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

Area of Science:

• Bioanalysis
• Metabolomics
• Analytical Chemistry

Background:

• Absolute quantification of endogenous metabolites is vital but challenging in bioanalysis.
• External calibration is complicated by the lack of true blank matrices.
• Homologous stable isotope-labeled standards (SILs) for multi-analyte internal calibration (IC) are often unavailable or costly.

Purpose of the Study:

• To evaluate the efficacy of heterologous internal calibration (H-IC) as an alternative to homologous IC for metabolite quantification.
• To assess the performance of H-IC in an LC-MS/MS method for chronic kidney disease (CKD) research.
• To determine if H-IC can reduce the number of SILs required and lower analytical costs.

Main Methods:

• Developed an LC-MS/MS method for quantifying 18 metabolites in human plasma.
• Generated reference values using individual homologous SILs for each metabolite.
• Compared quantitative results from H-IC using surrogate SILs against homologous IC.
• Validated the H-IC approach using 30 patient plasma samples.

Main Results:

• H-IC demonstrated satisfactory accuracy (trueness within 70-130%) and precision (below 10%) for most metabolites.
• Minor deviations were observed for a few low-abundance metabolites.
• Quantification using five heterologous SILs for 18 analytes yielded results comparable to using individual homologous SILs.

Conclusions:

• Heterologous internal calibration (H-IC) is a viable strategy for accurate and precise metabolite quantification in human plasma.
• H-IC significantly reduces the need for expensive homologous SILs, thereby lowering overall analytical costs.
• This method offers a practical and economical solution for bioanalytical quantification, particularly in metabolomics research and clinical diagnostics.