News from LabRulezLCMS Library - Week 46, 2024

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 11th November 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 you applications and other documents by SIELC, Thermo Fisher Scientific, Shimadzu, Waters Corporation, and Agilent Technologies!

1. SIELC: Samplify from SIELC Technologies

• Brochure

What is Samplify?

Samplify is an innovative new technology from SIELC, specifically designed for unattended, routine, periodic sampling of virtually any liquid source. Each sample is transferred automatically into an individual destination vial or 96-well plate. Vial trays or plates may then be loaded directly into a variety of analytical systems for further processing.

The Samplify system consists of two principal components:

1.  a sophisticated electro-mechanical sample probe constructed of materials providing chemical and temperature resistance, and
2. SIELC’s renowned and highly flexible liquid handler (LHS) for pulling samples from the liquid source and distributing samples into destination locations.

The LHS offers many additional capabilities; for example, the addition of quenching reagents into sample vials immediately after acquisition, if required, and thorough probe cleaning to guard against cross-sample contamination. The entire system may be controlled with SIELC’s Samplify Windows app, or via serial port using provided commands.

Samplify Specifications

Samplify Probe

• Connection: requires Samplify LHS
• Small dimensions: 11 x 1 inch, DIA 1/4 inch. Immersion length up to 6”
• Contact with liquid: Stainless Steel 316 (316L), PEEK & PTFE
• Variable sample volume: from 5 to 500 μL
• Max. working pressure: 120 psi (8 bar)
• Max. working temperature: 200 °C

Samplify LHS

• Instrument Size: 6 x 6.5 x 7 inch (15 x 16 x 17 cm)
• Weight: 5 lb (2.3 kg)
• Collection Options: 48 vial plate (2 mL vials); 96 well plate
• Pressure Max: 250 psi/ 14 bar (4000 μL volume)
• Syringe Capacity Options: 4000 μL
• Valve Options: 7x6
• Volume Accuracy: +/-1 μL (4000 μL volume)
• Probe dead volume: 767 μL
• Syringe draw/refill rates: 48,000 μL/min
• Communication: USB (type B) COM port
• Power: 24 V DC
• Contact with liquid: Stainless Steel 316 (316L), PEEK, PTFE, Vespel

2. Agilent Technologies: Ultrafast Quantitation of Immunosuppressant Drugs in Whole Blood by Agilent 6475 LC/MS

• Application

Abstract

An ultrafast, 2.5-minute quantitation method using liquid chromatography/mass spectrometry (LC/MS) for four immunosuppressant drugs was established using an Agilent 6475 triple quadrupole LC/MS with the RECIPE therapeutic drug monitoring (TDM) kit. This application note emphasizes validation of an ultrafast measurement of tacrolimus, sirolimus, everolimus, and cyclosporine A in whole blood with outstanding sensitivity and interday reliability.

Introduction

TDM is essential for ensuring efficacy and minimizing the adverse effects of drug delivery. The need for immunosuppressant drugs for organ and bone marrow transplant patients has been growing annually. Administration of immunosuppressants is strictly monitored as many side effects, including nephrotoxicity and neurotoxicity, must be considered. LC/MS is considered the gold standard for measurement of immunosuppressants due to results
showing remarkable sensitivity and specificity.

The RECIPE ClinMass TDM kit system for immunosuppressants in whole blood (Munich, Germany) was used on the 6475 triple quadrupole LC/MS for analysis of tacrolimus, sirolimus, everolimus, and cyclosporine A. Validation of the RECIPE TDM kit was implemented to evaluate sensitivity, linearity, and interday repeatability across three consecutive days (n = 3). Sample preparation is straightforward without the need for complicated cleanup or filtration.

Conclusion

A rapid analysis of tacrolimus, sirolimus, everolimus, and cyclosporine A in whole blood was established on an Agilent 6475 triple quadrupole LC/MS. Together with straightforward sample preparation, the method demonstrates robust measurement of immunosuppressant drugs in biological samples. Ultrafast acquisition times allow the high-throughput analysis of more than 500 samples per day. By coupling with the ready-to-use RECIPE ClinMass TDM kit system, Agilent provides a comprehensive end-to-end solution for therapeutic drug monitoring (TDM) of immunosuppressants.

3. Thermo Fisher Scientific: In-depth characterization of monoclonal antibodies

• Application

Goal

To assess the performance of the Thermo Scientific™ Orbitrap™ Ascend BioPharma Tribrid™ mass spectrometer for comprehensive monoclonal antibody (mAb) characterization using intact mass analysis and middle-down mass spectrometry (MD-MS) approaches. The MD-MS methods were developed using a combination of powerful ion activation techniques including electron transfer dissociation (ETD), electron transfer higher energy collision dissociation (EThcD), ultraviolet photodissociation (UVPD), and ion manipulation by proton-transfer charge reduction (PTCR).

Introduction

mAbs can display structural heterogeneity due to the presence of various post-translational modifications (PTMs), such as glycosylation, oxidation, and deamidation. These modifications can affect their structure, stability, and function. Therefore, in-depth characterization of mAb heterogeneity is critical to ensure the safety, efficacy, and quality of biotherapeutics. The middle-down mass spectrometry (MD-MS) approach has emerged as a promising tool for therapeutic characterization.^1-4 It involves the formation of mAb subunits by reduction and/or digestion, followed by tandem MS (MS/MS) fragmentation of these subunits, offering high sequence coverage while involving a straightforward sample preparation.

The Orbitrap Ascend BioPharma Tribrid mass spectrometer offers many benefits for biopharmaceutical characterizations. The high mass accuracy and high resolution offered by the Orbitrap technology enable precise mass measurement of intact mAbs or subunits. The Orbitrap Ascend BioPharma MS provides multiple MS/MS fragmentation techniques, including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), ETD, EThcD, electron transfer and collision-induced dissociation (ETciD), and UVPD, to achieve a full characterization of mAbs. In addition, this powerful instrument offers the PTCR technology with extended mass ranges (up to 16,000 m/z with Native MS option enabled) that can be employed alone or combined with different fragmentation techniques for the characterization of complex biotherapeutics. The Orbitrap Ascend BioPharma MS also benefits from its unique revolutionary architecture by having a second ion-routing multipole (front HCD cell) in the system which improves the
transmission of large molecules and increases the sensitivity to detect low-abundance species in complex samples.

In this work, intact mass analysis and MD-MS approaches were employed to characterize trastuzumab and its subunits on an Orbitrap Ascend BioPharma Tribrid MS. All the analyses were performed in the intact protein mode at various pressure modes, and for intact trastuzumab the Native MS option was used. Figure 1 shows the main LC-MS workflow employed for intact and subunit analyses of trastuzumab.

Conclusion

In this work, intact and middle-down workflows were employed to obtain precise intact mass and high sequence coverage of mAb subunits using the Vanquish Flex UHPLC system and Orbitrap Ascend BioPharma Tribrid mass spectrometer. The Orbitrap Ascend BioPharma MS provides the following advantages for comprehensive biopharmaceutical characterization.

• The high mass accuracy and high resolution offered by the Orbitrap technology provides precise mass measurement for both intact mAbs (native and denaturing) and their subunits.
• The system provides multiple fragmentation techniques, such as ETD, EThcD, and UVPD, for achieving high sequence coverage for various biopharmaceutical molecules.
• Compared to ETD, EThcD and UVPD provide an improved sequence coverage of mAb subunits due to the formation of additional fragment types.
• The combination of PTCR with EThcD and UVPD led to further increase in sequence coverage of mAb subunits and the number of complementary ion pairs.
• The combination of EThcD, UVPD, and PTCR offers a nearly complete sequence coverage (>90%) for subunits in the size of 20–25 kDa (Fc/2, LC, and Fd’ subunits) and high sequence coverage (>70%) for the HC subunit (≈50 kDa).

4. Shimadzu: Automatic Optimization of Gradient Conditions by AI Algorithm on Synthetic Peptide and Impurities

• Application

User Benefits

• The AI algorithm of LabSolutions MD can automatically optimize gradient conditions to greatly reduce labor of LC method development.
• Anyone can optimize gradient conditions, regardless of their experience in chromatography.
• Gradient conditions that meet the resolution criteria for specified peaks are automatically searched (e.g., principal component and related impurities).

Introduction

In the typical LC method development, the process begins with “preparation” which includes mobile phase preparation, column installation, and creation of analysis schedules, then the analysis is started. After that, the acquired data is analyzed and “preparation” for the subsequent analysis is carried out, followed by starting the next analysis again. The method development progresses by repeating these processes, but in addition to the
significant time required to repeatedly create analysis schedules, expertise in chromatography is necessary to explore optimal conditions based on data analysis. In other words, typical method development requires “human intervention”. Therefore, eliminating human involvement and automating such method development processes would be desirable to improve labor efficiency. This article introduces an example of automatic optimization of gradient conditions to meet resolution criteria for synthetic peptide and related impurities using LabSolutions MD (Technical Report C190-E309), a dedicated software for supporting method development.

Conclusion

Automatic optimization of gradient conditions using AI algorithm of LabSolutions MD was applied to synthetic peptide and related impurities. As a result, gradient conditions that met the criteria (resolution for FLP > 2.0) were successfully explored. This result indicates that significant labor saving in method development can be expected by LabSolutions MD. This article introduces an automatic optimization of gradient conditions in method development while LabSolutions MD also supports a series of workflow of method development, including screening of mobile phases and columns. For details, please refer to the application news, “Efficient Method Development for Synthetic Peptide and Related Impurities (01-00780)”.

5. Waters Corporation / HPLC: COMPARISON OF MIXER PERFORMANCE FOR HPLC METHODS UTILIZING TFA GRADIENTS

• Poster (HPLC)

INTRODUCTION

Solvent mixing is critical to obtaining optimal LC separations. Currently, there are two commonly available pump mixing designs for reversed phase gradient HPLC separations - high pressure (typically binary) and low pressure (all quaternary) systems. High pressure systems use two independent pumps to deliver different solvents. Mixing occurs at high pressures with solvent composition controlled by the flow rates of the two 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. 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.

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 TFA - acetonitrile gradients are used at low wavelengths. These baseline ripples may make peak integration difficult, and can impact the sensitivity and retention time precision of the method.

Most modern HPLC systems include a standard mixer in the pump design. Additionally, different mixers may be available to improve mixing performance for specific applications. In this study, mixers of varying volumes and design were evaluated.

CONCLUSION

Mixing performance is critical to obtaining optimal LC separations. In this study, several HPLC systems and mixers were evaluated using a TFA-Acetonitrile gradient method at a low wavelength which is known to produce baseline ripples. The USP signal to noise ratio was used to determine the impact of the different mixers.

The results demonstrate that the sensitivity of the method is impacted by the system/mixer used, and consideration should be given to mixer selection, especially when running methods which are subject to increased baseline noise.