A novel result about the stability of aqueous solution of folic acid under UV-A radiation

URRUTIA, M. NOEL; THOMAS, ANDRÉS H.; DANTOLA, MARIA LAURA

Villa Carlos Paz, Córdoba

Congreso; XIII Encuentro Latinoamericano de Fotoquímica y Fotobiología (XIII ELAFOT); 2017

Folic acid or pteroyl-L-glutamicacid (PteGlu), a synthetic and oxidized form offolate, is a pterin derivative widespreadin biological systems, whose chemical structure is composed of three moieties:a6-methylpterin, a p-aminobenzoic acid (PABA), and a glutamic acid (Glu)residue[1].PteGlu is a precursor of coenzymes involved in the metabolism ofnucleotides and aminoacids. It plays an important role in the prevention of anumber of important health complications such as male infertility, neural tubedefects (NTD) in the developing foetus and some cancers [1]. Different studieshave suggested that photolysis of PteGlu lead to increased risk of NTD, andthat skin pigmentation is an effective mechanism of protection against folatedepletion. Many reports have studied the photodegradation of aqueous solutionsof PteGlu under UV-A radiation.Nevertheless, no one has investigated theinfluence of the presence of pterins impurities on the photostability ofPteGlu. In order to study this, air equilibrated acidic aqueous solutions ofpurified PteGlu (pH 6.0±0.1), wereirradiated under UV-A radiation (350 nm) fordifferent periods of time. Thesamples were analyzed by UV-vis spectrophotometryand High Perfomance Liquid Cromatographyphotodiode (HPLC-PDA) and fluorescence(HPLC-RF) array detectors. The results werecompared with those obtained fromsolutions of unpurified PteGlu from differentcommercial solids. In order tocharacterize the impurities present in the commercial sample of PteGlu(SchircksLaboratories;purity > 98.5%), an acid aqueous solution was prepared in theabsence oflight and was analyzed by HPLC and HPLC coupled to a MassSpectrometry detector(HPLC-MS). The analysis using the HPLC-PDA detector showeda chromatogram with only one peak which corresponds to PteGlu. However,fluorescence chromatogram obtained by HPLC-FL analysis showed new peaks,suggesting that another compounds are present in the sample. The spectralfeatures of the impurities are similar to those reported for PABA-Glu,6-carboxypterin (Cap) and6-formylpterin (Fop), and the presence of thesecompounds was confirmed using the corresponding standard. The amount of eachimpurity was determined byi ntegration of each peak in the fluorescencechromatograms using the calibration curves of each compound. A Synergi Polar-RPcolumn (ether-linked phenyl phase with polar endcapping, 150 x4.6 mm, 4 mm, Phenomenex) was used forisolation of PteGlu from HPLC runs (preparative HPLC), by collecting the mobilephase after passing though the RF detector. Solutions containing 100 % NH4Ac (1mM, pH 6.5 ± 0.1) were used as mobilephase. The same column and runs conditionswere used to analyze the purity of the isolated PteGlu sample. The analysis ofthe collected fraction suggested that the isolation of PteGlu reducedsignificantly the amount of each impurities present in the sample. Airequilibrated acidic aqueous solutions of purified PteGlu were irradiated underUV-A radiation for different periods of time and the samples were analyzed byHPLC. The results were compared with those obtained from an unpurifiedsolution. The results indicated that the rate of photooxidation of purifiedPteGlu decreased significantly in comparison with the unpurified compound. PteGluwas rapidly oxidized into Fop and PABA-Glu when the percentage of pterinsimpurities increases. This result surprisingly suggests that the photodegradationof PteGlu could be due to the presence of impurities and not due to itsphotooxidation itself.