Analysis of 5-methyltetrahydrofolate in human blood, serum and urine by on-line coupling of capillary isotachophoresis and zone electrophoresis
Résumé
The predominant circulating folate coenzyme in plasma/serum, 5-methyltetrahydrofolate (5-MTHF) was determined in human blood, serum and urine using a method coupling capillary isotachophoresis and zone electrophoresis. Measurements were done with a commercially available instrument for capillary isotachophoresis equipped with a column-switching system. The choice of electrolytes was limited by the instability of 5-MTHF and volatility of electrolytes for the potential coupling of the instrumentation with MS detector. To get an insight into the separability of sample components in an isotachophoretic analysis we constructed zone existence diagrams for isotachophoretic electrolyte systems having a leading electrolyte composed of acetate and ammonium, hydrocarbonate and ammonium, chloride and ammonium, and chloride and creatinine, with hydroxide ion as the terminator. For isotachophoretic preseparation, the non-volatile leading electrolyte with good buffering capacity composed of 1x10-2 M HCl and 2.5 x 10-2 M creatinine, pH 5, and terminating electrolyte 1x10-2 M MES were selected as the most suitable. The optimum background electrolyte for CZE analysis from the standpoint of analyte stability, separability and volatility for MS coupling was 1x10-2 M acetate with 3.5 x 10-2 M ammonium, pH 4.5. Using this combination of electrolytes, LODs reached with optical detection at 220 nm were 1.6 x 10-7 M in human blood, 1.1 x 10-7 M in human serum, and 4.7 x 10-6 M in human urine. Estimated content of 5-MTHF in blood and serum samples of women following oral daily administration of 0.8 mg of folic acid was 1.2 x 10-5 M and 5.8 x 10-6 M, resp.
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