Abstract
BackgroundAnalysis of folic acid (FA) is not an easy task because of its presence in lower concentrations, its lower stability under acidic conditions, and its sensitiveness against light and high temperature. The present study is concerned with the development and validation of an automated environmentally friendly pre-column derivatization combined by solid-phase enrichment (SPEn) to determine low levels of FA.ResultsCerium (IV) trihydroxyhydroperoxide (CTH) as a packed oxidant reactor has been used for oxidative cleavage of FA into highly fluorescent product, 2-amino-4-hydroxypteridine-6-carboxylic acid. FA was injected into a carrier stream of 0.04 M phosphate buffer, pH 3.4 at a flow-rate of 0.25 mL/min. The sample zone containing the analyte was passed through the CTH reactor thermostated at 40°C, and the fluorescent product was trapped and enriched on a head of small ODS column (10 mm x 4.6 mm i.d., 5 μm particle size). The enriched product was then back-flush eluted by column-switching from the small ODS column to the detector with a greener mobile phase consisting of ethanol and phosphate buffer (0.04M, pH 3.4) in the ratio of 5:95 (v/v). The eluent was monitored fluorimetrically at emission and excitation wavelengths of 463 and 367 nm, respectively. The calibration graph was linear over concentrations of FA in the range of 1.25-50 ng/mL, with a detection limit of 0.49 ng/mL.ConclusionA new simple and sensitive green analytical procedure including on-line pre-column derivatization combined by SPEn has been developed for the routine quality control and dosage form assay of FA at very low concentration level. The method was a powerful analytical technique that had excellent sensitivity, sufficient accuracy and required relatively simple and inexpensive instrumentation.
Highlights
Analysis of folic acid (FA) is not an easy task because of its presence in lower concentrations, its lower stability under acidic conditions, and its sensitiveness against light and high temperature
The method was based on oxidative cleavage of FA into highly fluorescent 2-amino-4-hydroxypteridine-6-carboxylic acid (Figure 1) using a packed reactor of cerium (IV) trihydroxyhydroperoxide (CTH) and the reaction product was enriched on small column
The results showed that the reaction of FA with Cerium (IV) trihydroxyhydroperoxide (CTH) could be proceeded at room temperature (25°C) while the detector response quickly increased along with an increase in the temperature, which might mean that high temperature accelerates the oxidation reaction of FA with CTH (Figure 5)
Summary
Analysis of folic acid (FA) is not an easy task because of its presence in lower concentrations, its lower stability under acidic conditions, and its sensitiveness against light and high temperature. Several analytical methods have been developed for the determination of FA, including spectrophotometry [2,3], fluorimetry [4,5,6,7], chemiluminescence [8], enzyme assay [9], capillary electrophoresis [10,11] and high-performance liquid chromatography (HPLC) [12,13,14]. More strict regulation related to the quality control of pharmaceuticals led to increasing demands on automation of the analytical assays carried out in appropriate control laboratories. The flow injection analysis (FIA) technique became a versatile instrumental
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