¹H-qNMR analysis of human urine: Validation of an external standard approach for absolute metabolite quantitation

Quantitative nuclear magnetic resonance (qNMR) spectroscopy is a powerful tool for metabolite analysis, offering reproducibility, minimal sample preparation, and non-destructive measurements. In this study, a ¹H-qNMR method employing external standard calibration was developed and validated for the absolute quantification of 14 representative metabolites in human urine. Metabolite selection was based on their consistent occurrence in the Human Urine Metabolome and supported by literature frequency, resulting in a chemically diverse set including amino acids, organic acids, carbohydrates, and amines. The method was validated according to EUROLAB and ENFSI guidelines, assessing specificity, linearity, precision, accuracy, sensitivity, robustness, and matrix effects. The results demonstrated excellent analytical performance: calibration curves were highly linear (R² ≥ 0.9987), accuracy and precision met acceptance criteria (% error <10%, %RSD <15%), and recoveries ranged from 87% to 103%. Limits of detection and quantification were suitable for physiological urine concentrations, while matrix effects remained within the accepted ±20% range, except for one analyte. Robustness testing confirmed method stability under minor variations, with only aromatic resonances requiring control of relaxation delays. Application to urine samples from healthy individuals confirmed the suitability of the approach for quantifying both low- and high-abundance metabolites. Importantly, the inclusion of structurally diverse metabolites enabled different quantification strategies, such as spectral deconvolution in crowded regions and signal summation for conformers. Overall, the validated method provides a robust and flexible platform for absolute metabolite quantification in urine by ¹H-qNMR. This framework enhances the reliability of NMR-based metabolomics and supports its application in clinical research.