Heterologous internal calibration for multiplexed internal quantification in targeted LC–MS/MS bioanalysis

Absolute quantification is crucial in bioanalysis for both clinical and non-clinical applications but remains challenging, especially for endogenous metabolites. In external calibration, the absence of a true blank matrix complicates calibration curve preparation. Multi-targeted internal calibration (IC) using stable isotope-labeled standards (SILs) as one-point calibrants offers an attractive alternative, yet its use is limited by the availability and cost of homologous SILs. To address this, heterologous SILs can serve as surrogate calibrants for multiple analytes, relying on response factors (RFs) established between each analyte and selected SILs in the matrix of interest. This study assessed the suitability of such an alternative isotopic-dilution strategy, referred to as heterologous internal calibration (H-IC), within an LC-MS/MS method developed for chronic kidney disease (CKD) research. A panel of 18 metabolites and their corresponding SILs was initially analyzed to generate reference values, and the performance of various analyte-SIL combinations was compared with that of the original homologous internal calibration procedure. Validation indicated satisfactory accuracy and precision for most analytes, with trueness generally within 70-130% and precision below 10%. Only a few low-abundance metabolites showed minor deviations. Finally, reducing the calibration panel to five heterologous SILs for quantifying all 18 analytes in 30 patient samples produced results comparable to those obtained using individual homologous SILs. These findings demonstrate that H-IC can substantially reduce SIL requirements and analytical costs while maintaining acceptable quantitative performance for metabolite measurement in human plasma.