(T1430-06-39) A Process of Method Development for Tissue Homogenization and Analysis to Eliminate Binding and Matrix Effects Using Surrogate Matrices

Purpose: The analysis and distribution of pharmaceuticals in tissue is increasingly important in understanding the full action of drugs in vivo. Across molecular types from very small molecules to larger peptides and oligonucleotides, bioanalysis of tissues presents unique challenges inherent in a mixed matrix assay. In this work, homogenization methods for tissue samples are assessed for non-specific binding and analytical congruency of surrogate and tissue homogenates across species and tissue types. Generation of a broadly applicable method of sample processing has resulted in streamlined method development and provided robust conditions applicable to a variety of matrices. Assessment of processes are also described in optimizing the final method to individual analytical needs for compound types in specific tissues. Accurate and precise results in multiple tissue types across multiple species are obtained with reduced time spent in development using a baseline set of experiments and set points for optimization of parameters.

Methods: Fit-for-purpose methods were generated independently to quantify a small molecule in rat tissues and oligonucleotide in monkey tissues using LC/MS/MS detection. Tissues are homogenized using a Bullet Blender homogenizer and either steel or ceramic beads. Comparison of homogenate methods can be made to determine processing stability and non-specific binding and optimized into a final processing method that is tissue type and analyte specific using a set battery of tests. Common tissue types are diluted in same species plasma to eliminate binding effects and plasma surrogate control experiment is conducted to determine the acceptability of plasma quantification of the limited quantity tissue matrices. Rare tissue types are homogenized in appropriate buffer solutions then non-specific binding and surrogate parallelism experiments are conducted to determine the optimal buffer solution and tissue weight to volume dilution ratio. Extraction methods were developed to analyte specific requirements and employ both protein precipitations for small molecule method described and SPE methodology common to various oligonucleotides. LC/MS/MS methods are optimized for signal, mass/charge and interference contribution using a Sciex API 5000 mass spectrometer and UPLC separations using Shimadzu Nexera integrated dual system LC setup.

Results: Similar methodology was used the initial tissue assessment of both molecule classes to determine a standard set of best starting conditions and experiments for optimization of final methods. Buffer homogenate diluents and plasma were both tested and compared for NSB and accuracy of homogenate QCs against a diluent only calibration curve to conserve rare matrices. Analysis of a small molecule using the optimized method in rat liver and spleen tissues showed < 4.6% CV and -5.2 to 5.1 %RE for Liver QC samples and < 5.0% CV and -4.7 to 5.1 %RE for Spleen QC samples in a mixed tissue run using a plasma standard curve method derived in the optimization process. Analysis of a small molecule in rat skin homogenate was acceptable with >6.5% CV and -9.8 to 14.4% RE. Analysis of an oligonucleotide in monkey brain, liver and kidney demonstrated acceptable accuracy and precision utilizing a buffer homogenate and surrogate control matrix follow the same development protocol. Brain homogenate samples were analyzed with < 4.3 %CV and -3.5 to 3.6% RE in a fit-for purpose assay against the surrogate curve. Liver and Kidney homogenates were run in a mixed matrix analysis with < 11.3%CV and -10.0 to 11.2% RE for kidney samples and < 7.6% CV and -5.0 to 7.4 % RE for liver samples in fit-for purpose assay against the surrogate curve. The similarity of the data across species and tissue types is demonstrating acceptance of the mixed tissue method for samples analysis of multiple tissue types in a single run to expedite analysis and conserve project material resources.

Conclusion: An approach was found to determine levels of non-specific binding and to mitigate matrix effects in the process of homogenization and sample prep. This approach can be broadly applied to a variety of tissue homogenates and other matrices. A workflow approach of systematic optimization experiments allows for methods to be generated quickly for a variety of molecules and drug classes. Comparison of single tissue QCs against single tissue samples using surrogate matrix standard controls for acceptance provided a more robust comparison in a fit-for-purpose analysis while conserving rare and costly matrices.