Absolute quantitation of neopterin as an endogenous pharmacodynamic Biomarker: The successful method development, validation, and use of a surrogate matrix for clinical sample analysis

Abstract

Biomarkers are playing an increasing role in the drug discovery and drug development process. Molecular biomarkers pose a bioanalytical challenge due to their low concentrations and endogenous presence. Inaccurate quantitation could lead to biased study results. Surrogate analyte and surrogate matrix approaches can be used to overcome the lack of a blank matrix and provide accurate quantitation. However, the suitability of the surrogate analyte or matrix must be established during method validation. Here we describe the development and validation of a surrogate matrix approach for the absolute quantitation of neopterin as a PD biomarker for use in an FDA-sponsored clinical study (NCT04183491). Matrix suitability was established through parallelism, precision and accuracy, and internal standard response. Parallelism experiments showed FBS, and serum had identical slopes 0.0145. Additionally, the difference in the X-intercepts was able to accurately predict the amount of endogenous neopterin (1.0 ng/mL). Inter-day accuracy across four surrogate QC levels ranged from 92.08 to 109.06 % while precision ranged from 3.36 to 16.00 %. Inter-day accuracy for the QCs in study matrix ranged from 96.81 to 108.86 % and precision ranged from 4.13 to 6.01 %. The internal standard response in FBS was only 6.9 % different from serum. Additionally, there was no matrix effect, injection carryover, or cross-analyte interference observed. The method was then qualified for automated sample processing.