As drug candidates become increasingly diverse, bioanalytical laboratories must adapt workflows to accommodate molecules with a wide range of physicochemical properties while maintaining the sensitivity, reproducibility, and throughput required for preclinical research. Advances in liquid chromatography-mass spectrometry (LC-MS) instrumentation and assay development help scientists meet these demands, enabling more efficient analyses without compromising data quality.
Meet the QPS Subject Matter Expert
Lata Venkatarangan, PhD
Director of Bioanalysis and Head of DMPK at QPS
She has more than two decades of experience in bioanalysis and LC-MS/MS-based drug development support, with expertise in small molecule quantitation, method development, and analytical workflows for emerging therapeutic modalities. At QPS, she leads teams supporting preclinical drug development, including LC-MS assay development for small and large molecules such as ASOs, siRNA, and Oligonucleotides. Dr. Venkatarangan completed postdoctoral training in Biological Engineering at the Massachusetts Institute of Technology, earned a Ph.D. in Bioorganic Chemistry from the University of Connecticut, and received an M.Sc. in Organic Chemistry from the University of Mumbai.
In this Innovation Spotlight, Lata Venkatarangan, the director of bioanalytical at QPS, discusses best practices for developing LC-MS assays for small molecules and emerging drug modalities and explains how advances in ultra-high-performance liquid chromatography (UHPLC) technology improve throughput and reduce carryover.
1. What is the role of QPS as a contract research and development organization (CRDO) in the pharmaceutical industry?
As pharmaceutical and biotechnology companies pursue increasingly complex drug candidates, the role of CRDOs has become more central to successful drug development. Sponsors rely on experienced partners not only to provide specialized technologies and scientific expertise, but also to help them adapt quickly, manage regulatory risk, and generate the high-quality data needed to make confident development decisions.
QPS operates within this evolving environment as a global contract research organization focused on helping sponsors move promising therapies from discovery toward the clinic. By combining scientific rigor, advanced analytical capabilities, and flexible study execution, QPS supports the industry’s need for faster, more efficient development without compromising data quality. This is especially important in bioanalysis, where new modalities and increasingly diverse molecular properties require laboratories to continuously refine their methods, instrumentation, and workflows.
2. How does LC-MS fit into your team’s workflows?
My team is responsible for the identification and quantitation of drug products in complex biological mixtures for a variety of absorption, distribution, metabolism, and excretion (ADME) studies, including pharmacokinetics, drug-drug interactions (DDIs), protein binding, and biotransformation studies. This entails implementing an extraction procedure to clean up the biological sample, followed by liquid chromatography (LC) to separate endogenous interference peaks from the test articles of interest. The eluent is then analyzed using a mass spectrometer, which measures the specified mass-to-charge ratio of the test article. This allows for quantification of the test article in the biological matrix against calibration standards prepared identically in the same matrix or, in some cases, a surrogate matrix. Accurate bioanalytical quantitation is hence critical to ensure correct interpretation of data from ADME studies to guide decisions for the next steps.
3. What analytical challenges does your group experience when working with small molecules and new drug modalities?
Some of the analytical challenges overlap between small molecules and new drug modalities, mainly pertaining to the separation of analytes of interest from interferences in biological matrices. This can be achieved using both extraction methodologies and chromatographic separation. The choice of extraction method, including protein precipitation extraction (PPE), liquid-liquid extraction (LLE), and solid-phase extraction (SPE), is directed by the chemical structure of the analytes of interest, in addition to the complexity of the biological matrix. The stationary and mobile phases are also critical elements to further drive separation and achieve robust methods with targeted lower limits of quantitation.
New drug modalities, such as antisense oligonucleotides, siRNA, antibody-drug conjugates, and antibody-oligonucleotide conjugates, present additional analytical challenges because they are comprised of multiple components. As a result, extraction techniques and LC methods are driven by the components selected for monitoring, which serve as surrogates for the whole molecule. Because these new drug modalities tend to be multiply charged and, in some cases, highly polar, the choice of the stationary phase of the analytical column and the pH of the mobile phase is critical to ensure adequate retention and separation from components of the extracted biological matrix.
4. How have Shimadzu’s LC systems evolved since your team started working with them, and how has that changed your workflows?
My team has worked with several iterations of the Shimadzu LC systems over the past two decades, culminating in the current Nexera X3 LC-40 system. Over the years, improvements have been made to the hardware, including faster injection speeds to support high-throughput sample analysis, increased sample capacity through the use of a plate changer unit to aid sample loading, innovative injection port designs to reduce autosampler carryover, and additional wash solvent options to provide effective countermeasures for carryover. These improvements have allowed for very fast separations with good resolution of complex samples. Shorter runtimes have allowed for a greener UHPLC platform by reducing solvent usage. Substantial cost savings are also associated with reduced solvent purchase and waste disposal costs.
5. What advantages does the latest UHPLC system bring to your method development and high-throughput analysis?
The Nexera X3 SIL-40 series autosamplers are designed for high-throughput analysis with low system carryover. These systems offer fast injection speeds with minimal non-data acquisition time. With the introduction of a plate changer unit, their increased sample capacity allows for loading of long or multiple analytical sequences with minimal human intervention. In addition, Nexera’s open access system allows for sample plate addition without interrupting ongoing analyses. The SIL-40 series autosamplers have a needle-in-flow design, which reduces carryover from chemical adsorption. This type of injection allows for faster and cleaner LC methods.
These advantages have helped streamline our workflows for established DDI assays, where high-throughput analysis is crucial for maintaining our targeted data turnaround times. Additionally, larger molecules, especially peptides, have historically faced carryover issues arising from non-specific adsorption to components of the LC system. With the design changes to the injection port and additional washing program options available on the SIL-40 autosamplers, carryover issues have been successfully resolved without adding too much additional time to the overall sample-to-sample run time.
6. How do you balance fast analysis times with the need for reliable, reproducible data?
High throughput should never come at the cost of quality. Once a reliable extraction methodology has been established for consistent recovery of the analyte of interest with minimal matrix interferences, LC method development is the next crucial step. This ensures optimal separation and elution of the test article from other components in the biological sample. The use of sub-2µm full porous particles (FPPs) or sub-3µm superficially porous particles (SPPs) in 2.1mm or smaller internal diameter columns, combined with the high operating pressures in UHPLC systems, allows for fast resolution of closely eluting peaks. This also generates narrower peaks and improved signal-to-noise ratios, enabling detection of lower concentrations of test articles of interest.
7. For researchers who may be newer to LC-MS or working with novel biomolecules, what best practices would you recommend when developing assays for preclinical applications?
All assay development has two main components: sample extraction and LC-MS method development. Both are critical to ensure the development of a robust and reproducible assay.
Best practices include the following.
- Robust sample preparation: Utilization of appropriate internal standards, preferably stable labeled or structurally similar analog compounds, is important to correct for matrix effects and variability in extraction recovery. Appropriate extraction techniques, such as PPE, LLE, and SPE, are crucial for sample preparation to maximize recovery and minimize interferences.
- Optimization of chromatography: This starts with the selection of an appropriate stationary phase based on the chemical nature of the test article. FPPs or SPPs allow scientists to fully utilize the advantages of high-pressure UHPLC systems to improve sensitivity and throughput. SPPs can also enhance peak shape and efficiency without concerns of excessive back pressure. The use of mass spectrometry (MS)-compatible modifiers can aid ionization. Optimizing elution profiles using either gradient or isocratic methods can help flush out unretained, highly polar matrix interferences, followed by elution of the test article. Suitable wash protocols can minimize carryover, and post-column divert valves can help keep the MS clean.
- MS optimization: Optimization includes tuning source and multiple reaction monitoring-specific parameters to maximize ionization efficiency and minimize baseline noise. Selecting at least two transitions per analyte when possible, one as the quantitation ion and one as the qualifier ion, can improve method reliability, among other considerations.
- System testing: Ensuring sufficient control samples are added to the analytical sequence, such as quality controls, double blanks, blanks with internal standard only, and blanks with test article only, helps monitor assay performance.
Biomolecules bring additional challenges to LC-MS assay development. They are usually multiply charged and very polar. Hence, the choice of stationary phase and mobile phase is critical to ensure good retention and chromatography. Sample preparation is another important consideration since multistep procedures are usually required, either employing LLE or SPE, to ensure consistent and high recovery of the analyte of interest and its separation from interferences present in biological matrices. Biomolecules also tend to have high adsorption to the surfaces of the LC system, causing carryover. Hence, targeted wash protocols often need to be employed to minimize carryover.
