Method development and validation for quantitative determination of urinary biomarkers of food intake for multiple foods

Biomarkers of food intake (BFIs) have emerged as a promising objective tool to complement traditional self-reported dietary assessment in nutritional research, with the potential to reduce systematic errors and improve accuracy. The development of comprehensive and robust quantification methods for BFIs is essential for widespread application. However, existing methods typically cover only a moderate number of BFIs per method, hindering their wide application in the field. In this study, we present the development and validation of a method for simultaneous quantification of 80 BFIs in urine reflecting 27 foods. The method utilizes a simple sample preparation procedure, followed by separation using both high-performance liquid chromatography (HPLC) on a C18 column and a hydrophilic interaction chromatography (HILIC) column, combined with tandem mass spectrometry in positive and negative mode (HPLC-MS/MS) (individual runs: 6 min). The working range for each analyte was determined in urine samples from a non-randomized, non-blinded nutritional intervention study. The method was validated with respect to selectivity, linearity, robustness, matrix effects, recovery, accuracy, and precision. In total, 44 BFIs could be absolutely quantified without or with only limitations at low concentrations, while 36 BFIs could only be measured semi-quantitatively, including 16 BFIs with limited validation data due to uncertainties. The 80 BFIs represent 27 foods (6 semi-quantitative) frequently consumed in European diets, including 24 plant-derived and 3 animal-derived items. The future implementation of this large-scale BFI quantification method in nutritional studies is expected to demonstrate the benefits of routinely measuring BFIs to improve the accuracy of dietary assessment.