Unlocking the Power of Ultrasensitive Bioanalysis
🔬 How do you measure complex modality molecules at the lowest possible concentrations?
For modern therapies like antibody-drug conjugates (ADCs) and inhzlation drugs, precise bioanalysis at picogram levels is essential to ensure both efficacy and safety. At Resolian, we specialize in ultrasensitive assays that push the boundaries of detection using:
✅ Advanced sample preparation techniques to refine analyte extraction
✅ Optimized chromatography (including 2D-LC) for maximum separation and reduced noise
✅ Cutting-edge mass spectrometry for ultra-low detection limits
From chemical structure analysis to high-sensitivity LC-MS, our expert team ensures accurate quantification of even the most challenging analytes.
🎥 Watch the video interview here 👉
📖 Read the full article below to explore our approach in detail. 👇
Ultrasensitive Assays for Bioanalysis: Resolian’s Approach
For targeted and high potency therapies, ultrasensitive bioanalytical assays are essential to monitor low-concentration analytes accurately. In this article we explore the methods used by Resolian’s team, led by Executive Director of Bioanalysis Aihua Liu, we detail critical steps in assay development, including understanding chemical structures, optimizing sample preparation, chromatographic separation, coupled with mass spectrometry detection, allowing for precise measurement even at picogram levels.
The Importance of Ultrasensitive Assays in Modern Therapies
Therapies such as antibody-drug conjugates (ADCs) and inhalation drugs require ultrasensitive assays due to their low-dose delivery mechanisms and the high potency of active ingredients. These therapies, designed to minimize impact on healthy cells while delivering targeted effects, require exact quantification at trace levels to ensure both efficacy and safety.
Understanding the Chemical Structure
The process begins with an in-depth understanding of the target analyte’s chemical structure, which informs all subsequent steps. Knowing the structural details enables analysts to predict sensitivity, identify functional groups for sample preparation, and optimize LC-MS conditions. For instance, analytes with specific groups, like secondary amines e.g. Formoterol, can achieve picogram sensitivity through the use of low pH mobile phases to increase ionization efficiency.
Challenges Without Structural Information: Confidentiality concerns sometimes limit access to the analyte’s chemical structure. Even in the absence of that information, perhaps with only a molecular weight, our team can adapt their approach, typically employing a larger sample volume and a smaller reconstituted solvent volume, and also the large injection volume to proceed effectively. This approach then relies on an optimized sample preparation protocol.
Optimizing Sample Preparation: The AMOEBA Protocol
Sample preparation is crucial for minimizing matrix effects and enhancing analyte sensitivity. Resolian has implemented a standardized protocol known as AMOEBA, which streamlines method development through the systematic testing of techniques, including protein precipitation extraction (PPE), liquid-liquid extraction (LLE), supported liquid extraction (SLE), and solid-phase extraction (SPE). SPE is often necessary for ultrasensitive assays, allowing Resolian to effectively isolate analytes from interferences and achieve desired sensitivity levels.
Combined Techniques for Maximum Sensitivity: In cases requiring enhanced extraction, Resolian combines multiple methods, such as PPE+SPE or LLE+SPE, to refine analyte concentration. The systematic approach is efficient and eliminates the need to re-evaluate the sample prep protocol later.
Chromatographic Optimization for Efficient Separation
For known analyte structures, the column and mobile phase selection can be tailored to structural characteristics. When chemical structure details are lacking, Resolian uses a design-of-experiments (DoE) approach to screen column types (e.g., C4, C8, C18, phenol) and mobile phases, optimizing conditions rapidly. The company employs a pre-prepared set of columns and mobile phases to complete these screens in under a day.
Two-Dimensional Chromatography (2D-LC): For ultrasensitive assays, Resolian often utilizes two-dimensional chromatography, which enhances separation by using two columns with differing mechanisms. This technique lowers background noise and improves signal quality, essential for distinguishing low-level analytes from complex matrices.
Maximizing Detection Sensitivity Through Triple Quadrupole Mass Spectrometry
Triple quadrupole mass spectrometry (MS) is critical in achieving the detection sensitivity necessary for ultrasensitive assays. Resolian uses advanced instruments like the Sciex 7500 mass spectrometer, which enhances signal intensities and reduces noise levels, essential for detecting analytes at trace concentrations.
Ionization Techniques, Collision Energy, and Peak Summing
Effective ionization is key to improving sensitivity in mass spectrometry. Choosing the right ionization technique (e.g. electrospray ionization (ESI) for polar analytes or atmospheric pressure chemical ionization (APCI) for less polar compounds) and the correct ionization mode (positive or negative mode) — is crucial for minimizing matrix effects. Collision energy, tailored to the analyte’s structure, optimizes the fragmentation process, further enhancing detection precision.
Peak Summing for Increased Signal: For labs without high-sensitivity instruments, peak summing offers an alternative, where multiple MRM transitions are combined to improve overall signal-to-noise ratio, thus increasing detection sensitivity without noise interference.
Conclusion:
By optimizing each stage of the assay process, Resolian ensures accurate monitoring of low-concentration analytes, supporting the development of life-saving therapies that meet exacting safety and efficacy standards.
