(M1430-06-34) Validated Biomarker Assay for the Analysis of Coproporphyrin I and Coproporphyrin III in Human Plasma

Purpose: Coproporphyrin I (CP‐I) and Coproporphyrin III (CP‐III) are potential endogenous biomarkers for hepatic organic anion transporting polypeptide (OATP)1B1/1B3 function. We developed and validated a bioanalytical assay for monitoring these biomarkers to assess OATP1B1/1B3 inhibition in place of a standalone prospective clinical drug-drug interaction (DDI) study. Currently, investigational drugs that alter the pharmacokinetics of other medications are subject to additional testing to understand how to manage a DDI risk safely. By monitoring the effect of an investigation drug on the levels of these endogenous substrates of OATP1B1/1B3 in early clinical development, the potential need for a dedicated clinical DDI study could be avoided.

Methods: Coproporphyrin I (CP‐I) and Coproporphyrin III (CP‐III) are extracted from human K2EDTA plasma using a supported liquid extraction (SLE) methodology. After recovering the analytes with ethyl acetate, the samples were evaporation and reconstitution with formic acid and EDTA solution. The resulting extracts were analyzed by LC-MS/MS detection. The analytes were separated from potential interferences using a Water Acquity Premier BEH C18 (2.1 x 100mm) column and gradient conditions. A Sciex 6500+ mass spectrometer operating in positive electrospray (ESI) mode was used to detect CP-I, CP-III, and the labeled internal standards. Because CP-I and CP-III are endogenous compounds, calibrators and quality control samples were prepared in charcoal-stripped human K2EDTA plasma. The analytical range of the assay was 50.0 to 5000 pg/mL for both biomarkers. To achieve appropriate sensitivity, 200 microliters of sample volume was required.

Results: The method of analysis for CP-I and CP-III biomarkers was validated to support the needs of regulated clinical studies. Method validation experiments for these biomarkers were pre-established using the criteria in the FDA M10 bioanalytical method validation guidance and from the AAPS “Biomarker Assay Validation by Mass Spectrometry” white paper. Three accuracy and precision runs were conducted over three different days. The accuracy and precision of endogenous QC levels ranged from 98% to 104% and 2.4% to 3.63%. Parallelism was conducted by diluting endogenous QCs at 2X, 5X, and 20X with a surrogate matrix and determining the impact on the back-calculated accuracy and precision. The accuracy and precision of this experiment ranged from 91% to 111% and 1.0 to 2.4%, respectively, and demonstrated that endogenous samples can be quantitated against a calibration curve prepared in a surrogate matrix without affecting the calculated results. The recovery of CP-I and CP-III averaged 68% and 39%, respectively, over three different concentration levels. Recovery was performed by comparing the response of the analytes when added before and after extraction. Method validation experiments also included stability assessments. These experiments were conducted in the authentic matrix and with QCs prepared in charcoal-stripped human plasma. They included four freeze-thaw cycles, twenty-five hours of short-term stability at ambient temperatures, and eighty-three days of long-term matrix stability at negative eighty degrees Celsius. All experiments met the pre-established criteria, and the assay was considered appropriately validated to support regulated clinical studies.

Conclusion: Understanding the dose-dependent changes in these endogenous substrates of OATP1B1/1B3 makes it possible to identify investigation drugs with a potential DDI risk before conducting clinical DDI studies in patients. To aid in this decision-making process, a method of analysis for coproporphyrin I and coproporphyrin III in human plasma was developed and validated to support regulated clinical studies. This method was validated following the principles outlined in the FDA M10 bioanalytical method validation guidance and from the AAPS “Biomarker Assay Validation by Mass Spectrometry” white paper. The validation included experiments to measure accuracy and precision in an authentic and surrogate matrix, parallelism, and stability experiments.