Determination of alloxan by fluorometric high-performance liquid chromatography

Abstract

A fluorometric, reversed-phase high-performance liquid chromatography (RP-HPLC) method that allows quantitation of low levels of alloxan has been described. The method involved derivatization of alloxan with 500-200,000-fold excess of 1, 2-phenylenediamine (PD) in 0.1 M acetate buffer, pH 4.5 for 15 min at room temperature. The fluorescent product alloxazine (excitation: 382 nm; emission: 435 nm) was then analyzed by RP-HPLC using an Eclipse XDB-C18 (4.6 x 150 mm) column and a mobile phase consisting of 0.1% trifluoroacetic acid in 15/85 (v/v) acetonitrile/water at a flow of 1 mL/min (injection volume: 20 microL). The method is robust, and as low as 0.1 pmol of the analyte could be successfully detected and quantified. Following a minimal pre-treatment such as ultrafiltration (molecular weight cut-off 5000 Da) or protein precipitation using perchloric acid, acetonitrile, or phosphotungstic acid, the method is suitable for analysis of alloxan in complex physiological fluids (e.g. fetal bovine serum) and tissue homogenates (e.g. heart and kidney). The method has been rigorously evaluated and adapted in the laboratory for routine analysis and determination of alloxan added to cell cultures.

First report on the presence of Alloxan in bleached flour by LC-MS/MS method

“…For these compounds, the fragmentation is difficult due to unstable transitions, so the ionization efficiency in ESI is low (Santa, 2011). The pre-column derivatization with ophenylenediamine, which produces Alloxazine, has provided a better way to detect the analyte in question, due to a greater stability (Raghavamenon et al, 2009). Alloxazine is the result of reaction between a primary amine and carbonyl groups with formation of a product containing carbon nitrogen double bonds (Fig.…”

Simple, Sensitive, and Rapid Voltammetric Detection of Alloxan on Glassy Carbon Electrodes

“…reported the determination of alloxan by the flow injection method, while Raghavamenon et al. reported the fluorometric reversed-phase high-performance liquid chromatography (HPLC) method to detect alloxan at low levels . Electrochemical detection of alloxan was carried out on a modified electrode surface by Paramasivam et al and Monnappa et al …”

“…Shpigun et al reported the determination of alloxan by the flow injection method, 9 while Raghavamenon et al reported the fluorometric reversed-phase high-performance liquid chromatography (HPLC) method to detect alloxan at low levels. 10 Electrochemical detection of alloxan was carried out on a modified electrode surface by Paramasivam et al 11 and Monnappa et al 12 Like any other method, the rarely used electrochemical techniques for the quantification of alloxan also deal with the detection of less stable alloxan, resulting in poor accuracy. To The electrochemical methods are developed using glassy carbon electrodes (GCEs) without further modification, making it simple, which could later be extended to screenprinted electrodes in the future.…”

Design of a Sensitive and Selective Voltammetric Sensor Based on a Cationic Surfactant-Modified Carbon Paste Electrode for the Determination of Alloxan

“…Research enthusiasts have given ample consideration for developing methods to quantitatively determine bioactive substances such as AL in food products. Goyal et al 11 used HPLC, Raghavamenon et al 12 used fluorometric high performance liquid chromatography, Zheng 13 developed spectrophotometric method while Shpigun et al 14 used flow injection method and Murthy et al 15 used electrochemical techniques. Electrochemical techniques prove to be the most optimistic approach because of its ability to determine analyte levels up to the picomolar range and its automation at nominal cost in comparison with other instruments.…”