Quadruple isotope dilution versus conventional calibration methods for accurate urinary hippuric acid quantification with a synthesized isotopically labelled material

Hippuric acid is a key biomarker in toxicological and metabolic investigations. This study presents an accurate UHPLC-MS/MS method for the quantification of hippuric acid in urine matrix. A quadruple isotope dilution (ID 4 ) strategy was utilized to compensate possible matrix effects and variations in calibration and increase accuracy. Hippuric acid– d₅ was synthesized in-house from glycine and benzoyl chloride– d₅ , lowering material costs to easily affordable levels. The mass spectrometry and chromatographic parameters were optimized for improved sensitivity and separation. After the optimization experiments, the analytical performance of the UHPLC-MS/MS system was evaluated. The developed method exhibited a wide linear range from 55.80 ng/kg to 530.10 µg/kg, with limit of detection (LOD) of 15.6 ng/kg and limit of quantification (LOQ) of 52.1 ng/kg. The method was validated with a certified reference material and gradient spiked recovery runs in the same matrix, resulting in excellent agreement with the certified value and consistent recoveries. Results were put into comparison with more conventional techniques of external standard (ES) calibration and single–point isotopically labelled standard addition which demonstrated the superiority of the ID 4 method. The proposed method is intended as a candidate reference procedure for hippuric acid in urine, and it can be used to assign values to quality control and calibration materials for routine LC-MS/MS methods. In addition, the in-house synthesis of hippuric acid- d 5 makes this approach affordable for laboratories outside metrology institutes.