ascorbic-acid and 4-5-dimethyl-1-2-phenylenediamine

A reversed-phase ion pair liquid chromatographic method (RP-LC) for the determination of dehydroascorbic acid (DHA) and ascorbic acid (AA) and also acetaminophen, which is combined in pharmaceuticals, is proposed and validated. AA and acetaminophen were analyzed directly, while DHA was determined after pre-column derivatization with 4,5-dimethyl-1,2-phenylenediamine (DMPD). The derivatization reaction was carried out under mild conditions (10min at ambient temperature) in the dark in sodium acetate buffer (80mM; pH 3.7) solution containing EDTA as metal scavenger. The chromatographic separations were performed on a Phenomenex Synergi 4u hydro-RP (150mmx4.6mm) under isocratic elution conditions, using cetyltrimethylammonium bromide (CTAB) as ion-pairing reagent in the mobile phase. Linear responses were observed for each compound. The intra-day precision (R.S.D.) was < or =1.40% and there was no significant difference between intra- and inter-day data. Recovery studies showed good results for all compounds (99.7-101.8%) with R.S.D. ranging from 0.56 to 1.82%. The limits of quantitation were about 40, 50 and 140pmol for acetaminophen, AA and DHA, respectively. The DHA impurity values found in dosage forms were < or =0.2% of AA.

Topics: Acetaminophen; Ascorbic Acid; Chromatography, Liquid; Dehydroascorbic Acid; Phenylenediamines

A simple and accurate method for determination of vitamin C (ascorbic acid (AsA) and dehydroascorbic acid (DHA)) using 4,5-dimethyl-o-phenylenediamine (DMPD) was investigated. It was found that DMPD is a useful fluorescent reagent. The reaction product of DMPD with DHA showed strong and stable fluorescence (Ex; 360 nm, Em; 440 nm). Fluorometric derivatives were extracted with isobutanol or n-butanol. Extraction with isobutanol was superior to that with n-butanol in terms of specificity, since fluorometric derivatives of keto acids were extracted with n-butanol, together with DHA. The fluorescence intensity of DMPD derivatives was absolutely stable in isobutanol for at least 24 h. The sensitivity of determination of vitamin C was improved by removing several non-fluorometric compounds coexisting in the samples. The derivative derived from AsA was easily separated from those of keto acids by an HPLC method. The determination of vitamin C in natural products was thus improved by extraction and the HPLC method.

Topics: 1-Butanol; Ascorbic Acid; Butanols; Chromatography, High Pressure Liquid; Dehydroascorbic Acid; Phenylenediamines; Reproducibility of Results; Spectrometry, Fluorescence