ABSTRACT

Objectives

Ethylmalonic acid (EMA) is a key urinary biomarker of ethylmalonic encephalopathy (EE), but routine gas chromatography–mass spectrometry (GC–MS) is time- and resource-intensive. This study aimed to develop and validate a simple, rapid capillary electrophoresis (CE) method with indirect ultraviolet (UV) detection for the quantitative determination of EMA in urine.

Materials and Methods

A fused-silica capillary and a background electrolyte containing 5 mmol/L 2,6-pyridinedicarboxylic acid (PDCA) and 0.1 mmol/L cetyltrimethylammonium bromide (CTAB) (pH 5.74) were used. Separation was performed at 28 kV and monitored at 200 nm. Fresh human urine was diluted 1:20, filtered, and used to prepare matrix-matched calibrators (10–750 µmol/L). Linearity, precision, sensitivity, and accuracy were assessed according to International Council for Harmonisation validation guidelines, using spiked urine samples. Electropherograms from healthy control urine, EMA-spiked urine, and urine from a patient with EE illustrated the applicability.

Results

Under optimized conditions, EMA migrated at approximately 2.0 min with baseline resolution from endogenous components. Calibration was linear between 10 and 750 µmol/L (R² = 0.99920). Within-run precision, expressed as relative standard deviation (%), was 0.17% for migration time and 0.89% for corrected peak area, while between-day precision for peak area was 1.91%. The limits of detection and quantification were 11.78 and 39.3 µmol/L, respectively. Recovery in spiked urine ranged from 96 ± 2% to 98 ± 2%.

Conclusion

The CE–UV method enables rapid, reliable quantification of urinary EMA with minimal sample preparation. Its short analysis time and modest instrumentation requirements support its use for first-line screening and monitoring of EE, with GC–MS reserved for confirmatory analyses.