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Development and Validation of a Quantitative Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight (UPLC-QTof) Method for Urine Organic Acid Analysis

Biochemical/Metabolic and Therapeutics
  • Primary Categories:
    • Metabolic Genetics
  • Secondary Categories:
    • Metabolic Genetics
Introduction:
Urine organic acid (UOA) analysis is essential for diagnosing inborn errors of metabolism (IEMs). UOA analysis is commonly performed with gas chromatography-mass spectrometry (GC-MS) and requires time-consuming sample preparation procedures including liquid-liquid extraction and derivatization. We describe the development and validation of a quantitative ultra-performance liquid chromatography quadrupole time-of-flight (UPLC-QTof) method for UOA analysis with a “dilute-and-shoot” approach.

Methods:
Urine specimens were normalized to creatinine concentrations of 1 mmol/L. 20 µL of each urine specimen (normalized), calibrator, or quality control (QC) material was mixed with 400 µL of mobile phase A (0.05% formic acid in water) and a mixture of isotope-labeled internal standards. After centrifugation, 10 µL of the supernatant was analyzed using negative electrospray ionization (ESI) mode on a Xevo G3 QTof mass spectrometer (Waters) using the MSE method to produce fragment ions when applicable, after chromatographic separation with a ACQUITYTM Premier HSS T3 1.8 µm VanGuardTM FIT 2.1 x 150 mm column (Waters). Repeatability, reproducibility, and carryover were assessed using the QC materials. The analytical measuring range (AMR) was assessed using synthetic urine spiked with increasing concentrations of each organic acid. Accuracy was assessed by comparing results with the UOA test performed at a reference laboratory and by spike-recovery studies using pooled urine specimens. Matrix effect was evaluated by matrix dilution study. Strategies to obtain linear rather than quadratic calibration curves were also explored.  

Results:
An optimized UPLC method was used to enable high-resolution separation of 29 UOAs and isomers. Total analytical time was 20 min per injection. Both linear and quadratic regressions were used to build the calibration curves. AMR and correlation coefficients of a few representative UOAs were: orotic acid (3.4 to 214.2 mmol/mol creatinine, R2 = 0.99, linear regression); 2-methylcitric acid (4 to 189 mmol/mol creatinine, R2 = 0.99, linear regression); 3-methylcrotonylglycine (0.3 to 18.0 mmol/mol creatinine, R2 = 0.99, linear regression). Repeatability and reproducibility were <=11% CV and no carryover was observed. Spike-recovery study demonstrated recoveries between 80% and 120%, and method comparison studies demonstrated no discrepancies with results from a reference laboratory. 

Conclusion:
We have developed and validated a novel UPLC-QTof method for UOA analysis to support the diagnosis of IEMs with acceptable analytical and clinical performances. Compared with GC-MS methods, the UPLC-QTof method requires very small specimen volume and does not require laborious and time-consuming sample preparation procedures. Continued optimization of the method will be pursued to measure more UOAs to support the diagnosis of a broader range of IEMs.

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