Separation and quantitation of fructose-6-phosphate and fructose-1,6-diphosphate by LC-ESI-MS for the evaluation of fructose-1,6-biphosphatase activity

✍ Scribed by Francesca Mancini; Jessica Fiori; Vanni Cavrini; Vincenza Andrisano

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 554 KB
• Volume: 29
• Category: Article
• ISSN: 1615-9306
• DOI: 10.1002/jssc.200600077

✦ Synopsis

Abstract

An LC‐ESI‐MS method was developed for the identification and quantification of fructose‐1,6‐biphosphate (F1,6BP) and fructose‐6‐phosphate (F6P), respectively the substrate and the product of the enzymatic reaction catalysed by fructose‐1,6‐bisphosphatase (F1,6BPase). F1,6BPase, expressed predominantly in liver and kidney, is one of the rate‐limiting enzymes of hepatic gluconeogenesis and has become a target for the development of new drugs for type 2 diabetes. The two sugar phosphates were separated on a Phenomenex Luna NH~2~ column (150 mm×2.0 mm id) using the following mobile phase: 5 mM triethylamine acetate buffer/ACN (80 : 20) v/v in a linear pH gradient (from pH = 9 to 10 in 15 min) at the flow rate of 0.3 mL/min. The detection was performed with an IT mass spectrometer in negative polarity (full scan 100–450 m/z) and in SIM mode on the generated anions at m/z = 339 (F1,6BP) and m/z = 259 (F6P). Under the optimised final conditions, the method was validated for accuracy, specificity, precision (inter‐ and intradays RSD comprised between 1.0 and 6.3% over the range of concentrations used), linearity (50–400 μM), LODs (0.44 μM) and LOQs (1.47 μM), and the method was applied to F6P determination in the F1,6BPase catalysed hydrolysis of F1,6BP.