Developed an LC-MS/MS method to quantify small molecules in surrogate matrix, validated by ICH M10

Dr. Prajita Pandey, Assistant Director of Chemistry at Emery Pharma, presents an approach to LC-MS/MS method development for quantitating a small molecule in vitreous humor using human plasma as a surrogate matrix. The study follows ICH M10 bioanalytical method validation guidelines to ensure the method's accuracy, reliability, and reproducibility. Several challenges in bioanalysis, such as interference from endogenous analytes and rare matrices, are addressed using a surrogate matrix.
A surrogate matrix mimics the biological matrix of interest, offering advantages like reducing matrix effects, enhancing method robustness, and minimizing interference from endogenous components, thus improving detection limits. This approach is particularly useful when natural matrices are unstable or rare.
The study focuses on vitreous humor as the biological matrix, with human plasma used as the surrogate matrix. The overall workflow involves spiking the test compound X in both human plasma and vitreous humor, adding an internal standard, and conducting protein precipitation with methanol. The supernatant is then analyzed using LC-MS/MS.
The method validation includes evaluating specificity, selectivity, recovery, and carryover. Specificity and selectivity were confirmed by comparing chromatograms from blank human plasma and vitreous humor, ensuring that test compound X did not interfere with retention times. Recovery, which ranged between 80-98%, was evaluated by comparing pre- and post-spiked samples, demonstrating acceptable recovery rates.
Carryover, a common challenge in bioanalysis, was assessed to ensure that analyte response in blank samples was below 20% of the lower limit of quantitation (LLOQ), meeting ICH M10 guidelines. Linearity and range were validated using a calibration curve from 2.5 to 5000 ng/mL, with accuracy and precision confirmed through multiple runs and analyses over several days. Accuracy values fell within the acceptable range of 85-115%.
Parallelism studies were conducted to confirm that the surrogate matrix was suitable for quantifying the analyte. These studies compared quality control samples prepared in human plasma and vitreous humor, with results demonstrating acceptable accuracy and precision. Pooling vitreous humor with human plasma further validated the surrogate matrix’s applicability.
The dilution integrity study addressed the challenge of unknown analyte concentrations above the calibration curve. Two stocks were created at 3000 and 150,000 ng/mL, and dilution to target concentrations demonstrated that accuracy and precision were maintained even after dilution.
Stability studies were crucial to assess the effects of freeze-thaw cycles, room temperature stability, and auto-sampler conditions. Test compound X was spiked in vitreous humor and underwent three freeze-thaw cycles at -80°C, with accuracy values remaining within the acceptable range. Bench-top stability was confirmed by keeping samples at room temperature for four hours, and auto-sampler stability was ensured for samples analyzed after 15 hours. Long-term storage stability at -80°C was monitored for up to 9 weeks, with accuracy maintained throughout.
In summary, this method development approach demonstrates how a surrogate matrix can be effectively used to address bioanalytical challenges. Validating this method per ICH M10 guidelines ensures its robustness and reproducibility, streamlining future bioanalysis studies while offering potential cost savings.
About Emery Pharma:
Emery Pharma is deeply committed to advancing public health and exclusively focuses on projects that enhance the well-being of both individuals and the environment. As a comprehensive contract research laboratory, Emery Pharma provides extensive support across the entire spectrum of pharmaceutical development, covering small molecule drugs, biologics, biosimilars, gene therapy, botanicals, and more.
Emery Pharma's array of premium services includes analytical and bioanalytical solutions, microbiology and cell biology services, drug and impurity characterization, and comprehensive R&D with cGMP/GLP support. The Emery Pharma team excels in customizing innovative solutions tailored to the unique requirements of each client. They seamlessly oversee the entire process, from initial drug discovery to thorough data analysis and insights generation. Their expertise allows them to tackle scientific challenges proactively, ensuring clients avoid unnecessary setbacks.
Emery Pharma's accomplished chemists and biologists harness state-of-the-art technology, encompassing cutting-edge tools such as high-resolution mass spectrometry (Orbitrap), triple quad LC-MS/MS, GC-MS, preparative HPLC, and 400 MHz multinuclear NMR.
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