Development and validation of a quantitative ultra-performance liquid chromatography quadrupole time-of-flight method...

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.

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.

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.

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.