Development and validation of a quantitative ultra-performance liquid chromatography quadrupole time-of-flight method for urine organic acid analysis

OBJECTIVES: Urine organic acid analysis is essential for diagnosing inborn errors of metabolism and is conventionally performed using multistep, labor-intensive sample preparation and gas chromatography-mass spectrometry (GC-MS). We sought to develop and validate a quantitative ultra-performance liquid chromatography quadrupole time-of-flight (UPLC-QTOF) method with a "dilute-and-shoot" approach. METHODS: 20 µL of calibrator, quality control material, or urine specimen, normalized by creatinine concentration, was mixed with mobile phase A (0.05% formic acid in water) and internal standards to a final volume of 440 µL. The supernatant was injected onto a Waters ACUITY Premier HSS T3 UPLC column, with data acquired in MSE mode on a Waters Xevo G3 QTOF mass spectrometer and quantification achieved using both linear and quadratic regressions. RESULTS: The method quantifies 27 analytes and separates diagnostically important isomers in 20 minutes. Repeatability and reproducibility were 12% or less coefficient of variation, with no carryover observed. Spike-recovery studies demonstrated recoveries between 85% and 115%, and concordant results were obtained from 51 urine specimens vs the conventional GC-MS method. No matrix effect was identified except for 3-hydroxyglutaric acid. Compared with other UPLC-QTOF methods, improved chromatographic performance was achieved with the Premier HSS T3 column, while MSE high-resolution MS data provided fragmentation information to support higher-confidence compound identification. Compared with conventional GC-MS methods, this method requires substantially lower specimen volume and simplified sample preparation. CONCLUSIONS: This UPLC-QTOF dilute-and-shoot urine organic acid method demonstrated acceptable analytical and clinical performance. Continued optimization will be pursued to expand the panel and support the diagnosis of a broader range of inborn errors of metabolism.