NOVÉ POSTUPY DIAGNOSTIKY DĚDIČNÝCH PORUCH PURINOVÉHO A PYREVIIDINOVÉHO METABOLISMU POMOCÍ KAPILÁRNÍ ELEKTROFORÉZY

Importance of the Topic

Inherited disorders of purine and pyrimidine metabolism lead to accumulation of diagnostic metabolites in urine and blood. Reliable, rapid and cost-effective analytical approaches are essential for screening and diagnosis of these conditions. Capillary electrophoresis (CE) offers new opportunities for separation and identification of a wide range of nucleobases and nucleosides in clinical samples.

Objectives and Study Overview

This study aimed to develop and compare two CE separation modes—alkaline anionic and acidic cationic—for comprehensive profiling of purine and pyrimidine metabolites in urine. Methods were optimized on healthy individuals and validated on patients with known enzyme defects (OTC, PNP, APRT). A comparison with conventional HPLC methods evaluated performance advantages of CE in clinical diagnostics.

Methodology and Instrumentation Used

Sample Preparation and Conditions

• Urine samples sonicated 15 min and centrifuged at 3 000 g for 5 min.
• Daily capillary conditioning: rinse with water, 0.1 M NaOH, water and background electrolyte (5 min each).
• Between runs: 0.1 M NaOH (0.5 min) and background electrolyte (1 min).

Separation Modes

• Anionic mode: 0.015 M sodium tetraborate + 0.08 M SDS, pH 9.5, 15 kV, detection 190–300 nm.
• Cationic mode: 0.200 M sodium phosphate, pH 1.8, 18.5 kV, detection at 190, 260 and 300 nm.

Instrumentation

• Capillary Electrophoresis: Beckman P/ACE 5510 with diode array detector; fused-silica capillary (75 µm ID, 47 cm total, 40 cm effective length).
• High-Performance Liquid Chromatography: Millipore-Waters system with dual-wavelength UV detector; Hichrom Spherisorb ODS-2 column (12.5 cm × 4.6 mm, 5 µm).

Main Results and Discussion

Anionic mode achieved separation of over 60 urinary components with 230 000 theoretical plates for hypoxanthine and migration time reproducibility around 1%. Cyclodextrin additives enabled discrimination between nucleosides and bases via cis-diol complexation. Cationic mode provided 108 000 theoretical plates and simple interpretation with fewer interfering peaks, although some analytes (xanthine, uric acid) did not migrate under acidic conditions. Combined use of both modes allowed unambiguous detection of diagnostic markers in patients with OTC, PNP and APRT deficiencies. CE outperformed HPLC by approximately 300-fold in efficiency, threefold in speed and offered significantly lower per-sample costs.

Benefits and Practical Applications of the Method

• High separation efficiency and reproducibility enable reliable identification of a broad panel of purine/pyrimidine metabolites.
• Fast analysis times (minutes per run) support high-throughput diagnostics.
• Lower operating costs compared to HPLC improve accessibility for routine screening.
• Flexible use of anionic and cationic modes adapts to diverse analyte properties.

Future Trends and Potential Applications

Integration of mass spectrometric detection with CE could further enhance specificity and sensitivity. Development of automated sample handling and on-capillary derivatization may expand the clinical scope to quantitative biomarker profiling. Miniaturized CE platforms have the potential for point-of-care testing in metabolic disorders.

Conclusion

The combined anionic and cationic CE methods provide a robust, rapid and cost-effective tool for comprehensive screening of inherited purine and pyrimidine metabolic disorders. The approach surpasses traditional HPLC in efficiency and throughput, offering significant advantages for clinical laboratories.

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