Small Molecule Bioanalysis

When searching for the hard-to-find molecule, gaining insights and expertise from the DMPK community is critical. Browse these resources to be even more effective in quantifying small molecules for drug efficacy.

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Improved Analytical Sensitivity and Chromatographic Peak Shape for the Quantification of TCA Cycle Analytes in Human Plasma using the ACQUITY PREMIER System Solution
February 2021

In this application note, we have demonstrated that the analysis of the TCA cycle and related metabolites in human plasma can be achieved with great analytical sensitivity when incorporating MaxPeak HPS Technology into the liquid chromatograph as well as the analytical column. The ACQUITY PREMIER System Solution mitigates analyte interactions with metal to improve peak shape as well as analytical sensitivity without full system passivation with strong acids or chelating additives in the mobile phase. The simple mixed-mode anion exchange separation using the ACQUITY PREMIER CSH Phenyl-Hexyl Column allows for fast separations and easy adaptation.

Improvements in Sensitivity for Quantification of Steroid Phosphate Drugs Using ACQUITY PREMIER and MaxPeak HPS Columns
February 2021

In this application note, we show the impact of using this novel surface for quantifying hydrocortisone phosphate and dexamethasone phosphate in extracts of human plasma.

Demonstrating Improved Sensitivity and Dynamic Range with MaxPeak High Performance Surface (HPS) Technology: A Case Study on the Detection of Nucleotides
February 2021

Analyzing adenosine nucleotides, such as ATP, ADP, and ADP, was found to be difficult using standard LC systems and columns made of stainless-steel components. The challenge can be attributed to the strong affinity of these compounds towards exposed metal surfaces. The analytes with two or three phosphate groups showed the worst effects. With the standard LC and column, ATP and ADP were completely lost at and below 2 ng/µL sample concentrations. AMP, with a single phosphate group, showed comparatively minor losses. Nonetheless, it suffered from compromised assay sensitivity.

As shown with this case study, the ACQUITY PREMIER System can be effectively used with an ACQUITY PREMIER Column to overcome some metal adsorption challenges that can interfere with achieving robust quantitation. With this technology, new levels of performance will now become possible for the quantitation of nucleotides, whether the employed technique be based on a fit-for-purpose RPLC method, HILIC, or mixed mode chromatography.

Advantages of using ACQUITY PREMIER System and Columns with MaxPeak HPS Technology for Bioanalysis of Gefitinib – an EGFR Inhibitor
February 2021

In this application brief, we investigate the effect of these novel hybrid barrier surface technologies on metal insensitive compounds. Gefitinib (Iressa) was chosen as a candidate molecule for this study. Gefitinib is an epidermal growth factor receptor (EGFR) inhibitor and is indicated in non-small cell lung cancer (NSCLC) and certain breast cancer patients. There are no known reports of metal sensitivity exhibited by Gefitinib, however previous reported bioanalytical methods for this compound have all employed buffers in the mobile phase to improve chromatographic peak shape. Here, we show that using ACQUITY PREMIER and MaxPeak HPS Columns improves the robustness and reproducibility for bioanalysis of gefitinib.

PREMIER Standards to Investigate the Inertness of Chromatographic Surfaces
February 2021

Confirming that instruments are properly functioning is a critical aspect of any analytical measurement. System suitability testing helps add certainty to results. In this work, we demonstrated the ability to assess the inertness of the recently introduced ACQUITY PREMIER System, which has been designed to eliminate metal-analyte secondary interaction. For this, a non-hydrolyzable analog of ADP, known as AMPcP, has been used in the form of new PREMIER standards. The highly stable molecule has been formulated into the PREMIER AMPcP Standard as well as the PREMIER AMPcP and Adenosine Standard. Upon using the AMPcP-only standard with a series of no column injections, it was possible to observe more consistent recoveries from the ACQUITY PREMIER System versus a metal alloy-based ACQUITY UPLC H-Class PLUS Bio System. Meanwhile, with a chromatographic separation using an ACQUITY PREMIER Column and the PREMIER AMPcP and Adenosine Standard, it was possible to detect differences in relative recovery between the two types of LC instruments. These examples of improved performance for AMPcP measurement demonstrate the benefits ACQUITY PREMIER technology will bring to analyses of other compounds that interact with metals.

Comprehending COVID-19: LC-MS/MS Analysis of Small Molecule Anti-Viral and Anti-Inflammatory Drugs in Plasma in Clinical Research
August 2020

A simple and rapid LC-MS/MS method has been developed for the analysis of anti-viral and anti-inflammatory drugs in plasma for clinical research using the ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System.

The method uses a simple protein precipitation to extract the drugs from plasma and chromatography with a CORTECS T3 Column to enable selective and rapid separation of the drugs in the extracted sample. Analytical sensitivity, linearity, precision, and accuracy are excellent with reproducible extraction efficiency and matrix effects.

Clinical research into the impact of these drugs and their metabolites on SARS-CoV-2 is still ongoing. Phosphorylated metabolites of the RNA polymerase inhibitors haven’t been evaluated here and they may be of additional interest, with nucleoside triphosphate of remdesivir representing the predominant metabolite in Peripheral Blood Mononuclear Cells (PBMCs).3 Phosphorylated molecules bind to metals and represent an analytical challenge for robust and reproducible quantification in LC-MS/MS. Chelator additives or the use of high performance surface (HPS) technologies can help bridge the gap in providing a method for robust quantification of phosphorylated molecules in future studies using LC-MS/MS.

QuanOptimize: A Software Tool that Enables Rapid, Consistent, and Accurate MRM Method Development for Large Numbers of Small Molecule Analytes
September 2020

Discovery bioanalytical laboratories routinely develop methods for hundreds of compounds per week to support the various programs in their pipeline. Developing methods for each of these compounds individually can be time-consuming and tedious. Since the approach to small molecule method development is well understood, automation of this process is desirable. QuanOptimize automates the MRM method development for large sets of compounds. It can store the final method parameters to a database, automatically run sample lists using the optimized method, create data processing methods, and generate quantitative result with the click of a single button. QuanOptimize ensures consistent quality of methods across multiple users, reduces sample consumption and saves time by up to 5-fold.

Here, we discuss the use of QuanOptimize to develop methods for a set of 18 small molecule compounds using generic tune page and LC methods.

Development of a Bioanalytical SPE LC-MS/MS Assay for Oligonucleotides
January 2021

Over the past decade, there has been increased research oligonucleotides therapies (ONTs). With improved target specificity of next-generation oligonucleotides, demand for LC-MS bioanalytical assays in support of their research and development has also increased.

Developing robust, sensitive and selective sample preparation and LC-MS methods for ONTs remains quite challenging, due to their size, poly-anionic nature, physiochemical diversity, and stability, which often results in poor sample recovery from biomatrices, poor MS sensitivity due to limited ionization/fragmentation, and resolution from endogenous matrix interferences due to poor RP chromatographic retention.

This work described herein provides a single, simple method for the quantification of various oligonucleotides (15-35mer) from serum, using RP and mixed-mode µElution SPE sample preparation and analytical scale sub-2µm LC. This method achieves high recovery and low matrix effects, while achieving LODs as low as 0.5-1.0 ng/mL extracted from plasma/sera.

CDMO Pushes Bioanalytical Boundaries with Innovative Topical and Transdermal Services

In this case study learn how MedPharm is successful in its innovative approach to topical and transdermal product design and development services by utilizing Waters bioanalytical instrumentation and software solutions.

A Rapid Method for The Ultra-Sensitive Quantification of Fluticasone Propionate and Salmeterol Xinafoate from Human Plasma

This high-throughput method achieves the lowest published LLOQ's for fluticasone propinate (0.1 pg/mL) and salmeterol (0.05 pg/mL).