium species at µg/L levels in environmental waters. Although not with the same sensitivity as IC-ICP-MS, it offers the advantage of selectively identifying selenium species through their m/z ratio. Moreover, this method enables determining whether drinking water exceeds the EPA's outlined MCL of 50 ppb for selenium contamination.

7. Thermo Fisher Scientific: Size-exclusion chromatography of adeno-associated viruses with the SurePac Bio 550 SEC MDi column

• Application note

Keywords: Biopharmaceutical, size-exclusion liquid chromatography, SEC, adeno-associated virus, AAV, monomeric capsids, high molecular weight species, HMWS, 3 µm SurePac Bio 550 SEC MDi column, monodisperse

Key benefits

• High-resolution separation of monomeric AAV and aggregates by size-exclusion liquid chromatography within 4 minutes
• Utilizing UV and fluorescence detection for easy and conclusive confirmation of peak identities
• Resolution of aggregate and monomer peaks preserved even for injection of large volumes
• Consistent lot-to-lot and column-to-column performance

Goal: To demonstrate baseline separation of adeno-associated virus (AAV) monomeric capsids and high molecular weight species (HMWS) using a 4.6 × 150 mm sizeexclusion liquid chromatography column packed with 3 µm monodisperse silica particles with a 550 Å pore size.

Conclusions

• The SurePac Bio 550 SEC MDi column delivers baseline resolution of AAV monomer and aggregate peaks in less than 4 minutes.
• The SurePac Bio 550 SEC MDi column consistently delivers reliable separation of AAV across different lots.
• Careful optimization of flow rate, sample loading amounts, and buffer conditions is crucial for achieving accurate and consistent results in SEC AAV separation.
• The SurePac Bio 550 SEC MDi column outperforms other commercially available columns in AAV analysis, displaying advantages such as sharper peaks, improved resolution, and lower operational pressure