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 × 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 μL). 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.

Keywords::

• Alloxan
• Alloxazine
• α,β-Dicarbonyls
• 1,2,3-Tricarbonyls
• Reversed-phase HPLC/fluorescence detection

Acknowledgements

This publication was made possible by National Institutes of Health (NIH) grant ES10018 (from the ARCH program of the National Institute of Health Sciences) and P20 RR16456 (from the BRIN program of the National Center for Research Resources), and US Department of Education grant PO31B040030 (Title III, Part B - Strengthening Historically Black Graduate Institutions). The contents of this publication are solely the responsibility of authors and do not necessarily represent the official views of the NSF, NIH, or US Department of Education. We thank Vinitha Achuthan for critical reading of the manuscript. The assistance of Mr Joseph Allison during the initial stages of setting up RP-HPLC analysis is acknowledged.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.